articles

Initial

sequencing

and

analysis

of

the

human

genome

International

Human

Genome

Sequencing

Consortium

A

partial

list

of

authors

appears

on

the

opposite

page

Af

liations

are

listed

at

the

end

of

the

paper

The

human

genome

holds

an

extraordinary

trove

of

information

about

human

development

physiology

medicine

and

evolution

Here

we

report

the

results

of

an

international

collaboration

to

produce

and

make

freely

available

a

draft

sequence

of

the

human

genome

We

also

present

an

initial

analysis

of

the

data

describing

some

of

the

insights

that

can

be

gleaned

from

the

sequence

The

rediscovery

of

Mendel

's

laws

of

heredity

in

the

opening

weeks

of

the

sparked

a

scienti

c

quest

to

understand

the

nature

and

content

of

genetic

information

that

has

propelled

biology

for

the

last

hundred

years

The

scienti

c

progress

made

falls

naturally

into

four

main

phases

corresponding

roughly

to

the

four

quarters

of

the

century

The

rst

established

the

cellular

basis

of

heredity

the

chromosomes

The

second

de

ned

the

molecular

basis

of

heredity

the

DNA

double

helix

The

third

unlocked

the

informational

basis

of

heredity

with

the

discovery

of

the

biological

mechanism

by

which

cells

read

the

information

contained

in

genes

and

with

the

invention

of

the

recombinant

DNA

technologies

of

cloning

and

sequencing

by

which

scientists

can

do

the

same

The

last

quarter

of

a

century

has

been

marked

by

a

relentless

drive

to

decipher

rst

genes

and

then

entire

genomes

spawning

the

eld

of

genomics

The

fruits

of

this

work

already

include

the

genome

sequences

of

viruses

and

viroids

naturally

occurring

plasmids

organelles

eubacteria

seven

archaea

one

fungus

two

animals

and

one

plant

Here

we

report

the

results

of

a

collaboration

involving

groups

from

the

United

States

the

United

Kingdom

Japan

France

Germany

and

China

to

produce

a

draft

sequence

of

the

human

genome

The

draft

genome

sequence

was

generated

from

a

physical

map

covering

more

than

of

the

euchromatic

part

of

the

human

genome

and

together

with

additional

sequence

in

public

databases

it

covers

about

of

the

human

genome

The

sequence

was

produced

over

a

relatively

short

period

with

coverage

rising

from

about

to

more

than

over

roughly

fteen

months

The

sequence

data

have

been

made

available

without

restriction

and

updated

daily

throughout

the

project

The

task

ahead

is

to

produce

a

nished

sequence

by

closing

all

gaps

and

resolving

all

ambiguities

Already

about

one

billion

bases

are

in

nal

form

and

the

task

of

bringing

the

vast

majority

of

the

sequence

to

this

standard

is

now

straightforward

and

should

proceed

rapidly

The

sequence

of

the

human

genome

is

of

interest

in

several

respects

It

is

the

largest

genome

to

be

extensively

sequenced

so

far

being

times

as

large

as

any

previously

sequenced

genome

and

eight

times

as

large

as

the

sum

of

all

such

genomes

It

is

the

rst

vertebrate

genome

to

be

extensively

sequenced

And

uniquely

it

is

the

genome

of

our

own

species

Much

work

remains

to

be

done

to

produce

a

complete

nished

sequence

but

the

vast

trove

of

information

that

has

become

available

through

this

collaborative

effort

allows

a

global

perspective

on

the

human

genome

Although

the

details

will

change

as

the

sequence

is

nished

many

points

are

already

clear

X

The

genomic

landscape

shows

marked

variation

in

the

distribution

of

a

number

of

features

including

genes

transposable

elements

GC

content

CpG

islands

and

recombination

rate

This

gives

us

important

clues

about

function

For

example

the

developmentally

important

HOX

gene

clusters

are

the

most

repeat-poor

regions

of

the

human

genome

probably

re

ecting

the

very

complex

coordinate

regulation

of

the

genes

in

the

clusters

X

There

appear

to

be

about

protein-coding

genes

in

the

human

genome

only

about

twice

as

many

as

in

worm

or

y.

However

the

genes

are

more

complex

with

more

alternative

splicing

generating

a

larger

number

of

protein

products

X

The

full

set

of

proteins

-LRB-

the

proteome

-RRB-

encoded

by

the

human

genome

is

more

complex

than

those

of

invertebrates

This

is

due

in

part

to

the

presence

of

vertebrate-speci

c

protein

domains

and

motifs

-LRB-

an

estimated

of

the

total

-RRB-

but

more

to

the

fact

that

vertebrates

appear

to

have

arranged

pre-existing

components

into

a

richer

collection

of

domain

architectures

X

Hundreds

of

human

genes

appear

likely

to

have

resulted

from

horizontal

transfer

from

bacteria

at

some

point

in

the

vertebrate

lineage

Dozens

of

genes

appear

to

have

been

derived

from

transposable

elements

X

Although

about

half

of

the

human

genome

derives

from

transposable

elements

there

has

been

a

marked

decline

in

the

overall

activity

of

such

elements

in

the

hominid

lineage

DNA

transposons

appear

to

have

become

completely

inactive

and

long-terminal

repeat

-LRB-

LTR

-RRB-

retroposons

may

also

have

done

so

X

The

pericentromeric

and

subtelomeric

regions

of

chromosomes

are

lled

with

large

recent

segmental

duplications

of

sequence

from

elsewhere

in

the

genome

Segmental

duplication

is

much

more

frequent

in

humans

than

in

yeast

y

or

worm

X

Analysis

of

the

organization

of

Alu

elements

explains

the

longstanding

mystery

of

their

surprising

genomic

distribution

and

suggests

that

there

may

be

strong

selection

in

favour

of

preferential

retention

of

Alu

elements

in

GC-rich

regions

and

that

these

sel

sh

elements

may

bene

t

their

human

hosts

X

The

mutation

rate

is

about

twice

as

high

in

male

as

in

female

meiosis

showing

that

most

mutation

occurs

in

males

X

Cytogenetic

analysis

of

the

sequenced

clones

con

rms

suggestions

that

large

GC-poor

regions

are

strongly

correlated

with

dark

G-bands

in

karyotypes

X

Recombination

rates

tend

to

be

much

higher

in

distal

regions

-LRB-

around

megabases

-LRB-

Mb

-RRB-

-RRB-

of

chromosomes

and

on

shorter

chromosome

arms

in

general

in

a

pattern

that

promotes

the

occurrence

of

at

least

one

crossover

per

chromosome

arm

in

each

meiosis

X

More

than

million

single

nucleotide

polymorphisms

-LRB-

SNPs

-RRB-

in

the

human

genome

have

been

identi

ed

This

collection

should

allow

the

initiation

of

genome-wide

linkage

disequilibrium

mapping

of

the

genes

in

the

human

population

In

this

paper

we

start

by

presenting

background

information

on

the

project

and

describing

the

generation

assembly

and

evaluation

of

the

draft

genome

sequence

We

then

focus

on

an

initial

analysis

of

the

sequence

itself

the

broad

chromosomal

landscape

the

repeat

elements

and

the

rich

palaeontological

record

of

evolutionary

and

biological

processes

that

they

provide

the

human

genes

and

proteins

and

their

differences

and

similarities

with

those

of

other

NATURE

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Ltd

articles

Genome

Sequencing

Centres

-LRB-

Listed

in

order

of

total

genomic

sequence

contributed

with

a

partial

list

of

personnel

A

full

list

of

contributors

at

each

centre

is

available

as

Supplementary

Information

-RRB-

Whitehead

Institute

for

Biomedical

Research

Center

for

Genome

Research

Eric

S.

Lauren

M.

Bruce

Chad

Michael

C.

Jennifer

Keri

Ken

Michael

William

Roel

Diane

Katrina

Andrew

John

Lisa

Jessica

Rosie

Paul

Kevin

James

Jill

P.

Cher

William

Jerome

Christina

Mark

Ralph

Andrew

Carrie

Nicole

Nikola

Aravind

Dudley

The

Sanger

Centre

Jane

John

Rachael

Stephan

David

John

Christopher

Nigel

Alan

Rebecca

Panos

Andrew

Ian

Richard

Lisa

Darren

Simon

Tim

Sean

Adrienne

Matthew

Christine

Amanda

Lucy

Simon

Sarah

James

C.

Andrew

Robert

Mark

Ratna

Sarah

Washington

University

Genome

Sequencing

Center

Robert

H.

Richard

K.

LaDeana

W.

John

D.

Marco

A.

Elaine

R.

Lucinda

A.

Asif

T.

Kymberlie

H.

Warren

R.

Stephanie

L.

Michael

C.

Kim

D.

Tracie

L.

Andrew

Jason

B.

Lisa

L.

Robert

S.

Douglas

L.

Patrick

J.

Sandra

W.

US

DOE

Joint

Genome

Institute

Trevor

Elbert

Paul

Paul

Sarah

Tom

Norman

Jan-Fang

Anne

Susan

Christopher

Edward

Marvin

Baylor

College

of

Medicine

Human

Genome

Sequencing

Center

Richard

A.

Donna

M.

Steven

E.

John

B.

Erica

J.

Kim

C.

Catherine

M.

James

H.

Michael

L.

Susan

L.

Raju

S.

David

L.

Nelson

George

M.

RIKEN

Genomic

Sciences

Center

Yoshiyuki

Asao

Masahira

Tetsushi

Atsushi

Takehiko

Chiharu

Hidemi

Yasushi

Todd

Genoscope

and

CNRS

Jean

Roland

William

Francois

Philippe

Thomas

Eric

Catherine

Patrick

GTC

Sequencing

Center

Douglas

R.

Lynn

Marc

Ruben

Keith

Hong

Mei

JoAnn

Department

of

Genome

Analysis

Institute

of

Molecular

Biotechnology

Andre

Matthias

Gerald

Stefan

Andreas

Beijing

Genomics

Institute/Human

Genome

Center

Huanming

Jun

Jian

Guyang

Jun

Multimegabase

Sequencing

Center

The

Institute

for

Systems

Biology

Leroy

Lee

Anup

Shizen

Stanford

Genome

Technology

Center

Ronald

W.

Nancy

A.

A.

Pia

Michael

J.

Stanford

Human

Genome

Center

Richard

M.

Jeremy

Mark

Jane

David

R.

University

of

Washington

Genome

Center

Maynard

V.

Rajinder

Christopher

Department

of

Molecular

Biology

Keio

University

School

of

Medicine

Nobuyoshi

Kazuhiko

Shinsei

University

of

Texas

Southwestern

Medical

Center

at

Dallas

Glen

A.

Maria

Roger

University

of

Oklahoma

's

Advanced

Center

for

Genome

Technology

Bruce

A.

Feng

Huaqin

Max

Planck

Institute

for

Molecular

Genetics

Juliane

Hans

Richard

Cold

Spring

Harbor

Laboratory

Lita

Annenberg

Hazen

Genome

Center

W.

Richard

Melissa

de

la

Neilay

GBF

German

Research

Centre

for

Biotechnology

Helmut

Klaus

Gabriele

Genome

Analysis

Group

-LRB-

listed

in

alphabetical

order

also

includes

individuals

listed

under

other

headings

-RRB-

Richa

L.

Jeffrey

A.

Alex

Sera

m

Ewan

Peer

Daniel

G.

Christopher

B.

Lorenzo

Hsiu-Chuan

Deanna

Michele

Richard

R.

Tobias

Sean

R.

Evan

E.

Terrence

S.

James

James

G.

R.

Cyrus

Yoshihide

David

Henning

Karsten

Wonhee

L.

Steven

Thomas

A.

Simon

Arek

Scot

W.

James

Paul

Eugene

V.

Ian

David

Doron

Todd

M.

Aoife

Tarjei

John

V.

Nicola

Victor

J.

Chris

P.

Greg

Jorg

Guy

Arian

F.

A.

Elia

Joseph

Danielle

Jean

Lukas

John

Raymond

Alan

Yuri

I.

Kenneth

H.

Shiaw-Pyng

Ru-Fang

Scienti

c

management

National

Human

Genome

Research

Institute

US

National

Institutes

of

Health

Francis

Mark

S.

Jane

Adam

Kris

A.

Of

ce

of

Science

US

Department

of

Energy

Aristides

The

Wellcome

Trust

Michael

J.

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

organisms

and

the

history

of

genomic

segments

-LRB-

Comparisons

are

drawn

throughout

with

the

genomes

of

the

budding

yeast

Saccharomyces

cerevisiae

the

nematode

worm

Caenorhabditis

elegans

the

fruit

y

Drosophila

melanogaster

and

the

mustard

weed

Arabidopsis

thaliana

we

refer

to

these

for

convenience

simply

as

yeast

worm

y

and

mustard

weed

-RRB-

Finally

we

discuss

applications

of

the

sequence

to

biology

and

medicine

and

describe

next

steps

in

the

project

A

full

description

of

the

methods

is

provided

as

Supplementary

Information

on

Nature

's

web

site

-LRB-

nature.com

-RRB-

We

recognize

that

it

is

impossible

to

provide

a

comprehensive

analysis

of

this

vast

dataset

and

thus

our

goal

is

to

illustrate

the

range

of

insights

that

can

be

gleaned

from

the

human

genome

and

thereby

to

sketch

a

research

agenda

for

the

future

-LRB-

-RRB-

The

development

of

random

shotgun

sequencing

of

complementary

DNA

fragments

for

high-throughput

gene

discovery

by

and

Schimmel

and

later

dubbed

expressed

sequence

tags

-LRB-

ESTs

-RRB-

and

pursued

with

automated

sequencing

by

Venter

and

The

idea

of

sequencing

the

entire

human

genome

was

rst

proposed

in

discussions

at

scienti

c

meetings

organized

by

the

US

Department

of

Energy

and

others

from

to

-LRB-

refs

-RRB-

A

committee

appointed

by

the

US

National

Research

Council

endorsed

the

concept

in

its

but

recommended

a

broader

programme

to

include

the

creation

of

genetic

physical

and

sequence

maps

of

the

human

genome

parallel

efforts

in

key

model

organisms

such

as

bacteria

yeast

worms

ies

and

mice

the

development

of

technology

in

support

of

these

objectives

and

research

into

the

ethical

legal

and

social

issues

raised

by

human

genome

research

The

programme

was

launched

in

the

US

as

a

joint

effort

of

the

Department

of

Energy

and

the

National

Institutes

of

Health

In

other

countries

the

UK

Medical

Research

Council

and

the

Wellcome

Trust

supported

genomic

research

in

Britain

the

Centre

d'Etude

du

Polymorphisme

Humain

and

the

French

Muscular

Dystrophy

Association

launched

mapping

efforts

in

France

government

agencies

including

the

Science

and

Technology

Agency

and

the

Ministry

of

Education

Science

Sports

and

Culture

supported

genomic

research

efforts

in

Japan

and

the

European

Community

helped

to

launch

several

international

efforts

notably

the

programme

to

sequence

the

yeast

genome

By

late

the

Human

Genome

Project

had

been

launched

with

the

creation

of

genome

centres

in

these

countries

Additional

participants

subsequently

joined

the

effort

notably

in

Germany

and

China

In

addition

the

Human

Genome

Organization

-LRB-

HUGO

-RRB-

was

founded

to

provide

a

forum

for

international

coordination

of

genomic

research

Several

provide

a

more

comprehensive

discussion

of

the

genesis

of

the

Human

Genome

Project

Through

work

progressed

rapidly

on

two

fronts

-LRB-

Fig.

-RRB-

The

rst

was

construction

of

genetic

and

physical

maps

of

the

human

and

mouse

providing

key

tools

for

identi

cation

of

disease

genes

and

anchoring

points

for

genomic

sequence

The

second

was

sequencing

of

the

and

genomes

as

1995 1996 1997 1998

1999 2000 2001

Background

to

the

Human

Genome

Project

The

Human

Genome

Project

arose

from

two

key

insights

that

emerged

in

the

early

that

the

ability

to

take

global

views

of

genomes

could

greatly

accelerate

biomedical

research

by

allowing

researchers

to

attack

problems

in

a

comprehensive

and

unbiased

fashion

and

that

the

creation

of

such

global

views

would

require

a

communal

effort

in

infrastructure

building

unlike

anything

previously

attempted

in

biomedical

research

Several

key

projects

helped

to

crystallize

these

insights

including

-LRB-

-RRB-

The

sequencing

of

the

bacterial

viruses

and

the

animal

virus

and

the

human

between

and

These

projects

proved

the

feasibility

of

assembling

small

sequence

fragments

into

complete

genomes

and

showed

the

value

of

complete

catalogues

of

genes

and

other

functional

elements

-LRB-

-RRB-

The

programme

to

create

a

human

genetic

map

to

make

it

possible

to

locate

disease

genes

of

unknown

function

based

solely

on

their

inheritance

patterns

launched

by

Botstein

and

colleagues

in

-LRB-

ref

-RRB-

-LRB-

-RRB-

The

programmes

to

create

physical

maps

of

clones

covering

the

and

genomes

to

allow

isolation

of

genes

and

regions

based

solely

on

their

chromosomal

position

launched

by

Olson

and

Sulston

in

the

1984 1990 1991 1992

Discussion

and

debate

in

scientific

community

NRC

report

Bacterial

genome

sequencing

Other

organisms

H.

flu

S.

cerevisiae

sequencing

E.

coli

species

C.

elegans

sequencing

D.

melanogaster

sequencing

A.

thaliana

sequencing

Genetic

maps

Microsatellites

SNPs

Mouse

Physical

maps

cDNA

sequencing

Genomic

sequencing

Genetic

maps

Microsatellites

ESTs

Pilot

sequencing

Full

length

SNPs

Human

Physical

maps

cDNA

sequencing

Genomic

sequencing

ESTs

Pilot

project

Full

length

Working

draft

Finishing

Chromosome

Chromosome

Figure

Timeline

of

large-scale

genomic

analyses

Shown

are

selected

components

of

work

on

several

non-vertebrate

model

organisms

-LRB-

red

-RRB-

the

mouse

-LRB-

blue

-RRB-

and

the

human

-LRB-

green

-RRB-

from

earlier

projects

are

described

in

the

text

SNPs

single

nucleotide

polymorphisms

ESTs

expressed

sequence

tags

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

well

as

targeted

regions

of

mammalian

These

projects

showed

that

large-scale

sequencing

was

feasible

and

developed

the

two-phase

paradigm

for

genome

sequencing

In

the

rst

shotgun

phase

the

genome

is

divided

into

appropriately

sized

segments

and

each

segment

is

covered

to

a

high

degree

of

redundancy

-LRB-

typically

eight

to

tenfold

-RRB-

through

the

sequencing

of

randomly

selected

subfragments

The

second

is

a

nishing

phase

in

which

sequence

gaps

are

closed

and

remaining

ambiguities

are

resolved

through

directed

analysis

The

results

also

showed

that

complete

genomic

sequence

provided

information

about

genes

regulatory

regions

and

chromosome

structure

that

was

not

readily

obtainable

from

cDNA

studies

alone

In

genome

scientists

considered

a

that

would

have

involved

producing

a

draft

genome

sequence

of

the

human

genome

in

a

rst

phase

and

then

returning

to

nish

the

sequence

in

a

second

phase

After

vigorous

debate

it

was

decided

that

such

a

plan

was

premature

for

several

reasons

These

included

the

need

rst

to

prove

that

high-quality

long-range

nished

sequence

could

be

produced

from

most

parts

of

the

complex

repeat-rich

human

genome

the

sense

that

many

aspects

of

the

sequencing

process

were

still

rapidly

evolving

and

the

desirability

of

further

decreasing

costs

Instead

pilot

projects

were

launched

to

demonstrate

the

feasibility

of

cost-effective

large-scale

sequencing

with

a

target

completion

date

of

March

The

projects

successfully

produced

nished

sequence

with

accuracy

and

no

They

also

introduced

bacterial

arti

cial

chromosomes

-LRB-

BACs

-RRB-

a

new

largeinsert

cloning

system

that

proved

to

be

more

stable

than

the

cosmids

and

yeast

arti

cial

chromosomes

-LRB-

YACs

-RRB-

that

had

been

used

previously

The

pilot

projects

drove

the

maturation

and

convergence

of

sequencing

strategies

while

producing

of

the

human

genome

sequence

With

successful

completion

of

this

phase

the

human

genome

sequencing

effort

moved

into

full-scale

production

in

March

The

idea

of

rst

producing

a

draft

genome

sequence

was

revived

at

this

time

both

because

the

ability

to

nish

such

a

sequence

was

no

longer

in

doubt

and

because

there

was

great

hunger

in

the

scienti

c

community

for

human

sequence

data

In

addition

some

scientists

favoured

prioritizing

the

production

of

a

draft

genome

sequence

over

regional

nished

sequence

because

of

concerns

about

commercial

plans

to

generate

proprietary

databases

of

human

sequence

that

might

be

subject

to

undesirable

restrictions

on

The

consortium

focused

on

an

initial

goal

of

producing

in

a

rst

production

phase

lasting

until

June

a

draft

genome

sequence

covering

most

of

the

genome

Such

a

draft

genome

sequence

although

not

completely

nished

would

rapidly

allow

investigators

to

begin

to

extract

most

of

the

information

in

the

human

sequence

Experiments

showed

that

sequencing

clones

covering

about

of

the

human

genome

to

a

redundancy

of

about

four

to

vefold

-LRB-

halfshotgun

coverage

see

Box

-RRB-

would

accomplish

The

draft

genome

sequence

goal

has

been

achieved

as

described

below

The

second

sequence

production

phase

is

now

under

way

Its

aims

are

to

achieve

full-shotgun

coverage

of

the

existing

clones

during

to

obtain

clones

to

ll

the

remaining

gaps

in

the

physical

map

and

to

produce

a

nished

sequence

-LRB-

apart

from

regions

that

can

not

be

cloned

or

sequenced

with

currently

available

techniques

-RRB-

no

later

than

libraries

with

more

uniform

representation

The

practice

of

sequencing

from

both

ends

of

double-stranded

clones

-LRB-

double-barrelled

shotgun

sequencing

-RRB-

was

introduced

by

Ansorge

and

in

allowing

the

use

of

linking

information

between

sequence

fragments

The

application

of

shotgun

sequencing

was

also

extended

by

applying

it

to

larger

and

larger

DNA

molecules

from

plasmids

-LRB-

kilobases

-LRB-

kb

-RRB-

-RRB-

to

cosmid

-LRB-

kb

-RRB-

to

arti

cial

chromosomes

cloned

in

bacteria

and

-LRB-

kb

-RRB-

and

bacterial

-LRB-

megabases

-LRB-

Mb

-RRB-

-RRB-

In

principle

a

genome

of

arbitrary

size

may

be

directly

sequenced

by

the

shotgun

method

provided

that

it

contains

no

repeated

sequence

and

can

be

uniformly

sampled

at

random

The

genome

can

then

be

assembled

using

the

simple

computer

science

technique

of

hashing

-LRB-

in

which

one

detects

overlaps

by

consulting

an

alphabetized

look-up

table

of

all

k-letter

words

in

the

data

-RRB-

Mathematical

analysis

of

the

expected

number

of

gaps

as

a

function

of

coverage

is

similarly

Practical

dif

culties

arise

because

of

repeated

sequences

and

cloning

bias

Small

amounts

of

repeated

sequence

pose

little

problem

for

shotgun

sequencing

For

example

one

can

readily

assemble

typical

bacterial

genomes

-LRB-

about

repeat

-RRB-

or

the

euchromatic

portion

of

the

y

genome

-LRB-

about

repeat

-RRB-

By

contrast

the

human

genome

is

lled

-LRB-

-RRB-

with

repeated

sequences

including

interspersed

repeats

derived

from

transposable

elements

and

long

genomic

regions

that

have

been

duplicated

in

tandem

palindromic

or

dispersed

fashion

-LRB-

see

below

-RRB-

These

include

large

duplicated

segments

-LRB-

kb

-RRB-

with

high

sequence

identity

-LRB-

-RRB-

at

which

mispairing

during

recombination

creates

deletions

responsible

for

genetic

syndromes

Such

features

complicate

the

assembly

of

a

correct

and

nished

genome

sequence

There

are

two

approaches

for

sequencing

large

repeat-rich

genomes

The

rst

is

a

whole-genome

shotgun

sequencing

approach

as

has

been

used

for

the

repeat-poor

genomes

of

viruses

bacteria

and

ies

using

linking

information

and

computational

Hierarchical

shotgun

sequencing

Genomic

DNA

BAC

library

Organized

mapped

large

clone

contigs

BAC

to

be

sequenced

Shotgun

clones

Shotgun

ACCGTAAATGGGCTGATCATGCTTAAA

TGATCATGCTTAAACCCTGTGCATCCTACTG

sequence

Assembly

ACCGTAAATGGGCTGATCATGCTTAAACCCTGTGCATCCTACTG

Strategic

issues

Hierarchical

shotgun

sequencing

Soon

after

the

invention

of

DNA

sequencing

the

shotgun

sequencing

strategy

was

it

has

remained

the

fundamental

method

for

large-scale

genome

for

the

past

years

The

approach

has

been

re

ned

and

extended

to

make

it

more

ef

cient

For

example

improved

protocols

for

fragmenting

and

cloning

DNA

allowed

construction

of

shotgun

NATURE

VOL

FEBRUARY

Figure

Idealized

representation

of

the

hierarchical

shotgun

sequencing

strategy

A

library

is

constructed

by

fragmenting

the

target

genome

and

cloning

it

into

a

largefragment

cloning

vector

here

BAC

vectors

are

shown

The

genomic

DNA

fragments

represented

in

the

library

are

then

organized

into

a

physical

map

and

individual

BAC

clones

are

selected

and

sequenced

by

the

random

shotgun

strategy

Finally

the

clone

sequences

are

assembled

to

reconstruct

the

sequence

of

the

genome

Macmillan

Magazines

Ltd

articles

analysis

to

attempt

to

avoid

misassemblies

The

second

is

the

hierarchical

shotgun

sequencing

approach

-LRB-

Fig.

-RRB-

also

referred

to

as

map-based

BAC-based

or

clone-by-clone

This

approach

involves

generating

and

organizing

a

set

of

large-insert

clones

-LRB-

typically

kb

each

-RRB-

covering

the

genome

and

separately

performing

shotgun

sequencing

on

appropriately

chosen

clones

Because

the

sequence

information

is

local

the

issue

of

long-range

misassembly

is

eliminated

and

the

risk

of

short-range

misassembly

is

reduced

One

caveat

is

that

some

large-insert

clones

may

suffer

rearrangement

although

this

risk

can

be

reduced

by

appropriate

quality-control

measures

involving

clone

ngerprints

-LRB-

see

below

-RRB-

The

two

methods

are

likely

to

entail

similar

costs

for

producing

nished

sequence

of

a

mammalian

genome

The

hierarchical

approach

has

a

higher

initial

cost

than

the

whole-genome

approach

owing

to

the

need

to

create

a

map

of

clones

-LRB-

about

of

the

total

cost

of

sequencing

-RRB-

and

to

sequence

overlaps

between

clones

On

the

other

hand

the

whole-genome

approach

is

likely

to

require

much

greater

work

and

expense

in

the

nal

stage

of

producing

a

nished

sequence

because

of

the

challenge

of

resolving

misassemblies

Both

methods

must

also

deal

with

cloning

biases

resulting

in

under-representation

of

some

regions

in

either

large-insert

or

small-insert

clone

libraries

There

was

lively

scienti

c

debate

over

whether

the

human

genome

sequencing

effort

should

employ

whole-genome

or

hierarchical

shotgun

sequencing

Weber

and

stimulated

these

discussions

with

a

speci

c

proposal

for

a

whole-genome

shotgun

approach

together

with

an

analysis

suggesting

that

the

method

could

work

and

be

more

ef

cient

challenged

these

conclusions

and

argued

that

the

potential

bene

ts

did

not

outweigh

the

likely

risks

In

the

end

we

concluded

that

the

human

genome

sequencing

effort

should

employ

the

hierarchical

approach

for

several

reasons

First

it

was

prudent

to

use

the

approach

for

the

rst

project

to

sequence

a

repeat-rich

genome

With

the

hierarchical

approach

the

ultimate

frequency

of

misassembly

in

the

nished

product

would

probably

be

lower

than

with

the

whole-genome

approach

in

which

it

would

be

more

dif

cult

to

identify

regions

in

which

the

assembly

was

incorrect

Second

it

was

prudent

to

use

the

approach

in

dealing

with

an

outbred

organism

such

as

the

human

In

the

whole-genome

shotgun

method

sequence

would

necessarily

come

from

two

different

copies

of

the

human

genome

Accurate

sequence

assembly

could

be

complicated

by

sequence

variation

between

these

two

copies

both

SNPs

-LRB-

which

occur

at

a

rate

of

per

bases

-RRB-

and

larger-scale

structural

heterozygosity

-LRB-

which

has

been

documented

in

human

chromosomes

-RRB-

In

the

hierarchical

shotgun

method

each

largeinsert

clone

is

derived

from

a

single

haplotype

Third

the

hierarchical

method

would

be

better

able

to

deal

with

inevitable

cloning

biases

because

it

would

more

readily

allow

targeting

of

additional

sequencing

to

under-represented

regions

And

fourth

it

was

better

suited

to

a

project

shared

among

members

of

a

diverse

international

consortium

because

it

allowed

work

and

responsibility

to

be

easily

distributed

As

the

ultimate

goal

has

always

been

to

create

a

high-quality

nished

sequence

to

serve

as

a

foundation

for

biomedical

research

we

reasoned

that

the

advantages

of

this

more

conservative

approach

outweighed

the

additional

cost

if

any

A

biotechnology

company

Celera

Genomics

has

chosen

to

incorporate

the

whole-genome

shotgun

approach

into

its

own

efforts

to

sequence

the

human

genome

Their

uses

a

mixed

strategy

involving

combining

some

coverage

with

wholegenome

shotgun

data

generated

by

the

company

together

with

the

publicly

available

hierarchical

shotgun

data

generated

by

the

International

Human

Genome

Sequencing

Consortium

If

the

raw

sequence

reads

from

the

whole-genome

shotgun

component

are

made

available

it

may

be

possible

to

evaluate

the

extent

to

which

the

sequence

of

the

human

genome

can

be

assembled

without

the

need

for

clone-based

information

Such

analysis

may

help

to

re

ne

sequencing

strategies

for

other

large

genomes

Technology

for

large-scale

sequencing

Sequencing

the

human

genome

depended

on

many

technological

improvements

in

the

production

and

analysis

of

sequence

data

Key

innovations

were

developed

both

within

and

outside

the

Human

Genome

Project

Laboratory

innovations

included

four-colour

uorescence-based

sequence

improved

uorescent

dye-labelled

polymerases

speci

cally

designed

for

cycle

and

capillary

gel

These

studies

contributed

to

substantial

improvements

in

the

automation

quality

and

throughput

of

collecting

raw

DNA

There

were

also

important

advances

in

the

development

of

software

packages

for

the

analysis

of

sequence

data

The

PHRED

software

introduced

the

concept

of

assigning

a

base-quality

score

to

each

base

on

the

basis

of

the

probability

of

an

erroneous

call

These

quality

scores

make

it

possible

to

monitor

raw

data

quality

and

also

assist

in

determining

whether

two

similar

sequences

truly

overlap

The

PHRAP

computer

package

-LRB-

html

-RRB-

then

systematically

assembles

the

sequence

data

using

the

base-quality

scores

The

program

assigns

assembly-quality

scores

to

each

base

in

the

assembled

sequence

providing

an

objective

criterion

to

guide

sequence

nishing

The

quality

scores

were

based

on

and

validated

by

extensive

experimental

data

Another

key

innovation

for

scaling

up

sequencing

was

the

development

by

several

centres

of

automated

methods

for

sample

preparation

This

typically

involved

creating

new

biochemical

protocols

suitable

for

automation

followed

by

construction

of

appropriate

robotic

systems

Coordination

and

public

data

sharing

The

Human

Genome

Project

adopted

two

important

principles

with

regard

to

human

sequencing

The

rst

was

that

the

collaboration

would

be

open

to

centres

from

any

nation

Although

potentially

less

ef

cient

in

a

narrow

economic

sense

than

a

centralized

approach

involving

a

few

large

factories

the

inclusive

approach

was

strongly

favoured

because

we

felt

that

the

human

genome

sequence

is

the

common

heritage

of

all

humanity

and

the

work

should

transcend

national

boundaries

and

we

believed

that

scienti

c

progress

was

best

assured

by

a

diversity

of

approaches

The

collaboration

was

coordinated

through

periodic

international

meetings

-LRB-

referred

to

as

Bermuda

meetings

after

the

venue

of

the

rst

three

gatherings

-RRB-

and

regular

telephone

conferences

Work

was

shared

exibly

among

the

centres

with

some

groups

focusing

on

particular

chromosomes

and

others

contributing

in

a

genome-wide

fashion

The

second

principle

was

rapid

and

unrestricted

data

release

The

centres

adopted

a

policy

that

all

genomic

sequence

data

should

be

made

publicly

available

without

restriction

within

hours

of

Pre-publication

data

releases

had

been

pioneered

in

mapping

projects

in

the

and

mouse

and

were

prominently

adopted

in

the

sequencing

of

the

worm

providing

a

direct

model

for

the

human

sequencing

efforts

We

believed

that

scienti

c

progress

would

be

most

rapidly

advanced

by

immediate

and

free

availability

of

the

human

genome

sequence

The

explosion

of

scienti

c

work

based

on

the

publicly

available

sequence

data

in

both

academia

and

industry

has

con

rmed

this

judgement

Generating

the

draft

genome

sequence

Generating

a

draft

sequence

of

the

human

genome

involved

three

steps

selecting

the

BAC

clones

to

be

sequenced

sequencing

them

and

assembling

the

individual

sequenced

clones

into

an

overall

draft

genome

sequence

A

glossary

of

terms

related

to

genome

sequencing

and

assembly

is

provided

in

Box

The

draft

genome

sequence

is

a

dynamic

product

which

is

regularly

updated

as

additional

data

accumulate

en

route

to

the

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

ultimate

goal

of

a

completely

nished

sequence

The

results

below

are

based

on

the

map

and

sequence

data

available

on

October

except

as

otherwise

noted

At

the

end

of

this

section

we

provide

a

brief

update

of

key

data

Clone

selection

The

hierarchical

shotgun

method

involves

the

sequencing

of

overlapping

large-insert

clones

spanning

the

genome

For

the

Human

Genome

Project

clones

were

largely

chosen

from

eight

large-insert

libraries

containing

BAC

or

arti

cial

chromosome

-LRB-

PAC

-RRB-

clones

-LRB-

Table

refs

-RRB-

The

libraries

were

made

by

Box

Genome

glossary

partial

digestion

of

genomic

DNA

with

restriction

enzymes

Together

they

represent

around

coverage

-LRB-

redundant

sampling

-RRB-

of

the

genome

Libraries

based

on

other

vectors

such

as

cosmids

were

also

used

in

early

stages

of

the

project

The

libraries

-LRB-

Table

-RRB-

were

prepared

from

DNA

obtained

from

anonymous

human

donors

in

accordance

with

US

Federal

Regulations

for

the

Protection

of

Human

Subjects

in

Research

-LRB-

-RRB-

and

following

full

review

by

an

Institutional

Review

Board

Brie

y

the

opportunity

to

donate

DNA

for

this

purpose

was

broadly

advertised

near

the

two

laboratories

engaged

in

library

Sequence

Raw

sequence

Individual

unassembled

sequence

reads

produced

by

sequencing

of

clones

containing

DNA

inserts

Paired-end

sequence

Raw

sequence

obtained

from

both

ends

of

a

cloned

insert

in

any

vector

such

as

a

plasmid

or

bacterial

arti

cial

chromosome

Finished

sequence

Complete

sequence

of

a

clone

or

genome

with

an

accuracy

of

at

least

and

no

gaps

Coverage

-LRB-

or

depth

-RRB-

The

average

number

of

times

a

nucleotide

is

represented

by

a

high-quality

base

in

a

collection

of

random

raw

sequence

Operationally

a

high-quality

base

is

de

ned

as

one

with

an

accuracy

of

at

least

-LRB-

corresponding

to

a

PHRED

score

of

at

least

-RRB-

Full

shotgun

coverage

The

coverage

in

random

raw

sequence

needed

from

a

large-insert

clone

to

ensure

that

it

is

ready

for

nishing

this

varies

among

centres

but

is

typically

Clones

with

full

shotgun

coverage

can

usually

be

assembled

with

only

a

handful

of

gaps

per

kb

Half

shotgun

coverage

Half

the

amount

of

full

shotgun

coverage

-LRB-

typically

random

coverage

-RRB-

Sequenced-clone

contigs

Contigs

produced

by

merging

overlapping

sequenced

clones

Sequenced-clone-contig

scaffolds

Scaffolds

produced

by

joining

sequenced-clone

contigs

on

the

basis

of

linking

information

Draft

genome

sequence

The

sequence

produced

by

combining

the

information

from

the

individual

sequenced

clones

-LRB-

by

creating

merged

sequence

contigs

and

then

employing

linking

information

to

create

scaffolds

-RRB-

and

positioning

the

sequence

along

the

physical

map

of

the

chromosomes

length

A

measure

of

the

contig

length

-LRB-

or

scaffold

length

-RRB-

containing

a

typical

nucleotide

Speci

cally

it

is

the

maximum

length

L

such

that

of

all

nucleotides

lie

in

contigs

-LRB-

or

scaffolds

-RRB-

of

size

at

least

L.

Computer

programs

and

databases

Clones

BAC

clone

Bacterial

arti

cial

chromosome

vector

carrying

a

genomic

DNA

insert

typically

kb

Most

of

the

large-insert

clones

sequenced

in

the

project

were

BAC

clones

Finished

clone

A

large-insert

clone

that

is

entirely

represented

by

nished

sequence

Full

shotgun

clone

A

large-insert

clone

for

which

full

shotgun

sequence

has

been

produced

Draft

clone

A

large-insert

clone

for

which

roughly

half-shotgun

sequence

has

been

produced

Operationally

the

collection

of

draft

clones

produced

by

each

centre

was

required

to

have

an

average

coverage

of

fourfold

for

the

entire

set

and

a

minimum

coverage

of

threefold

for

each

clone

Predraft

clone

A

large-insert

clone

for

which

some

shotgun

sequence

is

available

but

which

does

not

meet

the

standards

for

inclusion

in

the

collection

of

draft

clones

PHRED

A

widely

used

computer

program

that

analyses

raw

sequence

to

produce

a

base

call

with

an

associated

quality

score

for

each

position

in

the

sequence

A

PHRED

quality

score

of

X

corresponds

to

an

error

probability

of

approximately

Thus

a

PHRED

quality

score

of

corresponds

to

accuracy

for

the

base

call

in

the

raw

read

PHRAP

A

widely

used

computer

program

that

assembles

raw

sequence

into

sequence

contigs

and

assigns

to

each

position

in

the

sequence

an

associated

quality

score

on

the

basis

of

the

PHRED

scores

of

the

raw

sequence

reads

A

PHRAP

quality

score

of

X

corresponds

to

an

error

probability

of

approximately

Thus

a

PHRAP

quality

score

of

corresponds

to

accuracy

for

a

base

in

the

assembled

sequence

GigAssembler

A

computer

program

developed

during

this

project

for

merging

the

information

from

individual

sequenced

clones

into

a

draft

genome

sequence

Public

sequence

databases

The

three

coordinated

international

sequence

databases

GenBank

the

EMBL

data

library

and

DDBJ

Map

features

Contigs

and

scaffolds

Contig

The

result

of

joining

an

overlapping

collection

of

sequences

or

clones

Scaffold

The

result

of

connecting

contigs

by

linking

information

from

paired-end

reads

from

plasmids

paired-end

reads

from

BACs

known

messenger

RNAs

or

other

sources

The

contigs

in

a

scaffold

are

ordered

and

oriented

with

respect

to

one

another

Fingerprint

clone

contigs

Contigs

produced

by

joining

clones

inferred

to

overlap

on

the

basis

of

their

restriction

digest

ngerprints

Sequenced-clone

layout

Assignment

of

sequenced

clones

to

the

physical

map

of

ngerprint

clone

contigs

Initial

sequence

contigs

Contigs

produced

by

merging

overlapping

sequence

reads

obtained

from

a

single

clone

in

a

process

called

sequence

assembly

Merged

sequence

contigs

Contigs

produced

by

taking

the

initial

sequence

contigs

contained

in

overlapping

clones

and

merging

those

found

to

overlap

These

are

also

referred

to

simply

as

sequence

contigs

where

no

confusion

will

result

Sequence-contig

scaffolds

Scaffolds

produced

by

connecting

sequence

contigs

on

the

basis

of

linking

information

NATURE

VOL

FEBRUARY

STS

Sequence

tagged

site

corresponding

to

a

short

-LRB-

typically

less

than

bp

-RRB-

unique

genomic

locus

for

which

a

polymerase

chain

reaction

assay

has

been

developed

EST

Expressed

sequence

tag

obtained

by

performing

a

single

raw

sequence

read

from

a

random

complementary

DNA

clone

SSR

Simple

sequence

repeat

a

sequence

consisting

largely

of

a

tandem

repeat

of

a

speci

c

k-mer

-LRB-

such

as

-LRB-

CA

-RRB-

-RRB-

Many

SSRs

are

polymorphic

and

have

been

widely

used

in

genetic

mapping

SNP

Single

nucleotide

polymorphism

or

a

single

nucleotide

position

in

the

genome

sequence

for

which

two

or

more

alternative

alleles

are

present

at

appreciable

frequency

-LRB-

traditionally

at

least

-RRB-

in

the

human

population

Genetic

map

A

genome

map

in

which

polymorphic

loci

are

positioned

relative

to

one

another

on

the

basis

of

the

frequency

with

which

they

recombine

during

meiosis

The

unit

of

distance

is

centimorgans

-LRB-

cM

-RRB-

denoting

a

chance

of

recombination

Radiation

hybrid

-LRB-

RH

-RRB-

map

A

genome

map

in

which

STSs

are

positioned

relative

to

one

another

on

the

basis

of

the

frequency

with

which

they

are

separated

by

radiation-induced

breaks

The

frequency

is

assayed

by

analysing

a

panel

of

human

hamster

hybrid

cell

lines

each

produced

by

lethally

irradiating

human

cells

and

fusing

them

with

recipient

hamster

cells

such

that

each

carries

a

collection

of

human

chromosomal

fragments

The

unit

of

distance

is

centirays

-LRB-

cR

-RRB-

denoting

a

chance

of

a

break

occuring

between

two

loci

Macmillan

Magazines

Ltd

articles

construction

Volunteers

of

diverse

backgrounds

were

accepted

on

a

rst-come

rst-taken

basis

Samples

were

obtained

after

discussion

with

a

genetic

counsellor

and

written

informed

consent

The

samples

were

made

anonymous

as

follows

the

sampling

laboratory

stripped

all

identi

ers

from

the

samples

applied

random

numeric

labels

and

transferred

them

to

the

processing

laboratory

which

then

removed

all

labels

and

relabelled

the

samples

All

records

of

the

labelling

were

destroyed

The

processing

laboratory

chose

samples

at

random

from

which

to

prepare

DNA

and

immortalized

cell

lines

Around

samples

were

collected

for

every

one

that

was

eventually

used

Because

no

link

was

retained

between

donor

and

DNA

sample

the

identity

of

the

donors

for

the

libraries

is

not

known

even

by

the

donors

themselves

A

more

complete

description

can

be

found

at

During

the

pilot

phase

centres

showed

that

sequence-tagged

sites

-LRB-

STSs

-RRB-

from

previously

constructed

genetic

and

physical

maps

could

be

used

to

recover

BACs

from

speci

c

regions

As

sequencing

expanded

some

centres

continued

this

approach

augmented

with

additional

probes

from

ow

sorting

of

chromosomes

to

obtain

long-range

coverage

of

speci

c

chromosomes

or

chromosomal

For

the

large-scale

sequence

production

phase

a

genome-wide

physical

map

of

overlapping

clones

was

also

constructed

by

systematic

analysis

of

BAC

clones

representing

coverage

of

the

human

Most

clones

came

from

the

rst

three

sections

of

the

library

supplemented

with

clones

from

sections

of

the

Table

Key

large-insert

genome-wide

libraries

Library

name

GenBank

Vector

Source

DNA

abbreviation

type

Library

segment

or

plate

numbers

Number

of

BAC-end

Enzyme

Average

Total

number

Number

of

clones

in

sequence

digest

insert

size

of

clones

in

ngerprinted

-LRB-

ends/clones

genome

clones

library

-LRB-

kb

-RRB-

layout

clones

with

both

ends

sequenced

-RRB-

Sequenced

clones

used

in

construction

of

the

draft

genome

sequence

and

CalTech

D

libraries

-LRB-

Table

-RRB-

DNA

from

each

BAC

clone

was

digested

with

the

restriction

enzyme

HindIII

and

the

sizes

of

the

resulting

fragments

were

measured

by

agarose

gel

electrophoresis

The

pattern

of

restriction

fragments

provides

a

ngerprint

for

each

BAC

which

allows

different

BACs

to

be

distinguished

and

the

degree

of

overlaps

to

be

assessed

We

used

these

restrictionfragment

ngerprints

to

determine

clone

overlaps

and

thereby

assembled

the

BACs

into

ngerprint

clone

contigs

The

ngerprint

clone

contigs

were

positioned

along

the

chromosomes

by

anchoring

them

with

STS

markers

from

existing

genetic

and

physical

maps

Fingerprint

clone

contigs

were

tied

to

speci

c

STSs

initially

by

probe

hybridization

and

later

by

direct

search

of

the

sequenced

clones

To

localize

ngerprint

clone

contigs

that

did

not

contain

known

markers

new

STSs

were

generated

and

placed

onto

Representative

clones

were

also

positioned

by

uorescence

in

situ

hybridization

-LRB-

FISH

-RRB-

-LRB-

ref

and

C.

McPherson

unpublished

-RRB-

We

selected

clones

from

the

ngerprint

clone

contigs

for

sequencing

according

to

various

criteria

Fingerprint

data

were

to

evaluate

overlaps

and

to

assess

clone

delity

-LRB-

to

bias

against

rearranged

-RRB-

STS

content

information

and

BAC

end

sequence

information

were

also

Where

possible

we

tried

to

select

a

minimally

overlapping

set

spanning

a

region

However

because

the

genome-wide

physical

map

was

constructed

concurrently

with

the

sequencing

continuity

in

many

regions

was

low

in

early

stages

These

small

ngerprint

clone

contigs

were

nonetheless

useful

in

identifying

validated

nonredundant

clones

Numberk

Total

bases

Fraction

of

-LRB-

Mb

-RRB-

total

from

library

Caltech

B

Caltech

C

Caltech

-LRB-

-RRB-

Caltech

-LRB-

-RRB-

CTB

CTC

CTD

BAC

BAC

BAC

BAC

cells

Human

sperm

Human

sperm

Human

sperm

All

All

All

All

All

All

All

All

All

HindIII

HindIII

HindIII

120 125 129

PAC

PAC

PAC

PAC

BAC

Male

blood

Male

blood

Male

blood

Male

blood

Male

blood

EcoRI

EcoRI

EcoRI

MboI

MboI

MboI

MboI

202 182 142 110

115 116 115 178

164 168 181 183

Total

of

top

eight

libraries

Total

all

libraries

EcoRI

EcoRI

EcoRI

EcoRI

MboI

For

the

CalTech

see

for

RPCI

see

For

the

FPC

map

and

see

The

number

of

raw

BAC

end

sequences

-LRB-

clones/ends/clones

with

both

ends

sequenced

-RRB-

available

for

use

in

human

genome

sequencing

Typically

for

clones

in

which

sequence

was

obtained

from

both

ends

more

than

of

both

end

sequences

contained

at

least

bp

of

nonrepetitive

sequence

BAC-end

sequencing

of

and

of

the

CalTech

libraries

was

done

at

The

Institute

for

Genomic

Research

the

California

Institute

of

Technology

and

the

University

of

Washington

High

Throughput

Sequencing

Center

The

sources

for

the

Table

were

BESstat.shtml

and

refs

These

are

the

clones

in

the

sequenced-clone

layout

map

-LRB-

-RRB-

that

were

pre-draft

draft

or

nished

k

The

number

of

sequenced

clones

used

in

the

assembly

This

number

is

less

than

that

in

the

previous

column

owing

to

removal

of

a

small

number

of

obviously

contaminated

combined

or

duplicated

projects

in

addition

not

all

of

the

clones

from

completed

chromosomes

and

were

included

here

because

only

the

available

nished

sequence

from

those

chromosomes

was

used

in

the

assembly

The

number

reported

is

the

total

sequence

from

the

clones

indicated

in

the

previous

column

Potential

overlap

between

clones

was

not

removed

here

but

Ns

were

excluded

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

that

were

used

to

seed

the

sequencing

of

new

regions

The

small

ngerprint

clone

contigs

were

extended

or

merged

with

others

as

the

map

matured

The

clones

that

make

up

the

draft

genome

sequence

therefore

do

not

constitute

a

minimally

overlapping

set

there

is

overlap

and

redundancy

in

places

The

cost

of

using

suboptimal

overlaps

was

justi

ed

by

the

bene

t

of

earlier

availability

of

the

draft

genome

sequence

data

Minimizing

the

overlap

between

adjacent

clones

would

have

required

completing

the

physical

map

before

undertaking

large-scale

sequencing

In

addition

the

overlaps

between

BAC

clones

provide

a

rich

collection

of

SNPs

More

than

million

SNPs

have

already

been

identi

ed

from

clone

overlaps

and

other

sequence

Because

the

sequencing

project

was

shared

among

twenty

centres

in

six

countries

it

was

important

to

coordinate

selection

of

clones

across

the

centres

Most

centres

focused

on

particular

chromosomes

or

in

some

cases

larger

regions

of

the

genome

We

also

maintained

a

clone

registry

to

track

selected

clones

and

their

progress

In

later

phases

the

global

map

provided

an

integrated

view

of

the

data

from

all

centres

facilitating

the

distribution

of

effort

to

maximize

coverage

of

the

genome

Before

performing

extensive

sequencing

on

a

clone

several

centres

routinely

examined

an

initial

sample

of

raw

sequence

reads

from

each

subclone

library

to

evaluate

possible

overlap

with

previously

sequenced

clones

Sequencing

Figure

The

automated

production

line

for

sample

preparation

at

the

Whitehead

Institute

Center

for

Genome

Research

The

system

consists

of

custom-designed

factorystyle

conveyor

belt

robots

that

perform

all

functions

from

purifying

DNA

from

bacterial

cultures

through

setting

up

and

purifying

sequencing

reactions

Sequence

-LRB-

Mb

-RRB-

Finished

Unfinished

-LRB-

draft

and

pre-draft

-RRB-

The

selected

clones

were

subjected

to

shotgun

sequencing

Although

the

basic

approach

of

shotgun

sequencing

is

well

established

the

details

of

implementation

varied

among

the

centres

For

example

there

were

differences

in

the

average

insert

size

of

the

shotgun

libraries

in

the

use

of

single-stranded

or

double-stranded

cloning

vectors

and

in

sequencing

from

one

end

or

both

ends

of

each

insert

Centres

differed

in

the

uorescent

labels

employed

and

in

the

degree

to

which

they

used

dye-primers

or

dye-terminators

The

sequence

detectors

included

both

slab

gel

and

capillary-based

devices

Detailed

protocols

are

available

on

the

web

sites

of

many

of

the

individual

centres

-LRB-

URLs

can

be

found

at

-RRB-

The

extent

of

automation

also

varied

greatly

among

the

centres

with

the

most

aggressive

automation

efforts

resulting

in

factory-style

systems

able

to

process

more

than

sequencing

reactions

in

hours

-LRB-

Fig.

-RRB-

In

addition

centres

differed

in

the

amount

of

raw

sequence

data

typically

obtained

for

each

clone

-LRB-

so-called

half-shotgun

full

shotgun

and

nished

sequence

-RRB-

Sequence

information

from

the

different

centres

could

be

directly

integrated

despite

this

diversity

because

the

data

were

analysed

by

a

common

computational

procedure

Raw

sequence

traces

were

processed

and

assembled

with

the

PHRED

and

PHRAP

software

-LRB-

P.

Green

unpublished

-RRB-

All

assembled

contigs

of

more

than

kb

were

deposited

in

public

databases

within

hours

of

assembly

The

overall

sequencing

output

rose

sharply

during

production

-LRB-

Fig.

-RRB-

Following

installation

of

new

sequence

detectors

beginning

in

June

sequencing

capacity

and

output

rose

approximately

eightfold

in

eight

months

to

nearly

million

samples

processed

per

month

with

little

or

no

drop

in

success

rate

-LRB-

ratio

of

useable

reads

to

attempted

reads

-RRB-

By

June

the

centres

were

producing

raw

sequence

at

a

rate

equivalent

to

onefold

coverage

of

the

entire

human

genome

in

less

than

six

weeks

This

corresponded

to

a

continuous

throughput

exceeding

nucleotides

per

second

hours

per

day

seven

days

per

week

This

scale-up

resulted

in

a

concomitant

increase

in

the

sequence

available

in

the

public

databases

-LRB-

Fig.

-RRB-

A

version

of

the

draft

genome

sequence

was

prepared

on

the

basis

of

the

map

and

sequence

data

available

on

October

For

this

version

the

mapping

effort

had

assembled

the

ngerprinted

BACs

into

ngerprint

clone

contigs

The

sequencing

effort

had

sequenced

and

assembled

overlapping

BACs

and

other

largeinsert

clones

-LRB-

Table

-RRB-

comprising

a

total

length

of

gigabases

-LRB-

Gb

-RRB-

This

resulted

from

around

Gb

of

underlying

raw

shotgun

sequence

data

or

about

coverage

averaged

across

the

genome

-LRB-

including

both

draft

and

nished

sequence

-RRB-

The

various

contributions

to

the

total

amount

of

sequence

deposited

in

the

HTGS

division

of

GenBank

are

given

in

Table

Table

Total

genome

sequence

from

the

collection

of

sequenced

clones

by

sequence

status

Sequence

status

Number

of

clones

Total

clone

length

-LRB-

Mb

-RRB-

Average

number

of

sequence

reads

per

kb

Average

sequence

depth

Total

amount

of

raw

sequence

-LRB-

Mb

-RRB-

Finished

Draft

Predraft

Total

Month

Figure

Total

amount

of

human

sequence

in

the

High

Throughput

Genome

Sequence

-LRB-

HTGS

-RRB-

division

of

GenBank

The

total

is

the

sum

of

nished

sequence

-LRB-

red

-RRB-

and

un

nished

-LRB-

draft

plus

predraft

-RRB-

sequence

-LRB-

yellow

-RRB-

NATURE

VOL

FEBRUARY

The

average

number

of

reads

per

kb

was

estimated

based

on

information

provided

by

each

sequencing

centre

This

number

differed

among

sequencing

centres

based

on

the

actual

protocols

used

The

average

depth

in

high

quality

bases

-LRB-

accuracy

-RRB-

was

estimated

from

information

provided

by

each

sequencing

centre

The

average

varies

among

the

centres

and

the

number

may

vary

considerably

for

clones

with

the

same

sequencing

status

For

draft

clones

in

the

public

databases

-LRB-

keyword

-RRB-

the

number

can

be

computed

from

the

quality

scores

listed

in

the

database

entry

Macmillan

Magazines

Ltd

articles

By

agreement

among

the

centres

the

collection

of

draft

clones

produced

by

each

centre

was

required

to

have

fourfold

average

sequence

coverage

with

no

clone

below

threefold

-LRB-

For

this

purpose

sequence

coverage

was

de

ned

as

the

average

number

of

times

that

each

base

was

independently

read

with

a

base-quality

score

corresponding

to

at

least

accuracy

-RRB-

We

attained

an

overall

average

of

coverage

across

the

genome

for

draft

clones

A

few

of

the

sequenced

clones

fell

below

the

minimum

of

threefold

sequence

coverage

or

have

not

been

formally

designated

by

centres

as

meeting

draft

standards

these

are

referred

to

as

predraft

-LRB-

Table

-RRB-

Some

of

these

are

clones

that

span

remaining

gaps

in

the

draft

genome

sequence

and

were

in

the

process

of

being

sequenced

on

October

a

few

are

old

submissions

from

centres

that

are

no

longer

active

The

lengths

of

the

initial

sequence

contigs

in

the

draft

clones

vary

as

a

function

of

coverage

but

half

of

all

nucleotides

reside

in

initial

sequence

contigs

of

at

least

kb

-LRB-

see

below

-RRB-

Various

properties

of

the

draft

clones

can

be

assessed

from

instances

in

which

there

was

substantial

overlap

between

a

draft

clone

and

a

nished

-LRB-

or

nearly

nished

-RRB-

clone

By

examining

the

sequence

alignments

in

the

overlap

regions

we

estimated

that

the

initial

sequence

contigs

in

a

draft

sequence

clone

cover

an

average

of

about

of

the

clone

and

are

separated

by

gaps

with

an

average

size

of

about

bp

Although

the

main

emphasis

was

on

producing

a

draft

genome

sequence

the

centres

also

maintained

sequence

nishing

activities

during

this

period

leading

to

a

twofold

increase

in

nished

sequence

from

June

to

June

-LRB-

Fig.

-RRB-

The

total

amount

of

human

sequence

in

this

nal

form

stood

at

more

than

Mb

on

October

or

more

than

of

the

human

genome

This

includes

the

nished

sequences

of

chromosomes

and

-LRB-

refs

-RRB-

As

centres

have

begun

to

shift

from

draft

to

nished

sequencing

in

the

last

quarter

of

the

production

of

nished

sequence

has

increased

to

an

annualized

rate

of

Gb

per

year

and

is

continuing

to

rise

In

addition

to

sequencing

large-insert

clones

three

centres

generated

a

large

collection

of

random

raw

sequence

reads

from

whole-genome

shotgun

libraries

-LRB-

Table

ref

-RRB-

These

million

successful

sequences

contained

Gb

of

high-quality

bases

this

corresponds

to

about

coverage

and

would

be

statistically

expected

to

include

about

of

the

nucleotides

in

the

human

genome

-LRB-

data

available

at

-RRB-

The

primary

objective

of

this

work

was

to

discover

SNPs

by

comparing

these

random

raw

sequences

-LRB-

which

came

from

different

individuals

-RRB-

with

the

draft

genome

sequence

However

many

of

these

raw

sequences

were

obtained

from

both

ends

of

plasmid

clones

and

thereby

also

provided

valuable

linking

information

that

was

used

in

sequence

assembly

In

addition

the

random

raw

sequences

provide

sequence

coverage

of

about

half

of

the

nucleotides

not

yet

represented

in

the

sequenced

large-insert

clones

these

can

be

used

as

probes

for

portions

of

the

genome

not

yet

recovered

Assembly

of

the

draft

genome

sequence

Table

Total

human

sequence

deposited

in

the

HTGS

division

of

GenBank

Sequencing

centre

Whitehead

Institute

Center

for

Genome

Research

The

Sanger

Centre

Washington

University

Genome

Sequencing

Center

US

DOE

Joint

Genome

Institute

Baylor

College

of

Medicine

Human

Genome

Sequencing

Center

RIKEN

Genomic

Sciences

Center

Genoscope

GTC

Sequencing

Center

Department

of

Genome

Analysis

Institute

of

Molecular

Biotechnology

Beijing

Genomics

Institute/Human

Genome

Center

Multimegabase

Sequencing

Center

Institute

for

Systems

Biology

Stanford

Genome

Technology

Center

The

Stanford

Human

Genome

Center

and

Department

of

Genetics

University

of

Washington

Genome

Center

Keio

University

University

of

Texas

Southwestern

Medical

Center

at

Dallas

University

of

Oklahoma

Advanced

Center

for

Genome

Technology

Max

Planck

Institute

for

Molecular

Genetics

GBF

German

Research

Centre

for

Biotechnology

Cold

Spring

Harbor

Laboratory

Lita

Annenberg

Hazen

Genome

Center

Other

Total

Total

human

Finished

human

sequence

-LRB-

kb

-RRB-

sequence

-LRB-

kb

-RRB-

We

then

set

out

to

assemble

the

sequences

from

the

individual

largeinsert

clones

into

an

integrated

draft

sequence

of

the

human

genome

The

assembly

process

had

to

resolve

problems

arising

from

the

draft

nature

of

much

of

the

sequence

from

the

variety

of

clone

sources

and

from

the

high

fraction

of

repeated

sequences

in

the

human

genome

This

process

involved

three

steps

ltering

layout

and

merging

The

entire

data

set

was

ltered

uniformly

to

eliminate

contamination

from

nonhuman

sequences

and

other

artefacts

that

had

not

already

been

removed

by

the

individual

centres

-LRB-

Information

about

contamination

was

also

sent

back

to

the

centres

which

are

updating

the

individual

entries

in

the

public

databases

-RRB-

We

also

identi

ed

instances

in

which

the

sequence

data

from

one

BAC

clone

was

substantially

contaminated

with

sequence

data

from

another

-LRB-

human

or

nonhuman

-RRB-

clone

The

problems

were

resolved

in

most

instances

clones

remained

unresolved

and

these

were

eliminated

from

the

assembly

reported

here

Instances

of

lower

levels

of

cross-contamination

-LRB-

for

example

a

single

microplate

misassigned

to

the

wrong

BAC

-RRB-

are

more

dif

cult

to

detect

some

undoubtedly

remain

and

may

give

rise

to

small

spurious

sequence

contigs

in

the

draft

genome

sequence

Such

issues

are

readily

resolved

as

the

clones

progress

towards

nished

sequence

but

they

necessitate

some

caution

in

certain

applications

of

the

current

data

The

sequenced

clones

were

then

associated

with

speci

c

clones

on

the

physical

map

to

produce

a

layout

In

principle

sequenced

clones

that

correspond

to

ngerprinted

BACs

could

be

directly

assigned

by

name

to

ngerprint

clone

contigs

on

the

ngerprintbased

physical

map

In

practice

however

laboratory

mixups

occasionally

resulted

in

incorrect

assignments

To

eliminate

such

problems

sequenced

clones

were

associated

with

the

ngerprint

clone

contigs

in

the

physical

map

by

using

the

sequence

data

to

calculate

a

Table

Plasmid

paired-end

reads

Total

reads

deposited

Random-sheared

Enzyme

digest

Total

Read

pairs

Size

range

of

inserts

-LRB-

kb

-RRB-

Total

human

sequence

deposited

in

GenBank

by

members

of

the

International

Human

Genome

Sequencing

Consortium

as

of

October

The

amount

of

total

sequence

-LRB-

nished

plus

draft

plus

predraft

-RRB-

is

shown

in

the

second

column

and

the

amount

of

nished

sequence

is

shown

in

the

third

column

Total

sequence

differs

from

totals

in

Tables

and

because

of

inclusion

of

padding

characters

and

of

some

clones

not

used

in

assembly

HTGS

high

throughput

genome

sequence

These

three

centres

produced

an

additional

Gb

of

raw

plasmid

paired-end

reads

-LRB-

see

Table

-RRB-

consisting

of

Gb

from

Whitehead

Institute

Gb

from

The

Sanger

Centre

and

Gb

from

Washington

University

The

plasmid

paired-end

reads

used

a

mixture

of

DNA

from

a

set

of

samples

from

the

DNA

Polymorphism

Discovery

Resource

-LRB-

-RRB-

This

set

of

anonymous

US

residents

contains

samples

from

European-Americans

African-Americans

MexicanAmericans

Native

Americans

and

Asian-Americans

although

the

ethnicities

of

the

individual

samples

are

not

identi

ed

Informed

consent

to

contribute

samples

to

the

DNA

Polymorphism

Discovery

Resource

was

obtained

from

all

individuals

who

contributed

samples

Samples

from

the

European-American

African-American

and

Mexican-American

individuals

came

from

NHANES

-LRB-

-RRB-

individuals

were

recontacted

to

obtain

their

consent

for

the

Resource

project

New

samples

were

obtained

from

Asian-Americans

whose

ancestry

was

from

a

variety

of

East

and

South

Asian

countries

New

samples

were

also

obtained

for

the

Native

Americans

tribal

permission

was

obtained

rst

and

then

individual

consents

See

http

and

ref

Re

ects

data

deposited

with

and

released

by

The

SNP

Consortium

-LRB-

see

-RRB-

Read

pairs

represents

the

number

of

cases

in

which

sequence

from

both

ends

of

a

genomic

cloned

fragment

was

determined

and

used

in

this

study

as

linking

information

Macmillan

Magazines

Ltd

NATURE

VOL

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articles

partial

list

of

restriction

fragments

in

silico

and

comparing

that

list

with

the

experimental

database

of

BAC

ngerprints

The

comparison

was

feasible

because

the

experimental

sizing

of

restriction

fragments

was

highly

accurate

-LRB-

to

within

of

the

true

size

for

of

fragments

from

to

base

pairs

-LRB-

bp

-RRB-

-RRB-

Reliable

matching

scores

could

be

obtained

for

of

the

clones

The

remaining

sequenced

clones

could

not

be

placed

on

the

map

by

this

method

because

they

were

too

short

or

they

contained

too

many

small

initial

sequence

contigs

to

yield

enough

restriction

fragments

or

possibly

because

their

sequences

were

not

represented

in

the

ngerprint

database

An

independent

approach

to

placing

sequenced

clones

on

the

physical

map

used

the

database

of

end

sequences

from

ngerprinted

BACs

-LRB-

Table

-RRB-

Sequenced

clones

could

typically

be

reliably

mapped

if

they

contained

multiple

matches

to

BAC

ends

with

all

corresponding

to

clones

from

a

single

genomic

region

-LRB-

multiple

matches

were

required

as

a

safeguard

against

errors

known

to

exist

in

the

BAC

end

database

and

against

repeated

sequences

-RRB-

This

approach

provided

useful

placement

information

for

sequenced

clones

Altogether

we

could

assign

sequenced

clones

to

ngerprint

clone

contigs

by

combining

in

silico

digestion

and

BAC

end

sequence

match

data

To

place

most

of

the

remaining

sequenced

clones

we

exploited

information

about

sequence

overlap

or

BACend

paired

links

of

these

clones

with

already

positioned

clones

This

left

only

a

few

mostly

small

sequenced

clones

that

could

not

be

placed

-LRB-

sequenced

clones

containing

Mb

of

sequence

out

of

sequenced

clones

containing

more

than

Mb

of

sequence

-RRB-

these

are

being

localized

by

radiation

hybrid

mapping

of

STSs

derived

from

their

sequences

The

ngerprint

clone

contigs

were

then

mapped

to

chromosomal

locations

using

sequence

matches

to

mapped

STSs

from

four

human

radiation

hybrid

one

YAC

and

radiation

hybrid

and

two

genetic

together

with

data

from

The

mapping

was

iteratively

re

ned

by

comparing

the

order

and

orientation

of

the

STSs

in

the

ngerprint

clone

contigs

and

the

various

STS-based

maps

to

identify

and

re

ne

discrepancies

-LRB-

Fig.

-RRB-

Small

ngerprint

clone

contigs

-LRB-

Mb

-RRB-

were

dif

cult

to

orient

and

sometimes

to

order

using

these

methods

In

all

ngerprint

clone

contigs

contained

sequenced

clones

-LRB-

An

additional

of

the

ngerprint

clone

contigs

did

not

contain

sequenced

clones

but

these

tended

to

be

extremely

small

and

together

contain

less

than

of

the

mapped

clones

About

onethird

have

been

targeted

for

sequencing

A

few

derive

from

the

Y

chromosome

for

which

the

map

was

constructed

Most

of

the

remainder

are

fragments

of

other

larger

contigs

or

represent

other

artefacts

These

are

being

eliminated

in

subsequent

versions

of

the

database

-RRB-

Of

these

contigs

with

sequenced

clones

-LRB-

containing

of

the

total

sequence

-RRB-

were

localized

to

speci

c

chromosome

locations

in

this

way

An

additional

ngerprint

clone

contigs

containing

of

the

sequence

could

be

assigned

to

a

speci

c

chromosome

but

not

to

a

precise

position

a

a

Chromosome

Y

X

Map

location

Genethon

map

Gene

map

Marshfield

map

YAC

map

Chromosome

b

Chromosome

Y

X

Map

location

Genethon

map

Gene

map

Marshfield

map

YAC

map

Chromosome

Figure

Positions

of

markers

on

previous

maps

of

the

genome

-LRB-

the

genetic

map

and

Marsh

eld

genetic

map

-LRB-

-RRB-

the

radiation

hybrid

and

the

Whitehead

YAC

and

radiation

hybrid

-RRB-

plotted

against

their

derived

position

on

the

draft

sequence

for

chromosome

The

horizontal

units

are

Mb

but

the

vertical

units

of

each

map

vary

-LRB-

cM

cR

and

so

on

-RRB-

and

thus

all

were

scaled

so

that

the

entire

map

spans

the

full

vertical

range

Markers

that

map

to

other

chromosomes

are

shown

in

the

chromosome

lines

at

the

top.The

data

sets

generally

follow

the

diagonal

indicating

that

order

and

orientation

of

the

marker

sets

on

the

different

maps

largely

agree

-LRB-

note

that

the

two

genetic

maps

are

completely

superimposed

-RRB-

In

a

there

are

two

segments

-LRB-

bars

-RRB-

that

are

inverted

in

an

earlier

version

draft

sequence

relative

to

all

the

other

maps

b

The

same

chromosome

after

the

information

was

used

to

reorient

those

two

segments

b

c

end-to-end

alignment

OK

alignment

in

middle

only

not

OK

d

Figure

The

key

steps

-LRB-

a

d

-RRB-

in

assembling

individual

sequenced

clones

into

the

draft

genome

sequence

represent

initial

sequence

contigs

derived

from

shotgun

sequencing

of

clone

A

and

are

from

clone

B.

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

The

remaining

contigs

containing

of

the

sequence

were

not

positioned

at

all

We

then

merged

the

sequences

from

overlapping

sequenced

clones

-LRB-

Fig.

-RRB-

using

the

computer

program

The

program

considers

nearby

sequenced

clones

detects

overlaps

between

the

initial

sequence

contigs

in

these

clones

merges

the

overlapping

sequences

and

attempts

to

order

and

orient

the

sequence

contigs

It

begins

by

aligning

the

initial

sequence

contigs

from

one

clone

with

those

from

other

clones

in

the

same

ngerprint

clone

contig

on

the

basis

of

length

of

alignment

per

cent

identity

of

the

alignment

position

in

the

sequenced

clone

layout

and

other

factors

Alignments

are

limited

to

one

end

of

each

initial

sequence

contig

for

partially

overlapping

contigs

or

to

both

ends

of

an

initial

sequence

contig

contained

entirely

within

another

this

eliminates

internal

alignments

that

may

re

ect

repeated

sequence

or

possible

misassembly

-LRB-

Fig.

-RRB-

Beginning

with

the

highest

scoring

pairs

initial

sequence

contigs

are

then

integrated

to

produce

merged

sequence

contigs

-LRB-

usually

referred

to

simply

as

sequence

contigs

-RRB-

The

program

re

nes

the

arrangement

of

the

clones

within

the

ngerprint

clone

contig

on

the

basis

of

the

extent

of

sequence

overlap

between

them

and

then

rebuilds

the

sequence

contigs

Next

the

program

selects

a

sequence

path

through

the

sequence

contigs

-LRB-

Fig.

-RRB-

It

tries

to

use

the

highest

quality

data

by

preferring

longer

initial

sequence

contigs

and

avoiding

the

rst

and

last

bases

of

initial

sequence

contigs

where

possible

Finally

it

attempts

to

order

and

orient

the

sequence

contigs

by

using

additional

information

including

sequence

data

from

paired-end

plasmid

and

BAC

reads

known

messenger

RNAs

and

ESTs

as

well

as

additional

linking

information

provided

by

centres

The

sequence

contigs

are

thereby

linked

together

to

create

sequence-contig

scaffolds

-LRB-

Fig.

-RRB-

The

process

also

joins

overlapping

sequenced

clones

into

sequencedclone

contigs

and

links

sequenced-clone

contigs

to

form

sequencedclone-contig

scaffolds

A

ngerprint

clone

contig

may

contain

several

sequenced-clone

contigs

because

bridging

clones

remain

to

be

sequenced

The

assembly

contained

sequenced-clone

Fingerprint

clone

contig

Pick

clones

for

sequencing

A

B

Sequenced-clone

contig

Sequenced-clone-contig

scaffold

Sequence

to

at

least

draft

coverage

Sequenced

clone

A

Merge

data

Sequenced

clone

B

Initial

sequence

contig

Merged

sequence

contig

Order

and

orient

with

mRNA

paired

end

reads

other

information

Sequence-contig

scaffold

Figure

Levels

of

clone

and

sequence

coverage

A

ngerprint

clone

contig

is

assembled

by

using

the

computer

program

to

analyse

the

restriction

enzyme

digestion

patterns

of

many

large-insert

clones

Clones

are

then

selected

for

sequencing

to

minimize

overlap

between

adjacent

clones

For

a

clone

to

be

selected

all

of

its

restriction

enzyme

fragments

-LRB-

except

the

two

vector-insert

junction

fragments

-RRB-

must

be

shared

with

at

least

one

of

its

neighbours

on

each

side

in

the

contig

Once

these

overlapping

clones

have

been

sequenced

the

set

is

a

sequenced-clone

contig

When

all

selected

clones

from

a

ngerprint

clone

contig

have

been

sequenced

the

sequenced-clone

contig

will

be

the

same

as

the

ngerprint

clone

contig

Until

then

a

ngerprint

clone

contig

may

contain

several

sequenced-clone

contigs

After

individual

clones

-LRB-

for

example

A

and

B

-RRB-

have

been

sequenced

to

draft

coverage

and

the

clones

have

been

mapped

the

data

are

analysed

by

GigAssembler

-LRB-

Fig.

-RRB-

producing

merged

sequence

contigs

from

initial

sequence

contigs

and

linking

these

to

form

sequence-contig

scaffolds

-LRB-

see

Box

-RRB-

Table

The

draft

genome

sequence

Chromosome

Finished

clones

All

X

Y

UL

Sequence

from

clones

-LRB-

kb

-RRB-

Draft

clones

Pre-draft

clones

Contigs

containing

nished

clones

Sequence

from

contigs

-LRB-

kb

-RRB-

Deep

coverage

sequence

contigs

Draft/predraft

sequence

contigs

The

table

presents

summary

statistics

for

the

draft

genome

sequence

over

the

entire

genome

and

by

individual

chromosome

clones

that

could

not

be

placed

into

the

sequenced

clone

layout

UL

clones

that

could

be

placed

in

the

layout

but

that

could

not

reliably

be

placed

on

a

chromosome

First

three

columns

data

from

nished

clones

draft

clones

and

predraft

clones

The

last

three

columns

break

the

data

down

according

to

the

type

of

sequence

contig

Contigs

containing

nished

clones

represent

sequence

contigs

that

consist

of

nished

sequence

plus

any

-LRB-

small

-RRB-

extensions

from

merged

sequence

contigs

that

arise

from

overlap

with

anking

draft

clones

Deep

coverage

sequence

contigs

include

sequence

from

two

or

more

overlapping

un

nished

clones

they

consist

of

roughly

full

shotgun

coverage

and

thus

are

longer

than

the

average

un

nished

sequence

contig

Draft/predraft

sequence

contigs

are

all

of

the

other

sequence

contigs

in

un

nished

clones

Thus

the

draft

genome

sequence

consists

of

approximately

one-third

nished

sequence

one-third

deep

coverage

sequence

and

one-third

draft/pre-draft

coverage

sequence

In

all

of

the

statistics

we

count

only

nonoverlapping

bases

in

the

draft

genome

sequence

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

contigs

in

ngerprint

clone

contigs

The

hierarchy

of

contigs

is

summarized

in

Fig.

Initial

sequence

contigs

are

integrated

to

create

merged

sequence

contigs

which

are

then

linked

to

form

sequence-contig

scaffolds

These

scaffolds

reside

within

sequenced-clone

contigs

which

in

turn

reside

within

ngerprint

clone

contigs

The

draft

genome

sequence

Quality

assessment

The

result

of

the

assembly

process

is

an

integrated

draft

sequence

of

the

human

genome

Several

features

of

the

draft

genome

sequence

are

reported

in

Tables

including

the

proportion

represented

by

nished

draft

and

predraft

categories

The

Tables

also

show

the

numbers

and

lengths

of

different

types

of

contig

for

each

chromosome

and

for

the

genome

as

a

whole

The

contiguity

of

the

draft

genome

sequence

at

each

level

is

an

important

feature

Two

commonly

used

statistics

have

signi

cant

drawbacks

for

describing

contiguity

The

average

length

of

a

contig

is

de

ated

by

the

presence

of

many

small

contigs

comprising

only

a

small

proportion

of

the

genome

whereas

the

length-weighted

average

length

is

in

ated

by

the

presence

of

large

segments

of

nished

sequence

Instead

we

chose

to

describe

the

contiguity

as

a

property

of

the

typical

nucleotide

We

used

a

statistic

called

the

length

de

ned

as

the

largest

length

L

such

that

of

all

nucleotides

are

contained

in

contigs

of

size

at

least

L

The

continuity

of

the

draft

genome

sequence

reported

here

and

the

effectiveness

of

assembly

can

be

readily

seen

from

the

following

half

of

all

nucleotides

reside

within

an

initial

sequence

contig

of

at

least

kb

a

sequence

contig

of

at

least

kb

a

sequence-contig

scaffold

of

at

least

kb

a

sequenced-clone

contig

of

at

least

kb

and

a

ngerprint

clone

contig

of

at

least

Mb

-LRB-

Tables

-RRB-

The

cumulative

distributions

for

each

of

these

measures

of

contiguity

are

shown

in

Fig.

in

which

the

values

for

each

measure

can

be

seen

as

the

value

at

which

the

cumulative

distributions

cross

We

have

also

estimated

the

size

of

each

chromosome

by

estimating

the

gap

sizes

-LRB-

see

below

-RRB-

and

the

extent

of

missing

heterochromatic

-LRB-

Table

-RRB-

This

is

undoubtedly

an

oversimpli

cation

and

does

not

adequately

take

into

account

the

sequence

status

of

each

chromosome

Nonetheless

it

provides

a

useful

way

to

relate

the

draft

sequence

to

the

chromosomes

Table

Clone

level

contiguity

of

the

draft

genome

sequence

Chromosome

Sequenced-clone

contigs

Number

All

X

Y

UL

453 348 409 384

385 292 224 292

143 179 224 196

128 54 123 159

138 137 159

length

-LRB-

kb

-RRB-

672 606 623 814

797 620 831 709

The

draft

genome

sequence

already

covers

the

vast

majority

of

the

genome

but

it

remains

an

incomplete

intermediate

product

that

is

regularly

updated

as

we

work

towards

a

complete

nished

sequence

The

current

version

contains

many

gaps

and

errors

We

therefore

sought

to

evaluate

the

quality

of

various

aspects

of

the

current

draft

genome

sequence

including

the

sequenced

clones

themselves

their

assignment

to

a

position

in

the

ngerprint

clone

contigs

and

the

assembly

of

initial

sequence

contigs

from

the

individual

clones

into

sequence-contig

scaffolds

Nucleotide

accuracy

is

re

ected

in

a

PHRAP

score

assigned

to

each

base

in

the

draft

genome

sequence

and

available

to

users

through

the

Genome

Browsers

-LRB-

see

below

-RRB-

and

public

database

entries

A

summary

of

these

scores

for

the

un

nished

portion

of

the

genome

is

shown

in

Table

About

of

the

un

nished

draft

genome

sequence

has

an

error

rate

of

less

than

per

bases

-LRB-

PHRAP

score

-RRB-

and

about

has

an

error

rate

of

less

than

in

bases

-LRB-

PHRAP

-RRB-

These

values

are

based

only

on

the

quality

scores

for

the

bases

in

the

sequenced

clones

they

do

not

re

ect

additional

con

dence

in

the

sequences

that

are

represented

in

overlapping

clones

The

nished

portion

of

the

draft

genome

sequence

has

an

error

rate

of

less

than

per

bases

Individual

sequenced

clones

We

assessed

the

frequency

of

misassemblies

which

can

occur

when

the

assembly

program

PHRAP

joins

two

nonadjacent

regions

in

the

clone

into

a

single

initial

sequence

contig

The

frequency

of

misassemblies

depends

heavily

on

the

depth

and

quality

of

coverage

of

each

clone

and

the

nature

of

the

underlying

sequence

thus

it

may

vary

among

genomic

regions

and

among

individual

centres

Most

clone

misassemblies

are

readily

corrected

as

coverage

is

added

during

nishing

but

they

may

have

been

propagated

into

the

current

version

of

the

draft

genome

sequence

and

they

justify

caution

for

certain

applications

We

estimated

the

frequency

of

misassembly

by

examining

instances

in

which

there

was

substantial

overlap

between

a

draft

clone

and

a

nished

clone

We

studied

Mb

of

such

overlaps

involving

about

initial

sequence

contigs

We

found

instances

per

Mb

in

which

the

alignment

of

an

initial

sequence

contig

to

the

nished

sequence

failed

to

extend

to

within

bases

Sequenced-clone-contig

scaffolds

Number

197 127 201 163

length

-LRB-

kb

-RRB-

Fingerprint

clone

contigs

with

sequence

Number

length

-LRB-

kb

-RRB-

Number

and

size

of

sequenced-clone

contigs

sequenced-clone-contig

scaffolds

and

those

ngerprint

clone

contigs

-LRB-

see

Box

-RRB-

that

contain

sequenced

clones

some

small

ngerprint

clone

contigs

do

not

as

yet

have

associated

sequence

UL

ngerprint

clone

contigs

that

could

not

reliably

be

placed

on

a

chromosome

These

length

estimates

are

from

the

draft

genome

sequence

in

which

gaps

between

sequence

contigs

are

arbitrarily

represented

with

Ns

and

gaps

between

sequence

clone

contigs

with

Ns

for

bridged

gaps

and

Ns

for

unbridged

gaps

These

arbitrary

values

differ

minimally

from

empirical

estimates

of

gap

size

-LRB-

see

text

-RRB-

and

using

the

empirically

derived

estimates

would

change

the

lengths

presented

here

only

slightly

For

un

nished

chromosomes

the

length

ranges

from

to

times

the

arithmetic

mean

for

sequenced-clone

contigs

to

times

for

sequenced-clone-contig

scaffolds

and

to

times

for

ngerprint

clone

contigs

with

sequence

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

of

the

end

of

the

contig

suggesting

a

possible

false

join

in

the

assembly

of

the

initial

sequence

contig

In

about

half

of

these

cases

the

potential

misassembly

involved

fewer

than

bases

suggesting

that

a

single

raw

sequence

read

may

have

been

incorrectly

joined

We

found

instances

per

Mb

in

which

the

alignment

showed

an

internal

gap

again

suggesting

a

possible

misassembly

and

instances

per

Mb

in

which

the

alignment

indicated

that

two

initial

sequence

contigs

that

overlapped

by

at

least

bp

had

not

been

merged

by

PHRAP

Finally

there

were

another

instances

per

Mb

with

various

other

problems

This

gives

a

total

of

instances

per

Mb

of

possible

misassembly

with

about

half

being

relatively

small

issues

involving

a

few

hundred

bases

Some

of

the

potential

problems

might

not

result

from

misassembly

but

might

re

ect

sequence

polymorphism

in

the

population

a

Cumulative

percentage

Size

-LRB-

kb

-RRB-

Clone

level

continuity

Sequenced

clones

Sequenced-clone

contigs

Sequenced-clone-contig

scaffolds

Fingerprint

clone

contigs

small

rearrangements

during

growth

of

the

large-insert

clones

regions

of

low-quality

sequence

or

matches

between

segmental

duplications

Thus

the

frequency

of

misassemblies

may

be

overstated

On

the

other

hand

the

criteria

for

recognizing

overlap

between

draft

and

nished

clones

may

have

eliminated

some

misassemblies

Layout

of

the

sequenced

clones

We

assessed

the

accuracy

of

the

layout

of

sequenced

clones

onto

the

ngerprinted

clone

contigs

by

calculating

the

concordance

between

the

positions

assigned

to

a

sequenced

clone

on

the

basis

of

in

silico

digestion

and

the

position

assigned

on

the

basis

of

BAC

end

sequence

data

The

positions

agreed

in

of

cases

in

which

independent

assignments

could

be

made

by

both

methods

The

results

were

also

compared

with

well

studied

regions

containing

both

nished

and

draft

genome

sequence

These

results

indicated

that

sequenced

clone

order

in

the

ngerprint

map

was

reliable

to

within

about

half

of

one

clone

length

-LRB-

kb

-RRB-

A

direct

test

of

the

layout

is

also

provided

by

the

draft

genome

sequence

assembly

itself

With

extensive

coverage

of

the

genome

a

correctly

placed

clone

should

usually

-LRB-

although

not

always

-RRB-

show

sequence

overlap

with

its

neighbours

in

the

map

We

found

only

instances

of

singleton

clones

that

failed

to

overlap

a

neighbouring

clone

Close

examination

of

the

data

suggests

that

most

of

these

are

correctly

placed

but

simply

do

not

yet

overlap

an

adjacent

sequenced

clone

About

clones

appeared

to

be

candidates

for

being

incorrectly

placed

Alignment

of

the

ngerprint

clone

contigs

The

alignment

of

the

ngerprint

clone

contigs

with

the

chromosomes

was

based

on

the

radiation

hybrid

YAC

and

genetic

maps

of

STSs

The

positions

of

most

of

the

STSs

in

the

draft

genome

sequence

were

consistent

with

these

previous

maps

but

the

positions

of

about

differed

from

one

or

more

of

them

Some

of

these

disagreements

may

be

due

to

errors

in

the

layout

of

the

sequenced

clones

or

in

the

underlying

Figure

Overview

of

features

of

draft

human

genome

The

Figure

shows

the

Q

occurrences

of

twelve

important

types

of

feature

across

the

human

genome

Large

grey

blocks

represent

centromeres

and

centromeric

heterochromatin

-LRB-

size

not

precisely

to

scale

-RRB-

Each

of

the

feature

types

is

depicted

in

a

track

from

top

to

bottom

as

follows

-LRB-

-RRB-

Chromosome

position

in

Mb

-LRB-

-RRB-

The

approximate

positions

of

Giemsa-stained

chromosome

bands

at

the

band

resolution

-LRB-

-RRB-

Level

of

coverage

in

the

draft

genome

sequence

Red

areas

covered

by

nished

clones

yellow

areas

covered

by

predraft

sequence

Regions

covered

by

draft

sequenced

clones

are

in

orange

with

darker

shades

re

ecting

increasing

shotgun

sequence

coverage

-LRB-

-RRB-

GC

content

Percentage

of

bases

in

a

base

window

that

are

C

or

G.

-LRB-

-RRB-

Repeat

density

Red

line

density

of

SINE

class

repeats

in

a

window

blue

line

density

of

LINE

class

repeats

in

a

base

window

-LRB-

-RRB-

Density

of

SNPs

in

a

window

The

SNPs

were

detected

by

sequencing

and

alignments

of

random

genomic

reads

Some

of

the

heterogeneity

in

SNP

density

re

ects

the

methods

used

for

SNP

discovery

Rigorous

analysis

of

SNP

density

requires

comparing

the

number

of

SNPs

identi

ed

to

the

precise

number

of

bases

surveyed

-LRB-

-RRB-

Non-coding

RNA

genes

Brown

functional

RNA

genes

such

as

tRNAs

snoRNAs

and

rRNAs

light

orange

RNA

pseudogenes

-LRB-

-RRB-

CpG

islands

Green

ticks

represent

regions

of

bases

with

CpG

levels

signi

cantly

higher

than

in

the

genome

as

a

whole

and

GC

ratios

of

at

least

-LRB-

-RRB-

Exo

sh

ecores

Regions

of

homology

with

the

puffer

sh

T.

nigroviridis

are

blue

-LRB-

-RRB-

ESTs

with

at

least

one

intron

when

aligned

against

genomic

DNA

are

shown

as

black

tick

marks

-LRB-

-RRB-

The

starts

of

genes

predicted

by

Genie

or

Ensembl

are

shown

as

red

ticks

The

starts

of

known

genes

from

the

RefSeq

are

shown

in

blue

-LRB-

-RRB-

The

names

of

genes

that

have

been

uniquely

located

in

the

draft

genome

sequence

characterized

and

named

by

the

HGM

Nomenclature

Committee

Known

disease

genes

from

the

OMIM

database

are

red

other

genes

blue

This

Figure

is

based

on

an

earlier

version

of

the

draft

genome

sequence

than

analysed

in

the

text

owing

to

production

constraints

We

are

aware

of

various

errors

in

the

Figure

including

omissions

of

some

known

genes

and

misplacements

of

others

Some

genes

are

mapped

to

more

than

one

location

owing

to

errors

in

assembly

close

paralogues

or

pseudogenes

Manual

review

was

performed

to

select

the

most

likely

location

in

these

cases

and

to

correct

other

regions

For

updated

information

see

and

NATURE

VOL

FEBRUARY

b

Cumulative

percentage

100 200 300 400

Initial

sequence

contigs

Sequence

contigs

Sequence-contig

scaffolds

500 600 700

Size

-LRB-

kb

-RRB-

Sequence

level

continuity

Figure

Cumulative

distributions

of

several

measures

of

clone

level

contiguity

and

sequence

contiguity

The

gures

represent

the

proportion

of

the

draft

genome

sequence

contained

in

contigs

of

at

most

the

indicated

size

a

Clone

level

contiguity

The

clones

have

a

tight

size

distribution

with

an

of

kb

-LRB-

corresponding

to

on

the

cumulative

distribution

-RRB-

Sequenced-clone

contigs

represent

the

next

level

of

continuity

and

are

linked

by

mRNA

sequences

or

pairs

of

BAC

end

sequences

to

yield

the

sequenced-clone-contig

scaffolds

The

underlying

contiguity

of

the

layout

of

sequenced

clones

against

the

ngerprinted

clone

contigs

is

only

partially

shown

at

this

scale

b

Sequence

contiguity

The

input

fragments

have

low

continuity

-LRB-

kb

-RRB-

After

merging

the

sequence

contigs

grow

to

an

length

of

about

kb

After

linking

sequence-contig

scaffolds

with

an

length

of

about

kb

are

created

Macmillan

Magazines

Ltd

articles

Table

Sequence

level

contiguity

of

the

draft

genome

sequence

Chromosome

Initial

sequence

contigs

Number

All

X

Y

UL

length

-LRB-

kb

-RRB-

Number

Sequence

contigs

length

-LRB-

kb

-RRB-

Sequence-contig

scaffolds

Number

length

-LRB-

kb

-RRB-

This

Table

is

similar

to

Table

but

shows

the

number

and

length

for

various

types

of

sequence

contig

-LRB-

see

Box

-RRB-

See

legend

to

Table

concerning

treatment

of

gaps

For

sequence

contigs

in

the

draft

genome

sequence

the

length

ranges

from

to

times

the

arithmetic

mean

for

initial

sequence

contigs

to

times

for

merged

sequence

contigs

and

to

times

for

sequence-contig

scaffolds

Table

Chromosome

size

estimates

Chromosome

Sequenced

bases

-LRB-

Mb

-RRB-

FCC

gaps

SCC

gapsk

Sequence

gaps

Heterochromatin

and

short

arm

adjustments

-LRB-

Mb

-RRB-

Total

estimated

chromosome

size

-LRB-

including

artefactual

duplication

in

draft

genome

sequence

-RRB-

-LRB-

Mb

-RRB-

Previously

estimated

chromosome

size

-LRB-

Mb

-RRB-

Number

All

X

Y

UL

Total

bases

in

gaps

-LRB-

Mb

-RRB-

Number

347 296 336 343

337 275 195 249

122 163 193 168

115 40 104 102

95 113 108

Total

bases

in

gaps

-LRB-

Mb

-RRB-

Number

113 577 566

Total

bases

in

gapsI

-LRB-

Mb

-RRB-

sequenced

clones

that

could

not

be

associated

with

ngerprint

clone

contigs

UL

clone

contigs

that

could

not

be

reliably

placed

on

a

chromosome

Total

number

of

bases

in

the

draft

genome

sequence

excluding

gaps

Total

length

of

scaffold

-LRB-

including

gaps

contained

within

clones

-RRB-

is

Gb

Gaps

between

those

ngerprint

clone

contigs

that

contain

sequenced

clones

excluding

gaps

for

centromeres

For

un

nished

chromosomes

we

estimate

an

average

size

of

Mb

per

FCC

gap

based

on

retrospective

estimates

of

the

clone

coverage

of

chromosomes

and

Gap

estimates

for

chromosomes

and

are

taken

from

refs

k

Gaps

between

sequenced-clone

contigs

within

a

ngerprint

clone

contig

For

un

nished

chromosomes

we

estimate

sequenced

clone

gaps

at

Mb

each

based

on

evaluation

of

a

sample

of

these

gaps

Gaps

between

two

sequence

contigs

within

a

sequenced-clone

contig

I

We

estimate

the

average

number

of

bases

in

sequence

gaps

from

alignments

of

the

initial

sequence

contigs

of

un

nished

clones

-LRB-

see

text

-RRB-

and

extrapolation

to

the

whole

chromosome

Including

adjustments

for

estimates

of

the

sizes

of

the

short

arms

of

the

acrocentric

chromosomes

and

-LRB-

ref

-RRB-

estimates

for

the

centromere

and

heterochromatic

regions

of

chromosomes

and

-LRB-

refs

-RRB-

and

estimates

of

Mb

for

the

centromere

and

Mb

for

telomeric

heterochromatin

for

the

Y

The

sum

of

the

ve

lengths

in

the

preceding

columns

This

is

an

overestimate

because

the

draft

genome

sequence

contains

some

artefactual

sequence

owing

to

inability

to

correctly

to

merge

all

underlying

sequence

contigs

The

total

amount

of

artefactual

duplication

varies

among

chromosomes

the

overall

amount

is

estimated

by

computational

analysis

to

be

about

Mb

or

about

of

the

total

length

given

yielding

a

total

estimated

size

of

about

Mb

for

the

human

genome

Including

heterochromatic

regions

and

acrocentric

short

arm

-LRB-

s

-RRB-

279 251 221 197

198 176 163 148

140 143 148 142

118 107 100 104

263 255 214 203

194 183 171 155

145 144 144 143

114 109 106

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

ngerprint

map

However

many

involve

STSs

that

have

been

localized

on

only

one

or

two

of

the

previous

maps

or

that

occur

as

isolated

discrepancies

in

con

ict

with

several

anking

STSs

Many

of

these

cases

are

probably

due

to

errors

in

the

previous

maps

-LRB-

with

error

rates

for

individual

maps

estimated

at

-RRB-

Others

may

be

due

to

incorrect

assignment

of

the

STSs

to

the

draft

genome

sequence

-LRB-

by

the

electronic

polymerase

chain

reaction

-LRB-

e-PCR

-RRB-

computer

program

-RRB-

or

to

database

entries

that

contain

sequence

data

from

more

than

one

clone

-LRB-

owing

to

crosscontamination

-RRB-

Graphical

views

of

the

independent

data

sets

were

particularly

useful

in

detecting

problems

with

order

or

orientation

-LRB-

Fig.

-RRB-

Areas

of

con

ict

were

reviewed

and

corrected

if

supported

by

the

underlying

data

In

the

version

discussed

here

there

were

sequenced

clones

falling

in

sequenced-clone

contigs

with

STS

content

information

from

multiple

maps

that

disagreed

with

the

anking

clones

or

sequenced-clone

contigs

the

placement

of

these

clones

thus

remains

suspect

Four

of

these

instances

suggest

errors

in

the

ngerprint

map

whereas

the

others

suggest

errors

in

the

layout

of

sequenced

clones

These

cases

are

being

investigated

and

will

be

corrected

in

future

versions

Assembly

of

the

sequenced

clones

We

assessed

the

accuracy

of

the

assembly

by

using

a

set

of

draft

clones

comprising

Mb

for

which

nished

sequence

subsequently

became

The

initial

sequence

contigs

lack

information

about

order

and

orientation

and

GigAssembler

attempts

to

use

linking

data

to

infer

such

information

as

far

as

Starting

with

initial

sequence

contigs

that

were

unordered

and

unoriented

the

program

placed

of

the

initial

sequence

contigs

in

the

correct

orientation

and

in

the

correct

order

with

respect

to

one

another

In

a

separate

test

GigAssembler

was

tested

on

simulated

draft

data

produced

from

nished

sequence

on

chromosome

and

similar

results

were

obtained

Some

problems

remain

at

all

levels

First

errors

in

the

initial

sequence

contigs

persist

in

the

merged

sequence

contigs

built

from

them

and

can

cause

dif

culties

in

the

assembly

of

the

draft

genome

sequence

Second

GigAssembler

may

fail

to

merge

some

overlapping

sequences

because

of

poor

data

quality

allelic

differences

or

misassemblies

of

the

initial

sequence

contigs

this

may

result

in

apparent

local

duplication

of

a

sequence

We

have

estimated

by

various

methods

the

amount

of

such

artefactual

duplication

in

the

assembly

from

these

and

other

sources

to

be

about

Mb

On

the

other

hand

nearby

duplicated

sequences

may

occasionally

be

incorrectly

merged

Some

sequenced

clones

remain

incorrectly

placed

on

the

layout

as

discussed

above

and

others

-LRB-

-RRB-

remain

unplaced

The

ngerprint

map

has

undoubtedly

failed

to

resolve

some

closely

related

duplicated

regions

such

as

the

Williams

region

and

several

highly

repetitive

subtelomeric

and

pericentric

regions

-LRB-

see

below

-RRB-

Detailed

examination

and

sequence

nishing

may

be

required

to

sort

out

these

regions

precisely

as

has

been

done

with

chromosome

Finally

small

sequenced-clone

contigs

with

limited

or

no

STS

Table

Distribution

of

PHRAP

scores

in

the

draft

genome

sequence

PHRAP

score

Percentage

of

bases

in

the

draft

genome

sequence

PHRAP

scores

are

a

logarithmically

based

representation

of

the

error

probability

A

PHRAP

score

of

X

corresponds

to

an

error

probability

of

Thus

PHRAP

scores

of

and

correspond

to

accuracy

of

and

respectively

PHRAP

scores

are

derived

from

quality

scores

of

the

underlying

sequence

reads

used

in

sequence

assembly

See

landmark

content

remain

dif

cult

to

place

Full

utilization

of

the

higher

resolution

radiation

hybrid

map

-LRB-

the

TNG

map

-RRB-

may

help

in

Future

targeted

FISH

experiments

and

increased

map

continuity

will

also

facilitate

positioning

of

these

sequences

Genome

coverage

We

next

assessed

the

nature

of

the

gaps

within

the

draft

genome

sequence

and

attempted

to

estimate

the

fraction

of

the

human

genome

not

represented

within

the

current

version

Gaps

in

draft

genome

sequence

coverage

There

are

three

types

of

gap

in

the

draft

genome

sequence

gaps

within

un

nished

sequenced

clones

gaps

between

sequenced-clone

contigs

but

within

ngerprint

clone

contigs

and

gaps

between

ngerprint

clone

contigs

The

rst

two

types

are

relatively

straightforward

to

close

simply

by

performing

additional

sequencing

and

nishing

on

already

identi

ed

clones

Closing

the

third

type

may

require

screening

of

additional

large-insert

clone

libraries

and

possibly

new

technologies

for

the

most

recalcitrant

regions

We

consider

these

three

cases

in

turn

We

estimated

the

size

of

gaps

within

draft

clones

by

studying

instances

in

which

there

was

substantial

overlap

between

a

draft

clone

and

a

nished

clone

as

described

above

The

average

gap

size

in

these

draft

sequenced

clones

was

bp

although

the

precise

estimate

was

sensitive

to

certain

assumptions

in

the

analysis

Assuming

that

the

sequence

gaps

in

the

draft

genome

sequence

are

fairly

represented

by

this

sample

about

Mb

or

about

-LRB-

likely

range

-RRB-

of

sequence

may

lie

in

the

gaps

within

draft

sequenced

clones

The

gaps

between

sequenced-clone

contigs

but

within

ngerprint

clone

contigs

are

more

dif

cult

to

evaluate

directly

because

the

draft

genome

sequence

anking

many

of

the

gaps

is

often

not

precisely

aligned

with

the

ngerprinted

clones

However

most

are

much

smaller

than

a

single

BAC

In

fact

nearly

three-quarters

of

these

gaps

are

bridged

by

one

or

more

individual

BACs

as

indicated

by

linking

information

from

BAC

end

sequences

We

measured

the

sizes

of

a

subset

of

gaps

directly

by

examining

restriction

fragment

ngerprints

of

overlapping

clones

A

study

of

bridged

gaps

and

unbridged

gaps

gave

an

average

gap

size

of

kb

Allowing

for

the

possibility

that

these

gaps

may

not

be

fully

representative

and

that

some

restriction

fragments

are

not

included

in

the

calculation

a

more

conservative

estimate

of

gap

size

would

be

kb

This

would

indicate

that

about

Mb

or

of

the

human

genome

may

reside

in

the

gaps

between

sequenced-clone

contigs

This

sequence

should

be

readily

obtained

as

the

clones

spanning

them

are

sequenced

The

size

of

the

gaps

between

ngerprint

clone

contigs

was

estimated

by

comparing

the

ngerprint

maps

to

the

essentially

completed

chromosomes

and

The

analysis

shows

that

the

ngerprinted

BAC

clones

in

the

global

database

cover

of

the

sequenced

portions

of

those

The

published

sequences

of

these

chromosomes

also

contain

a

few

small

gaps

-LRB-

and

respectively

-RRB-

amounting

to

some

of

the

euchromatic

sequence

and

do

not

include

the

heterochromatic

portion

This

suggests

that

the

gaps

between

contigs

in

the

ngerprint

map

contain

about

of

the

euchromatic

genome

Experience

with

closure

of

such

gaps

on

chromosomes

and

suggests

that

many

of

these

gaps

are

less

than

one

clone

in

length

and

will

be

closed

by

clones

from

other

libraries

However

recovery

of

sequence

from

these

gaps

represents

the

most

challenging

aspect

of

producing

a

complete

nished

sequence

of

the

human

genome

As

another

measure

of

the

representation

of

the

BAC

libraries

has

found

BAC

or

cosmid

clones

that

link

to

telomeric

half-YACs

or

to

the

telomeric

sequence

itself

for

of

the

nonsatellite

telomeres

Thus

the

ngerprint

map

appears

to

have

no

substantial

gaps

in

these

regions

Many

of

the

pericentric

regions

are

also

represented

but

analysis

is

less

complete

here

-LRB-

see

below

-RRB-

Representation

of

random

raw

sequences

In

another

approach

to

measuring

coverage

we

compared

a

collection

of

random

raw

sequence

reads

to

the

existing

draft

genome

sequence

In

principle

NATURE

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Macmillan

Magazines

Ltd

articles

the

fraction

of

reads

matching

the

draft

genome

sequence

should

provide

an

estimate

of

genome

coverage

In

practice

the

comparison

is

complicated

by

the

need

to

allow

for

repeat

sequences

the

imperfect

sequence

quality

of

both

the

raw

sequence

and

the

draft

genome

sequence

and

the

possibility

of

polymorphism

Nonetheless

the

analysis

provides

a

reasonable

view

of

the

extent

to

which

the

genome

is

represented

in

the

draft

genome

sequence

and

the

public

databases

We

compared

the

raw

sequence

reads

against

both

the

sequences

used

in

the

construction

of

the

draft

genome

sequence

and

all

of

GenBank

using

the

BLAST

computer

program

Of

the

raw

sequence

reads

analysed

-LRB-

each

containing

at

least

bp

of

contiguous

non-repetitive

sequence

-RRB-

had

a

match

of

identity

with

a

sequenced

clone

indicating

that

of

the

genome

was

represented

in

sequenced

clones

The

estimate

is

subject

to

various

uncertainties

Most

serious

is

the

proportion

of

repeat

sequence

in

the

remainder

of

the

genome

If

the

unsequenced

portion

of

the

genome

is

unusually

rich

in

repeated

sequence

we

would

underestimate

its

size

-LRB-

although

the

excess

would

be

comprised

of

repeated

sequence

-RRB-

We

examined

those

raw

sequences

that

failed

to

match

by

comparing

them

to

the

other

publicly

available

sequence

resources

Fifty

-LRB-

-RRB-

had

matches

in

public

databases

containing

cDNA

sequences

STSs

and

similar

data

An

additional

-LRB-

or

of

the

remaining

raw

sequence

-RRB-

had

matches

to

the

whole-genome

shotgun

reads

discussed

above

-LRB-

consistent

with

the

idea

that

these

reads

cover

about

half

of

the

genome

-RRB-

We

also

examined

the

extent

of

genome

coverage

by

aligning

the

cDNA

sequences

for

genes

in

the

RefSeq

to

the

draft

genome

sequence

We

found

that

of

the

bases

of

these

cDNAs

could

be

aligned

to

the

draft

genome

sequence

at

high

stringency

-LRB-

at

least

identity

-RRB-

-LRB-

A

few

of

the

alignments

with

either

the

random

raw

sequence

reads

or

the

cDNAs

may

be

to

a

highly

similar

region

in

the

genome

but

such

matches

should

affect

the

estimate

of

genome

coverage

by

considerably

less

than

based

on

the

estimated

extent

of

duplication

within

the

genome

-LRB-

see

below

-RRB-

-RRB-

These

results

indicate

that

about

of

the

human

genome

is

represented

in

the

draft

genome

sequence

and

about

in

the

combined

publicly

available

sequence

databases

The

gure

of

agrees

well

with

our

independent

estimates

above

that

about

and

of

the

genome

reside

in

the

three

types

of

gap

in

the

draft

genome

sequence

Finally

a

small

experimental

check

was

performed

by

screening

a

large-insert

clone

library

with

probes

corresponding

to

of

the

whole

genome

shotgun

reads

that

failed

to

match

the

draft

genome

sequence

Five

hybridized

to

many

clones

from

different

ngerprint

clone

contigs

and

were

discarded

as

being

repetitive

Of

the

remaining

eleven

two

fell

within

sequenced

clones

-LRB-

presumably

within

sequence

gaps

of

the

rst

type

-RRB-

eight

fell

in

ngerprint

clone

contigs

but

between

sequenced

clones

-LRB-

gaps

of

the

second

type

-RRB-

and

one

failed

to

identify

clones

in

the

ngerprint

map

-LRB-

gaps

of

the

third

type

-RRB-

but

did

identify

clones

in

another

large-insert

library

Although

these

numbers

are

small

they

are

consistent

with

the

view

that

the

much

of

the

remaining

genome

sequence

lies

within

already

identi

ed

clones

in

the

current

map

Estimates

of

genome

and

chromosome

sizes

Informed

by

this

analysis

of

genome

coverage

we

proceeded

to

estimate

the

sizes

of

the

genome

and

each

of

the

chromosomes

-LRB-

Table

-RRB-

Beginning

with

the

current

assigned

sequence

for

each

chromosome

we

corrected

for

the

known

gaps

on

the

basis

of

their

estimated

sizes

-LRB-

see

above

-RRB-

We

attempted

to

account

for

the

sizes

of

centromeres

and

heterochromatin

neither

of

which

are

well

represented

in

the

draft

sequence

Finally

we

corrected

for

around

Mb

of

artefactual

duplication

in

the

assembly

We

arrived

at

a

total

human

genome

size

estimate

of

around

Mb

which

compares

favourably

with

previous

estimates

based

on

DNA

content

We

also

independently

estimated

the

size

of

the

euchromatic

portion

of

the

genome

by

determining

the

fraction

of

the

random

raw

sequences

that

matched

the

nished

portion

of

the

human

genome

-LRB-

whose

total

length

is

known

with

greater

precision

-RRB-

Twenty-nine

per

cent

of

these

raw

sequences

found

a

match

among

Mb

of

nonredundant

nished

sequence

This

leads

to

an

estimate

of

the

euchromatic

genome

size

of

Gb

This

agrees

reasonably

with

the

prediction

above

based

on

the

length

of

the

draft

genome

sequence

-LRB-

Table

-RRB-

Update

The

results

above

re

ect

the

data

on

October

New

data

are

continually

being

added

with

improvements

being

made

to

the

physical

map

new

clones

being

sequenced

to

close

gaps

and

draft

clones

progressing

to

full

shotgun

coverage

and

nishing

The

draft

genome

sequence

will

be

regularly

reassembled

and

publicly

released

Currently

the

physical

map

has

been

re

ned

such

that

the

number

of

ngerprint

clone

contigs

has

fallen

from

to

this

re

ects

the

elimination

of

some

artefactual

contigs

and

the

closure

of

some

gaps

The

sequence

coverage

has

risen

such

that

of

the

human

genome

is

now

represented

in

the

sequenced

clones

and

more

than

is

represented

in

the

combined

publicly

available

sequence

databases

The

total

amount

of

nished

sequence

is

now

around

Gb

Broad

genomic

landscape

What

biological

insights

can

be

gleaned

from

the

draft

sequence

In

this

section

we

consider

very

large-scale

features

of

the

draft

genome

sequence

the

distribution

of

GC

content

CpG

islands

and

recombination

rates

and

the

repeat

content

and

gene

content

of

the

human

genome

The

draft

genome

sequence

makes

it

possible

to

integrate

these

features

and

others

at

scales

ranging

from

individual

Figure

Screen

shot

from

UCSC

Draft

Human

Genome

Browser

See

NATURE

VOL

FEBRUARY

Figure

Screen

shot

from

the

Genome

Browser

of

Project

Ensembl

See

Macmillan

Magazines

Ltd

articles

nucleotides

to

collections

of

chromosomes

Unless

noted

all

analyses

were

conducted

on

the

assembled

draft

genome

sequence

described

above

Figure

provides

a

high-level

view

of

the

contents

of

the

draft

genome

sequence

at

a

scale

of

about

Mb

per

centimetre

Of

course

navigating

information

spanning

nearly

ten

orders

of

magnitude

requires

computational

tools

to

extract

the

full

value

We

have

created

and

made

freely

available

various

Genome

Browsers

Browsers

were

developed

and

are

maintained

by

the

University

of

California

at

Santa

Cruz

-LRB-

Fig.

-RRB-

and

the

EnsEMBL

project

of

the

European

Bioinformatics

Institute

and

the

Sanger

Centre

-LRB-

Fig.

-RRB-

Additional

browsers

have

been

created

URLs

are

listed

at

These

web-based

computer

tools

allow

users

to

view

an

annotated

display

of

the

draft

genome

sequence

with

the

ability

to

scroll

along

the

chromosomes

and

zoom

in

or

out

to

different

scales

They

include

the

nucleotide

sequence

sequence

contigs

clone

contigs

sequence

coverage

and

nishing

status

local

GC

content

CpG

islands

known

STS

markers

from

previous

genetic

and

physical

maps

families

of

repeat

sequences

known

genes

ESTs

and

mRNAs

predicted

genes

SNPs

and

sequence

similarities

with

other

organisms

-LRB-

currently

the

puffer

sh

Tetraodon

nigroviridis

-RRB-

These

browsers

will

be

updated

as

the

draft

genome

sequence

is

re

ned

and

corrected

as

additional

annotations

are

developed

In

addition

to

using

the

Genome

Browsers

one

can

download

Box

Sources

of

publicly

available

sequence

data

and

other

relevant

genomic

information

University

of

California

at

Santa

Cruz

Contains

the

assembly

of

the

draft

genome

sequence

used

in

this

paper

and

updates

human/Mapping

Washington

University

Contains

links

to

clone

and

accession

maps

of

the

human

genome

EBI/Sanger

Centre

Allows

access

to

DNA

and

protein

sequences

with

automatic

baseline

annotation

genome/guide

NCBI

Views

of

chromosomes

and

maps

and

loci

with

links

to

other

NCBI

resources

Gene

map

contains

data

and

viewers

for

radiation

hybrid

maps

of

EST-based

STSs

Oak

Ridge

National

Laboratory

from

these

sites

the

entire

draft

genome

sequence

together

with

the

annotations

in

a

computer-readable

format

The

sequences

of

the

underlying

sequenced

clones

are

all

available

through

the

public

sequence

databases

URLs

for

these

and

other

genome

websites

are

listed

in

Box

A

larger

list

of

useful

URLs

can

be

found

at

An

introduction

to

using

the

draft

genome

sequence

as

well

as

associated

databases

and

analytical

tools

is

provided

in

an

accompanying

In

addition

the

human

cytogenetic

map

has

been

integrated

with

the

draft

genome

sequence

as

part

of

a

related

project

The

BAC

Resource

established

dense

connections

between

the

maps

using

more

than

sequenced

large-insert

clones

that

had

been

cytogenetically

mapped

by

FISH

the

average

density

of

the

map

is

clones

per

Mb

Although

the

precision

of

the

integration

is

limited

by

the

resolution

of

FISH

the

links

provide

a

powerful

tool

for

the

analysis

of

cytogenetic

aberrations

in

inherited

diseases

and

cancer

These

cytogenetic

links

can

also

be

accessed

through

the

Genome

Browsers

Long-range

variation

in

GC

content

The

existence

of

GC-rich

and

GC-poor

regions

in

the

human

genome

was

rst

revealed

by

experimental

studies

involving

density

gradient

separation

which

indicated

substantial

variation

in

average

GC

content

among

large

fragments

Subsequent

studies

have

indicated

that

these

GC-rich

and

GC-poor

regions

may

have

different

biological

properties

such

as

gene

density

composition

of

repeat

sequences

correspondence

with

cytogenetic

bands

and

recombination

Many

of

these

studies

were

indirect

owing

to

the

lack

of

suf

cient

sequence

data

The

draft

genome

sequence

makes

it

possible

to

explore

the

variation

in

GC

content

in

a

direct

and

global

manner

Visual

inspection

-LRB-

Fig.

-RRB-

con

rms

that

local

GC

content

undergoes

substantial

long-range

excursions

from

its

genome-wide

average

of

If

the

genome

were

drawn

from

a

uniform

distribution

of

GC

content

the

local

GC

content

in

a

window

of

size

n

bp

should

be

-LRB-

-LRB-

-RRB-

-LRB-

-RRB-

n

-RRB-

Fluctuations

would

be

modest

with

the

standard

deviation

being

halved

as

the

window

size

is

quadrupled

for

example

and

for

windows

of

size

and

kb

The

draft

genome

sequence

however

contains

many

regions

with

much

more

extreme

variation

There

are

huge

regions

-LRB-

Mb

-RRB-

with

GC

content

far

from

the

average

For

example

the

most

distal

Mb

of

chromosome

-LRB-

from

the

telomere

to

about

STS

marker

-RRB-

has

an

average

GC

content

of

and

chromosome

has

a

region

-LRB-

roughly

between

STS

marker

and

Number

of

windows

Java

viewers

for

human

genome

data

RIKEN

and

the

University

of

Tokyo

Gives

an

overview

of

the

entire

human

genome

structure

The

SNP

Consortium

Includes

a

variety

of

ways

to

query

for

SNPs

in

the

human

genome

Online

Mendelian

Inheritance

in

Man

Contain

information

about

human

genes

and

disease

and

NHGRI

and

DOE

Contains

information

links

and

articles

on

a

wide

range

of

social

ethical

and

legal

issues

GC

content

Figure

Histogram

of

GC

content

of

windows

in

the

draft

genome

sequence

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Figure

Variation

in

GC

content

at

various

scales

The

GC

content

in

subregions

of

a

region

of

chromosome

is

plotted

starting

at

about

Mb

from

the

beginning

of

the

draft

genome

sequence

This

region

is

AT-rich

overall

Top

the

GC

content

of

the

entire

region

analysed

in

non-overlapping

windows

Middle

GC

content

of

the

rst

Mb

analysed

in

windows

Bottom

GC

content

of

the

rst

Mb

analysed

in

windows

At

this

scale

gaps

in

the

sequence

can

be

seen

-RRB-

with

only

GC

content

There

are

also

examples

of

large

shifts

in

GC

content

between

adjacent

multimegabase

regions

For

example

the

average

GC

content

on

chromosome

is

for

the

distal

Mb

but

drops

to

for

the

adjacent

Mb

There

are

regions

of

less

than

kb

with

even

wider

swings

in

GC

content

for

example

from

to

Long-range

variation

in

GC

content

is

evident

not

just

from

extreme

outliers

but

throughout

the

genome

The

distribution

of

average

GC

content

in

windows

across

the

draft

genome

sequence

is

shown

in

Fig.

The

spread

is

larger

than

predicted

by

a

uniform

process

Moreover

the

standard

deviation

barely

decreases

as

window

size

increases

by

successive

factors

of

four

and

for

windows

of

size

and

kb

The

distribution

is

also

notably

skewed

with

below

the

average

and

above

the

average

of

with

a

long

tail

of

GCrich

regions

Bernardi

and

proposed

that

the

long-range

variation

in

GC

content

may

re

ect

that

the

genome

is

composed

of

a

mosaic

of

compositionally

homogeneous

regions

that

they

dubbed

isochores

They

suggested

that

the

skewed

distribution

is

composed

of

ve

normal

distributions

corresponding

to

ve

distinct

types

of

isochore

-LRB-

and

with

GC

contents

of

and

respectively

-RRB-

We

studied

the

draft

genome

sequence

to

see

whether

strict

isochores

could

be

identi

ed

For

example

the

sequence

was

divided

into

windows

and

each

window

was

subdivided

into

subwindows

We

calculated

the

average

GC

content

for

each

window

and

subwindow

and

investigated

how

much

of

the

variance

in

the

GC

content

of

subwindows

across

the

genome

can

be

statistically

explained

by

the

average

GC

content

in

each

window

About

three-quarters

of

the

genome-wide

variance

among

windows

can

be

statistically

explained

by

the

average

GC

content

of

windows

that

contain

them

but

the

residual

variance

among

subwindows

-LRB-

standard

deviation

-RRB-

is

still

far

too

large

to

be

consistent

with

a

homogeneous

distribution

In

fact

the

hypothesis

of

homogeneity

could

be

rejected

for

each

window

in

the

draft

genome

sequence

Similar

results

were

obtained

with

other

window

and

subwindow

sizes

Some

of

the

local

heterogeneity

in

GC

content

is

attributable

to

transposable

element

insertions

-LRB-

see

below

-RRB-

Such

repeat

elements

typically

have

a

higher

GC

content

than

the

surrounding

sequence

with

the

effect

being

strongest

for

the

most

recent

insertions

These

results

rule

out

a

strict

notion

of

isochores

as

compositionally

homogeneous

Instead

there

is

substantial

variation

at

many

different

scales

as

illustrated

in

Fig.

Although

isochores

do

not

appear

to

merit

the

pre

x

iso

the

genome

clearly

does

contain

large

regions

of

distinctive

GC

content

and

it

is

likely

to

be

NATURE

VOL

FEBRUARY

worth

rede

ning

the

concept

so

that

it

becomes

possible

rigorously

to

partition

the

genome

into

regions

In

the

absence

of

a

precise

de

nition

we

will

loosely

refer

to

such

regions

as

GC

content

domains

in

the

context

of

the

discussion

below

Fickett

et

al.

have

explored

a

model

in

which

the

underlying

preference

for

a

particular

GC

content

drifts

continuously

throughout

the

genome

an

approach

that

bears

further

examination

has

proposed

that

the

boundaries

between

GC

content

domains

can

in

some

cases

be

predicted

by

a

hidden

Markov

model

with

one

state

representing

a

GC-rich

region

and

one

representing

an

AT-rich

region

We

found

that

this

approach

tended

to

identify

only

very

short

domains

of

less

than

a

kilobase

-LRB-

data

not

shown

-RRB-

but

variants

of

this

approach

deserve

further

attention

The

correlation

between

GC

content

domains

and

various

biological

properties

is

of

great

interest

and

this

is

likely

to

be

the

most

fruitful

route

to

understanding

the

basis

of

variation

in

GC

content

As

described

below

we

con

rm

the

existence

of

strong

correlations

with

both

repeat

content

and

gene

density

Using

the

integration

between

the

draft

genome

sequence

and

the

cytogenetic

map

described

above

it

is

possible

to

con

rm

a

statistically

signi

cant

correlation

between

GC

content

and

Giemsa

bands

-LRB-

Gbands

-RRB-

For

example

of

large-insert

clones

mapping

to

the

darkest

G-bands

are

in

regions

of

low

GC

content

-LRB-

average

-RRB-

whereas

more

than

of

clones

mapping

to

the

lightest

Gbands

are

in

regions

of

high

GC

content

-LRB-

average

-RRB-

Estimated

band

locations

can

be

seen

in

Fig.

and

viewed

in

the

context

of

other

genome

annotation

at

mapPlots

and

CpG

islands

A

related

topic

is

the

distribution

of

so-called

CpG

islands

across

the

genome

The

dinucleotide

CpG

is

notable

because

it

is

greatly

under-represented

in

human

DNA

occurring

at

only

about

one

fth

of

the

roughly

frequency

that

would

be

expected

by

simply

multiplying

the

typical

fraction

of

Cs

and

Gs

-LRB-

-RRB-

The

de

cit

occurs

because

most

CpG

dinucleotides

are

methylated

on

the

cytosine

base

and

spontaneous

deamination

of

methyl-C

residues

gives

rise

to

T

residues

-LRB-

Spontaneous

deamination

of

ordinary

cytosine

residues

gives

rise

to

uracil

residues

that

are

readily

recognized

and

repaired

by

the

cell

-RRB-

As

a

result

methylCpG

dinucleotides

steadily

mutate

to

TpG

dinucleotides

However

the

genome

contains

many

CpG

islands

in

which

CpG

dinucleotides

are

not

methylated

and

occur

at

a

frequency

closer

to

that

predicted

by

the

local

GC

content

CpG

islands

are

of

particular

interest

because

many

are

associated

with

the

ends

of

We

searched

the

draft

genome

sequence

for

CpG

islands

Ideally

they

should

be

de

ned

by

directly

testing

for

the

absence

of

cytosine

methylation

but

that

was

not

practical

for

this

report

There

are

Macmillan

Magazines

Ltd

articles

Table

Number

of

CpG

islands

by

GC

content

GC

content

of

island

Total

Number

of

islands

Percentage

of

islands

Nucleotides

in

islands

Percentage

of

nucleotides

in

islands

Potential

CpG

islands

were

identi

ed

by

searching

the

draft

genome

sequence

one

base

at

a

time

scoring

each

dinucleotide

-LRB-

for

GC

for

others

-RRB-

and

identifying

maximally

scoring

segments

Each

segment

was

then

evaluated

to

determine

GC

content

-LRB-

-RRB-

length

-LRB-

-RRB-

and

ratio

of

observed

proportion

of

GC

dinucleotides

to

the

expected

proportion

on

the

basis

of

the

GC

content

of

the

segment

-LRB-

-RRB-

using

a

modi

cation

of

a

program

developed

by

G.

Micklem

-LRB-

personal

communication

-RRB-

unusually

low

islands

per

Mb

and

chromosomes

and

have

islands

per

Mb

The

extreme

outlier

is

chromosome

with

islands

per

Mb

Similar

trends

are

seen

when

considering

the

percentage

of

bases

contained

in

CpG

islands

The

relative

density

of

CpG

islands

correlates

reasonably

well

with

estimates

of

relative

gene

density

on

these

chromosomes

based

both

on

previous

mapping

studies

involving

ESTs

-LRB-

Fig.

-RRB-

and

on

the

distribution

of

gene

predictions

discussed

below

Comparison

of

genetic

and

physical

distance

Number

of

genes

per

Mb

Distance

from

centromere

-LRB-

cM

-RRB-

various

computer

programs

that

attempt

to

identify

CpG

islands

on

the

basis

of

primary

sequence

alone

These

programs

differ

in

some

important

respects

-LRB-

such

as

how

aggressively

they

subdivide

long

CpG-containing

regions

-RRB-

and

the

precise

correspondence

with

experimentally

undermethylated

islands

has

not

been

validated

Nevertheless

there

is

a

good

correlation

and

computational

analysis

thus

provides

a

reasonable

picture

of

the

distribution

of

CpG

islands

in

the

genome

To

identify

CpG

islands

we

used

the

de

nition

proposed

by

Gardiner-Garden

and

and

embodied

in

a

computer

program

We

searched

the

draft

genome

sequence

for

CpG

islands

using

both

the

full

sequence

and

the

sequence

masked

to

eliminate

repeat

sequences

The

number

of

regions

satisfying

the

de

nition

of

a

CpG

island

was

in

the

full

sequence

and

in

the

repeat-masked

sequence

The

difference

re

ects

the

fact

that

some

repeat

elements

-LRB-

notably

Alu

-RRB-

are

GC-rich

Although

some

of

these

repeat

elements

may

function

as

control

regions

it

seems

unlikely

that

most

of

the

apparent

CpG

islands

in

repeat

sequences

are

functional

Accordingly

we

focused

on

those

in

the

non-repeated

sequence

The

count

of

CpG

islands

is

reasonably

close

to

the

previous

estimate

of

about

-LRB-

ref

as

modi

ed

by

ref

-RRB-

Most

of

the

islands

are

short

with

GC

content

-LRB-

Table

-RRB-

More

than

of

the

islands

are

less

than

bp

long

and

more

than

are

less

than

bp

The

longest

CpG

island

-LRB-

on

chromosome

-RRB-

is

bp

long

and

are

longer

than

bp

Some

of

the

larger

islands

contain

ribosomal

pseudogenes

although

RNA

genes

and

pseudogenes

account

for

only

a

small

proportion

of

all

islands

-LRB-

-RRB-

The

smaller

islands

are

consistent

with

their

previously

hypothesized

function

but

the

role

of

these

larger

islands

is

uncertain

The

density

of

CpG

islands

varies

substantially

among

some

of

the

chromosomes

Most

chromosomes

have

islands

per

Mb

with

a

mean

of

islands

per

Mb

However

chromosome

Y

has

an

The

draft

genome

sequence

makes

it

possible

to

compare

genetic

and

physical

distances

and

thereby

to

explore

variation

in

the

rate

of

recombination

across

the

human

chromosomes

We

focus

here

on

large-scale

variation

Finer

variation

is

examined

in

an

accompanying

The

genetic

and

physical

maps

are

integrated

by

polymorphic

loci

from

the

Marsh

eld

genetic

whose

positions

are

known

in

terms

of

centimorgans

-LRB-

cM

-RRB-

and

Mb

along

the

chromosomes

Figure

shows

the

comparison

of

the

draft

genome

sequence

for

chromosome

with

the

male

female

and

sex-averaged

maps

One

can

calculate

the

approximate

ratio

of

cM

per

Mb

across

a

chromosome

-LRB-

re

ected

in

the

slopes

in

Fig.

-RRB-

and

the

average

recombination

rate

for

each

chromosome

arm

Two

striking

features

emerge

from

analysis

of

these

data

First

the

average

recombination

rate

increases

as

the

length

of

the

chromosome

arm

decreases

-LRB-

Fig.

-RRB-

Long

chromosome

arms

have

an

average

recombination

rate

of

about

cM

per

Mb

whereas

the

shortest

arms

are

in

the

range

of

cM

per

Mb

A

similar

trend

has

been

seen

in

the

yeast

despite

the

fact

that

the

physical

scale

is

nearly

times

as

small

Moreover

experimental

studies

have

shown

that

lengthening

or

shortening

yeast

chromosomes

results

in

a

compensatory

change

in

recombination

The

second

observation

is

that

the

recombination

rate

tends

to

be

suppressed

near

the

centromeres

and

higher

in

the

distal

portions

of

most

chromosomes

with

the

increase

largely

in

the

terminal

140 130 120 110

X

Sex-averaged

Male

Female

80 90 100 110

120 130 140

Centromere

Position

-LRB-

Mb

-RRB-

Number

of

CpG

islands

per

Mb

Figure

Number

of

CpG

islands

per

Mb

for

each

chromosome

plotted

against

the

number

of

genes

per

Mb

-LRB-

the

number

of

genes

was

taken

from

-LRB-

ref

-RRB-

-RRB-

Chromosomes

and

particularly

are

clear

outliers

with

a

density

of

CpG

islands

that

is

even

greater

than

would

be

expected

from

the

high

gene

counts

for

these

four

chromosomes

Figure

Distance

in

cM

along

the

genetic

map

of

chromosome

plotted

against

position

in

Mb

in

the

draft

genome

sequence

Female

male

and

sex-averaged

maps

are

shown

Female

recombination

rates

are

much

higher

than

male

recombination

rates

The

increased

slopes

at

either

end

of

the

chromosome

re

ect

the

increased

rates

of

recombination

per

Mb

near

the

telomeres

Conversely

the

atter

slope

near

the

centromere

shows

decreased

recombination

there

especially

in

male

meiosis

This

is

typical

of

the

other

chromosomes

as

well

-LRB-

see

mapPlots

-RRB-

Discordant

markers

may

be

map

marker

placement

or

assembly

errors

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

Mb

The

increase

is

most

pronounced

in

the

male

meiotic

map

The

effect

can

be

seen

for

example

from

the

higher

slope

at

both

ends

of

chromosome

-LRB-

Fig.

-RRB-

Regional

and

sex-speci

c

effects

have

been

observed

for

chromosome

-LRB-

refs

-RRB-

Why

is

recombination

higher

on

smaller

chromosome

arms

A

higher

rate

would

increase

the

likelihood

of

at

least

one

crossover

during

meiosis

on

each

chromosome

arm

as

is

generally

observed

in

human

chiasmata

Crossovers

are

believed

to

be

necessary

for

normal

meiotic

disjunction

of

homologous

chromosome

pairs

in

eukaryotes

An

extreme

example

is

the

pseudoautosomal

regions

on

chromosomes

Xp

and

Yp

which

pair

during

male

meiosis

this

physical

region

of

only

Mb

has

a

genetic

length

of

cM

-LRB-

corresponding

to

cM

per

Mb

-RRB-

with

the

result

that

a

crossover

is

virtually

assured

Mechanistically

the

increased

rate

of

recombination

on

shorter

chromosome

arms

could

be

explained

if

once

an

initial

recombination

event

occurs

additional

nearby

events

are

blocked

by

positive

crossover

interference

on

each

arm

Evidence

from

yeast

mutants

in

which

interference

is

abolished

shows

that

interference

plays

a

key

role

in

distributing

a

limited

number

of

crossovers

among

the

various

chromosome

arms

in

An

alternative

possibility

is

that

a

checkpoint

mechanism

scans

for

and

enforces

the

presence

of

at

least

one

crossover

on

each

chromosome

arm

Variation

in

recombination

rates

along

chromosomes

and

between

the

sexes

is

likely

to

re

ect

variation

in

the

initiation

of

meiosis-induced

double-strand

breaks

-LRB-

DSBs

-RRB-

that

initiate

recombination

DSBs

in

yeast

have

been

associated

with

open

rather

than

with

speci

c

DNA

sequence

motifs

With

the

availability

of

the

draft

genome

sequence

it

should

be

possible

to

explore

in

an

analogous

manner

whether

variation

in

human

recombination

rates

re

ects

systematic

differences

in

chromosome

accessibility

during

meiosis

Amoeba

This

mystery

-LRB-

the

C-value

paradox

-RRB-

was

largely

resolved

with

the

recognition

that

genomes

can

contain

a

large

quantity

of

repetitive

sequence

far

in

excess

of

that

devoted

to

protein-coding

genes

-LRB-

reviewed

in

refs

-RRB-

In

the

human

coding

sequences

comprise

less

than

of

the

genome

-LRB-

see

below

-RRB-

whereas

repeat

sequences

account

for

at

least

and

probably

much

more

Broadly

the

repeats

fall

into

ve

classes

-LRB-

-RRB-

transposon-derived

repeats

often

referred

to

as

interspersed

repeats

-LRB-

-RRB-

inactive

-LRB-

partially

-RRB-

retroposed

copies

of

cellular

genes

-LRB-

including

protein-coding

genes

and

small

structural

RNAs

-RRB-

usually

referred

to

as

processed

pseudogenes

-LRB-

-RRB-

simple

sequence

repeats

consisting

of

direct

repetitions

of

relatively

short

k-mers

such

as

-LRB-

A

-RRB-

n

-LRB-

CA

-RRB-

n

or

-LRB-

CGG

-RRB-

n

-LRB-

-RRB-

segmental

duplications

consisting

of

blocks

of

around

kb

that

have

been

copied

from

one

region

of

the

genome

into

another

region

and

-LRB-

-RRB-

blocks

of

tandemly

repeated

sequences

such

as

at

centromeres

telomeres

the

short

arms

of

acrocentric

chromosomes

and

ribosomal

gene

clusters

-LRB-

These

regions

are

intentionally

under-represented

in

the

draft

genome

sequence

and

are

not

discussed

here

-RRB-

Repeats

are

often

described

as

junk

and

dismissed

as

uninteresting

However

they

actually

represent

an

extraordinary

trove

of

information

about

biological

processes

The

repeats

constitute

a

rich

palaeontological

record

holding

crucial

clues

about

evolutionary

events

and

forces

As

passive

markers

they

provide

assays

for

studying

processes

of

mutation

and

selection

It

is

possible

to

recognize

cohorts

of

repeats

born

at

the

same

time

and

to

follow

their

fates

in

different

regions

of

the

genome

or

in

different

species

As

active

agents

repeats

have

reshaped

the

genome

by

causing

ectopic

rearrangements

creating

entirely

new

genes

modifying

and

reshuf

ing

existing

genes

and

modulating

overall

GC

content

They

also

shed

light

on

chromosome

structure

and

dynamics

and

provide

tools

for

medical

genetic

and

population

genetic

studies

The

human

is

the

rst

repeat-rich

genome

to

be

sequenced

and

so

we

investigated

what

information

could

be

gleaned

from

this

majority

component

of

the

human

genome

Although

some

of

the

general

observations

about

repeats

were

suggested

by

previous

studies

the

draft

genome

sequence

provides

the

rst

comprehensive

view

allowing

some

questions

to

be

resolved

and

new

mysteries

to

emerge

Transposon-derived

repeats

Repeat

content

of

the

human

genome

A

puzzling

observation

in

the

early

days

of

molecular

biology

was

that

genome

size

does

not

correlate

well

with

organismal

complexity

For

example

Homo

sapiens

has

a

genome

that

is

times

as

large

as

that

of

the

yeast

S.

cerevisiae

but

times

as

small

as

that

of

Recombination

rate

-LRB-

cM

per

Mb

-RRB-

60 80 100 120

Length

of

chromosome

arm

-LRB-

Mb

-RRB-

Figure

Rate

of

recombination

averaged

across

the

euchromatic

portion

of

each

chromosome

arm

plotted

against

the

length

of

the

chromosome

arm

in

Mb

For

large

chromosomes

the

average

recombination

rates

are

very

similar

but

as

chromosome

arm

length

decreases

average

recombination

rates

rise

markedly

NATURE

VOL

FEBRUARY

Most

human

repeat

sequence

is

derived

from

transposable

We

can

currently

recognize

about

of

the

genome

as

belonging

to

this

class

Much

of

the

remaining

unique

DNA

must

also

be

derived

from

ancient

transposable

element

copies

that

have

diverged

too

far

to

be

recognized

as

such

To

describe

our

analyses

of

interspersed

repeats

it

is

necessary

brie

y

to

review

the

relevant

features

of

human

transposable

elements

Classes

of

transposable

elements

In

mammals

almost

all

transposable

elements

fall

into

one

of

four

types

-LRB-

Fig.

-RRB-

of

which

three

transpose

through

RNA

intermediates

and

one

transposes

directly

as

DNA

These

are

long

interspersed

elements

-LRB-

LINEs

-RRB-

short

interspersed

elements

-LRB-

SINEs

-RRB-

LTR

retrotransposons

and

DNA

transposons

LINEs

are

one

of

the

most

ancient

and

successful

inventions

in

eukaryotic

genomes

In

humans

these

transposons

are

about

kb

long

harbour

an

internal

polymerase

II

promoter

and

encode

two

open

reading

frames

-LRB-

ORFs

-RRB-

Upon

translation

a

LINE

RNA

assembles

with

its

own

encoded

proteins

and

moves

to

the

nucleus

where

an

endonuclease

activity

makes

a

single-stranded

nick

and

the

reverse

transcriptase

uses

the

nicked

DNA

to

prime

reverse

transcription

from

the

end

of

the

LINE

RNA

Reverse

transcription

frequently

fails

to

proceed

to

the

end

resulting

in

many

truncated

nonfunctional

insertions

Indeed

most

LINE-derived

repeats

are

short

with

an

average

size

of

bp

for

all

copies

and

a

median

size

of

bp

for

copies

of

the

currently

active

element

-LRB-

-RRB-

New

insertion

sites

are

anked

by

a

small

Macmillan

Magazines

Ltd

articles

Classes

of

interspersed

repeat

in

the

human

genome

Length

LINEs

SINEs

Retrovirus-like

elements

Autonomous

Non-autonomous

Autonomous

Non-autonomous

gag

AB

-LRB-

pol

-RRB-

AAA

Copy

number

Fraction

of

genome

kb

bp

AAA

pol

-LRB-

env

-RRB-

kb

kb

-LRB-

gag

-RRB-

DNA

transposon

fossils

Autonomous

Non-autonomous

transposase

kb

bp

Figure

Almost

all

transposable

elements

in

mammals

fall

into

one

of

four

classes

See

text

for

details

target

site

duplication

of

bp

The

LINE

machinery

is

believed

to

be

responsible

for

most

reverse

transcription

in

the

genome

including

the

retrotransposition

of

the

non-autonomous

and

the

creation

of

processed

Three

distantly

related

LINE

families

are

found

in

the

human

genome

and

Only

is

still

active

SINEs

are

wildly

successful

freeloaders

on

the

backs

of

LINE

elements

They

are

short

-LRB-

about

bp

-RRB-

harbour

an

internal

polymerase

III

promoter

and

encode

no

proteins

These

nonautonomous

transposons

are

thought

to

use

the

LINE

machinery

for

transposition

Indeed

most

SINEs

live

by

sharing

the

end

with

a

resident

LINE

The

promoter

regions

of

all

known

SINEs

are

derived

from

tRNA

sequences

with

the

exception

of

a

single

monophyletic

family

of

SINEs

derived

from

the

signal

recognition

particle

component

This

family

which

also

does

not

share

its

end

with

a

LINE

includes

the

only

active

SINE

in

the

human

genome

the

Alu

element

By

contrast

the

mouse

has

both

tRNA-derived

and

SINEs

The

human

genome

contains

three

distinct

monophyletic

families

of

SINEs

the

active

Alu

and

the

inactive

MIR

and

LTR

retroposons

are

anked

by

long

terminal

direct

repeats

that

contain

all

of

the

necessary

transcriptional

regulatory

elements

The

autonomous

elements

-LRB-

retrotransposons

-RRB-

contain

gag

and

pol

genes

which

encode

a

protease

reverse

transcriptase

RNAse

H

and

integrase

Exogenous

retroviruses

seem

to

have

arisen

from

endogenous

retrotransposons

by

acquisition

of

a

cellular

envelope

gene

-LRB-

env

-RRB-

Transposition

occurs

through

the

retroviral

mechanism

with

reverse

transcription

occurring

in

a

cytoplasmic

virus-like

particle

primed

by

a

tRNA

-LRB-

in

contrast

to

the

nuclear

location

and

chromosomal

priming

of

LINEs

-RRB-

Although

a

variety

of

LTR

retrotransposons

exist

only

the

vertebrate-speci

c

endogenous

retroviruses

-LRB-

ERVs

-RRB-

appear

to

have

been

active

in

the

mammalian

genome

Mammalian

retroviruses

fall

into

three

classes

-LRB-

I

III

-RRB-

each

comprising

many

families

with

independent

origins

Most

-LRB-

-RRB-

of

the

LTR

retroposon-derived

fossils

consist

only

of

an

isolated

LTR

with

the

internal

sequence

having

been

lost

by

homologous

recombination

between

the

anking

LTRs

DNA

transposons

resemble

bacterial

transposons

having

terminal

inverted

repeats

and

encoding

a

transposase

that

binds

near

the

inverted

repeats

and

mediates

mobility

through

a

cut-and-paste

mechanism

The

human

genome

contains

at

least

seven

major

classes

of

DNA

transposon

which

can

be

subdivided

into

many

families

with

independent

-LRB-

see

RepBase

girinst.org/,server/repbase.html

-RRB-

DNA

transposons

tend

to

have

short

life

spans

within

a

species

This

can

be

explained

by

contrasting

the

modes

of

transposition

of

DNA

transposons

and

LINE

elements

LINE

transposition

tends

to

involve

only

functional

elements

owing

to

the

cis-preference

by

which

LINE

proteins

assemble

with

the

RNA

from

which

they

were

translated

By

contrast

DNA

transposons

can

not

exercise

a

cis-preference

the

encoded

transposase

is

produced

in

the

cytoplasm

and

when

it

returns

to

the

nucleus

it

can

not

distinguish

active

from

inactive

elements

As

inactive

copies

accumulate

in

the

genome

transposition

becomes

less

ef

cient

This

checks

the

expansion

of

any

DNA

transposon

family

and

in

due

course

causes

it

to

die

out

To

survive

DNA

transposons

must

eventually

move

by

horizontal

transfer

to

virgin

genomes

and

there

is

considerable

evidence

for

such

Transposable

elements

employ

different

strategies

to

ensure

their

evolutionary

survival

LINEs

and

SINEs

rely

almost

exclusively

on

vertical

transmission

within

the

host

-LRB-

but

see

refs

-RRB-

DNA

transposons

are

more

promiscuous

requiring

relatively

frequent

horizontal

transfer

LTR

retroposons

use

both

strategies

with

some

being

long-term

active

residents

of

the

human

genome

-LRB-

such

as

members

of

the

ERVL

family

-RRB-

and

others

having

only

short

residence

times

Table

Number

of

copies

and

fraction

of

genome

for

classes

of

interspersed

repeat

Number

of

copies

-LRB-

-RRB-

Total

number

of

bases

in

the

draft

genome

sequence

-LRB-

Mb

-RRB-

Fraction

of

the

draft

genome

sequence

-LRB-

-RRB-

Number

of

families

-LRB-

subfamilies

-RRB-

SINEs

Alu

MIR

LINEs

LTR

elements

ERV-class

I

ERV

-LRB-

K

-RRB-

class

II

ERV

-LRB-

L

-RRB-

class

III

MaLR

DNA

elements

hAT

group

Zaphod

group

Mariner

PiggyBac-like

Unclassi

ed

Unclassi

ed

Total

interspersed

repeats

393 75 868 516

315 37 443 112

83 240 294 182

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

number

of

copies

and

base

pair

contributions

of

the

major

classes

and

subclasses

of

transposable

elements

in

the

human

genome

Data

extracted

from

a

RepeatMasker

analysis

of

the

draft

genome

sequence

-LRB-

RepeatMasker

version

sensitive

settings

using

RepBase

Update

-RRB-

In

calculating

percentages

RepeatMasker

excluded

the

runs

of

Ns

linking

the

contigs

in

the

draft

genome

sequence

In

the

last

column

separate

consensus

sequences

in

the

repeat

databases

are

considered

subfamilies

rather

than

families

when

the

sequences

are

closely

related

or

related

through

intermediate

subfamilies

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

Census

of

human

repeats

We

began

by

taking

a

census

of

the

transposable

elements

in

the

draft

genome

sequence

using

a

recently

updated

version

of

the

RepeatMasker

program

-LRB-

version

-RRB-

run

under

sensitive

settings

-LRB-

see

genome.washington.edu

-RRB-

This

program

scans

sequences

to

identify

full-length

and

partial

members

of

all

known

repeat

families

represented

in

RepBase

Update

-LRB-

version

see

girinst.org/,server/repbase.html

and

ref

-RRB-

Table

shows

the

number

of

copies

and

fraction

of

the

draft

genome

sequence

occupied

by

each

of

the

four

major

classes

and

the

main

subclasses

The

precise

count

of

repeats

is

obviously

underestimated

because

the

genome

sequence

is

not

nished

but

their

density

and

other

properties

can

be

stated

with

reasonable

con

dence

Currently

recognized

SINEs

LINEs

LTR

retroposons

and

DNA

transposon

copies

comprise

and

of

the

sequence

respectively

We

expect

these

densities

to

grow

as

more

repeat

families

are

recognized

among

which

will

be

lower

copy

number

LTR

elements

and

DNA

transposons

and

possibly

high

copy

number

ancient

-LRB-

highly

diverged

-RRB-

repeats

Age

distribution

The

age

distribution

of

the

repeats

in

the

human

genome

provides

a

rich

fossil

record

stretching

over

several

hundred

million

years

The

ancestry

and

approximate

age

of

each

fossil

can

be

inferred

by

exploiting

the

fact

that

each

copy

is

derived

from

and

therefore

initially

carried

the

sequence

of

a

then-active

transposon

and

being

generally

under

no

functional

constraint

has

accumulated

mutations

randomly

and

independently

of

other

copies

We

can

infer

the

sequence

of

the

ancestral

active

elements

by

clustering

the

modern

derivatives

into

phylogenetic

trees

and

building

a

consensus

based

on

the

multiple

sequence

alignment

of

a

cluster

of

copies

Using

available

consensus

sequences

for

known

repeat

subfamilies

we

calculated

the

per

cent

divergence

from

the

inferred

ancestral

active

transposon

for

each

of

three

million

interspersed

repeats

in

the

draft

genome

sequence

The

percentage

of

sequence

divergence

can

be

converted

into

an

approximate

age

in

millions

of

years

-LRB-

Myr

-RRB-

on

the

basis

of

evolutionary

information

Care

is

required

in

calibrating

the

clock

because

the

rate

of

sequence

divergence

may

not

be

constant

over

time

or

between

The

relative-rate

can

be

used

to

calculate

the

sequence

divergence

that

accumulated

in

a

lineage

after

a

given

timepoint

on

the

basis

of

comparison

with

a

sibling

species

that

diverged

at

that

time

and

an

outgroup

species

For

example

the

substitution

rate

over

roughly

the

last

Myr

in

the

human

lineage

can

be

calculated

by

using

old

world

monkeys

-LRB-

which

diverged

about

Myr

ago

-RRB-

as

a

sibling

species

and

new

world

monkeys

as

an

outgroup

We

have

used

currently

available

calibrations

for

the

human

lineage

but

the

issue

should

be

revisited

as

sequence

information

becomes

available

from

different

mammals

Figure

shows

the

representation

of

various

classes

of

trans

a

b

Fraction

of

human

genome

comprised

by

repeat

class

-LRB-

-RRB-

Fraction

of

human

genome

comprised

by

repeat

class

-LRB-

-RRB-

Per

cent

substitution

from

consensus

sequences

Ranges

of

substitution

level

representing

million

years

each

d

Fraction

of

mouse

genome

comprised

by

repeat

class

-LRB-

-RRB-

c

Fraction

of

mouse

genome

comprised

by

repeat

class

-LRB-

-RRB-

Per

cent

substitution

from

consensus

sequences

SINE/Alu

SINE/MIR

SINE/other

LTR

DNA

Ranges

of

substitution

level

representing

million

years

each

Figure

Age

distribution

of

interspersed

repeats

in

the

human

and

mouse

genomes

Bases

covered

by

interspersed

repeats

were

sorted

by

their

divergence

from

their

consensus

sequence

-LRB-

which

approximates

the

repeat

's

original

sequence

at

the

time

of

insertion

-RRB-

The

average

number

of

substitutions

per

bp

-LRB-

substitution

level

K

-RRB-

was

calculated

from

the

mismatch

level

p

assuming

equal

frequency

of

all

substitutions

-LRB-

the

one-parameter

Jukes

Cantor

model

K

-LRB-

-RRB-

-RRB-

This

model

tends

to

underestimate

higher

substitution

levels

CpG

dinucleotides

in

the

consensus

were

excluded

from

the

substitution

level

calculations

because

the

C!T

transition

rate

in

CpG

pairs

is

about

tenfold

higher

than

other

transitions

and

causes

distortions

in

comparing

NATURE

VOL

FEBRUARY

transposable

elements

with

high

and

low

CpG

content

a

The

distribution

for

the

human

genome

in

bins

corresponding

to

increments

in

substitution

levels

b

The

data

grouped

into

bins

representing

roughly

equal

time

periods

of

Myr

c

d

Equivalent

data

for

available

mouse

genomic

sequence

There

is

a

different

correspondence

between

substitution

levels

and

time

periods

owing

to

different

rates

of

nucleotide

substitution

in

the

two

species

The

correspondence

between

substitution

levels

and

time

periods

was

largely

derived

from

three-way

species

comparisons

-LRB-

relative

rate

-RRB-

with

the

age

estimates

based

on

fossil

data

Human

divergence

from

gibbon

Myr

old

world

monkey

Myr

prosimians

Myr

eutherian

mammalian

radiation

Myr

Macmillan

Magazines

Ltd

articles

posable

elements

in

categories

re

ecting

equal

amounts

of

sequence

divergence

In

Fig.

the

data

are

grouped

into

four

bins

corresponding

to

successive

periods

on

the

basis

of

an

approximate

clock

Figure

shows

the

mean

ages

of

various

subfamilies

of

DNA

transposons

Several

facts

are

apparent

from

these

graphs

First

most

interspersed

repeats

in

the

human

genome

predate

the

eutherian

radiation

This

is

a

testament

to

the

extremely

slow

rate

with

which

nonfunctional

sequences

are

cleared

from

vertebrate

genomes

-LRB-

see

below

concerning

comparison

with

the

y

-RRB-

Second

LINE

and

SINE

elements

have

extremely

long

lives

The

monophyletic

and

Alu

lineages

are

at

least

and

Myr

old

respectively

In

earlier

times

the

reigning

transposons

were

and

The

SINE

MIR

was

perfectly

adapted

for

reverse

transcription

by

as

it

carried

the

same

sequence

at

its

end

When

became

extinct

Myr

ago

it

spelled

the

doom

of

MIR

Third

there

were

two

major

peaks

of

DNA

transposon

activity

-LRB-

Fig.

-RRB-

The

rst

involved

Charlie

elements

and

occurred

long

before

the

eutherian

radiation

the

second

involved

Tigger

elements

and

occurred

after

this

radiation

Because

DNA

transposons

can

produce

large-scale

chromosome

it

is

possible

that

widespread

activity

could

be

involved

in

speciation

events

Fourth

there

is

no

evidence

for

DNA

transposon

activity

in

the

past

Myr

in

the

human

genome

The

youngest

two

DNA

transposon

families

that

we

can

identify

in

the

draft

genome

sequence

-LRB-

and

-RRB-

show

divergence

from

their

respective

consensus

sequences

representing

the

ancestral

element

-LRB-

Fig.

-RRB-

indicating

that

they

were

active

before

the

divergence

of

humans

and

new

world

monkeys

Moreover

these

elements

were

relatively

unsuccessful

together

contributing

just

kb

to

the

draft

genome

sequence

Charlie

Number

of

kb

covered

by

family

PiggyBac-like

Mariner

Tigger

Unclassified

Zaphod

Average

nucleotide

substitution

level

for

family

Figure

Median

ages

and

per

cent

of

the

genome

covered

by

subfamilies

of

DNA

transposons

The

Charlie

and

Zaphod

elements

were

-LRB-

hAT

-RRB-

DNA

transposons

Mariner

and

Tigger

were

elements

Unlike

retroposons

DNA

transposons

are

thought

to

have

a

short

life

span

in

a

genome

Thus

the

average

or

median

divergence

of

copies

from

the

consensus

is

a

particularly

accurate

measure

of

the

age

of

the

DNA

transposon

copies

Table

Number

and

nature

of

interspersed

repeats

in

eukaryotic

genomes

Human

Percentage

of

bases

LINE/SINE

LTR

DNA

Total

Approximate

number

of

families

Percentage

of

bases

Fly

Finally

LTR

retroposons

appear

to

be

teetering

on

the

brink

of

extinction

if

they

have

not

already

succumbed

For

example

the

most

proli

c

elements

-LRB-

ERVL

and

MaLRs

-RRB-

ourished

for

more

than

Myr

but

appear

to

have

died

out

about

Myr

Only

a

single

LTR

retroposon

family

-LRB-

-RRB-

is

known

to

have

transposed

since

our

divergence

from

the

chimpanzee

Myr

ago

with

only

one

known

copy

-LRB-

in

the

HLA

region

-RRB-

that

is

not

shared

between

all

In

the

draft

genome

sequence

we

can

identify

only

three

full-length

copies

with

all

ORFs

intact

-LRB-

the

nal

total

may

be

slightly

higher

owing

to

the

imperfect

state

of

the

draft

genome

sequence

-RRB-

More

generally

the

overall

activity

of

all

transposons

has

declined

markedly

over

the

past

Myr

with

the

possible

exception

of

-LRB-

Fig.

-RRB-

Indeed

apart

from

an

exceptional

burst

of

activity

of

Alus

peaking

around

Myr

ago

there

would

appear

to

have

been

a

fairly

steady

decline

in

activity

in

the

hominid

lineage

since

the

mammalian

radiation

The

extent

of

the

decline

must

be

even

greater

than

it

appears

because

old

repeats

are

gradually

removed

by

random

deletion

and

because

old

repeat

families

are

harder

to

recognize

and

likely

to

be

under-represented

in

the

repeat

databases

-LRB-

We

con

rmed

that

the

decline

in

transposition

is

not

an

artefact

arising

from

errors

in

the

draft

genome

sequence

which

in

principle

could

increase

the

divergence

level

in

recent

elements

First

the

sequence

error

rate

-LRB-

Table

-RRB-

is

far

too

low

to

have

a

signi

cant

effect

on

the

apparent

age

of

recent

transposons

and

second

the

same

result

is

seen

if

one

considers

only

nished

sequence

-RRB-

What

explains

the

decline

in

transposon

activity

in

the

lineage

leading

to

humans

We

return

to

this

question

below

in

the

context

of

the

observation

that

there

is

no

similar

decline

in

the

mouse

genome

Comparison

with

other

organisms

We

compared

the

complement

of

transposable

elements

in

the

human

genome

with

those

of

the

other

sequenced

eukaryotic

genomes

We

analysed

the

y

worm

and

mustard

weed

genomes

for

the

number

and

nature

of

repeats

-LRB-

Table

-RRB-

and

the

age

distribution

-LRB-

Fig.

-RRB-

-LRB-

For

the

y

we

analysed

the

Mb

of

un

nished

large

contigs

produced

by

the

whole-genome

shotgun

which

are

reported

to

represent

euchromatic

sequence

Similar

results

were

obtained

by

analysing

Mb

of

nished

euchromatic

sequence

-RRB-

The

human

genome

stands

in

stark

contrast

to

the

genomes

of

the

other

organisms

-LRB-

-RRB-

The

euchromatic

portion

of

the

human

genome

has

a

much

higher

density

of

transposable

element

copies

than

the

euchromatic

DNA

of

the

other

three

organisms

The

repeats

in

the

other

organisms

may

have

been

slightly

underestimated

because

the

repeat

databases

for

the

other

organisms

are

less

complete

than

for

the

human

especially

with

regard

to

older

elements

on

the

other

hand

recent

additions

to

these

databases

appear

to

increase

the

repeat

content

only

marginally

-LRB-

-RRB-

The

human

genome

is

lled

with

copies

of

ancient

transposons

whereas

the

transposons

in

the

other

genomes

tend

to

be

of

more

recent

origin

The

difference

is

most

marked

with

the

y

but

is

clear

for

the

other

genomes

as

well

The

accumulation

of

old

repeats

is

likely

to

be

determined

by

the

rate

at

which

organisms

engage

in

housecleaning

through

genomic

deletion

Studies

of

pseudogenes

Worm

Approximate

number

of

families

Percentage

of

bases

Approximate

number

of

families

Mustard

weed

Percentage

of

bases

Approximate

number

of

families

The

complete

genomes

of

y

worm

and

chromosomes

and

of

mustard

weed

-LRB-

as

deposited

at

ncbi.nlm.nih.gov

genbank/genomes

-RRB-

were

screened

against

the

repeats

in

RepBase

Update

-LRB-

September

-RRB-

with

RepeatMasker

at

sensitive

settings

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

have

suggested

that

small

deletions

occur

at

a

rate

that

is

higher

in

ies

than

in

mammals

the

half-life

of

such

nonfunctional

DNA

is

estimated

at

Myr

for

ies

and

Myr

for

The

rate

of

large

deletions

has

not

been

systematically

compared

but

seems

likely

also

to

differ

markedly

-LRB-

-RRB-

Whereas

in

the

human

two

repeat

families

-LRB-

and

Alu

-RRB-

account

for

of

all

interspersed

repeat

sequence

the

other

organisms

have

no

dominant

families

Instead

the

worm

y

and

mustard

weed

genomes

all

contain

many

transposon

families

each

consisting

of

typically

hundreds

to

thousands

of

elements

This

difference

may

be

explained

by

the

observation

that

the

vertically

transmitted

long-term

residential

LINE

and

SINE

elements

represent

of

interspersed

repeats

in

the

human

genome

but

only

in

the

other

genomes

In

contrast

the

horizontally

transmitted

and

shorter-lived

DNA

transposons

represent

only

a

small

portion

of

all

interspersed

repeats

in

humans

-LRB-

-RRB-

but

a

much

larger

fraction

in

y

mustard

weed

and

worm

-LRB-

and

respectively

-RRB-

These

features

of

the

human

genome

are

probably

general

to

all

mammals

The

relative

lack

of

horizontally

transmitted

elements

may

have

its

origin

in

the

well

developed

immune

system

of

mammals

as

horizontal

transfer

requires

infectious

vectors

such

as

viruses

against

which

the

immune

system

guards

We

also

looked

for

differences

among

mammals

by

comparing

the

transposons

in

the

human

and

mouse

genomes

As

with

the

human

genome

care

is

required

in

calibrating

the

substitution

clock

for

the

mouse

genome

There

is

considerable

evidence

that

the

rate

of

substitution

per

Myr

is

higher

in

rodent

lineages

than

in

the

hominid

In

fact

we

found

clear

evidence

for

different

rates

of

substitution

by

examining

families

of

transposable

elements

whose

insertions

predate

the

divergence

of

the

human

and

mouse

lineages

In

an

analysis

of

such

families

we

found

that

the

substitution

level

was

an

average

of

higher

in

mouse

than

human

-LRB-

not

shown

-RRB-

-LRB-

This

is

likely

to

be

an

underestimate

because

of

an

ascertainment

bias

against

the

most

diverged

copies

-RRB-

The

faster

clock

in

mouse

is

also

evident

from

the

fact

that

the

ancient

and

MIR

elements

which

transposed

before

the

mammalian

radiation

and

are

readily

detectable

in

the

human

genome

can

not

be

readily

identi

ed

in

available

mouse

genomic

sequence

-LRB-

Fig.

-RRB-

We

used

the

best

available

estimates

to

calibrate

substitution

levels

and

The

ratio

of

substitution

rates

varied

from

about

higher

over

the

past

Myr

to

about

higher

over

the

past

Myr

The

analysis

shows

that

although

the

overall

density

of

the

four

transposon

types

in

human

and

mouse

is

similar

the

age

distribution

is

strikingly

different

-LRB-

Fig.

-RRB-

Transposon

activity

in

the

mouse

genome

has

not

undergone

the

decline

seen

in

humans

and

proceeds

at

a

much

higher

rate

In

contrast

to

their

possible

extinction

in

humans

LTR

retroposons

are

alive

and

well

in

the

Proportion

of

interspersed

repeats

-LRB-

-RRB-

Oldest

mouse

with

such

representatives

as

the

active

IAP

family

and

putatively

active

members

of

the

long-lived

ERVL

and

MaLR

families

and

a

variety

of

SINEs

are

quite

active

These

evolutionary

ndings

are

consistent

with

the

empirical

observations

that

new

spontaneous

mutations

are

times

more

likely

to

be

caused

by

LINE

insertions

in

mouse

than

in

human

-LRB-

versus

-RRB-

and

times

more

likely

to

be

caused

by

transposable

elements

in

general

It

is

estimated

that

around

in

mutations

in

human

are

due

to

transpositions

whereas

of

mutations

in

mouse

are

due

to

transpositions

-LRB-

mostly

IAP

insertions

-RRB-

The

contrast

between

human

and

mouse

suggests

that

the

explanation

for

the

decline

of

transposon

activity

in

humans

may

lie

in

some

fundamental

difference

between

hominids

and

rodents

Population

structure

and

dynamics

would

seem

to

be

likely

suspects

Rodents

tend

to

have

large

populations

whereas

hominid

populations

tend

to

be

small

and

may

undergo

frequent

bottlenecks

Evolutionary

forces

affected

by

such

factors

include

inbreeding

and

genetic

drift

which

might

affect

the

persistence

of

active

transposable

Studies

in

additional

mammalian

lineages

may

shed

light

on

the

forces

responsible

for

the

differences

in

the

activity

of

transposable

Variation

in

the

distribution

of

repeats

We

next

explored

variation

in

the

distribution

of

repeats

across

the

draft

genome

sequence

by

calculating

the

repeat

density

in

windows

of

various

sizes

across

the

genome

There

is

striking

variation

at

smaller

scales

Some

regions

of

the

genome

are

extraordinarily

dense

in

repeats

The

prizewinner

appears

to

be

a

region

on

chromosome

with

an

overall

transposable

element

density

of

This

region

contains

a

segment

with

density

as

well

as

a

segment

of

kb

in

which

sequences

alone

comprise

of

the

sequence

In

addition

there

are

regions

of

more

than

kb

with

extremely

high

densities

of

Alu

-LRB-

at

three

loci

including

one

on

with

a

stretch

of

Alu

-RRB-

and

the

ancient

transposons

MIR

-LRB-

on

chromosome

-RRB-

and

-LRB-

on

chromosome

-RRB-

In

contrast

some

genomic

regions

are

nearly

devoid

of

repeats

The

absence

of

repeats

may

be

a

sign

of

large-scale

cis-regulatory

elements

that

can

not

tolerate

being

interrupted

by

insertions

The

four

regions

with

the

lowest

density

of

interspersed

repeats

in

the

human

genome

are

the

four

homeobox

gene

clusters

HOXA

HOXB

HOXC

and

HOXD

-LRB-

Fig.

-RRB-

Each

locus

contains

regions

of

around

kb

containing

less

than

interspersed

repeats

Ongoing

sequence

analysis

of

the

four

HOX

clusters

in

mouse

rat

and

baboon

shows

a

similar

absence

of

transposable

elements

and

reveals

a

high

density

of

conserved

noncoding

elements

-LRB-

K.

Dewar

and

B.

Birren

manuscript

in

preparation

-RRB-

The

presence

of

a

complex

collection

of

regulatory

regions

may

explain

why

individual

HOX

genes

carried

in

transgenic

mice

fail

to

show

proper

regulation

It

may

be

worth

investigating

other

repeat-poor

regions

such

as

a

region

on

chromosome

-LRB-

repeat

over

kb

-RRB-

containing

a

gene

encoding

a

homeodomain

zinc

nger

protein

-LRB-

homologous

to

mouse

pID

-RRB-

a

region

on

chromosome

-LRB-

repeat

over

kb

-RRB-

with

no

obvious

genes

and

a

region

on

chromosome

-LRB-

over

kb

-RRB-

containing

three

genes

of

unknown

function

-LRB-

among

which

is

-RRB-

It

will

be

interesting

to

see

whether

the

homologous

regions

in

the

mouse

genome

have

chr

chr

Youngest

or

m

us

w

tard

ee

d

um

kb

Fl

HoxD

cluster

an

y

H

W

M

Figure

Comparison

of

the

age

of

interspersed

repeats

in

eukaryotic

genomes

The

copies

of

repeats

were

pooled

by

their

nucleotide

substitution

level

from

the

consensus

NATURE

VOL

FEBRUARY

Figure

Two

regions

of

about

Mb

on

chromosomes

and

Red

bars

interspersed

repeats

blue

bars

exons

of

known

genes

Note

the

de

cit

of

repeats

in

the

HoxD

cluster

which

contains

a

collection

of

genes

with

complex

interrelated

regulation

Macmillan

Magazines

Ltd

articles

Proportion

of

genome

comprised

by

each

class

-LRB-

-RRB-

SSR

MIR

ALU

DNA

LTR

GC

content

-LRB-

-RRB-

Figure

Density

of

the

major

repeat

classes

as

a

function

of

local

GC

content

in

windows

of

kb

Frequency

of

Alu

class

relative

to

its

average

density

in

the

genome

Proportion

of

DNA

transposons

comprised

by

each

age

group

-LRB-

-RRB-

Nucleotide

subsitution

level

GC

content

bins

-LRB-

-RRB-

GC

content

bins

-LRB-

-RRB-

AluY

-LRB-

Myr

-RRB-

AluY

-LRB-

Myr

-RRB-

AluY

-LRB-

Myr

-RRB-

AluSc

-LRB-

Myr

-RRB-

AluS

-LRB-

Myr

-RRB-

AluJ

FAM

-LRB-

Myr

-RRB-

Figure

DNA

transposon

copies

in

AT-rich

DNA

tend

to

be

younger

than

those

in

more

GC-rich

DNA

DNA

transposon

families

were

grouped

into

ve

age

categories

by

their

median

substitution

level

-LRB-

see

Fig.

-RRB-

The

proportion

attributed

to

each

age

class

is

shown

as

a

function

of

GC

content

Similar

patterns

are

seen

for

and

LTR

elements

Figure

Alu

elements

target

AT-rich

DNA

but

accumulate

in

GC-rich

DNA

This

graph

shows

the

relative

distribution

of

various

Alu

cohorts

as

a

function

of

local

GC

content

The

divergence

levels

-LRB-

including

CpG

sites

-RRB-

and

ages

of

the

cohorts

are

shown

in

the

key

similarly

resisted

the

insertion

of

transposable

elements

during

rodent

evolution

Distribution

by

GC

content

We

next

focused

on

the

correlation

between

the

nature

of

the

transposons

in

a

region

and

its

GC

content

We

calculated

the

density

of

each

repeat

type

as

a

function

of

the

GC

content

in

windows

-LRB-

Fig.

-RRB-

As

has

been

LINE

sequences

occur

at

much

higher

density

in

AT-rich

regions

-LRB-

roughly

fourfold

enriched

-RRB-

whereas

SINEs

-LRB-

MIR

Alu

-RRB-

show

the

opposite

trend

-LRB-

for

Alu

up

to

vefold

lower

in

ATrich

DNA

-RRB-

LTR

retroposons

and

DNA

transposons

show

a

more

uniform

distribution

dipping

only

in

the

most

GC-rich

regions

The

preference

of

LINEs

for

AT-rich

DNA

seems

like

a

reasonable

way

for

a

genomic

parasite

to

accommodate

its

host

by

targeting

gene-poor

AT-rich

DNA

and

thereby

imposing

a

lower

mutational

burden

Mechanistically

selective

targeting

is

nicely

explained

by

the

fact

that

the

preferred

cleavage

site

of

the

LINE

endonuclease

is

TTTT/A

-LRB-

where

the

slash

indicates

the

point

of

cleavage

-RRB-

which

is

used

to

prime

reverse

transcription

from

the

poly

-LRB-

A

-RRB-

tail

of

LINE

The

contrary

behaviour

of

SINEs

however

is

baf

ing

How

do

SINEs

accumulate

in

GC-rich

DNA

particularly

if

they

depend

on

the

LINE

transposition

Notably

the

same

pattern

is

seen

for

the

Alu-like

and

the

tRNA-derived

SINEs

in

mouse

and

for

MIR

in

One

possibility

is

that

SINEs

somehow

target

GC-rich

DNA

for

insertion

The

alternative

is

that

SINEs

initially

insert

with

the

same

proclivity

for

AT-rich

DNA

as

LINEs

but

that

the

distribution

is

subsequently

reshaped

by

evolutionary

We

used

the

draft

genome

sequence

to

investigate

this

mystery

by

comparing

the

proclivities

of

young

adolescent

middle-aged

and

old

Alus

-LRB-

Fig.

-RRB-

Strikingly

recent

Alus

show

a

preference

for

ATrich

DNA

resembling

that

of

LINEs

whereas

progressively

older

Alus

show

a

progressively

stronger

bias

towards

GC-rich

DNA

These

results

indicate

that

the

GC

bias

must

result

from

strong

pressure

Fig.

shows

that

a

enrichment

of

Alus

in

GC-rich

DNA

has

occurred

within

the

last

Myr

and

possibly

more

recently

These

results

raise

a

new

mystery

What

is

the

force

that

produces

the

great

and

rapid

enrichment

of

Alus

in

GC-rich

DNA

One

explanation

may

be

that

deletions

are

more

readily

tolerated

in

gene-poor

AT-rich

regions

than

in

gene-rich

GC-rich

regions

resulting

in

older

elements

being

enriched

in

GC-rich

regions

Such

an

enrichment

is

seen

for

transposable

elements

such

as

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

DNA

transposons

-LRB-

Fig.

-RRB-

However

this

effect

seems

too

slow

and

too

small

to

account

for

the

observed

remodelling

of

the

Alu

distribution

This

can

be

seen

by

performing

a

similar

analysis

for

LINE

elements

-LRB-

Fig.

-RRB-

There

is

no

signi

cant

change

in

the

LINE

distribution

over

the

past

Myr

in

contrast

to

the

rapid

change

seen

for

Alu

There

is

an

eventual

shift

after

more

than

Myr

although

its

magnitude

is

still

smaller

than

seen

for

Alus

These

observations

indicate

that

there

may

be

some

force

acting

particularly

on

Alus

This

could

be

a

higher

rate

of

random

loss

of

Alus

in

AT-rich

DNA

negative

selection

against

Alus

in

AT-rich

DNA

or

positive

selection

in

favour

of

Alus

in

GC-rich

DNA

The

rst

two

possibilities

seem

unlikely

because

AT-rich

DNA

is

Frequency

of

class

relative

to

its

average

density

in

the

genome

GC

content

bins

-LRB-

-RRB-

-LRB-

Myr

-RRB-

-LRB-

Myr

-RRB-

-LRB-

Myr

-RRB-

-LRB-

Myr

-RRB-

-LRB-

Myr

-RRB-

Figure

Distribution

of

various

LINE

cohorts

as

a

function

of

local

GC

content

The

divergence

levels

and

ages

of

the

cohorts

are

shown

in

the

key

-LRB-

The

divergence

levels

were

measured

for

the

UTR

of

the

element

only

which

is

best

characterized

evolutionarily

This

region

contains

almost

no

CpG

sites

and

thus

divergence

level

corresponds

to

a

much

longer

time

than

for

CpG-rich

Alu

copies

-RRB-

gene-poor

and

tolerates

the

accumulation

of

other

transposable

elements

The

third

seems

more

feasible

in

that

it

involves

selecting

in

favour

of

the

minority

of

Alus

in

GC-rich

regions

rather

than

against

the

majority

that

lie

in

AT-rich

regions

But

positive

selection

for

Alus

in

GC-rich

regions

would

imply

that

they

bene

t

the

organism

has

proposed

such

a

function

for

SINEs

This

hypothesis

is

based

on

the

observation

that

in

many

species

SINEs

are

transcribed

under

conditions

of

stress

and

the

resulting

RNAs

speci

cally

bind

a

particular

protein

kinase

-LRB-

PKR

-RRB-

and

block

its

ability

to

inhibit

protein

SINE

RNAs

would

thus

promote

protein

translation

under

stress

SINE

RNA

may

be

well

suited

to

such

a

role

in

regulating

protein

translation

because

it

can

be

quickly

transcribed

in

large

quantities

from

thousands

of

elements

and

it

can

function

without

protein

translation

Under

this

theory

there

could

be

positive

selection

for

SINEs

in

readily

transcribed

open

chromatin

such

as

is

found

near

genes

This

could

explain

the

retention

of

Alus

in

gene-rich

GC-rich

regions

It

is

also

consistent

with

the

observation

that

SINE

density

in

AT-rich

DNA

is

higher

near

Further

insight

about

Alus

comes

from

the

relationship

between

Alu

density

and

GC

content

on

individual

chromosomes

-LRB-

Fig.

-RRB-

There

are

two

outliers

Chromosome

is

even

richer

in

Alus

than

predicted

by

its

-LRB-

high

-RRB-

GC

content

the

chromosome

comprises

of

the

genome

but

contains

of

Alus

On

the

other

hand

chromosome

Y

shows

the

lowest

density

of

Alus

relative

to

its

GC

content

being

higher

than

average

for

GC

content

less

than

and

lower

than

average

for

GC

content

over

Even

in

AT-rich

DNA

Alus

are

under-represented

on

chromosome

Y

compared

with

other

young

interspersed

repeats

-LRB-

see

below

-RRB-

These

phenomena

may

be

related

to

an

unusually

high

gene

density

on

chromosome

and

an

unusually

low

density

of

somatically

active

genes

on

chromosome

Y

-LRB-

both

relative

to

GC

content

-RRB-

This

would

be

consistent

with

the

idea

that

Alu

correlates

not

with

GC

content

but

with

actively

transcribed

genes

Our

results

may

support

the

controversial

idea

that

SINEs

actually

earn

their

keep

in

the

genome

Clearly

much

additional

work

will

be

needed

to

prove

or

disprove

the

hypothesis

that

SINEs

are

genomic

symbionts

Biases

in

human

mutation

Indirect

studies

have

suggested

that

nucleotide

substitution

is

not

uniform

across

mammalian

ChrX

ChrY

Proportion

comprised

by

Alu

GC

content

bins

-LRB-

-RRB-

Figure

Comparison

of

the

Alu

density

of

each

chromosome

as

a

function

of

local

GC

content

At

higher

GC

levels

the

Alu

density

varies

widely

between

chromosomes

with

chromosome

being

a

particular

outlier

In

contrast

the

density

pattern

is

quite

NATURE

VOL

FEBRUARY

uniform

for

most

chromosomes

with

the

exception

of

a

to

over-representation

in

AT-rich

regions

of

the

X

and

Y

chromosomes

-LRB-

not

shown

-RRB-

Macmillan

Magazines

Ltd

articles

By

studying

sets

of

repeat

elements

belonging

to

a

common

cohort

one

can

directly

measure

nucleotide

substitution

rates

in

different

regions

of

the

genome

We

nd

strong

evidence

that

the

pattern

of

neutral

substitution

differs

as

a

function

of

local

GC

content

-LRB-

Fig.

-RRB-

Because

the

results

are

observed

in

repetitive

elements

throughout

the

genome

the

variation

in

the

pattern

of

nucleotide

substitution

seems

likely

to

be

due

to

differences

in

the

underlying

mutational

process

rather

than

to

selection

The

effect

can

be

seen

most

clearly

by

focusing

on

the

substitution

process

g

a

where

g

denotes

GC

or

CG

base

pairs

and

a

denotes

ATor

TA

base

pairs

If

K

is

the

equilibrium

constant

in

the

direction

of

a

base

pairs

-LRB-

de

ned

by

the

ratio

of

the

forward

and

reverse

rates

-RRB-

then

the

equilibrium

GC

content

should

be

-LRB-

K

-RRB-

Two

observations

emerge

First

there

is

a

regional

bias

in

substitution

patterns

The

equilibrium

constant

varies

as

a

function

of

local

GC

content

g

base

pairs

are

more

likely

to

mutate

towards

a

base

pairs

in

AT-rich

regions

than

in

GC-rich

regions

For

the

analysis

in

Fig.

the

equilibrium

constant

K

is

and

when

the

draft

genome

sequence

is

partitioned

into

three

bins

with

average

GC

content

of

and

respectively

This

bias

could

be

due

to

a

reported

tendency

for

GC-rich

regions

to

replicate

earlier

in

the

cell

cycle

than

AT-rich

regions

and

for

guanine

pools

which

are

limiting

for

Substitution

level

in

diverged

sequence

-LRB-

-RRB-

G

C

A

T

A

T

G

C

G

C

T

A

A

T

C

G

A

T

T

A

G

C

C

G

Average

background

nucleotide

composition

GC

GC

GC

DNA

replication

to

become

depleted

late

in

the

cell

cycle

thereby

resulting

in

a

small

but

signi

cant

shift

in

substitution

towards

a

base

Another

theory

proposes

that

many

substitutions

are

due

to

differences

in

DNA

repair

mechanisms

possibly

related

to

transcriptional

activity

and

thereby

to

gene

density

and

GC

There

is

also

an

absolute

bias

in

substitution

patterns

resulting

in

directional

pressure

towards

lower

GC

content

throughout

the

human

genome

The

genome

is

not

at

equilibrium

with

respect

to

the

pattern

of

nucleotide

substitution

the

expected

equilibrium

GC

content

corresponding

to

the

values

of

K

above

is

and

for

regions

with

average

GC

contents

of

and

respectively

Recent

observations

on

con

rm

that

the

mutation

pattern

in

GC-rich

DNA

is

biased

towards

a

base

pairs

it

should

be

possible

to

perform

similar

analyses

throughout

the

genome

with

the

availability

of

million

On

the

basis

solely

of

nucleotide

substitution

patterns

the

GC

content

would

be

expected

to

be

about

lower

throughout

the

genome

What

accounts

for

the

higher

GC

content

One

possible

explanation

is

that

in

GC-rich

regions

a

considerable

fraction

of

the

nucleotides

is

likely

to

be

under

functional

constraint

owing

to

the

high

gene

density

Selection

on

coding

regions

and

regulatory

CpG

islands

may

maintain

the

higher-than-predicted

GC

content

Another

is

that

throughout

the

rest

of

the

genome

a

constant

in

ux

of

transposable

elements

tends

to

increase

GC

content

-LRB-

Fig.

-RRB-

Young

repeat

elements

clearly

have

a

higher

GC

content

than

their

surrounding

regions

except

in

extremely

GC-rich

regions

Moreover

repeat

elements

clearly

shift

with

age

towards

a

lower

GC

content

closer

to

that

of

the

neighbourhood

in

which

they

reside

Much

of

the

non-repeat

DNA

in

AT-rich

regions

probably

consists

of

ancient

repeats

that

are

not

detectable

by

current

methods

and

that

have

had

more

time

to

approach

the

local

equilibrium

value

The

repeats

can

also

be

used

to

study

how

the

mutation

process

is

affected

by

the

immediately

adjacent

nucleotide

Such

context

effects

will

be

discussed

elsewhere

-LRB-

A.

Kas

and

A.

F.

A.

Smit

unpublished

results

-RRB-

Fast

living

on

chromosome

Y

The

pattern

of

interspersed

repeats

can

be

used

to

shed

light

on

the

unusual

evolutionary

history

of

chromosome

Y

Our

analysis

shows

that

the

genetic

material

on

All

DNA

Young

interspersed

repeats

-LRB-

divergence

-RRB-

All

interspersed

repeats

Non-repeat

DNA

Type

of

substitution

Figure

Substitution

patterns

in

interspersed

repeats

differ

as

a

function

of

GC

content

We

collected

all

copies

of

ve

DNA

transposons

-LRB-

and

-RRB-

chosen

for

their

high

copy

number

and

well

de

ned

consensus

sequences

DNA

transposons

are

optimal

for

the

study

of

neutral

substitutions

they

do

not

segregate

into

subfamilies

with

diagnostic

differences

presumably

because

they

are

short-lived

and

new

active

families

do

not

evolve

in

a

genome

-LRB-

see

text

-RRB-

Duplicates

and

close

paralogues

resulting

from

duplication

after

transposition

were

eliminated

The

copies

were

grouped

on

the

basis

of

GC

content

of

the

anking

bp

on

both

sides

and

aligned

to

the

consensus

sequence

-LRB-

representing

the

state

of

the

copy

at

integration

-RRB-

Recursive

efforts

using

parameters

arising

from

this

study

did

not

change

the

alignments

signi

cantly

Alignments

were

inspected

by

hand

and

obvious

misalignments

caused

by

insertions

and

duplications

were

eliminated

Substitutions

-LRB-

n

-RRB-

were

counted

for

each

position

in

the

consensus

excluding

those

in

CpG

dinucleotides

and

a

substitution

frequency

matrix

was

de

ned

From

the

matrices

for

each

repeat

-LRB-

which

corresponded

to

different

ages

-RRB-

a

single

rate

matrix

was

calculated

for

these

bins

of

GC

content

-LRB-

GC

GC

and

GC

-RRB-

Data

are

shown

for

a

repeat

with

an

average

divergence

-LRB-

in

non-CpG

sites

-RRB-

of

in

GC

content

-LRB-

the

repeat

has

slightly

higher

divergence

in

AT-rich

DNA

and

lower

in

GC-rich

DNA

-RRB-

From

the

rate

matrix

we

calculated

log-likelihood

matrices

with

different

entropies

-LRB-

divergence

levels

-RRB-

which

are

theoretically

optimal

for

alignments

of

neutrally

diverged

copies

to

their

common

ancestral

state

-LRB-

A.

Kas

and

A.

F.

A.

Smit

unpublished

-RRB-

These

matrices

are

in

use

by

the

RepeatMasker

program

GC

content

of

feature

-LRB-

-RRB-

GC

content

of

genome

DNA

-LRB-

-RRB-

Figure

Interspersed

repeats

tend

to

diminish

the

differences

between

GC

bins

despite

the

fact

that

GC-rich

transposable

elements

-LRB-

speci

cally

Alu

-RRB-

accumulate

in

GC-rich

DNA

and

AT-rich

elements

-LRB-

-RRB-

in

AT-rich

DNA

The

GC

content

of

particular

components

of

the

sequence

-LRB-

repeats

young

repeats

and

non-repeat

sequence

-RRB-

was

calculated

as

a

function

of

overall

GC

content

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

chromosome

Y

is

unusually

young

probably

owing

to

a

high

tolerance

for

gain

of

new

material

by

insertion

and

loss

of

old

material

by

deletion

Several

lines

of

evidence

support

this

picture

For

example

LINE

elements

on

chromosome

Yare

on

average

much

younger

than

those

on

autosomes

-LRB-

not

shown

-RRB-

Similarly

MaLRfamily

retroposons

on

chromosome

Y

are

younger

than

those

on

autosomes

with

the

representation

of

subfamilies

showing

a

strong

inverse

correlation

with

the

age

of

the

subfamily

Moreover

chromosome

Y

has

a

relative

over-representation

of

the

younger

retroviral

class

II

-LRB-

ERVK

-RRB-

and

a

relative

under-representation

of

the

primarily

older

class

III

-LRB-

ERVL

-RRB-

compared

with

other

chromosomes

Overall

chromosome

Y

seems

to

maintain

a

youthful

appearance

by

rapid

turnover

Interspersed

repeats

on

chromosome

Y

can

also

be

used

to

estimate

the

relative

mutation

rates

am

and

af

in

the

male

and

female

germlines

Chromosome

Y

always

resides

in

males

whereas

chromosome

X

resides

in

females

twice

as

often

as

in

males

The

substitution

rates

mY

and

mX

on

these

two

chromosomes

should

thus

be

in

the

ratio

mY

mX

-LRB-

am

-RRB-

-LRB-

am

-RRB-

provided

that

one

considers

equivalent

neutral

sequences

Several

authors

have

estimated

the

mutation

rate

in

the

male

germline

to

be

vefold

higher

than

in

the

female

germline

by

comparing

the

rates

of

evolution

of

X

and

Y-linked

genes

in

humans

and

primates

However

Page

and

have

challenged

these

estimates

as

too

high

They

studied

a

region

that

is

apparently

devoid

of

genes

and

resides

within

a

large

segmental

duplication

from

X

to

Y

that

occurred

Myr

ago

in

the

human

lineage

On

the

basis

of

phylogenetic

analysis

of

the

sequence

on

human

Y

and

human

chimp

and

gorilla

X

they

obtained

a

much

lower

estimate

of

mY

mX

corresponding

to

am

af

They

suggested

that

the

other

estimates

may

have

been

higher

because

they

were

based

on

much

longer

evolutionary

periods

or

because

the

genes

studied

may

have

been

under

selection

Our

database

of

human

repeats

provides

a

powerful

resource

for

addressing

this

question

We

identi

ed

the

repeat

elements

from

recent

subfamilies

-LRB-

effectively

birth

cohorts

dating

from

the

past

Myr

-RRB-

and

measured

the

substitution

rates

for

subfamily

members

on

chromosomes

X

and

Y

-LRB-

Fig.

-RRB-

There

is

a

clear

linear

relationship

with

a

slope

of

mY

mX

corresponding

to

am

af

The

estimate

is

in

reasonable

agreement

with

that

of

Page

et

al.

although

it

is

based

on

much

more

total

sequence

-LRB-

kb

on

Y

Mb

on

X

-RRB-

and

a

much

longer

time

period

In

particular

the

discrepancy

with

earlier

reports

is

not

explained

by

recent

changes

in

the

human

lineage

Various

theories

have

been

proposed

for

the

higher

mutation

rate

in

the

male

germline

including

the

greater

number

of

cell

divisions

in

the

formation

of

sperm

than

eggs

and

different

repair

mechanisms

in

sperm

and

eggs

Active

transposons

We

were

interested

in

identifying

the

youngest

retrotransposons

in

the

draft

genome

sequence

This

set

should

contain

the

currently

active

retrotransposons

as

well

as

the

insertion

sites

that

are

still

polymorphic

in

the

human

population

The

youngest

branch

in

the

phylogenetic

tree

of

human

elements

is

called

-LRB-

ref

-RRB-

it

differs

in

its

untranslated

region

-LRB-

UTR

-RRB-

by

diagnostic

substitutions

from

the

next

oldest

subfamily

-LRB-

-RRB-

Within

the

family

there

are

two

subsets

referred

to

as

Ta

and

pre-Ta

de

ned

by

a

diagnostic

All

active

elements

are

thought

to

belong

to

these

two

subsets

because

they

account

for

all

known

cases

of

human

disease

arising

from

new

transposition

-LRB-

with

belonging

to

the

Ta

subset

and

one

to

the

pre-Ta

subset

-RRB-

These

subsets

are

also

of

great

interest

for

population

genetics

because

at

least

are

still

segregating

as

polymorphisms

in

the

human

they

provide

powerful

markers

for

tracing

population

history

because

they

represent

unique

-LRB-

non-recurrent

and

non-revertible

-RRB-

genetic

events

that

can

be

used

-LRB-

along

with

similarly

polymorphic

Alus

-RRB-

for

reconstructing

human

migrations

elements

that

are

retrotransposition-competent

should

consist

of

a

full-length

sequence

and

should

have

both

ORFs

intact

Eleven

such

elements

from

the

Ta

subset

have

been

identi

ed

including

the

likely

progenitors

of

mutagenic

insertions

into

the

factor

VIII

and

dystrophin

A

cultured

cell

retrotransposition

assay

has

revealed

that

eight

of

these

elements

remain

We

searched

the

draft

genome

sequence

and

identi

ed

LINEs

belonging

to

the

Ta

subset

and

belonging

to

the

pre-Ta

subset

These

elements

provide

a

large

collection

of

tools

for

probing

human

population

history

We

also

identi

ed

those

consisting

of

full-length

elements

with

intact

ORFs

which

are

candidate

active

LINEs

We

found

such

elements

belonging

to

the

Ta

subset

and

belonging

to

the

pre-Ta

subset

this

substantially

increases

the

number

in

the

rst

category

and

provides

the

rst

known

examples

in

the

second

category

These

elements

can

now

be

tested

for

retrotransposition

competence

in

the

cell

culture

assay

Preliminary

analysis

resulted

in

the

identi

cation

of

two

of

these

elements

as

the

likely

progenitors

of

mutagenic

insertions

into

the

b-globin

and

genes

-LRB-

R.

Badge

and

J.

V.

Moran

unpublished

data

-RRB-

Similar

analyses

should

allow

the

identi

cation

of

the

progenitors

of

most

if

not

all

other

known

mutagenic

insertions

elements

can

carry

extra

DNA

if

transcription

extends

through

the

native

transcriptional

termination

site

into

anking

genomic

DNA

This

process

termed

transduction

provides

a

means

for

the

mobilization

of

DNA

sequences

around

the

genome

and

may

be

a

mechanism

for

exon

shuf

ing

Twenty-one

per

cent

of

the

full-length

analysed

contained

sequences

before

the

target-site

duplication

site

in

cases

in

which

the

site

was

unambiguously

recognizable

The

length

of

the

transduced

sequence

was

bp

supporting

the

suggestion

that

of

the

human

genome

may

have

arisen

by

LINE-based

transduction

of

anking

Our

analysis

also

turned

up

two

instances

of

transduction

-LRB-

bp

and

bp

-RRB-

Although

this

possibility

had

been

suggested

on

the

basis

of

cell

culture

these

are

the

rst

documented

examples

Such

events

may

arise

from

transcription

initiating

in

a

cellular

promoter

upstream

of

the

elements

transcription

is

generally

con

ned

to

the

but

transcription

from

other

promoters

could

explain

a

somatic

retrotransposition

event

that

resulted

in

colon

Transposons

as

a

creative

force

The

primary

force

for

the

origin

and

expansion

of

most

transposons

has

been

selection

for

their

ability

to

create

progeny

and

not

a

selective

advantage

for

the

host

However

these

sel

sh

pieces

of

DNA

have

been

responsible

for

important

innovations

in

many

genomes

for

example

by

contributing

regulatory

elements

and

even

new

genes

Twenty

human

genes

have

been

recognized

as

probably

derived

Median

substitution

level

of

repeat

subfamily

on

Y

-LRB-

-RRB-

Median

substitution

level

of

repeat

subfamily

on

X

-LRB-

-RRB-

Figure

Higher

substitution

rate

on

chromosome

Y

than

on

chromosome

X

We

calculated

the

median

substitution

level

-LRB-

excluding

CpG

sites

-RRB-

for

copies

of

the

most

recent

subfamilies

-LRB-

-RRB-

on

the

X

and

Y

chromosomes

Only

the

UTR

of

the

element

was

considered

because

its

consensus

sequence

is

best

established

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articles

from

These

include

the

and

recombinases

and

the

major

centromere-binding

protein

CENPB

We

scanned

the

draft

genome

sequence

and

identi

ed

another

cases

bringing

the

total

to

-LRB-

Table

refs

-RRB-

All

but

four

are

derived

from

DNA

transposons

which

give

rise

to

only

a

small

proportion

of

the

interspersed

repeats

in

the

genome

Why

there

are

so

many

DNA

transposase-like

genes

many

of

which

still

contain

the

critical

residues

for

transposase

activity

is

a

mystery

To

illustrate

this

concept

we

describe

the

discovery

of

one

of

the

new

examples

We

searched

the

draft

genome

sequence

to

identify

the

autonomous

DNA

transposon

responsible

for

the

distribution

of

the

non-autonomous

element

one

of

the

most

recently

-LRB-

Myr

ago

-RRB-

active

DNA

transposons

Most

non-autonomous

elements

are

internal

deletion

products

of

a

DNA

transposon

We

identi

ed

one

instance

of

a

large

-LRB-

bp

-RRB-

ORF

anked

by

the

and

halves

of

a

element

The

ORF

encodes

a

novel

protein

-LRB-

partially

published

as

pID

-RRB-

whose

closest

homologue

is

the

transposase

of

the

piggyBac

DNA

transposon

which

is

found

in

insects

and

has

the

same

characteristic

TTAA

target-site

as

The

ORF

is

actively

transcribed

in

fetal

brain

and

in

cancer

cells

That

it

has

not

been

lost

to

mutation

in

Myr

of

evolution

-LRB-

whereas

the

anking

noncoding

Human

genes

derived

from

transposable

elements

GenBank

ID

nID

pID

pID

pID

pID

pID

EST

PID

EST

pID

pID

EST

EST

EST

EST

EST

EST

PID

PID

PID

EST

PID

PID

PID

PID

PID

PID

PID

PID

EST

pID

EST

EST

pID

pID

pID

pID

EST

pID

BAC

pID

EST

pID

EST

BAC

EST

EST

Gene

name

Telomerase

env

Syncytin

and

CENP-B

Related

transposon

family

FLAM

Alu

non-LTR

retrotransposon

Retroviridae/HERV-R

Retroviridae/HERV-W

hAT/Hobo

hAT/Charlie

hAT/Charlie

hAT/Charlie

hAT/Charlie

hAT/Charlie

hAT/Charlie

piggyBac

Possible

fusion

gene

Newly

recognized

derivationk

like

termini

show

the

typical

divergence

level

of

such

elements

-RRB-

and

is

actively

transcribed

provides

strong

evidence

that

it

has

been

adopted

by

the

human

genome

as

a

gene

Its

function

is

unknown

activity

clearly

has

also

had

fringe

bene

ts

We

mentioned

above

the

possibility

of

exon

reshuf

ing

by

cotranscription

of

neighbouring

DNA

The

machinery

can

also

cause

reverse

transcription

of

genic

mRNAs

which

typically

results

in

nonfunctional

processed

pseudogenes

but

can

occasionally

give

rise

to

functional

processed

genes

There

are

at

least

eight

human

and

eight

mouse

genes

for

which

evidence

strongly

supports

such

an

-LRB-

see

tables.html

-RRB-

Many

other

intronless

genes

may

have

been

created

in

the

same

way

Transposons

have

made

other

creative

contributions

to

the

genome

A

few

hundred

genes

for

example

use

transcriptional

terminators

donated

by

LTR

retroposons

-LRB-

data

not

shown

-RRB-

Other

genes

employ

regulatory

elements

derived

from

repeat

Simple

sequence

repeats

Simple

sequence

repeats

-LRB-

SSRs

-RRB-

are

a

rather

different

type

of

repetitive

structure

that

is

common

in

the

human

genome

perfect

or

slightly

imperfect

tandem

repeats

of

a

particular

k-mer

SSRs

with

a

short

repeat

unit

-LRB-

n

bases

-RRB-

are

often

termed

microsa

Jerky

JRKL

Sancho

Tramp

The

Table

lists

human

genes

with

a

likely

origin

in

up

to

different

transposon

copies

Where

available

the

GenBank

ID

numbers

are

given

for

proteins

otherwise

a

representative

EST

or

a

clone

name

is

shown

Six

groups

-LRB-

two

or

three

genes

each

-RRB-

have

similarity

at

the

DNA

level

well

beyond

that

observed

between

different

DNA

transposon

families

in

the

genome

they

are

indicated

in

italics

with

all

but

the

initial

member

of

each

group

indented

This

could

be

explained

if

the

genes

were

paralogous

-LRB-

derived

from

a

single

inserted

transposon

and

subsequently

duplicated

-RRB-

Classi

cation

of

the

transposon

Indicates

that

the

transposon

from

which

the

gene

is

derived

is

precisely

known

Proteins

probably

formed

by

fusion

of

a

cellular

and

transposon

gene

many

have

acquired

zinc

nger

domains

k

Not

previously

reported

as

being

derived

from

transposable

element

genes

The

remaining

genes

can

be

found

in

refs

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

Table

SSR

content

of

the

human

genome

Length

of

repeat

unit

Average

bases

per

Mb

Average

number

of

SSR

elements

per

Mb

the

population

In

fact

analysis

of

the

draft

genome

sequence

shows

that

chromosome

X

has

the

same

density

of

-LRB-

CA

-RRB-

n

repeats

per

Mb

as

the

autosomes

-LRB-

data

not

shown

-RRB-

Thus

the

de

cit

of

polymorphic

markers

relative

to

autosomes

results

from

population

genetic

forces

Possible

explanations

include

that

chromosome

X

has

a

smaller

effective

population

size

experiences

more

frequent

selective

sweeps

reducing

diversity

-LRB-

owing

to

its

hemizygosity

in

males

-RRB-

or

has

a

lower

mutation

rate

-LRB-

owing

to

its

more

frequent

passage

through

the

less

mutagenic

female

germline

-RRB-

The

availability

of

the

draft

genome

sequence

should

provide

ways

to

test

these

alternative

explanations

Segmental

duplications

SSRs

were

identi

ed

by

using

the

computer

program

Tandem

Repeat

Finder

with

the

following

parameters

match

score

mismatch

score

indel

minimum

alignment

maximum

repeat

length

minimum

repeat

length

tellites

whereas

those

with

longer

repeat

units

-LRB-

n

bases

-RRB-

are

often

termed

minisatellites

With

the

exception

of

poly

-LRB-

A

-RRB-

tails

from

reverse

transcribed

messages

SSRs

are

thought

to

arise

by

slippage

during

DNA

We

compiled

a

catalogue

of

all

SSRs

over

a

given

length

in

the

human

draft

genome

sequence

and

studied

their

properties

-LRB-

Table

-RRB-

SSRs

comprise

about

of

the

human

genome

with

the

greatest

single

contribution

coming

from

dinucleotide

repeats

-LRB-

-RRB-

-LRB-

The

precise

criteria

for

the

number

of

repeat

units

and

the

extent

of

divergence

allowed

in

an

SSR

affect

the

exact

census

but

not

the

qualitative

conclusions

-RRB-

There

is

approximately

one

SSR

per

kb

-LRB-

the

number

of

nonoverlapping

tandem

repeats

is

per

Mb

-RRB-

The

catalogue

con

rms

various

properties

of

SSRs

that

have

been

inferred

from

sampling

approaches

-LRB-

Table

-RRB-

The

most

frequent

dinucleotide

repeats

are

AC

and

AT

-LRB-

and

of

dinucleotide

repeats

respectively

-RRB-

whereas

AG

repeats

-LRB-

-RRB-

are

less

frequent

and

GC

repeats

-LRB-

-RRB-

are

greatly

under-represented

The

most

frequent

trinucleotides

are

AAT

and

AAC

-LRB-

and

respectively

-RRB-

whereas

ACC

-LRB-

-RRB-

AGC

-LRB-

-RRB-

ACT

-LRB-

-RRB-

and

ACG

-LRB-

-RRB-

are

relatively

rare

Overall

trinucleotide

SSRs

are

much

less

frequent

than

dinucleotide

SSRs

have

been

extremely

important

in

human

genetic

studies

because

they

show

a

high

degree

of

length

polymorphism

in

the

human

population

owing

to

frequent

slippage

by

DNA

polymerase

during

replication

Genetic

markers

based

on

SSRs

particularly

-LRB-

CA

-RRB-

n

repeats

have

been

the

workhorse

of

most

human

diseasemapping

The

availability

of

a

comprehensive

catalogue

of

SSRs

is

thus

a

boon

for

human

genetic

studies

The

SSR

catalogue

also

allowed

us

to

resolve

a

mystery

regarding

mammalian

genetic

maps

Such

genetic

maps

in

rat

mouse

and

human

have

a

de

cit

of

polymorphic

-LRB-

CA

-RRB-

n

repeats

on

chromosome

There

are

two

possible

explanations

for

this

de

cit

There

may

simply

be

fewer

-LRB-

CA

-RRB-

n

repeats

on

chromosome

X

or

-LRB-

CA

-RRB-

n

repeats

may

be

as

dense

on

chromosome

X

but

less

polymorphic

in

Table

SSRs

by

repeat

unit

Repeat

unit

AC

AT

AG

GC

AAT

AAC

AGG

AAG

ATG

CGG

ACC

AGC

ACT

ACG

Number

of

SSRs

per

Mb

A

remarkable

feature

of

the

human

genome

is

the

segmental

duplication

of

portions

of

genomic

Such

duplications

involve

the

transfer

of

blocks

of

genomic

sequence

to

one

or

more

locations

in

the

genome

The

locations

of

both

donor

and

recipient

regions

of

the

genome

are

often

not

tandemly

arranged

suggesting

mechanisms

other

than

unequal

crossing-over

for

their

origin

They

are

relatively

recent

inasmuch

as

strong

sequence

identity

is

seen

in

both

exons

and

introns

-LRB-

in

contrast

to

regions

that

are

considered

to

show

evidence

of

ancient

duplications

characterized

by

similarities

only

in

coding

regions

-RRB-

Indeed

many

such

duplications

appear

to

have

arisen

in

very

recent

evolutionary

time

as

judged

by

high

sequence

identity

and

by

their

absence

in

closely

related

species

Segmental

duplications

can

be

divided

into

two

categories

First

interchromosomal

duplications

are

de

ned

as

segments

that

are

duplicated

among

nonhomologous

chromosomes

For

example

a

genomic

segment

of

the

adrenoleukodystrophy

locus

from

has

been

duplicated

to

regions

near

the

centromeres

of

chromosomes

and

-LRB-

refs

-RRB-

Anecdotal

observations

suggest

that

many

interchromosomal

duplications

map

near

the

centromeric

and

telomeric

regions

of

human

The

second

category

is

intrachromosomal

duplications

which

occur

within

a

particular

chromosome

or

chromosomal

arm

This

category

includes

several

duplicated

segments

also

known

as

low

copy

repeat

sequences

that

mediate

recurrent

chromosomal

structural

rearrangements

associated

with

genetic

Examples

on

chromosome

include

three

copies

of

a

roughly

repeat

separated

by

around

Mb

and

two

copies

of

a

roughly

repeat

separated

by

Mb

The

copies

are

so

similar

-LRB-

identity

-RRB-

that

paralogous

recombination

events

can

occur

giving

rise

to

contiguous

gene

syndromes

Smith

Magenis

syndrome

and

Charcot

Marie

Tooth

syndrome

Several

other

examples

are

known

and

are

also

suspected

to

be

responsible

for

recurrent

microdeletion

syndromes

-LRB-

for

example

Prader

Willi/Angelman

SSRs

were

identi

ed

as

in

Table

Figure

Duplication

landscape

of

chromosome

The

size

and

location

of

intrachromosomal

-LRB-

blue

-RRB-

and

interchromosomal

-LRB-

red

-RRB-

duplications

are

depicted

for

chromosome

using

the

PARASIGHT

computer

program

-LRB-

Bailey

and

Eichler

unpublished

-RRB-

Each

horizontal

line

represents

Mb

-LRB-

ticks

intervals

-RRB-

The

chromosome

sequence

is

oriented

from

centromere

-LRB-

top

left

-RRB-

to

telomere

-LRB-

bottom

right

-RRB-

Pairwise

alignments

with

nucleotide

identity

and

kb

long

are

shown

Gaps

within

the

chromosomal

sequence

are

of

known

size

and

shown

as

empty

space

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articles

velocardiofacial/DiGeorge

and

Williams

-RRB-

Until

now

the

identi

cation

and

characterization

of

segmental

duplications

have

been

based

on

anecdotal

reports

for

example

nding

that

certain

probes

hybridize

to

multiple

chromosomal

sites

or

noticing

duplicated

sequence

at

certain

recurrent

chromosomal

breakpoints

The

availability

of

the

entire

genomic

sequence

will

make

it

possible

to

explore

the

nature

of

segmental

duplications

more

systematically

This

analysis

can

begin

with

the

current

state

of

the

draft

genome

sequence

although

caution

is

required

because

some

apparent

duplications

may

arise

from

a

failure

to

merge

sequence

contigs

from

overlapping

clones

Alternatively

erroneous

assembly

of

closely

related

sequences

from

nonoverlapping

clones

may

underestimate

the

true

frequency

of

such

features

particularly

among

those

segments

with

the

highest

sequence

similarity

Accordingly

we

adopted

a

conservative

approach

for

estimating

such

duplication

from

the

available

draft

genome

sequence

Pericentromeres

and

subtelomeres

We

began

by

re-evaluating

the

nished

sequences

of

chromosomes

and

The

initial

papers

on

these

noted

some

instances

of

interchromosomal

duplication

near

each

centromere

With

the

ability

now

to

compare

these

chromosomes

to

the

vast

majority

of

the

genome

it

is

apparent

that

the

regions

near

the

centromeres

consist

almost

entirely

of

interchromosomal

duplicated

segments

with

little

or

no

unique

sequence

Smaller

regions

of

interchromosomal

duplication

are

also

observed

near

the

telomeres

Chromosome

contains

a

region

of

Mb

adjacent

to

the

centromere

in

which

of

sequence

can

now

be

recognized

to

consist

of

interchromosomal

duplication

-LRB-

Fig.

-RRB-

Conversely

of

the

interchromosomal

duplications

on

chromosome

were

located

in

this

region

which

comprises

only

of

the

chromosome

Also

the

subtelomeric

end

consists

of

a

region

consisting

almost

entirely

of

interchromosomal

duplications

Chromosome

presents

a

similar

landscape

-LRB-

Fig.

-RRB-

The

rst

Mb

after

the

centromere

is

composed

of

interchromosomal

repeats

as

well

as

the

largest

-LRB-

kb

-RRB-

block

of

intrachromosomally

duplicated

material

Again

most

interchromosomal

duplications

on

the

chromosome

map

to

this

region

and

the

most

subtelomeric

region

-LRB-

kb

-RRB-

shows

extensive

duplication

among

nonhomologous

chromosomes

c

Sequence

identity

-LRB-

-RRB-

100 200 300 400

Figure

Duplication

landscape

of

chromosome

The

size

and

location

of

intrachromosomal

-LRB-

blue

-RRB-

and

interchromosomal

-LRB-

red

-RRB-

duplications

are

depicted

along

the

sequence

of

the

long

arm

of

chromosome

Gaps

between

nished

sequence

are

denoted

by

empty

space

but

do

not

represent

actual

gap

size

a

Sequence

identity

-LRB-

-RRB-

Sequence

identity

-LRB-

-RRB-

Sequence

identity

-LRB-

-RRB-

b

CTR

CMD

ALD

400 420 440

d

Sequence

identity

-LRB-

-RRB-

100 200 300 400

Sequence

identity

Sequence

identity

-LRB-

-RRB-

Sequence

identity

-LRB-

-RRB-

Figure

Mosaic

patterns

of

duplications

Panels

depict

various

patterns

of

duplication

within

the

human

genome

-LRB-

PARASIGHT

-RRB-

For

each

region

a

segment

of

draft

genome

sequence

-LRB-

kb

-RRB-

is

shown

with

both

interchromosomal

-LRB-

red

-RRB-

and

intrachromosomal

-LRB-

blue

-RRB-

duplications

displayed

along

the

horizontal

line

Below

the

line

each

separate

sequence

duplication

is

indicated

-LRB-

with

a

distinct

colour

-RRB-

relative

to

per

cent

nucleotide

identity

for

the

duplicated

segment

-LRB-

y

axis

-RRB-

Black

bars

show

the

relative

locations

of

large

blocks

of

heterochromatic

sequences

-LRB-

alpha

gamma

and

HSAT

sequence

-RRB-

a

An

active

pericentromeric

region

on

chromosome

b

An

ancestral

region

from

that

has

contributed

various

genic

segments

to

pericentromeric

regions

c

A

pericentromeric

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Sequence

identity

-LRB-

-RRB-

articles

The

pericentromeric

regions

are

structurally

very

complex

as

illustrated

for

chromosome

in

Fig.

The

pericentromeric

regions

appear

to

have

been

bombarded

by

successive

insertions

of

duplications

the

insertion

events

must

be

fairly

recent

because

the

degree

of

sequence

conservation

with

the

genomic

source

loci

is

fairly

high

-LRB-

with

an

apparent

peak

around

-RRB-

Distinct

insertions

are

typically

separated

by

AT-rich

or

GC-rich

minisatellite-like

repeats

that

have

been

hypothesized

to

have

a

functional

role

in

targeting

duplications

to

these

A

single

genomic

source

locus

often

gives

rise

to

pericentromeric

copies

on

multiple

chromosomes

with

each

having

essentially

the

same

breakpoints

and

the

same

degree

of

divergence

An

example

of

such

a

source

locus

on

is

shown

in

Fig.

Phylogenetic

analysis

has

suggested

a

two-step

mechanism

for

the

origin

and

dispersal

of

these

segments

whereby

an

initial

segmental

duplication

in

the

pericentromeric

region

of

one

chromosome

occurs

and

is

then

redistributed

as

part

of

a

larger

cassette

to

other

such

A

comprehensive

analysis

for

all

chromosomes

will

have

to

await

complete

sequencing

of

the

genome

but

the

evidence

from

the

draft

genome

sequence

indicates

that

the

same

picture

is

likely

to

be

seen

throughout

the

genome

Several

papers

have

analysed

nished

segments

within

pericentromeric

regions

of

chromosomes

-LRB-

kb

-RRB-

-LRB-

kb

-RRB-

and

-LRB-

kb

-RRB-

all

of

which

show

extensive

interchromosomal

segmental

An

example

from

another

pericentromeric

region

on

chromosome

is

shown

in

Fig.

Interchromosomal

duplications

in

subtelomeric

regions

also

appear

to

be

a

fairly

general

phenomenon

as

illustrated

by

a

large

tract

-LRB-

kb

-RRB-

of

complex

duplication

on

chromosome

-LRB-

Fig.

-RRB-

The

explanation

for

the

clustering

of

segmental

duplications

may

be

that

the

genome

has

a

damage-control

mechanism

whereby

chromosomal

breakage

products

are

preferentially

inserted

into

pericentromeric

and

to

a

lesser

extent

subtelomeric

regions

The

possibility

of

a

speci

c

mechanism

for

the

insertion

of

these

sequences

has

been

suggested

on

the

basis

of

the

unusual

sequences

found

anking

the

insertions

Although

it

is

also

possible

that

these

regions

simply

have

greater

tolerance

for

large

insertions

many

large

gene-poor

deserts

have

been

identi

and

there

is

no

accumulation

of

duplicated

segments

within

these

regions

Along

with

the

fact

that

transitions

between

duplicons

-LRB-

from

different

regions

of

the

genome

-RRB-

occur

at

speci

c

sequences

this

suggests

that

active

recruitment

of

duplications

to

such

regions

may

occur

In

any

case

the

duplicated

regions

are

in

general

young

-LRB-

with

many

duplications

showing

nucleotide

divergence

from

their

source

loci

-RRB-

and

in

constant

ux

both

through

additional

duplications

and

by

large-scale

exchange

among

similar

chromosomal

environments

There

is

evidence

of

structural

polymorphism

in

the

human

population

such

as

the

presence

or

absence

of

olfactory

receptor

segments

located

within

the

telomeric

regions

of

several

human

Genome-wide

analysis

of

segmental

duplications

We

also

performed

a

global

genome-wide

analysis

to

characterize

the

amount

of

segmental

duplication

in

the

genome

We

repeat-masked

the

known

interspersed

repeats

in

the

draft

genome

sequence

and

compared

the

remaining

draft

genomic

sequence

with

itself

in

a

massive

all-by-all

BLASTN

similarity

search

We

excluded

matches

in

which

the

sequence

identity

was

so

high

that

it

might

re

ect

artefactual

duplications

resulting

from

a

failure

to

overlap

sequence

contigs

correctly

in

assembling

the

draft

genome

sequence

Speci

cally

we

considered

only

matches

with

less

than

identity

for

nished

sequence

and

less

than

identity

for

un

nished

sequence

We

took

several

approaches

to

avoid

counting

artefactual

duplications

in

the

sequence

In

the

rst

approach

we

studied

only

nished

sequence

We

compared

the

nished

sequence

with

itself

to

identify

segments

of

at

least

kb

and

sequence

identity

This

analysis

will

underestimate

the

extent

of

segmental

NATURE

VOL

FEBRUARY

duplication

because

it

requires

that

at

least

two

copies

of

the

segment

are

present

in

the

nished

sequence

and

because

some

true

duplications

have

over

identity

The

nished

sequence

consists

of

at

least

segmental

duplication

-LRB-

Table

-RRB-

Interchromosomal

duplication

accounts

for

about

and

intrachromosomal

duplication

for

about

with

some

overlap

-LRB-

-RRB-

between

these

categories

We

analysed

the

lengths

and

divergence

of

the

segmental

duplications

-LRB-

Fig.

-RRB-

The

duplications

tend

to

be

large

-LRB-

kb

-RRB-

and

highly

homologous

especially

for

the

interchromosomal

segments

The

sequence

divergence

for

the

interchromosomal

duplications

appears

to

peak

between

and

This

may

indicate

that

interchromosomal

duplications

occurred

in

a

punctuated

manner

It

will

be

intriguing

to

investigate

whether

such

genomic

upheaval

has

a

role

in

speciation

events

In

a

second

approach

we

compared

the

entire

human

draft

genome

sequence

-LRB-

nished

and

un

nished

-RRB-

with

itself

to

identify

duplications

with

sequence

identity

-LRB-

Table

-RRB-

The

draft

genome

sequence

contains

at

least

segmental

duplication

The

actual

proportion

will

be

signi

cantly

higher

because

we

excluded

many

true

matches

with

more

than

sequence

identity

-LRB-

at

least

of

the

nished

sequence

-RRB-

Although

exact

measurement

must

await

a

nished

sequence

the

human

genome

seems

likely

to

contain

about

segmental

duplication

with

most

of

this

sequence

in

large

blocks

-LRB-

kb

-RRB-

Such

a

high

proportion

of

large

duplications

clearly

distinguishes

the

human

genome

from

other

sequenced

genomes

such

as

the

y

and

worm

-LRB-

Table

-RRB-

The

structure

of

large

highly

paralogous

regions

presents

one

of

the

serious

and

unanticipated

challenges

to

producing

a

nished

sequence

of

the

The

absence

of

unique

STS

or

ngerprint

signatures

over

large

genomic

distances

-LRB-

Mb

-RRB-

and

the

high

degree

of

sequence

similarity

makes

the

distinction

between

paralogous

sequence

variation

and

allelic

polymorphism

problematic

Furthermore

the

fact

that

such

regions

frequently

harbour

intron

exon

structures

of

genuine

unique

sequence

will

complicate

efforts

to

generate

a

genome-wide

SNP

map

The

data

indicate

that

a

modest

portion

of

the

human

genome

may

be

relatively

recalcitrant

to

genomic-based

methods

for

SNP

detection

Owing

to

their

repetitive

nature

and

their

location

in

the

genome

segmental

Table

Fraction

of

nished

sequence

in

inter

and

intrachromosomal

duplications

Chromosome

X

Y

Un

Total

Intrachromosomal

-LRB-

-RRB-

Interchromosomal

-LRB-

-RRB-

All

-LRB-

-RRB-

Excludes

duplications

with

identities

to

avoid

artefactual

duplication

due

to

incomplete

merger

in

the

assembly

process

Calculation

was

performed

on

the

nished

sequence

available

in

September

and

re

ects

the

duplications

found

within

the

total

amount

of

nished

sequence

then

Note

that

there

is

some

overlap

between

the

interchromosomal

and

intrachromosomal

sets

Macmillan

Magazines

Ltd

articles

a

Sum

of

aligned

bases

-LRB-

kb

-RRB-

Intrachromosomal

b

Sum

of

aligned

bases

-LRB-

kb

-RRB-

Interchromosomal

c

Sum

of

aligned

bases

-LRB-

kb

-RRB-

Similarity

-LRB-

-RRB-

d

Sum

of

aligned

bases

-LRB-

kb

-RRB-

Similarity

-LRB-

-RRB-

Length

of

alignment

-LRB-

kb

-RRB-

Length

of

alignment

-LRB-

kb

-RRB-

Figure

a

d

Sequence

properties

of

segmental

duplications

Distributions

of

length

and

per

cent

nucleotide

identity

for

segmental

duplications

are

shown

as

a

function

of

the

number

of

aligned

bp

for

the

subset

of

nished

genome

sequence

Intrachromosomal

red

interchromosomal

blue

duplications

may

well

be

underestimated

by

the

current

analysis

An

understanding

of

the

biology

pathology

and

evolution

of

these

duplications

will

require

specialized

efforts

within

these

exceptional

regions

of

the

human

genome

The

presence

and

distribution

of

such

segments

may

provide

evolutionary

fodder

for

processes

of

exon

shuf

ing

and

a

general

increase

in

protein

diversity

associated

with

domain

accretion

It

will

be

important

to

consider

both

genome-wide

duplication

events

and

more

restricted

punctuated

events

of

genome

duplication

as

forces

in

the

evolution

of

vertebrate

genomes

Gene

content

of

the

human

genome

Genes

-LRB-

or

at

least

their

coding

regions

-RRB-

comprise

only

a

tiny

fraction

of

human

DNA

but

they

represent

the

major

biological

function

of

the

genome

and

the

main

focus

of

interest

by

biologists

They

are

also

the

most

challenging

feature

to

identify

in

the

human

genome

sequence

The

ultimate

goal

is

to

compile

a

complete

list

of

all

human

genes

and

their

encoded

proteins

to

serve

as

a

periodic

table

for

biomedical

But

this

is

a

dif

cult

task

In

organisms

with

small

genomes

it

is

straightforward

to

identify

most

genes

by

the

presence

of

long

ORFs

In

contrast

human

genes

tend

to

have

small

exons

-LRB-

encoding

an

average

of

only

codons

-RRB-

separated

by

long

introns

-LRB-

some

exceeding

kb

-RRB-

This

creates

a

signal-to-noise

problem

with

the

result

that

computer

programs

for

direct

gene

prediction

have

only

limited

accuracy

Instead

computational

prediction

of

human

genes

must

rely

largely

on

the

availability

of

cDNA

sequences

or

on

sequence

conservation

with

genes

and

proteins

from

other

organisms

This

approach

is

adequate

for

strongly

conserved

genes

-LRB-

such

as

histones

or

ubiquitin

-RRB-

but

may

be

less

sensitive

to

rapidly

evolving

genes

-LRB-

including

many

crucial

to

speciation

sex

determination

and

fertilization

-RRB-

Here

we

describe

our

efforts

to

recognize

both

the

RNA

genes

and

protein-coding

genes

in

the

human

genome

We

also

study

the

properties

of

the

predicted

human

protein

set

attempting

to

discern

how

the

human

proteome

differs

from

those

of

invertebrates

such

as

worm

and

y.

Noncoding

RNAs

Table

Fraction

of

the

draft

genome

sequence

in

inter

and

intrachromosomal

duplications

Chromosome

X

Y

UL

Total

Intrachromosomal

-LRB-

-RRB-

Interchromosomal

-LRB-

-RRB-

All

-LRB-

-RRB-

Although

biologists

often

speak

of

a

tight

coupling

between

genes

Table

Cross-species

comparison

for

large

highly

homologous

segmental

duplications

Percentage

of

genome

-LRB-

-RRB-

Fly

kb

kb

kb

Worm

Human

-LRB-

nished

-RRB-

Excludes

duplications

with

identities

to

avoid

artefactual

duplication

due

to

incomplete

merger

in

the

assembly

process

Calculation

was

performed

on

an

earlier

version

of

the

draft

genome

sequence

based

on

data

available

in

July

and

re

ects

the

duplications

found

within

the

total

amount

of

nished

sequence

then

Note

that

there

is

some

overlap

between

the

interchromosomal

and

intrachromosomal

sets

This

is

an

underestimate

of

the

total

amount

of

segmental

duplication

in

the

human

genome

because

it

only

re

ects

duplication

detectable

with

available

nished

sequence

The

proportion

of

segmental

duplications

of

kb

is

probably

about

-LRB-

see

text

-RRB-

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

and

their

encoded

protein

products

it

is

important

to

remember

that

thousands

of

human

genes

produce

noncoding

RNAs

-LRB-

ncRNAs

-RRB-

as

their

ultimate

There

are

several

major

classes

of

ncRNA

-LRB-

-RRB-

Transfer

RNAs

-LRB-

tRNAs

-RRB-

are

the

adapters

that

translate

the

triplet

nucleic

acid

code

of

RNA

into

the

amino-acid

sequence

of

proteins

-LRB-

-RRB-

ribosomal

RNAs

-LRB-

rRNAs

-RRB-

are

also

central

to

the

translational

machinery

and

recent

X-ray

crystallography

results

strongly

indicate

that

peptide

bond

formation

is

catalysed

by

rRNA

not

-LRB-

-RRB-

small

nucleolar

RNAs

-LRB-

snoRNAs

-RRB-

are

required

for

rRNA

processing

and

base

modi

cation

in

the

and

-LRB-

-RRB-

small

nuclear

RNAs

-LRB-

snRNAs

-RRB-

are

critical

components

of

spliceosomes

the

large

ribonucleoprotein

-LRB-

RNP

-RRB-

complexes

that

splice

introns

out

of

pre-mRNAs

in

the

nucleus

Humans

have

both

a

major

snRNA-dependent

spliceosome

that

splices

most

introns

and

a

minor

snRNA-dependent

spliceosome

that

splices

a

rare

class

of

introns

that

often

have

AT/AC

dinucleotides

at

the

splice

sites

instead

of

the

canonical

GT/AG

splice

site

Other

ncRNAs

include

both

RNAs

of

known

biochemical

function

-LRB-

such

as

telomerase

RNA

and

the

signal

recognition

particle

RNA

-RRB-

and

ncRNAs

of

enigmatic

function

-LRB-

such

as

the

large

Xist

transcript

implicated

in

X

dosage

or

the

small

vault

RNAs

found

in

the

bizarre

vault

ribonucleoprotein

which

is

three

times

the

mass

of

the

ribosome

but

has

unknown

function

-RRB-

ncRNAs

do

not

have

translated

ORFs

are

often

small

and

are

not

polyadenylated

Accordingly

novel

ncRNAs

can

not

readily

be

found

by

computational

gene

nding

techniques

-LRB-

which

search

for

features

such

as

ORFs

-RRB-

or

experimental

sequencing

of

cDNA

or

EST

libraries

-LRB-

most

of

which

are

prepared

by

reverse

transcription

using

a

primer

complementary

to

a

poly

-LRB-

A

-RRB-

tail

-RRB-

Even

if

the

complete

nished

sequence

of

the

human

genome

were

available

discovering

novel

ncRNAs

would

still

be

challenging

We

can

however

identify

genomic

sequences

that

are

homologous

to

known

ncRNA

genes

using

BLASTN

or

in

some

cases

more

specialized

methods

It

is

sometimes

dif

cult

to

tell

whether

such

homologous

genes

are

orthologues

paralogues

or

closely

related

pseudogenes

-LRB-

because

inactivating

mutations

are

much

less

obvious

than

for

proteincoding

genes

-RRB-

For

tRNA

there

is

suf

ciently

detailed

information

about

the

cloverleaf

secondary

structure

to

allow

true

genes

and

pseudogenes

to

be

distinguished

with

high

sensitivity

For

many

other

ncRNAs

there

is

much

less

structural

information

and

so

we

employ

an

operational

criterion

of

high

sequence

similarity

-LRB-

sequence

identity

and

full

length

-RRB-

to

distinguish

true

genes

from

pseudogenes

These

assignments

will

eventually

need

to

be

reconciled

with

experimental

data

Transfer

RNA

genes

The

classical

experimental

estimate

of

the

number

of

human

tRNA

genes

is

-LRB-

ref

-RRB-

In

the

draft

genome

sequence

we

nd

only

human

tRNA

genes

-LRB-

Tables

-RRB-

How

do

we

account

for

this

discrepancy

We

believe

that

the

original

estimate

is

likely

to

have

been

in

ated

in

two

respects

First

it

came

from

a

hybridization

experiment

that

probably

counted

closely

related

pseudogenes

by

analysis

of

the

draft

genome

sequence

there

are

in

fact

tRNA-derived

putative

pseudogenes

Table

Number

of

tRNA

genes

in

various

organisms

Organism

Human

Worm

Fly

Yeast

Methanococcus

jannaschii

Escherichia

coli

Number

of

canonical

tRNAs

497 584 284 273

SeCys

tRNA

Number

of

tRNA

genes

in

each

of

six

genome

sequences

according

to

analysis

by

the

computer

program

tRNAscan-SE

Canonical

tRNAs

read

one

of

the

standard

sense

codons

this

category

excludes

pseudogenes

undetermined

anticodons

putative

supressors

and

selenocysteine

tRNAs

Most

organisms

have

a

selenocysteine

-LRB-

SeCys

-RRB-

tRNA

species

but

some

unicellular

eukaryotes

do

not

-LRB-

such

as

the

yeast

S.

cerevisiae

-RRB-

-LRB-

Table

-RRB-

Second

the

earlier

estimate

assumed

too

high

a

value

for

the

size

of

the

human

genome

repeating

the

calculation

using

the

correct

value

yields

an

estimate

of

about

tRNA-related

loci

which

is

in

reasonable

accord

with

our

count

of

tRNA

genes

and

pseudogenes

in

the

draft

genome

sequence

The

human

tRNA

gene

set

predicted

from

the

draft

genome

sequence

appears

to

include

most

of

the

known

human

tRNA

species

The

draft

genome

sequence

contains

of

human

tRNA

species

listed

in

a

tRNA

allowing

for

up

to

one

mismatch

This

includes

one

copy

of

the

known

gene

for

a

specialized

selenocysteine

tRNA

one

of

several

components

of

a

baroque

translational

mechanism

that

reads

UGA

as

a

selenocysteine

codon

in

certain

rare

mRNAs

that

carry

a

speci

c

cis-acting

RNA

regulatory

site

-LRB-

a

so-called

SECIS

element

-RRB-

in

their

UTRs

The

one

tRNA

gene

in

the

database

not

found

in

the

draft

genome

sequence

is

a

tRNAGlu

species

which

differs

in

two

positions

from

the

most

related

tRNA

gene

in

the

human

genome

Possible

explanations

are

that

the

database

version

of

this

tRNA

contains

two

errors

the

gene

is

polymorphic

or

this

is

a

genuine

functional

tRNA

that

is

missing

from

the

draft

genome

sequence

-LRB-

The

database

also

lists

one

additional

tRNA

gene

-LRB-

-RRB-

but

this

is

apparently

a

contaminant

most

similar

to

bacterial

tRNAs

the

parent

entry

-LRB-

-RRB-

was

withdrawn

from

the

DNA

database

but

the

tRNA

entry

has

not

yet

been

removed

from

the

tRNA

database

-RRB-

Although

the

human

set

appears

substantially

complete

by

this

test

the

tRNA

gene

numbers

in

Table

should

be

considered

tentative

and

used

with

caution

The

human

and

y

-LRB-

but

not

the

worm

-RRB-

are

known

to

be

missing

signi

cant

amounts

of

heterochromatic

DNA

and

additional

tRNA

genes

could

be

located

there

With

this

caveat

the

results

indicate

that

the

human

has

fewer

tRNA

genes

than

the

worm

but

more

than

the

y

This

may

seem

surprising

but

tRNA

gene

number

in

metazoans

is

thought

to

be

related

not

to

organismal

complexity

but

more

to

idiosyncrasies

of

the

demand

for

tRNA

abundance

in

certain

tissues

or

stages

of

embryonic

development

For

example

the

frog

Xenopus

laevis

which

must

load

each

oocyte

with

a

remarkable

ng

of

tRNA

has

thousands

of

tRNA

The

degeneracy

of

the

genetic

code

has

allowed

an

inspired

economy

of

tRNA

anticodon

usage

Although

sense

codons

need

to

be

decoded

not

all

different

anticodons

are

present

in

tRNAs

Rather

tRNAs

generally

follow

stereotyped

and

conserved

wobble

Wobble

reduces

the

number

of

required

anticodons

substantially

and

provides

a

connection

between

the

genetic

code

and

the

hybridization

stability

of

modi

ed

and

unmodi

ed

RNA

bases

In

eukaryotes

the

rules

proposed

by

Guthrie

and

predict

that

about

tRNA

species

will

be

suf

cient

to

read

the

sense

codons

-LRB-

counting

the

initiator

and

elongator

methionine

tRNAs

as

two

species

-RRB-

According

to

these

rules

in

the

codon

's

third

-LRB-

wobble

-RRB-

position

U

and

C

are

generally

decoded

by

a

single

tRNA

species

whereas

A

and

G

are

decoded

by

two

separate

tRNA

species

In

two-codon

boxes

of

the

genetic

code

-LRB-

where

codons

ending

with

U/C

encode

a

different

amino

acid

from

those

ending

with

A/G

-RRB-

the

U/C

wobble

position

should

be

decoded

by

a

G

at

position

in

the

tRNA

anticodon

Thus

in

the

top

left

of

Fig.

there

is

no

tRNA

with

an

AAA

anticodon

for

Phe

but

the

GAA

anticodon

can

recognize

both

UUU

and

UUC

codons

in

the

mRNA

In

fourcodon

boxes

of

the

genetic

code

-LRB-

where

U

C

A

and

G

in

the

wobble

position

all

encode

the

same

amino

acid

-RRB-

the

U/C

wobble

position

is

almost

always

decoded

by

-LRB-

inosine

-RRB-

in

the

tRNA

where

the

inosine

is

produced

by

post-transcriptional

modi

cation

of

an

adenine

-LRB-

A

-RRB-

In

the

bottom

left

of

Fig.

for

example

the

GUU

and

GUC

codons

of

the

four-codon

Val

box

are

decoded

by

a

tRNA

with

an

anticodon

of

AAC

which

is

no

doubt

modi

ed

to

IAC

Presumably

this

pattern

which

is

strikingly

conserved

in

eukaryotes

has

to

do

with

the

fact

that

IA

base

pairs

are

also

possible

thus

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

the

IAC

anticodon

for

a

Val

tRNA

could

recognize

GUU

GUC

and

even

GUA

codons

Were

this

same

to

be

utilized

in

two-codon

boxes

however

misreading

of

the

NNA

codon

would

occur

resulting

in

translational

havoc

Eukaryotic

glycine

tRNAs

represent

a

conserved

exception

to

this

last

rule

they

use

a

GCC

anticodon

to

decode

GGU

and

GGC

rather

than

the

expected

ICC

anticodon

Satisfyingly

the

human

tRNA

set

follows

these

wobble

rules

almost

perfectly

-LRB-

Fig.

-RRB-

Only

three

unexpected

tRNA

species

are

found

single

genes

for

a

tRNATyr-AUA

tRNAIle-GAU

and

tRNAAsn-AUU

Perhaps

these

are

pseudogenes

but

they

appear

to

be

plausible

tRNAs

We

also

checked

the

possibility

of

sequencing

errors

in

their

anticodons

but

each

of

these

three

genes

is

in

a

region

of

high

sequence

accuracy

with

PHRAP

quality

scores

higher

than

for

every

base

in

their

anticodons

As

in

all

other

organisms

human

protein-coding

genes

show

codon

bias

preferential

use

of

one

synonymous

codon

over

-LRB-

Fig.

-RRB-

In

less

complex

organisms

such

as

yeast

or

bacteria

highly

expressed

genes

show

the

strongest

codon

bias

Cytoplasmic

abundance

of

tRNA

species

is

correlated

with

both

codon

bias

and

overall

amino-acid

frequency

-LRB-

for

example

tRNAs

for

preferred

codons

and

for

more

common

amino

acids

are

more

abundant

-RRB-

This

is

presumably

driven

by

selective

pressure

for

ef

cient

or

accurate

In

many

organisms

tRNA

abundance

in

turn

appears

to

be

roughly

correlated

with

tRNA

gene

copy

number

so

tRNA

gene

copy

number

has

been

used

as

a

proxy

for

tRNA

In

vertebrates

however

codon

bias

is

not

so

obviously

correlated

with

gene

expression

level

Differing

codon

biases

between

human

genes

is

more

a

function

of

their

location

in

regions

of

different

GC

In

agreement

with

the

literature

we

see

only

a

very

rough

correlation

of

human

tRNA

gene

number

with

either

amino-acid

frequency

or

codon

bias

-LRB-

Fig.

-RRB-

The

most

obvious

outliers

in

these

weak

correlations

are

UUU

Phe

UUC

UUA

Leu

UUG

CAA

UCG

CGA

stop

UAG

CUA

Trp

UGG

CCA

GAA

Ser

UAA

UCA

UGA

UCC

GGA

stop

AAA

UCU

AGA

Tyr

UAC

UAA

GUA

UUA

stop

the

strongly

preferred

CUG

leucine

codon

with

a

mere

six

tRNALeu-CAG

genes

producing

a

tRNA

to

decode

it

and

the

relatively

rare

cysteine

UGU

and

UGC

codons

with

tRNA

genes

to

decode

them

The

tRNA

genes

are

dispersed

throughout

the

human

genome

However

this

dispersal

is

nonrandom

tRNA

genes

have

sometimes

been

seen

in

clusters

at

small

but

we

can

now

see

striking

clustering

on

a

genome-wide

scale

More

than

of

the

tRNA

genes

-LRB-

-RRB-

are

found

in

a

region

of

only

about

Mb

on

chromosome

This

small

region

only

about

of

the

genome

contains

an

almost

suf

cient

set

of

tRNA

genes

all

by

itself

The

tRNA

genes

contain

a

representative

for

of

the

anticodons

found

in

the

complete

set

and

of

the

isoacceptor

types

only

tRNAs

to

decode

Asn

Cys

Glu

and

selenocysteine

are

missing

Many

of

these

tRNA

genes

meanwhile

are

clustered

elsewhere

of

the

Cys

tRNAs

are

found

in

a

stretch

of

chromosome

and

many

of

the

Asn

and

Glu

tRNA

genes

are

loosely

clustered

on

chromosome

More

than

half

of

the

tRNA

genes

-LRB-

out

of

-RRB-

reside

on

either

chromosome

or

chromosome

Chromosomes

and

X

appear

to

have

fewer

than

tRNA

genes

each

and

chromosomes

and

Y

have

at

all

-LRB-

each

has

a

single

pseudogene

-RRB-

Ribosomal

RNA

genes

The

ribosome

the

protein

synthetic

machine

of

the

cell

is

made

up

of

two

subunits

and

contains

four

rRNA

species

and

many

proteins

The

large

ribosomal

subunit

contains

and

S

rRNAs

-LRB-

collectively

called

large

subunit

-LRB-

LSU

-RRB-

rRNA

-RRB-

and

also

a

rRNA

The

small

ribosomal

subunit

contains

rRNA

-LRB-

small

subunit

-LRB-

SSU

-RRB-

rRNA

-RRB-

The

genes

for

LSU

and

SSU

rRNA

occur

in

the

human

genome

as

a

tandem

repeat

There

are

thought

to

be

about

copies

of

this

repeat

unit

arrayed

on

the

short

arms

of

acrocentric

chromosomes

and

-LRB-

refs

-RRB-

There

are

no

true

complete

UAU

AUA

Cys

UGC

UGA

GCA

UCA

UGU

ACA

CUU

CUC

Leu

CUA

CUG

AAG

GAG

Pro

UAG

CAG

CCU

CCC

CCA

CCG

AGG

His

GGG

UGG

Gln

CGG

CAU

CAC

CAA

CAG

AUG

GUG

Arg

UUG

CUG

CGU

CGC

CGA

CGG

ACG

GCG

UCG

CCG

AUU

Ile

AUC

AUA

Met

AUG

AAU

GAU

Thr

UAU

CAU

ACU

ACC

ACA

ACG

AGU

Asn

GGU

UGU

Lys

CGU

AAU

AAC

AAA

AAG

AUU

Ser

GUU

UUU

Arg

CUU

AGU

AGC

AGA

AGG

ACU

GCU

UCU

CCU

GUU

GUC

Val

GUA

GUG

AAC

GAC

Ala

UAC

CAC

GCU

GCC

GCA

GCG

AGC

Asp

GGC

UGC

Glu

CGC

GAU

GAC

GAA

GAG

AUC

GUC

Gly

UUC

CUC

GGU

GGC

GGA

GGG

ACC

GCC

UCC

CCC

Figure

The

human

genetic

code

and

associated

tRNA

genes

For

each

of

the

codons

we

show

the

corresponding

amino

acid

the

observed

frequency

of

the

codon

per

codons

the

codon

predicted

wobble

pairing

to

a

tRNA

anticodon

-LRB-

black

lines

-RRB-

an

unmodi

ed

tRNA

anticodon

sequence

and

the

number

of

tRNA

genes

found

with

this

anticodon

For

example

phenylalanine

is

encoded

by

UUU

or

UUC

UUC

is

seen

more

frequently

to

occurrences

per

total

codons

both

codons

are

expected

to

be

decoded

by

a

single

tRNA

anticodon

type

GAA

using

a

G/U

wobble

and

there

are

tRNA

genes

found

with

this

anticodon

The

modi

ed

anticodon

sequence

in

the

mature

tRNA

is

not

shown

even

where

post-transcriptional

modi

cations

can

be

con

dently

predicted

-LRB-

for

example

when

an

A

is

used

to

decode

a

U/C

third

position

the

A

is

almost

certainly

an

inosine

in

the

mature

tRNA

-RRB-

The

Figure

also

does

not

show

the

number

of

distinct

tRNA

species

-LRB-

such

as

distinct

sequence

families

-RRB-

for

each

anticodon

often

there

is

more

than

one

species

for

each

anticodon

Macmillan

Magazines

Ltd

NATURE

VOL

FEBRUARY

articles

copies

of

the

rDNA

tandem

repeats

in

the

draft

genome

sequence

owing

to

the

deliberate

bias

in

the

initial

phase

of

the

sequencing

effort

against

sequencing

BAC

clones

whose

restriction

fragment

ngerprints

showed

them

to

contain

primarily

tandemly

repeated

sequence

Sequence

similarity

analysis

with

the

BLASTN

computer

program

does

however

detect

hundreds

of

rDNA-derived

sequence

fragments

dispersed

throughout

the

complete

genome

including

one

full-length

copy

of

an

individual

S

rRNA

gene

not

associated

with

a

true

tandem

repeat

unit

-LRB-

Table

-RRB-

The

rDNA

genes

also

occur

in

tandem

arrays

the

largest

of

which

is

on

chromosome

between

and

close

to

the

There

are

true

genes

in

these

The

number

of

sequences

in

the

genome

including

numerous

dispersed

pseudogenes

is

classically

cited

as

-LRB-

refs

-RRB-

The

long

tandem

array

on

chromosome

is

not

yet

present

in

the

draft

genome

sequence

because

there

are

no

EcoRI

or

HindIII

sites

present

and

thus

it

was

not

cloned

in

the

most

heavily

utilized

BAC

libraries

-LRB-

Table

-RRB-

We

expect

to

recover

it

during

the

nishing

stage

We

do

detect

four

individual

copies

of

rDNA

by

our

search

criteria

-LRB-

identity

and

full

length

-RRB-

We

also

nd

many

more

distantly

related

dispersed

sequences

-LRB-

at

P

-RRB-

which

we

interpret

as

probable

pseudogenes

-LRB-

Table

-RRB-

Small

nucleolar

RNA

genes

Eukaryotic

rRNA

is

extensively

processed

and

modi

ed

in

the

nucleolus

Much

of

this

activity

is

directed

by

numerous

snoRNAs

These

come

in

two

families

C/D

box

snoRNAs

-LRB-

mostly

involved

in

guiding

site-speci

c

methylations

of

other

RNAs

-RRB-

and

H/ACA

snoRNAs

-LRB-

mostly

involved

in

guiding

site-speci

c

pseudouridylations

-RRB-

We

compiled

a

set

of

known

human

snoRNA

gene

sequences

of

these

-LRB-

-RRB-

have

at

least

one

copy

in

the

draft

genome

sequence

-LRB-

Table

-RRB-

almost

all

as

single-copy

genes

It

is

thought

that

all

methylations

and

pseudouridylations

in

eukaryotic

rRNA

are

guided

by

snoRNAs

There

are

methylations

and

around

pseudouridylations

in

human

Table

Known

non-coding

RNA

genes

in

the

draft

genome

sequence

RNA

gene

tRNA

SSU

-LRB-

-RRB-

rRNA

S

rRNA

LSU

-LRB-

-RRB-

rRNA

rRNA

SRP

-LRB-

-RRB-

RNA

RNAse

P

RNAse

MRP

Telomerase

RNA

-LRB-

S

RNA

-RRB-

Vault

RNAs

Xist

Known

C/D

snoRNAs

Known

H/ACA

snoRNAs

Number

expected

Number

found

Number

of

related

genes

40 11 181 520

353 414 115

Function

Protein

synthesis

Protein

synthesis

Protein

synthesis

Protein

synthesis

Protein

synthesis

Spliceosome

component

Spliceosome

component

Spliceosome

component

Component

of

minor

-LRB-

-RRB-

spliceosome

Spliceosome

component

Spliceosome

component

Component

of

minor

-LRB-

-RRB-

spliceosome

Histone

mRNA

processing

Component

of

minor

-LRB-

-RRB-

spliceosome

Component

of

minor

-LRB-

-RRB-

spliceosome

Component

of

signal

recognition

particle

-LRB-

protein

secretion

-RRB-

tRNA

end

processing

rRNA

processing

Template

for

addition

of

telomeres

Component

of

Ro

RNP

function

unknown

Component

of

Ro

RNP

function

unknown

Component

of

Ro

RNP

function

unknown

Component

of

Ro

RNP

function

unknown

Component

of

vault

RNP

function

unknown

Unknown

Unknown

Initiation

of

X

chromosome

inactivation

-LRB-

dosage

compensation

-RRB-

Pre-rRNA

processing

or

site-speci

c

ribose

methylation

of

rRNA

Pre-rRNA

processing

or

site-speci

c

pseudouridylation

of

rRNA

Only

about

half

of

these

have

been

tentatively

assigned

to

known

guide

snoRNAs

There

are

also

snoRNA-directed

modi

cations

on

other

stable

RNAs

such

as

-LRB-

ref

-RRB-

and

the

extent

of

this

is

just

beginning

to

be

explored

Sequence

similarity

has

so

far

proven

insuf

cient

to

recognize

all

snoRNA

genes

We

therefore

expect

that

there

are

many

unrecognized

snoRNA

genes

that

are

not

detected

by

BLAST

queries

Spliceosomal

RNAs

and

other

ncRNA

genes

We

also

looked

for

copies

of

other

known

ncRNA

genes

We

found

at

least

one

copy

of

-LRB-

-RRB-

of

known

ncRNAs

including

the

spliceosomal

snRNAs

There

were

multiple

copies

for

several

ncRNAs

as

expected

for

example

we

nd

dispersed

genes

for

snRNA

and

for

snRNA

-LRB-

Table

-RRB-

For

some

of

these

RNA

genes

homogeneous

multigene

families

that

occur

in

tandem

arrays

are

again

under-represented

owing

to

the

restriction

enzymes

used

in

constructing

the

BAC

libraries

and

in

some

instances

the

decision

to

delay

the

sequencing

of

BAC

clones

with

low

complexity

ngerprints

indicative

of

tandemly

repeated

DNA

The

RNA

genes

are

located

at

the

locus

a

tandem

array

of

copies

of

nearly

identical

units

at

-LRB-

refs

-RRB-

Similarly

the

snoRNA

genes

-LRB-

included

in

the

aggregate

count

of

C/D

snoRNAs

in

Table

-RRB-

are

clustered

at

the

locus

at

not

in

a

tandem

array

but

in

a

complex

inverted

repeat

structure

of

about

copies

per

haploid

The

RNA

genes

are

clustered

with

about

copies

at

the

locus

at

but

this

cluster

is

thought

to

be

loose

and

irregularly

organized

no

two

genes

have

been

cloned

on

the

same

In

the

draft

genome

sequence

we

see

six

copies

of

RNA

that

meet

our

criteria

for

true

genes

three

of

which

appear

to

be

in

the

expected

position

on

chromosome

For

so

far

we

see

one

true

copy

at

the

correct

place

on

chromosome

For

we

see

true

genes

of

which

are

loosely

clustered

within

Mb

at

and

another

are

elsewhere

on

chromosome

Again

these

and

other

clusters

will

be

a

matter

for

the

nishing

process

Known

ncRNA

genes

-LRB-

or

gene

families

such

as

the

C/D

and

H/ACA

snoRNA

families

-RRB-

reference

sequences

were

extracted

from

GenBank

and

used

to

probe

the

draft

genome

sequence

Number

of

genes

that

were

expected

in

the

human

genome

based

on

previous

literature

-LRB-

note

that

earlier

experimental

techniques

probably

tend

to

overestimate

copy

number

by

counting

closely

related

pseudogenes

-RRB-

The

copy

number

of

true

full-length

genes

identi

ed

in

the

draft

genome

sequence

The

copy

number

of

other

signi

cantly

related

copies

-LRB-

pseudogenes

fragments

paralogues

-RRB-

found

Except

for

the

true

tRNA

genes

all

sequence

similarities

were

identi

ed

by

WashU

BLASTN

MP

-LRB-

W.

Gish

unpublished

-RRB-

with

parameters

kap

wordmask

seg

B

W

and

the

default

DNA

scoring

matrix

True

genes

were

operationally

de

ned

as

BLAST

hits

with

identity

over

of

the

length

of

the

query

Related

sequences

were

operationally

de

ned

as

all

other

BLAST

hits

with

P-values

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

Table

Characteristics

of

human

genes

Median

Internal

exon

Exon

number

Introns

UTR

UTR

Coding

sequence

-LRB-

CDS

-RRB-

Genomic

extent

bp

bp

bp

bp

bp

aa

kb

Mean

bp

bp

bp

bp

bp

aa

kb

Sample

-LRB-

size

-RRB-

RefSeq

alignments

to

draft

genome

sequence

with

con

rmed

intron

boundaries

-LRB-

exons

-RRB-

RefSeq

alignments

to

nished

sequence

-LRB-

genes

-RRB-

RefSeq

alignments

to

nished

sequence

-LRB-

introns

-RRB-

Con

rmed

by

mRNA

or

EST

on

chromosome

-LRB-

-RRB-

Con

rmed

by

mRNA

or

EST

on

chromosome

-LRB-

-RRB-

Selected

RefSeq

entries

-LRB-

-RRB-

Selected

RefSeq

entries

-LRB-

-RRB-

Median

and

mean

values

for

a

number

of

properties

of

human

protein-coding

genes

The

selected

RefSeq

entries

were

those

that

could

be

unambiguously

aligned

to

nished

sequence

over

their

entire

length

Percentage

of

exons

Protein-coding

genes

Our

observations

also

con

rm

the

striking

proliferation

of

ncRNA-derived

pseudogenes

-LRB-

Table

-RRB-

There

are

hundreds

or

thousands

of

sequences

in

the

draft

genome

sequence

related

to

some

of

the

ncRNA

genes

The

most

proli

c

pseudogene

counts

generally

come

from

RNA

genes

transcribed

by

RNA

polymerase

III

promoters

including

the

hY

RNAs

and

SRP-RNA

These

ncRNA

pseudogenes

presumably

arise

through

reverse

transcription

The

frequency

of

such

events

gives

insight

into

how

ncRNA

genes

can

evolve

into

SINE

retroposons

such

as

the

tRNA-derived

SINEs

found

in

many

vertebrates

and

the

SRP-RNA-derived

Alu

elements

found

in

humans

underestimates

In

particular

the

UTRs

given

in

the

RefSeq

database

are

likely

to

be

incomplete

they

are

considerably

shorter

for

example

than

those

derived

from

careful

reconstructions

on

chromosome

Intron

sizes

were

measured

only

for

genes

in

nished

genomic

sequence

to

mitigate

the

bias

arising

from

the

fact

that

a

100 200 300 400

Exon

length

-LRB-

bp

-RRB-

700 800 900

Human

Worm

Fly

Identifying

the

protein-coding

genes

in

the

human

genome

is

one

of

the

most

important

applications

of

the

sequence

data

but

also

one

of

the

most

dif

cult

challenges

We

describe

below

our

efforts

to

create

an

initial

human

gene

and

protein

index

Exploring

properties

of

known

genes

Before

attempting

to

identify

new

genes

we

explored

what

could

be

learned

by

aligning

the

cDNA

sequences

of

known

genes

to

the

draft

genome

sequence

Genomic

alignments

allow

one

to

study

exon

intron

structure

and

local

GC

content

and

are

valuable

for

biomedical

studies

because

they

connect

genes

with

the

genetic

and

cytogenetic

map

link

them

with

regulatory

sequences

and

facilitate

the

development

of

polymerase

chain

reaction

-LRB-

PCR

-RRB-

primers

to

amplify

exons

Until

now

genomic

alignment

was

available

for

only

about

a

quarter

of

known

genes

The

known

genes

studied

were

those

in

the

RefSeq

a

manually

curated

collection

designed

to

contain

nonredundant

representatives

of

most

full-length

human

mRNA

sequences

in

GenBank

-LRB-

RefSeq

intentionally

contains

some

alternative

splice

forms

of

the

same

genes

-RRB-

The

version

of

RefSeq

used

contained

mRNAs

The

RefSeq

genes

were

aligned

with

the

draft

genome

sequence

using

both

the

Spidey

-LRB-

S.

Wheelan

personal

communication

-RRB-

and

Acembly

-LRB-

D.

Thierry-Mieg

and

J.

Thierry-Mieg

unpublished

-RRB-

computer

programs

Because

this

sequence

is

incomplete

and

contains

errors

not

all

genes

could

be

fully

aligned

and

some

may

have

been

incorrectly

aligned

More

than

of

the

RefSeq

entries

could

be

aligned

at

high

stringency

over

at

least

part

of

their

length

and

could

be

aligned

over

more

than

half

of

their

length

Some

genes

-LRB-

-RRB-

had

high

stringency

alignments

to

more

than

one

location

in

the

draft

genome

sequence

owing

for

example

to

paralogues

or

pseudogenes

In

such

cases

we

considered

only

the

best

match

In

a

few

of

these

cases

the

assignment

may

not

be

correct

because

the

true

matching

region

has

not

yet

been

sequenced

Three

per

cent

of

entries

appeared

to

be

alternative

splice

products

of

the

same

gene

on

the

basis

of

their

alignment

to

the

same

location

in

the

draft

genome

sequence

In

all

we

obtained

at

least

partial

genomic

alignments

for

distinct

known

genes

and

essentially

complete

alignment

for

of

them

Previous

efforts

to

study

human

gene

have

been

hampered

by

limited

sample

sizes

and

strong

biases

in

favour

of

compact

genes

Table

gives

the

mean

and

median

values

of

some

basic

characteristics

of

gene

structures

Some

of

the

values

may

be

b

Percentage

of

introns

bp

bp

kb

kb

kb

Intron

length

kb

kb

Human

Worm

Fly

c

Percentage

of

introns

Human

Worm

Fly

60 80 100 120

Intron

length

-LRB-

bp

-RRB-

Figure

Size

distributions

of

exons

introns

and

short

introns

in

sequenced

genomes

a

Exons

b

introns

c

short

introns

-LRB-

enlarged

from

b

-RRB-

Con

rmed

exons

and

introns

for

the

human

were

taken

from

RefSeq

alignments

and

for

worm

and

y

from

Acembly

alignments

of

ESTs

-LRB-

J.

and

D.

Thierry-Mieg

and

for

worm

Y.

Kohara

unpublished

-RRB-

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

long

introns

are

more

likely

than

short

introns

to

be

interrupted

by

gaps

in

the

draft

genome

sequence

Nonetheless

there

may

be

some

residual

bias

against

long

genes

and

long

introns

There

is

considerable

variation

in

overall

gene

size

and

intron

size

with

both

distributions

having

very

long

tails

Many

genes

are

over

kb

long

the

largest

known

example

being

the

dystrophin

gene

-LRB-

DMD

-RRB-

at

Mb

The

variation

in

the

size

distribution

of

coding

sequences

and

exons

is

less

extreme

although

there

are

still

some

remarkable

outliers

The

titin

has

the

longest

currently

known

coding

sequence

at

bp

it

also

has

the

largest

number

of

exons

-LRB-

-RRB-

and

longest

single

exon

-LRB-

bp

-RRB-

It

is

instructive

to

compare

the

properties

of

human

genes

with

those

from

worm

and

y.

For

all

three

organisms

the

typical

length

of

a

coding

sequence

is

similar

-LRB-

bp

for

worm

bp

for

y

and

bp

for

human

-RRB-

and

most

internal

exons

fall

within

a

common

peak

between

and

bp

-LRB-

Fig.

-RRB-

However

the

worm

and

y

exon

distributions

have

a

fatter

tail

resulting

in

a

larger

mean

size

for

internal

exons

-LRB-

bp

for

worm

versus

bp

for

human

-RRB-

The

conservation

of

preferred

exon

size

across

all

three

species

supports

suggestions

of

a

conserved

exon-based

component

of

the

splicing

Intriguingly

the

few

extremely

short

human

exons

show

an

unusual

base

composition

In

detected

a

Relative

gene

density

Frequency

Genome

Genes

human

exons

of

less

than

bp

the

nucleotide

frequencies

of

A

G

T

and

C

are

and

respectively

showing

a

strong

purine

bias

Purine-rich

sequences

may

enhance

and

it

is

possible

that

such

sequences

are

required

or

strongly

selected

for

to

ensure

correct

splicing

of

very

short

exons

Previous

studies

have

shown

that

short

exons

require

intronic

but

not

exonic

splicing

In

contrast

to

the

exons

the

intron

size

distributions

differ

substantially

among

the

three

species

-LRB-

Fig.

c

-RRB-

The

worm

and

y

each

have

a

reasonably

tight

distribution

with

most

introns

near

the

preferred

minimum

intron

length

-LRB-

bp

for

worm

bp

for

y

-RRB-

and

an

extended

tail

-LRB-

overall

average

length

of

bp

for

worm

and

bp

for

y

-RRB-

Intron

size

is

much

more

variable

in

humans

with

a

peak

at

bp

but

a

very

long

tail

resulting

in

a

mean

of

more

than

bp

The

variation

in

intron

size

results

in

great

variation

in

gene

size

The

variation

in

gene

size

and

intron

size

can

partly

be

explained

by

the

fact

that

GC-rich

regions

tend

to

be

gene-dense

with

many

compact

genes

whereas

AT-rich

regions

tend

to

be

gene-poor

with

many

sprawling

genes

containing

large

introns

The

correlation

of

gene

density

with

GC

content

is

shown

in

Fig.

b

the

relative

density

increases

more

than

tenfold

as

GC

content

increases

from

b

GC

content

GC

content

c

Median

intron

length

Intron

Exon

GC

content

Figure

GC

content

a

Distribution

of

GC

content

in

genes

and

in

the

genome

For

known

genes

mapped

to

the

draft

genome

sequence

the

local

GC

content

was

calculated

in

a

window

covering

either

the

whole

alignment

or

bp

centred

around

the

midpoint

of

the

alignment

whichever

was

larger

Ns

in

the

sequence

were

not

counted

GC

content

for

the

genome

was

calculated

for

adjacent

nonoverlapping

bp

windows

across

the

sequence

Both

the

gene

and

genome

distributions

have

been

NATURE

VOL

FEBRUARY

normalized

to

sum

to

one

b

Gene

density

as

a

function

of

GC

content

obtained

by

taking

the

ratio

of

the

data

in

a.

Values

are

less

accurate

at

higher

GC

levels

because

the

denominator

is

small

c

Dependence

of

mean

exon

and

intron

lengths

on

GC

content

For

exons

and

introns

the

local

GC

content

was

derived

from

alignments

to

nished

sequence

only

and

were

calculated

from

windows

covering

the

feature

or

bp

centred

on

the

feature

whichever

was

larger

Macmillan

Magazines

Ltd

Median

exon

length

articles

to

The

correlation

appears

to

be

due

primarily

to

intron

size

which

drops

markedly

with

increasing

GC

content

-LRB-

Fig.

-RRB-

In

contrast

coding

properties

such

as

exon

length

-LRB-

Fig.

-RRB-

or

exon

number

-LRB-

data

not

shown

-RRB-

vary

little

Intergenic

distance

is

also

probably

lower

in

high-GC

areas

although

this

is

hard

to

prove

directly

until

all

genes

have

been

identi

ed

The

large

number

of

con

rmed

human

introns

allows

us

to

analyse

variant

splice

sites

con

rming

and

extending

recent

Intron

positions

were

con

rmed

by

applying

a

stringent

criterion

that

EST

or

mRNA

sequence

show

an

exact

match

of

bp

in

the

anking

exonic

sequence

on

each

side

Of

con

rmed

introns

use

the

canonical

dinucleotides

GT

at

the

splice

site

and

AG

at

the

site

-LRB-

GT

AG

pattern

-RRB-

Another

use

the

related

GC

AG

About

use

AT

AC

which

is

a

rare

alternative

pattern

primarily

recognized

by

the

variant

splicing

The

remaining

belong

to

types

some

of

which

undoubtedly

re

ect

sequencing

or

alignment

errors

Finally

we

looked

at

alternative

splicing

of

human

genes

Alternative

splicing

can

allow

many

proteins

to

be

produced

from

a

single

gene

and

can

be

used

for

complex

gene

regulation

It

appears

to

be

prevalent

in

humans

with

lower

estimates

of

about

of

human

genes

being

subject

to

alternative

These

studies

may

have

underestimated

the

prevalence

of

alternative

splicing

because

they

examined

only

EST

alignments

covering

only

a

portion

of

a

gene

To

investigate

the

prevalence

of

alternative

splicing

we

analysed

reconstructed

mRNA

transcripts

covering

the

entire

coding

regions

of

genes

on

chromosome

-LRB-

omitting

small

genes

with

coding

regions

of

less

than

bp

-RRB-

Potential

transcripts

identi

ed

by

alignments

of

ESTs

and

cDNAs

to

genomic

sequence

were

veri

ed

by

human

inspection

We

found

transcripts

covering

genes

-LRB-

average

of

distinct

transcripts

per

gene

-RRB-

Two

or

more

alternatively

spliced

transcripts

were

found

for

-LRB-

-RRB-

of

these

genes

A

similar

analysis

for

the

gene-rich

chromosome

gave

transcripts

corresponding

to

genes

-LRB-

average

distinct

transcripts

per

gene

-RRB-

Because

we

are

sampling

only

a

subset

of

all

transcripts

the

true

extent

of

alternative

splicing

is

likely

to

be

greater

These

gures

are

considerably

higher

than

those

for

worm

in

which

analysis

reveals

alternative

splicing

for

of

genes

for

which

ESTs

have

been

found

with

an

average

of

-LRB-

-RRB-

splice

variants

per

gene

-LRB-

The

apparently

higher

extent

of

alternative

splicing

seen

in

human

than

in

worm

was

not

an

artefact

resulting

from

much

deeper

coverage

of

human

genes

by

ESTs

and

mRNAs

Although

there

are

many

times

more

ESTs

available

for

human

than

worm

these

ESTs

tend

to

have

shorter

average

length

-LRB-

because

many

were

the

product

of

early

sequencing

efforts

-RRB-

and

many

match

no

human

genes

We

calculated

the

actual

coverage

per

bp

used

in

the

analysis

of

the

human

and

worm

genes

the

coverage

is

only

modestly

higher

-LRB-

about

-RRB-

for

the

human

with

a

strong

bias

towards

UTRs

which

tend

to

show

much

less

alternative

splicing

We

also

repeated

the

analysis

using

equal

coverage

for

the

two

organisms

and

con

rmed

that

higher

levels

of

alternative

splicing

were

still

seen

in

human

-RRB-

Seventy

per

cent

of

alternative

splice

forms

found

in

the

genes

on

chromosomes

and

affect

the

coding

sequence

rather

than

merely

changing

the

or

UTR

-LRB-

This

estimate

may

be

affected

by

the

incomplete

representation

of

UTRs

in

the

RefSeq

database

and

in

the

transcripts

studied

-RRB-

Alternative

splicing

of

the

terminal

exon

was

seen

for

of

mRNAs

that

were

aligned

to

the

draft

genome

sequence

and

correspond

to

con

rmed

EST

clusters

In

addition

to

alternative

splicing

we

found

evidence

of

the

terminal

exon

employing

alternative

polyadenylation

sites

-LRB-

separated

by

bp

-RRB-

in

of

cases

Towards

a

complete

index

of

human

genes

We

next

focused

on

creating

an

initial

index

of

human

genes

and

proteins

This

index

is

quite

incomplete

owing

to

the

dif

culty

of

gene

identi

cation

in

human

DNA

and

the

imperfect

state

of

the

draft

genome

sequence

Nonetheless

it

is

valuable

for

experimental

studies

and

provides

important

insights

into

the

nature

of

human

genes

and

proteins

The

challenge

of

identifying

genes

from

genomic

sequence

varies

greatly

among

organisms

Gene

identi

cation

is

almost

trivial

in

bacteria

and

yeast

because

the

absence

of

introns

in

bacteria

and

their

paucity

in

yeast

means

that

most

genes

can

be

readily

recognized

by

ab

initio

analysis

as

unusually

long

ORFs

It

is

not

as

simple

but

still

relatively

straightforward

to

identify

genes

in

animals

with

small

genomes

and

small

introns

such

as

worm

and

y

A

major

factor

is

the

high

signal-to-noise

ratio

coding

sequences

comprise

a

large

proportion

of

the

genome

and

a

large

proportion

of

each

gene

-LRB-

about

for

worm

and

y

-RRB-

and

exons

are

relatively

large

Gene

identi

cation

is

more

dif

cult

in

human

DNA

The

signalto-noise

ratio

is

lower

coding

sequences

comprise

only

a

few

per

cent

of

the

genome

and

an

average

of

about

of

each

gene

internal

exons

are

smaller

than

in

worms

and

genes

appear

to

have

more

alternative

splicing

The

challenge

is

underscored

by

the

work

on

human

chromosomes

and

Even

with

the

availability

of

nished

sequence

and

intensive

experimental

work

the

gene

content

remains

uncertain

with

upper

and

lower

estimates

differing

by

as

much

as

The

initial

report

of

the

nished

sequence

of

chromosome

-LRB-

ref

-RRB-

identi

ed

previously

known

genes

predicted

genes

con

rmed

by

sequence

homology

or

ESTs

and

ab

initio

predictions

without

additional

support

Many

of

the

con

rmed

predictions

represented

partial

genes

In

the

past

year

additional

exons

-LRB-

-RRB-

have

been

added

to

existing

gene

annotations

by

the

chromosome

annotation

group

although

the

number

of

con

rmed

genes

has

increased

by

only

and

some

previously

identi

ed

gene

predictions

have

been

Before

discussing

the

gene

predictions

for

the

human

genome

it

is

useful

to

consider

background

issues

including

previous

estimates

of

the

number

of

human

genes

lessons

learned

from

worms

and

ies

and

the

representativeness

of

currently

known

human

genes

Previous

estimates

of

human

gene

number

Although

direct

enumeration

of

human

genes

is

only

now

becoming

possible

with

the

advent

of

the

draft

genome

sequence

there

have

been

many

attempts

in

the

past

quarter

of

a

century

to

estimate

the

number

of

genes

indirectly

Early

estimates

based

on

reassociation

kinetics

estimated

the

mRNA

complexity

of

typical

vertebrate

tissues

to

be

and

were

extrapolated

to

suggest

around

for

the

entire

In

the

Gilbert

suggested

that

there

might

be

about

genes

based

on

the

approximate

ratio

of

the

size

of

a

typical

gene

-LRB-

bp

-RRB-

to

the

size

of

the

genome

-LRB-

bp

-RRB-

Although

this

was

intended

only

as

a

back-of-the-envelope

estimate

the

pleasing

roundness

of

the

gure

seems

to

have

led

to

it

being

widely

quoted

and

adopted

in

many

textbooks

-LRB-

W.

Gilbert

personal

communication

ref

-RRB-

An

estimate

of

genes

was

made

by

extrapolating

from

the

number

of

CpG

islands

and

the

frequency

of

their

association

with

known

As

human

sequence

information

has

accumulated

it

has

been

possible

to

derive

estimates

on

the

basis

of

sampling

Such

studies

have

sought

to

extrapolate

from

various

types

of

data

including

ESTs

mRNAs

from

known

genes

cross-species

genome

comparisons

and

analysis

of

nished

chromosomes

Estimates

based

on

have

varied

widely

from

-LRB-

ref

-RRB-

to

Some

of

the

discrepancy

lies

in

differing

estimates

of

the

amount

of

contaminating

genomic

sequence

in

the

EST

collection

and

the

extent

to

which

multiple

distinct

ESTs

correspond

to

a

single

gene

The

most

rigorous

exclude

as

spurious

any

ESTs

that

appear

only

once

in

the

data

set

and

carefully

calibrate

sensitivity

and

speci

city

Such

calculations

consistently

produce

low

estimates

in

the

region

of

Comparison

of

whole-genome

shotgun

sequence

from

the

puffer

sh

T.

nigroviridis

with

the

human

can

be

used

to

estimate

the

density

of

exons

-LRB-

detected

as

conserved

sequences

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

between

sh

and

human

-RRB-

These

analyses

also

suggest

around

human

genes

Extrapolations

have

also

been

made

from

the

gene

counts

for

chromosomes

and

-LRB-

refs

-RRB-

adjusted

for

differences

in

gene

densities

on

these

chromosomes

as

inferred

from

EST

mapping

These

estimates

are

between

and

depending

on

the

precise

assumptions

Insights

from

invertebrates

The

worm

and

y

genomes

contain

a

large

proportion

of

novel

genes

-LRB-

around

of

worm

genes

and

of

y

genes

-RRB-

in

the

sense

of

showing

no

signi

cant

similarity

to

organisms

outside

their

Such

genes

may

have

been

present

in

the

original

eukaryotic

ancestor

but

were

subsequently

lost

from

the

lineages

of

the

other

eukaryotes

for

which

sequence

is

available

they

may

be

rapidly

diverging

genes

so

that

it

is

dif

cult

to

recognize

homologues

solely

on

the

basis

of

sequence

they

may

represent

true

innovations

developed

within

the

lineage

or

they

may

represent

acquisitions

by

horizontal

transfer

Whatever

their

origin

these

genes

tend

to

have

different

biological

properties

from

highly

conserved

genes

In

particular

they

tend

to

have

low

expression

levels

as

assayed

both

by

direct

studies

and

by

a

paucity

of

corresponding

ESTs

and

are

less

likely

to

produce

a

visible

phenotype

in

loss-of-function

genetic

Gene

prediction

Current

gene

prediction

methods

employ

combinations

of

three

basic

approaches

direct

evidence

of

transcription

provided

by

ESTs

or

indirect

evidence

based

on

sequence

similarity

to

previously

identi

ed

genes

and

and

ab

initio

recognition

of

groups

of

exons

on

the

basis

of

hidden

Markov

models

-LRB-

HMMs

-RRB-

that

combine

statistical

information

about

splice

sites

coding

bias

and

exon

and

intron

lengths

-LRB-

for

example

and

-RRB-

The

rst

approach

relies

on

direct

experimental

data

but

is

subject

to

artefacts

arising

from

contaminating

ESTs

derived

from

unspliced

mRNAs

genomic

DNA

contamination

and

nongenic

transcription

-LRB-

for

example

from

the

promoter

of

a

transposable

element

-RRB-

The

rst

two

problems

can

be

mitigated

by

comparing

transcripts

with

the

genomic

sequence

and

using

only

those

that

show

clear

evidence

of

splicing

This

solution

however

tends

to

discard

evidence

from

genes

with

long

terminal

exons

or

single

exons

The

second

approach

tends

correctly

to

identify

gene-derived

sequences

although

some

of

these

may

be

pseudogenes

However

it

obviously

can

not

identify

truly

novel

genes

that

have

no

sequence

similarity

to

known

genes

The

third

approach

would

suf

ce

alone

if

one

could

accurately

de

ne

the

features

used

by

cells

for

gene

recognition

but

our

current

understanding

is

insuf

cient

to

do

so

The

sensitivity

and

speci

city

of

ab

initio

predictions

are

greatly

affected

by

the

signal-to-noise

ratio

Such

methods

are

more

accurate

in

the

y

and

worm

than

in

human

In

y

ab

initio

methods

can

correctly

predict

around

of

individual

exons

and

can

correctly

predict

all

coding

exons

of

a

gene

in

about

of

For

human

the

comparable

gures

are

only

about

and

These

estimates

may

be

optimistic

owing

to

the

design

of

the

tests

used

In

any

collection

of

gene

predictions

we

can

expect

to

see

various

errors

Some

gene

predictions

may

represent

partial

genes

because

of

inability

to

detect

some

portions

of

a

gene

-LRB-

incomplete

sensitivity

-RRB-

or

to

connect

all

the

components

of

a

gene

-LRB-

fragmentation

-RRB-

some

may

be

gene

fusions

and

others

may

be

spurious

predictions

-LRB-

incomplete

speci

city

-RRB-

resulting

from

chance

matches

or

pseudogenes

Creating

an

initial

gene

index

We

set

out

to

create

an

initial

integrated

gene

index

-LRB-

IGI

-RRB-

and

an

associated

integrated

protein

index

-LRB-

IPI

-RRB-

for

the

human

genome

We

describe

the

results

obtained

from

a

version

of

the

draft

genome

sequence

based

on

the

sequence

data

available

in

July

to

allow

time

for

detailed

analysis

of

the

gene

and

protein

content

The

additional

sequence

data

that

has

since

become

available

will

affect

the

results

quantitatively

but

are

unlikely

to

change

the

conclusions

qualitatively

NATURE

VOL

FEBRUARY

We

began

with

predictions

produced

by

the

Ensembl

Ensembl

starts

with

ab

initio

predictions

produced

by

and

then

attempts

to

con

rm

them

by

virtue

of

similarity

to

proteins

mRNAs

ESTs

and

protein

motifs

-LRB-

contained

in

the

Pfam

-RRB-

from

any

organism

In

particular

it

con

rms

introns

if

they

are

bridged

by

matches

and

exons

if

they

are

anked

by

con

rmed

introns

It

then

attempts

to

extend

protein

matches

using

the

GeneWise

computer

Because

it

requires

con

rmatory

evidence

to

support

each

gene

component

it

frequently

produces

partial

gene

predictions

In

addition

when

there

is

evidence

of

alternative

splicing

it

reports

multiple

overlapping

transcripts

In

total

Ensembl

produced

gene

predictions

with

transcripts

To

reduce

fragmentation

we

next

merged

Ensembl-based

gene

predictions

with

overlapping

gene

predictions

from

another

program

Genie

starts

with

mRNA

or

EST

matches

and

employs

an

HMM

to

extend

these

matches

by

using

ab

initio

statistical

approaches

To

avoid

fragmentation

it

attempts

to

link

information

from

and

ESTs

from

the

same

cDNA

clone

and

thereby

to

produce

a

complete

coding

sequence

from

an

initial

ATG

to

a

stop

codon

As

a

result

it

may

generate

complete

genes

more

accurately

than

Ensembl

in

cases

where

there

is

extensive

EST

support

-LRB-

Genie

also

generates

potential

alternative

transcripts

but

we

used

only

the

longest

transcript

in

each

group

-RRB-

We

merged

Ensembl

predictions

into

clusters

and

the

longest

transcript

in

each

cluster

-LRB-

from

either

Genie

or

Ensembl

-RRB-

was

taken

as

the

representative

Next

we

merged

these

results

with

known

genes

contained

in

the

RefSeq

-LRB-

version

of

September

-RRB-

SWISSPROT

-LRB-

release

of

August

-RRB-

and

TrEMBL

databases

-LRB-

TrEMBL

release

of

October

of

October

-RRB-

Incorporating

these

sequences

gave

rise

to

overlapping

sequences

because

of

alternative

splice

forms

and

partial

sequences

To

construct

a

nonredundant

set

we

selected

the

longest

sequence

from

each

overlapping

set

by

using

direct

protein

comparison

and

by

mapping

the

gene

predictions

back

onto

the

genome

to

construct

the

overlapping

sets

This

may

occasionally

remove

some

close

paralogues

in

the

event

that

the

correct

genomic

location

has

not

yet

been

sequenced

but

this

number

is

expected

to

be

small

Finally

we

searched

the

set

to

eliminate

any

genes

derived

from

contaminating

bacterial

sequences

recognized

by

virtue

of

near

identity

to

known

bacterial

plasmids

transposons

and

chromosomal

genes

Although

most

instances

of

such

contamination

had

been

removed

in

the

assembly

process

a

few

cases

had

slipped

through

and

were

removed

at

this

stage

The

process

resulted

in

version

of

the

IGI

-LRB-

IGI

-RRB-

The

composition

of

the

corresponding

IPI

protein

set

obtained

by

translating

IGI

is

given

in

Table

There

are

protein

predictions

with

from

known

genes

predictions

from

Ensembl

merged

with

Genie

and

predictions

from

Ensembl

alone

The

average

lengths

are

amino

acids

for

the

known

proteins

amino

acids

for

protein

predictions

from

the

Ensembl

Genie

merge

and

amino

acids

for

those

from

Ensembl

alone

-LRB-

The

smaller

average

size

for

the

predictions

from

Ensembl

alone

re

ects

its

tendency

to

predict

partial

genes

where

there

is

supporting

evidence

for

only

part

of

the

gene

the

remainder

of

the

gene

will

often

not

be

predicted

at

all

rather

than

included

as

part

of

another

prediction

Accordingly

the

smaller

size

can

not

be

used

to

estimate

the

rate

of

fragmentation

in

such

predictions

-RRB-

The

set

corresponds

to

fewer

than

actual

genes

because

some

genes

are

fragmented

into

more

than

one

partial

prediction

and

some

predictions

may

be

spurious

or

correspond

to

pseudogenes

As

discussed

below

our

best

estimate

is

that

IGI

includes

about

true

genes

Evaluation

of

IGI/IPI

We

used

several

approaches

to

evaluate

the

sensitivity

speci

city

and

fragmentation

of

the

IGI/IPI

set

Comparison

with

new

known

genes

One

approach

was

to

examine

Macmillan

Magazines

Ltd

articles

Table

Properties

of

the

IGI/IPI

human

protein

set

Source

RefSeq/SwissProt/TrEMBL

Ensembl

Genie

Ensembl

Total

Number

Average

length

-LRB-

amino

acids

-RRB-

469 443 187 352

Matches

to

nonhuman

proteins

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Matches

to

RIKEN

mouse

cDNA

set

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Matches

to

RIKEN

mouse

cDNA

set

but

not

to

nonhuman

proteins

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

matches

to

nonhuman

proteins

were

obtained

by

using

Smith-Waterman

sequence

alignment

with

an

E-value

threshold

of

and

the

matches

to

the

RIKEN

mouse

cDNAs

by

using

TBLASTN

with

an

E-value

threshold

of

The

last

column

shows

that

a

signi

cant

number

of

the

IGI

members

that

do

not

have

nonhuman

protein

matches

do

match

sequences

in

the

RIKEN

mouse

cDNA

set

suggesting

that

both

the

IGI

and

the

RIKEN

sets

contain

a

signi

cant

number

of

novel

proteins

newly

discovered

genes

arising

from

independent

work

that

were

not

used

in

our

gene

prediction

effort

We

identi

ed

such

genes

recent

entries

to

RefSeq

and

from

the

Sanger

Centre

's

gene

identi

cation

program

on

chromosome

X.

Of

these

were

contained

in

the

draft

genome

sequence

and

were

represented

in

the

IGI/IPI

This

suggests

that

the

gene

prediction

process

has

a

sensitivity

of

about

-LRB-

-RRB-

for

the

detection

of

novel

genes

in

the

draft

genome

sequence

and

that

the

current

IGI

contains

about

-LRB-

-RRB-

of

novel

genes

in

the

human

genome

On

average

of

each

gene

was

detected

The

extent

of

fragmentation

could

also

be

estimated

of

the

genes

corresponded

to

a

single

prediction

in

the

IGI/IPI

three

genes

corresponded

to

two

predictions

one

gene

to

three

predictions

and

one

gene

to

four

predictions

This

corresponds

to

a

fragmentation

rate

of

about

gene

predictions

per

true

gene

Comparison

with

RIKEN

mouse

cDNAs

In

a

less

direct

but

largerscale

approach

we

compared

the

IGI

gene

set

to

a

set

of

mouse

cDNAs

sequenced

by

the

Genome

Exploration

Group

of

the

RIKEN

Genomic

Sciences

This

set

of

cDNAs

subjected

to

full-insert

sequencing

was

enriched

for

novel

genes

by

selecting

cDNAs

with

novel

ends

from

a

collection

of

nearly

one

million

ESTs

from

diverse

tissues

and

developmental

timepoints

We

determined

the

proportion

of

the

RIKEN

cDNAs

that

showed

sequence

similarity

to

the

draft

genome

sequence

and

the

proportion

that

showed

sequence

similarity

to

the

IGI/IPI

Around

of

the

genes

in

the

RIKEN

mouse

set

showed

sequence

similarity

to

the

human

genome

sequence

whereas

showed

sequence

similarity

to

the

IGI/IPI

This

suggests

a

sensitivity

of

-LRB-

-RRB-

This

is

higher

than

the

sensitivity

estimate

above

perhaps

because

some

of

the

matches

may

be

due

to

paralogues

rather

than

orthologues

It

is

consistent

with

the

IGI/IPI

representing

a

substantial

fraction

of

the

human

proteome

Conversely

-LRB-

-RRB-

of

the

IGI

matches

the

RIKEN

cDNA

set

Table

shows

the

breakdown

of

these

matches

among

the

different

components

of

the

IGI

This

is

lower

than

the

proportion

of

matches

among

known

proteins

although

this

is

expected

because

known

proteins

tend

to

be

more

highly

conserved

-LRB-

see

above

-RRB-

and

because

the

predictions

are

on

average

shorter

than

known

proteins

Table

also

shows

the

numbers

of

matches

to

the

RIKEN

cDNAs

among

IGI

members

that

do

not

match

known

proteins

The

results

indicate

that

both

the

IGI

and

the

RIKEN

set

contain

a

signi

cant

number

of

genes

that

are

novel

in

the

sense

of

not

having

known

protein

homologues

Comparison

with

genes

on

chromosome

We

also

compared

the

IGI/IPI

with

the

gene

annotations

on

chromosome

to

assess

the

proportion

of

gene

predictions

corresponding

to

pseudogenes

and

to

estimate

the

rate

of

overprediction

We

compared

IGI

gene

predictions

to

con

rmed

genes

and

pseudogenes

on

chromosome

-LRB-

with

the

immunoglobulin

lambda

locus

excluded

owing

to

its

highly

atypical

gene

structure

-RRB-

Of

these

hit

annotated

pseudogenes

This

suggests

that

of

the

IGI

predictions

may

correspond

to

pseudogenes

and

also

suggests

a

fragmentation

rate

of

gene

predictions

per

gene

Of

the

remaining

hits

did

not

overlap

with

any

current

annotations

This

would

suggest

a

rate

of

spurious

predictions

of

about

-LRB-

-RRB-

although

the

true

rate

is

likely

to

be

much

lower

because

many

of

these

may

correspond

to

unannotated

portions

of

existing

gene

predictions

or

to

currently

unannotated

genes

-LRB-

of

which

there

are

estimated

to

be

about

on

this

-RRB-

Chromosomal

distribution

Finally

we

examined

the

chromosomal

distribution

of

the

IGI

gene

set

The

average

density

of

gene

predictions

is

per

Mb

across

the

genome

with

the

extremes

being

chromosome

at

per

Mb

and

chromosome

Y

at

per

Mb

It

is

likely

that

a

signi

cant

number

of

the

predictions

on

chromosome

Y

are

pseudogenes

-LRB-

this

chromosome

is

known

to

be

rich

in

pseudogenes

-RRB-

and

thus

that

the

density

for

chromosome

Y

is

an

overestimate

The

density

of

both

genes

and

Alus

on

chromosome

is

much

higher

than

expected

even

accounting

for

the

high

GC

content

of

the

chromosome

this

supports

the

idea

that

Alu

density

is

more

closely

correlated

with

gene

density

than

with

GC

content

itself

Summary

We

are

clearly

still

some

way

from

having

a

complete

set

of

human

genes

The

current

IGI

contains

signi

cant

numbers

of

partial

genes

fragmented

and

fused

genes

pseudogenes

and

spurious

predictions

and

it

also

lacks

signi

cant

numbers

of

true

genes

This

re

ects

the

current

state

of

gene

prediction

methods

in

vertebrates

even

in

nished

sequence

as

well

as

the

additional

challenges

related

to

the

current

state

of

the

draft

genome

sequence

Nonetheless

the

gene

predictions

provide

a

valuable

starting

point

for

a

wide

range

of

biological

studies

and

will

be

rapidly

re

ned

in

the

coming

year

The

analysis

above

allows

us

to

estimate

the

number

of

distinct

genes

in

the

IGI

as

well

as

the

number

of

genes

in

the

human

genome

The

IGI

set

contains

about

known

genes

and

about

gene

predictions

Assuming

that

the

gene

predictions

are

subject

to

a

rate

of

overprediction

-LRB-

spurious

predictions

and

pseudogenes

-RRB-

of

and

a

rate

of

fragmentation

of

the

IGI

would

be

estimated

to

contain

about

actual

human

genes

Assuming

that

the

gene

predictions

contain

about

of

previously

unknown

human

genes

the

total

number

of

genes

in

the

human

genome

would

be

estimated

to

be

about

This

is

consistent

with

most

recent

estimates

based

on

sampling

which

suggest

a

gene

number

of

If

there

are

genes

with

an

average

coding

length

of

about

bp

and

average

genomic

extent

of

about

kb

then

about

of

the

human

genome

would

consist

of

coding

sequence

and

one-third

of

the

genome

would

be

transcribed

in

genes

The

IGI/IPI

was

constructed

primarily

on

the

basis

of

gene

predictions

from

Ensembl

However

we

also

generated

an

expanded

set

-LRB-

IGI

-RRB-

by

including

additional

predictions

from

two

other

gene

prediction

programs

Genie

and

GenomeScan

-LRB-

C.

Burge

personal

communication

-RRB-

These

predictions

were

not

included

in

the

core

IGI

set

because

of

the

concern

that

each

additional

set

will

provide

diminishing

returns

in

identifying

true

genes

while

contributing

its

own

false

positives

-LRB-

increased

sensitivity

at

the

expense

of

speci

city

-RRB-

Genie

produced

an

additional

gene

predictions

not

overlapping

the

IGI

and

GenomeScan

produced

such

gene

predictions

If

all

of

these

gene

predictions

were

included

in

the

IGI

the

number

of

the

new

known

genes

-LRB-

see

above

-RRB-

contained

in

the

IGI

would

rise

from

to

This

would

amount

to

an

increase

of

about

in

sensitivity

at

the

expense

of

increasing

the

number

of

predicted

genes

-LRB-

excluding

knowns

-RRB-

by

Allowing

a

higher

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

overprediction

rate

of

for

gene

predictions

in

this

expanded

set

the

analysis

above

suggests

that

IGI

set

contains

about

true

genes

and

yields

an

estimate

of

about

human

genes

We

are

investigating

ways

to

lter

the

expanded

set

to

produce

an

IGI

with

the

advantage

of

the

increased

sensitivity

resulting

from

combining

multiple

gene

prediction

programs

without

the

corresponding

loss

of

speci

city

Meanwhile

the

IGI

set

can

be

used

by

researchers

searching

for

genes

that

can

not

be

found

in

the

IGI

Some

classes

of

genes

may

have

been

missed

by

all

of

the

gene

nding

methods

Genes

could

be

missed

if

they

are

expressed

at

low

levels

or

in

rare

tissues

-LRB-

being

absent

or

very

under-represented

in

EST

and

mRNA

databases

-RRB-

and

have

sequences

that

evolve

rapidly

-LRB-

being

hard

to

detect

by

protein

homology

and

genome

comparison

-RRB-

Both

the

worm

and

y

gene

sets

contain

a

substantial

number

of

such

Single-exon

genes

encoding

small

proteins

may

also

have

been

missed

because

EST

evidence

that

supports

them

can

not

be

distinguished

from

genomic

contamination

in

the

EST

dataset

and

because

homology

may

be

hard

to

detect

for

small

The

human

thus

appears

to

have

only

about

twice

as

many

genes

as

worm

or

y.

However

human

genes

differ

in

important

respects

from

those

in

worm

and

y

They

are

spread

out

over

much

larger

regions

of

genomic

DNA

and

they

are

used

to

construct

more

alternative

transcripts

This

may

result

in

perhaps

ve

times

as

many

primary

protein

products

in

the

human

as

in

the

worm

or

y

The

predicted

gene

and

protein

sets

described

here

are

clearly

far

from

nal

Nonetheless

they

provide

a

valuable

starting

point

for

experimental

and

computational

research

The

predictions

will

improve

progressively

as

the

sequence

is

nished

as

further

con

rmatory

evidence

becomes

available

-LRB-

particularly

from

other

vertebrate

genome

sequences

such

as

those

of

mouse

and

T.

nigroviridis

-RRB-

and

as

computational

methods

improve

We

intend

to

create

and

release

updated

versions

of

the

IGI

and

IPI

regularly

until

they

converge

to

a

nal

accurate

list

of

every

human

gene

The

gene

predictions

will

be

linked

to

RefSeq

HUGO

and

SWISSPROT

identi

ers

where

available

and

tracking

identi

ers

between

versions

will

be

included

so

that

individual

genes

under

study

can

be

traced

forwards

as

the

human

sequence

is

completed

Comparative

proteome

analysis

Knowledge

of

the

human

proteome

will

provide

unprecedented

opportunities

for

studies

of

human

gene

function

Often

clues

will

be

provided

by

sequence

similarity

with

proteins

of

known

function

in

model

organisms

Such

initial

observations

must

then

be

followed

up

by

detailed

studies

to

establish

the

actual

function

of

these

molecules

in

humans

For

example

proteins

are

known

to

be

involved

in

the

vacuolar

protein-sorting

machinery

in

yeast

Human

genes

encoding

homologues

can

be

found

in

the

draft

human

sequence

for

of

these

yeast

proteins

but

precise

relationships

are

not

always

clear

In

nine

cases

there

appears

to

be

a

single

clear

human

orthologue

-LRB-

a

gene

that

arose

as

a

consequence

of

speciation

-RRB-

in

cases

there

are

matches

to

a

family

of

human

paralogues

-LRB-

genes

that

arose

owing

to

intra-genome

duplication

-RRB-

and

in

cases

there

are

matches

to

speci

c

protein

Hundreds

of

similar

stories

emerge

from

the

draft

sequence

but

each

merits

a

detailed

interpretation

in

context

To

treat

these

subjects

properly

there

will

be

many

following

studies

the

rst

of

which

appear

in

accompanying

Here

we

aim

to

take

a

more

global

perspective

on

the

content

of

the

human

proteome

by

comparing

it

with

the

proteomes

of

yeast

worm

y

and

mustard

weed

Such

comparisons

shed

useful

light

on

the

commonalities

and

differences

among

these

The

analysis

is

necessarily

preliminary

because

of

the

imperfect

nature

of

the

human

sequence

uncertainties

in

the

gene

and

protein

sets

for

all

of

the

multicellular

organisms

considered

and

our

incomplete

knowledge

of

protein

structures

Nonetheless

some

general

patterns

emerge

These

include

insights

into

fundamental

NATURE

VOL

FEBRUARY

mechanisms

that

create

functional

diversity

including

invention

of

protein

domains

expansion

of

protein

and

domain

families

evolution

of

new

protein

architectures

and

horizontal

transfer

of

genes

Other

mechanisms

such

as

alternative

splicing

post-translational

modi

cation

and

complex

regulatory

networks

are

also

crucial

in

generating

diversity

but

are

much

harder

to

discern

from

the

primary

sequence

We

will

not

attempt

to

consider

the

effects

of

alternative

splicing

on

proteins

we

will

consider

only

a

single

splice

form

from

each

gene

in

the

various

organisms

even

when

multiple

splice

forms

are

known

Functional

and

evolutionary

classi

cation

We

began

by

classifying

the

human

proteome

on

the

basis

of

functional

categories

and

evolutionary

conservation

We

used

the

InterPro

annotation

protocol

to

identify

conserved

biochemical

and

cellular

processes

InterPro

is

a

tool

for

combining

sequence-pattern

information

from

four

databases

The

rst

two

databases

-LRB-

and

-RRB-

primarily

contain

information

about

motifs

corresponding

to

speci

c

family

subtypes

such

as

type

II

receptor

tyrosine

kinases

-LRB-

RTK-II

-RRB-

in

particular

or

tyrosine

kinases

in

general

The

second

two

databases

-LRB-

and

Prosite

Pro

-RRB-

contain

information

-LRB-

in

the

form

of

pro

les

or

HMMs

-RRB-

about

families

of

structural

domains

for

example

protein

kinase

domains

InterPro

integrates

the

motif

and

domain

assignments

into

a

hierarchical

classi

cation

system

so

a

protein

might

be

classi

ed

at

the

most

detailed

level

as

being

an

RTK-II

at

a

more

general

level

as

being

a

kinase

speci

c

for

tyrosine

and

at

a

still

more

general

level

as

being

a

protein

kinase

The

complete

hierarchy

of

InterPro

entries

is

described

at

We

collapsed

the

InterPro

entries

into

broad

categories

each

re

ecting

a

set

of

cellular

functions

The

InterPro

families

are

partly

the

product

of

human

judgement

and

re

ect

the

current

state

of

biological

and

evolutionary

knowledge

The

system

is

a

valuable

way

to

gain

insight

into

large

collections

of

proteins

but

not

all

proteins

can

be

classi

ed

at

present

The

proportions

of

the

yeast

worm

y

and

mustard

weed

protein

sets

that

are

assigned

to

at

least

one

InterPro

family

is

for

each

organism

about

-LRB-

Table

refs

-RRB-

About

of

the

predicted

human

proteins

in

the

IPI

could

be

assigned

to

InterPro

entries

and

functional

categories

On

the

basis

of

these

assignments

we

could

compare

organisms

according

to

the

number

of

proteins

in

each

category

-LRB-

Fig.

-RRB-

Compared

with

the

two

invertebrates

humans

appear

to

have

many

proteins

involved

in

cytoskeleton

defence

and

immunity

and

transcription

and

translation

These

expansions

are

clearly

related

to

aspects

of

vertebrate

physiology

Humans

also

have

many

more

proteins

that

are

classi

ed

as

falling

into

more

than

one

functional

category

-LRB-

in

human

versus

in

worm

and

in

y

data

not

shown

-RRB-

Interestingly

of

these

are

transmembrane

receptors

We

obtained

further

insight

into

the

evolutionary

conservation

of

proteins

by

comparing

each

sequence

to

the

complete

nonredundant

database

of

protein

sequences

maintained

at

NCBI

using

the

BLASTP

computer

and

then

breaking

down

the

matches

according

to

organismal

taxonomy

-LRB-

Fig.

-RRB-

Overall

of

the

proteins

had

signi

cant

matches

to

known

proteins

Such

classi

cations

are

based

on

the

presence

of

clearly

detectable

homologues

in

existing

databases

Many

of

these

genes

have

surely

evolved

from

genes

that

were

present

in

common

ancestors

but

have

since

diverged

substantially

Indeed

one

can

detect

more

distant

relationships

by

using

sensitive

computer

programs

that

can

recognize

weakly

conserved

features

Using

PSI-BLAST

we

can

recognize

probable

nonvertebrate

homologues

for

about

of

the

vertebrate-speci

c

set

Nonetheless

the

classi

cation

is

useful

for

gaining

insights

into

the

commonalities

and

differences

among

the

proteomes

of

different

organisms

Probable

horizontal

transfer

An

interesting

category

is

a

set

of

proteins

that

have

signi

cant

similarity

to

proteins

from

bacteria

but

no

comparable

similarity

to

proteins

from

yeast

worm

y

and

Macmillan

Magazines

Ltd

articles

Table

Properties

of

genome

and

proteome

in

essentially

completed

eukaryotic

proteomes

Human

Number

of

identi

ed

genes

with

InterPro

matches

Number

of

annotated

domain

families

Number

of

InterPro

entries

per

gene

Number

of

distinct

domain

architectures

Percentage

of

Signal

sequences

Transmembrane

proteins

Repeat-containing

Coiled-coil

Fly

Worm

Yeast

Mustard

weed

The

numbers

of

distinct

architectures

were

calculated

using

and

the

percentages

of

repeat-containing

proteins

were

estimated

using

and

a

P-value

threshold

of

The

protein

sets

used

in

the

analysis

were

taken

from

for

yeast

worm

and

y

The

proteins

from

mustard

weed

were

taken

from

the

TAIR

website

-LRB-

http

-RRB-

on

September

The

protein

set

was

searched

against

the

InterPro

database

-LRB-

-RRB-

using

the

InterProscan

software

Comparison

of

protein

sequences

with

the

InterPro

database

allows

prediction

of

protein

families

domain

and

repeat

families

and

sequence

motifs

The

searches

used

Pfam

release

Prints

release

Prosite

release

and

Prosite

preliminary

pro

les

InterPro

analysis

results

are

available

as

Supplementary

Information

The

fraction

of

is

the

percentage

of

the

genome

that

falls

into

orthologous

groups

composed

of

only

one

member

each

in

human

y

worm

and

yeast

The

gene

number

for

the

human

is

still

uncertain

-LRB-

see

text

-RRB-

Table

is

based

on

known

genes

and

gene

predictions

mustard

weed

or

indeed

from

any

other

-LRB-

nonvertebrate

-RRB-

eukaryote

These

sequences

should

not

represent

bacterial

contamination

in

the

draft

human

sequence

because

we

ltered

the

sequence

to

eliminate

sequences

that

were

essentially

identical

to

known

bacterial

plasmid

transposon

or

chromosomal

DNA

-LRB-

such

as

the

host

strains

for

the

large-insert

clones

-RRB-

To

investigate

whether

these

were

genuine

human

sequences

we

designed

PCR

primers

for

of

these

genes

and

con

rmed

that

most

could

be

readily

detected

directly

in

human

genomic

DNA

-LRB-

Table

-RRB-

Orthologues

of

many

of

these

genes

have

also

been

detected

in

other

vertebrates

-LRB-

Table

-RRB-

A

more

detailed

computational

analysis

indicated

that

at

least

of

these

genes

are

widespread

among

bacteria

but

among

eukaryotes

appear

to

be

present

only

in

vertebrates

It

is

possible

that

the

genes

encoding

these

proteins

were

present

in

both

early

prokaryotes

and

eukaryotes

but

were

lost

in

each

of

the

lineages

of

yeast

worm

y

mustard

weed

and

possibly

from

other

nonvertebrate

eukaryote

lineages

A

more

parsimonious

explanation

is

that

these

genes

entered

the

vertebrate

-LRB-

or

prevertebrate

-RRB-

lineage

by

horizontal

transfer

from

bacteria

Many

of

these

genes

contain

introns

which

presumably

were

acquired

after

the

putative

horizontal

transfer

event

Similar

observations

indicating

probable

lineage-speci

c

horizontal

gene

transfers

as

well

as

intron

insertion

in

the

acquired

genes

have

been

made

in

the

worm

Yeast

We

can

not

formally

exclude

the

possibility

that

gene

transfer

occurred

in

the

opposite

direction

that

is

that

the

genes

were

invented

in

the

vertebrate

lineage

and

then

transferred

to

bacteria

However

we

consider

this

less

likely

Under

this

scenario

the

broad

distribution

of

these

genes

among

bacteria

would

require

extensive

horizontal

dissemination

after

their

initial

acquisition

In

addition

the

functional

repertoire

of

these

genes

which

largely

encode

intracellular

enzymes

-LRB-

Table

-RRB-

is

uncharacteristic

of

vertebratespeci

c

evolutionary

innovations

-LRB-

which

appear

to

be

primarily

extracellular

proteins

see

below

-RRB-

We

did

not

identify

a

strongly

preferred

bacterial

source

for

the

putative

horizontally

transferred

genes

indicating

the

likelihood

of

multiple

independent

gene

transfers

from

different

bacteria

-LRB-

Table

-RRB-

Notably

several

of

the

probable

recent

acquisitions

have

established

-LRB-

or

likely

-RRB-

roles

in

metabolism

of

xenobiotics

or

stress

response

These

include

several

hydrolases

of

different

speci

cities

including

epoxide

hydrolase

and

several

dehydrogenases

-LRB-

Table

-RRB-

Of

particular

interest

is

the

presence

of

two

paralogues

of

monoamine

oxidase

-LRB-

MAO

-RRB-

an

enzyme

of

the

mitochondrial

outer

membrane

that

is

central

in

the

metabolism

of

neuromediators

and

is

a

target

of

important

psychiatric

This

example

shows

that

at

least

some

of

the

genes

thought

to

be

horizontally

transferred

into

the

vertebrate

lineage

appear

to

be

involved

in

important

physiological

functions

and

so

probably

have

been

xed

and

maintained

during

evolution

because

Number

of

proteins

Mustard

weed

Worm

Fly

Human

Prokaryotes

only

Vertebrate

only

Eukaryote

and

prokaryote

ss

es

io

eta

bo

n

an

m

lis

sc

m

rip

odi

fic

tio

In

at

n

tra

io

n

ce

tran

C

Pr

llu

el

sl

ot

at

l-c

la

ei

io

rs

el

n

n

l

ig

fo

ld

com

in

llin

m

g

g

un

an

ic

d

de

ati

o

gr

ad

n

M

ul

at

tif

io

un

Tr

n

an

ct

C

io

sp

yt

os

or

lp

k

t

r

De

ele

ta

ote

fe

nc

l/st

ins

ru

e

M

ct

a

is

u

ce

nd

im

ral

lla

ne

m

un

ou

ity

s

fu

nc

tio

n

Vertebrates

and

other

animals

No

animal

homology

Animals

and

other

eukaryotes

rp

el

lu

la

C

ic

at

M

ro

ce

Figure

Functional

categories

in

eukaryotic

proteomes

The

classi

cation

categories

were

derived

from

functional

classi

cation

systems

including

the

top-level

biological

function

category

of

the

Gene

Ontology

project

-LRB-

GO

see

-RRB-

Figure

Distribution

of

the

homologues

of

the

predicted

human

proteins

For

each

protein

a

homologue

to

a

phylogenetic

lineage

was

considered

present

if

a

search

of

the

NCBI

nonredundant

protein

sequence

database

using

the

gapped

BLASTP

program

gave

a

random

expectation

-LRB-

E

-RRB-

value

of

Additional

searches

for

probable

homologues

with

lower

sequence

conservation

were

performed

using

the

PSI-BLAST

program

run

for

three

iterations

using

the

same

cut-off

for

inclusion

of

sequences

into

the

pro

NATURE

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pl

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articles

of

the

increased

selective

advantage

-LRB-

s

-RRB-

they

provide

Genes

shared

with

y

worm

and

yeast

IPI

contains

apparent

homologues

of

of

the

y

proteome

of

the

worm

proteome

and

of

the

yeast

proteome

We

next

considered

the

groups

of

proteins

containing

likely

orthologues

and

paralogues

-LRB-

genes

that

arose

from

intragenome

duplication

-RRB-

in

human

y

worm

and

yeast

Brie

y

we

performed

all-against-all

sequence

for

the

combined

protein

sets

of

human

yeast

y

and

worm

Pairs

of

sequences

that

were

one

another

's

best

matches

in

their

respective

genomes

were

considered

to

be

potential

orthologues

These

were

then

used

to

identify

orthologous

groups

across

three

Recent

species-speci

c

paralogues

were

de

ned

by

using

the

allagainst-all

sequence

comparison

to

cluster

the

protein

set

for

each

organism

For

each

sequence

found

in

an

orthologous

group

the

recent

paralogues

were

de

ned

to

be

the

largest

species-speci

c

cluster

including

it

The

set

of

paralogues

may

be

in

ated

by

unrecognized

splice

variants

and

by

fragmentation

Table

Probable

vertebrate-speci

c

acquisitions

of

bacterial

genes

Human

protein

-LRB-

accession

-RRB-

Predicted

function

Known

orthologues

in

other

vertebrates

Pig

rat

chicken

Rodents

ungulates

Bacterial

homologues

Range

Thermotoga

Thermoplasma

Methylobacter

Most

bacteria

Best

hit

Thermotoga

maritima

Vibrio

parahaemolyticus

Human

origin

con

rmed

by

PCR

Yes

Yes

-LRB-

-RRB-

NT

-LRB-

-RRB-

Yes

Yes

NT

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

We

identi

ed

groups

of

proteins

each

containing

at

least

one

predicted

orthologue

in

each

species

and

many

containing

additional

paralogues

The

groups

contained

human

proteins

y

proteins

worm

proteins

and

yeast

proteins

These

groups

represent

a

conserved

core

of

proteins

that

are

mostly

responsible

for

the

basic

housekeeping

functions

of

the

cell

including

metabolism

DNA

replication

and

repair

and

translation

In

of

the

groups

one

orthologue

-LRB-

and

no

additional

paralogues

-RRB-

could

be

unambiguously

assigned

for

each

of

human

y

worm

and

yeast

These

groups

will

be

referred

to

as

groups

More

than

half

-LRB-

-RRB-

of

these

groups

could

be

assigned

to

the

functional

categories

shown

in

Fig.

Within

these

functional

categories

the

numbers

of

groups

containing

single

orthologues

in

each

of

the

four

proteomes

was

for

cellular

processes

for

metabolism

for

DNA

replication

and

modi

cation

for

transcription/translation

for

intracellular

signalling

for

protein

folding

and

degradation

for

transport

for

Formiminotransferase

cyclodeaminase

Na/glucose

cotransporter

Mouse

Danio

fugu

sh

Protein-methionine-S-oxide

reductase

Cow

Hypertension-associated

protein

SA

Mouse

rat

cow

acetate-CoA

ligase

transporter

Mouse

rat

glycogen

storage

disease

type

protein

Monoamine

oxidase

Cow

rat

salmon

Acyl-CoA

dehydrogenase

mitochondrial

protein

Mouse

rat

pig

Pig

-LRB-

also

found

in

plants

-RRB-

Epoxide

hydrolase

-LRB-

a/b-hydrolase

-RRB-

Most

bacteria

Most

bacteria

Most

bacteria

Most

bacteria

Most

bacteria

Most

bacteria

Streptomyces

Bacillus

Pseudomonas

aeruginosa

Synechocystis

sp.

Bacillus

halodurans

Chlamydophila

pneumoniae

Mycobacterium

tuberculosis

P.

aeruginosa

Streptomyces

coelicolor

NT

not

tested

Representative

genes

con

rmed

by

PCR

to

be

present

in

the

human

genome

The

similarity

to

a

bacterial

homologue

was

considered

to

be

signi

cantly

greater

than

that

to

eukaryotic

homologues

if

the

difference

in

alignment

scores

returned

by

BLASTP

was

greater

than

bits

-LRB-

orders

of

magnitude

in

terms

of

E-value

-RRB-

A

complete

classi

ed

and

annotated

list

of

probable

vertebrate-speci

c

horizontal

gene

transfers

detected

in

this

analysis

is

available

as

Supplementary

Information

cDNA

sequences

for

each

protein

were

searched

using

the

SSAHA

algorithm

against

the

draft

genome

sequence

Primers

were

designed

and

PCR

was

performed

using

three

human

genomic

samples

and

a

random

BAC

clone

The

predicted

genes

were

considered

to

be

present

in

the

human

genome

if

a

band

of

the

expected

size

was

found

in

all

three

human

samples

but

not

in

the

control

clone

Predicted

carboxylase

-LRB-

C-terminal

Streptomyces

Rhizobium

S.

coelicolor

domain

N-terminal

domain

unique

-RRB-

Bacillus

Uncharacterized

protein

Gamma-proteobacteria

Escherichia

coli

Uncharacterized

protein

Most

bacteria

T.

maritima

b-Lactamase

superfamily

hydrolase

Most

bacteria

Synechocystis

sp.

Oxidoreductase

-LRB-

Rossmann

fold

-RRB-

-LRB-

several

human

Actinomycetes

Leptospira

S.

coelicolor

fused

to

a

six-transmembrane

protein

paralogues

of

both

more

distant

homologues

in

parts

-RRB-

other

bacteria

Oxidoreductase

-LRB-

Rossmann

fold

-RRB-

Synechocystis

Pseudomonas

Synechocystis

sp.

a/b-hydrolase

Rickettsia

more

distant

Rickettsia

prowazekii

homologues

in

other

bacteria

ADP-ribosylglycohydrolase

Streptomyces

Aquifex

S.

coelicolor

Archaeoglobus

-LRB-

archaeon

-RRB-

E.

coli

Thymidine

phosporylase/endothelial

Most

bacteria

Bacillus

stearothermophilus

cell

growth

factor

Ribosomal

protein

acid

Most

bacteria

and

archaea

Haemophilus

in

uenzae

ligase

Ribosomal

protein

acid

Most

bacteria

and

archaea

H.

in

uenzae

ligase

-LRB-

paralogue

of

the

above

-RRB-

Hydratase

Synechocystis

Synechocystis

sp.

Sphingomonas

Homologue

of

histone

C

-LRB-

several

human

Thermotoga

Alcaligenes

E.

T.

maritima

terminal

domain

predicted

paralogues

RNA

coli

more

distant

homologues

phosphatase

viruses

-RRB-

in

other

bacteria

Sugar

transporter

Most

bacteria

Synechocystis

sp.

Predicted

metal-binding

protein

Most

bacteria

Borrelia

burgdorferi

Pseudouridine

synthase

Most

bacteria

Zymomonas

mobilis

Surfactin

synthetase

domain

Gram-positive

bacteria

Bacillus

subtilis

Actinomycetes

Cyanobacteria

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articles

multifunctional

proteins

and

for

cytoskeletal/structural

No

such

groups

were

found

for

defence

and

immunity

or

cell

cell

communication

The

groups

probably

represent

key

functions

that

have

not

undergone

duplication

and

elaboration

in

the

various

lineages

They

include

many

anabolic

enzymes

responsible

for

such

functions

as

respiratory

chain

and

nucleotide

biosynthesis

In

contrast

there

are

few

catabolic

enzymes

As

anabolic

pathways

branch

less

frequently

than

catabolic

pathways

this

indicates

that

alternative

routes

and

displacements

are

more

frequent

in

catabolic

reactions

If

proteins

from

the

single-celled

yeast

are

excluded

from

the

analysis

there

are

groups

The

additional

groups

include

many

examples

of

more

complex

signalling

proteins

such

as

receptor-type

and

src-like

tyrosine

kinases

likely

to

have

arisen

early

in

the

metazoan

lineage

The

fact

that

this

set

comprises

only

a

small

proportion

of

the

proteome

of

each

of

the

animals

indicates

that

apart

from

a

modest

conserved

core

there

has

been

extensive

elaboration

and

innovation

within

the

protein

complement

Most

proteins

do

not

show

simple

orthologous

relationships

across

the

three

animals

To

illustrate

this

we

investigated

the

nuclear

hormone

receptor

family

In

the

human

proteome

this

family

consists

of

different

classical

members

each

with

a

zinc

nger

and

a

ligand-binding

domain

In

comparison

the

y

proteome

has

and

the

worm

proteome

has

As

shown

in

Fig.

few

simple

orthologous

relationships

can

be

derived

among

these

homologues

And

where

potential

subgroups

of

orthologues

and

paralogues

could

be

identi

ed

it

was

apparent

that

the

functions

of

the

subgroup

members

could

differ

signi

cantly

For

example

the

y

receptor

for

the

y-speci

c

hormone

ecdysone

and

the

human

retinoic

acid

receptors

cluster

together

on

the

basis

of

sequence

similarity

Such

examples

underscore

that

the

assignment

of

functional

similarity

on

the

basis

of

sequence

similarities

among

these

three

organisms

is

not

trivial

in

most

cases

New

vertebrate

domains

and

proteins

We

then

explored

how

the

proteome

of

vertebrates

-LRB-

as

represented

by

the

human

-RRB-

differs

from

those

of

the

other

species

considered

The

InterPro

families

were

scanned

to

identify

those

that

contain

only

vertebrate

proteins

Only

-LRB-

-RRB-

of

the

families

were

vertebrate-speci

c

These

represent

protein

families

and

domain

families

Only

one

of

the

families

represents

enzymes

which

is

consistent

with

the

ancient

origins

of

most

The

single

vertebrate-speci

c

enzyme

family

identi

ed

was

the

pancreatic

or

eosinophil-associated

ribonucleases

These

enzymes

evolved

rapidly

possibly

to

combat

vertebrate

The

relatively

small

proportion

of

vertebrate-speci

c

multicopy

families

suggests

that

few

new

protein

domains

have

been

invented

in

the

vertebrate

lineage

and

that

most

protein

domains

trace

at

least

as

far

back

as

a

common

animal

ancestor

This

conclusion

must

be

tempered

by

the

fact

that

the

InterPro

classi

cation

system

is

incomplete

additional

vertebrate-speci

c

families

undoubtedly

exist

that

have

not

yet

been

recognized

in

the

InterPro

system

The

vertebrate-speci

c

families

appear

to

re

ect

important

physiological

differences

between

vertebrates

and

other

eukaryotes

Defence

and

immunity

proteins

-LRB-

families

-RRB-

and

proteins

that

function

in

the

nervous

system

-LRB-

families

-RRB-

are

particularly

enriched

in

this

set

These

data

indicate

the

recent

emergence

or

rapid

divergence

of

these

proteins

Representative

human

proteins

were

previously

known

for

nearly

all

of

the

vertebrate-speci

c

families

This

was

not

surprising

given

the

anthropocentrism

of

biological

research

However

the

analysis

did

identify

the

rst

mammalian

proteins

belonging

to

two

of

these

families

Both

of

these

families

were

originally

de

ned

in

sh

The

rst

is

the

family

of

polar

sh

antifreeze

III

proteins

We

found

a

human

sialic

acid

synthase

containing

a

domain

homologous

to

polar

sh

antifreeze

III

protein

-LRB-

-RRB-

This

nding

suggests

that

sh

created

the

antifreeze

function

by

adaptation

of

this

domain

We

also

found

a

human

protein

-LRB-

-RRB-

homologous

to

the

ependymin

found

in

teleost

sh

Ependymins

are

major

glycoproteins

of

sh

brains

that

have

been

claimed

to

be

involved

in

long-term

memory

The

function

of

the

mammalian

ependymin

homologue

will

need

to

be

elucidated

New

architectures

from

old

domains

Whereas

there

appears

to

be

only

modest

invention

at

the

level

of

new

vertebrate

protein

domains

there

appears

to

be

substantial

innovation

in

the

creation

of

new

vertebrate

proteins

This

innovation

is

evident

at

the

level

of

domain

architecture

de

ned

as

the

linear

arrangement

of

domains

within

a

polypeptide

New

architectures

can

be

created

by

shuf

ing

adding

or

deleting

domains

resulting

in

new

proteins

from

old

parts

We

quanti

ed

the

number

of

distinct

protein

architectures

found

in

yeast

worm

y

and

human

by

using

the

SMART

annotation

-LRB-

Fig.

-RRB-

The

human

proteome

set

contained

times

as

many

protein

architectures

as

worm

or

y

and

times

as

many

as

yeast

This

difference

is

most

prominent

in

the

recent

evolution

of

novel

extracellular

and

transmembrane

architectures

in

the

human

lineage

Human

extracellular

proteins

show

the

greatest

innovation

the

human

has

times

as

many

extracellular

architectures

as

y

and

times

as

many

as

worm

The

larger

number

of

human

architectures

does

not

simply

re

ect

differences

in

the

number

of

domains

known

in

these

organisms

the

result

remains

qualitatively

the

same

even

if

the

number

of

architectures

in

each

organism

is

normalized

by

dividing

by

the

total

number

of

domains

-LRB-

not

shown

-RRB-

-LRB-

We

also

checked

that

the

larger

number

of

human

NATURE

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Human

-LRB-

-RRB-

Worm

Fly

-LRB-

-RRB-

-LRB-

-RRB-

Hepatocyte

nuclear

factors

-LRB-

-RRB-

-LRB-

-RRB-

Vitamin

Ecdysone

-LRB-

-RRB-

-LRB-

-RRB-

Thyroid

hormone

-LRB-

-RRB-

Retinoic

acids

-LRB-

-RRB-

Steroid

hormone

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Peroxisome

proliferator

activated

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Ecdysone

-LRB-

-RRB-

Retinoic

acids

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Apolipoprotein

regulatory

protein

Figure

Simpli

ed

cladogram

-LRB-

relationship

tree

-RRB-

of

the

many-to-many

relationships

of

classical

nuclear

receptors

Triangles

indicate

expansion

within

one

lineage

bars

represent

single

members

Numbers

in

parentheses

indicate

the

number

of

paralogues

in

each

group

Macmillan

Magazines

Ltd

articles

Number

of

distinct

domain

architectures

Intracellular

800 600 400 200

Human

Fly

Worm

Yeast

Transmembrane

Extracellular

Figure

Number

of

distinct

domain

architectures

in

the

four

eukaryotic

genomes

predicted

using

The

number

of

architectures

is

split

into

three

cellular

environments

intracellular

extracellular

and

membrane-associated

The

increase

in

architectures

for

the

human

relative

to

the

other

lineages

is

seen

when

these

numbers

are

normalized

with

respect

to

the

numbers

of

domains

predicted

in

each

phylum

To

avoid

artefactual

results

from

the

relatively

low

detection

rate

for

some

repeat

types

tandem

occurrences

of

tetratricopeptide

armadillo

EF-hand

leucine-rich

or

ankyrin

repeats

or

zinc

ngers

were

treated

as

single

occurrences

architectures

could

not

be

an

artefact

resulting

from

erroneous

gene

predictions

Three-quarters

of

the

architectures

can

be

found

in

known

genes

which

already

yields

an

increase

of

about

over

worm

and

y

We

expect

the

nal

number

of

human

architectures

to

grow

as

the

complete

gene

set

is

identi

ed

-RRB-

A

related

measure

of

proteome

complexity

can

be

obtained

by

considering

an

individual

domain

and

counting

the

number

of

different

domain

types

with

which

it

co-occurs

For

example

the

trypsin-like

serine

protease

domain

-LRB-

number

in

Fig.

-RRB-

co-occurs

with

domain

types

in

human

-LRB-

including

proteins

involved

in

the

mammalian

complement

system

blood

coagulation

and

brinolytic

and

related

systems

-RRB-

By

contrast

the

trypsin-like

serine

protease

domain

occurs

with

only

eight

other

domains

in

y

ve

in

worm

and

one

in

yeast

Similar

results

for

common

domains

are

shown

in

Fig.

In

general

there

are

more

different

co-occurring

domains

in

the

human

proteome

than

in

the

other

proteomes

One

mechanism

by

which

architectures

evolve

is

through

the

fusion

of

additional

domains

often

at

one

or

both

ends

of

the

proteins

Such

domain

accretion

is

seen

in

many

human

proteins

when

compared

with

proteins

from

other

eukaryotes

The

effect

is

illustrated

by

several

chromatin-associated

proteins

-LRB-

Fig.

-RRB-

In

these

examples

the

domain

architectures

of

human

proteins

differ

from

those

found

in

yeast

worm

and

y

proteins

only

by

the

addition

of

domains

at

their

termini

Among

chromatin-associated

proteins

and

transcription

factors

a

signi

cant

proportion

of

domain

architectures

is

shared

between

the

vertebrate

and

y

but

not

with

worm

-LRB-

Fig.

-RRB-

The

trend

was

even

more

prominent

in

architectures

of

proteins

involved

in

another

key

cellular

process

programmed

cell

death

-LRB-

Fig.

-RRB-

These

examples

might

seem

to

bear

upon

the

unresolved

issue

of

the

evolutionary

branching

order

of

worms

ies

and

humans

suggesting

that

worms

branched

off

rst

However

there

were

other

cases

in

which

worms

and

humans

shared

architectures

not

present

in

y

A

global

analysis

of

shared

architectures

could

not

conclusively

distinguish

between

the

two

models

given

the

possibility

of

lineage-speci

c

loss

of

architectures

Comparison

of

protein

architectures

may

help

to

resolve

the

evolutionary

issue

but

it

will

require

more

detailed

analyses

of

many

protein

families

New

physiology

from

old

proteins

An

important

aspect

of

NATURE

VOL

FEBRUARY

Number

of

co-occurring

domains

Mustard

weed

Yeast

Worm

Fly

Human

Domain

family

Figure

Number

of

different

Pfam

domain

types

that

co-occur

in

the

same

protein

for

each

of

the

most

common

domain

families

in

each

of

the

ve

eukaryotic

proteomes

Because

some

common

domain

families

are

shared

there

are

families

rather

than

The

data

are

ranked

according

to

decreasing

numbers

of

human

co-occurring

Pfam

domains

The

domain

families

are

-LRB-

-RRB-

eukaryotic

protein

kinase

-LSB-

-RSB-

-LRB-

-RRB-

immunoglobulin

domain

-LSB-

-RSB-

-LRB-

-RRB-

ankyrin

repeat

-LSB-

-RSB-

-LRB-

-RRB-

RING

nger

-LSB-

-RSB-

-LRB-

-RRB-

zinc

nger

-LSB-

-RSB-

-LRB-

-RRB-

ATP/GTP-binding

P-loop

-LSB-

-RSB-

-LRB-

-RRB-

reverse

transcriptase

-LRB-

RNA-dependent

DNA

polymerase

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

leucine-rich

repeat

-LSB-

-RSB-

-LRB-

-RRB-

G-proteinb

repeats

-LSB-

-RSB-

-LRB-

-RRB-

RNA-binding

region

-LRB-

RNA

recognition

motif

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

C-type

lectin

domain

-LSB-

-RSB-

-LRB-

-RRB-

serine

proteases

trypsin

family

-LSB-

-RSB-

-LRB-

-RRB-

helicase

C-terminal

domain

-LSB-

-RSB-

-LRB-

-RRB-

collagen

triple

helix

repeat

-LSB-

-RSB-

-LRB-

-RRB-

rhodopsin-like

GPCR

superfamily

-LSB-

-RSB-

-LRB-

-RRB-

esterase/lipase/thioesterase

-LSB-

-RSB-

-LRB-

-RRB-

Myb

DNA-binding

domain

-LSB-

-RSB-

-LRB-

-RRB-

F-box

domain

-LSB-

-RSB-

-LRB-

-RRB-

ATP-binding

transport

protein

P-loop

motif

-LSB-

-RSB-

-LRB-

-RRB-

homeobox

domain

-LSB-

-RSB-

-LRB-

-RRB-

steroid

receptor

zinc

nger

-LSB-

-RSB-

-LRB-

-RRB-

sugar

transporter

-LSB-

-RSB-

-LRB-

-RRB-

PPR

repeats

-LSB-

-RSB-

-LRB-

-RRB-

seven-helix

G-protein-coupled

receptor

worm

-LRB-

probably

olfactory

-RRB-

family

-LSB-

-RSB-

-LRB-

-RRB-

cytochrome

enzyme

-LSB-

-RSB-

-LRB-

-RRB-

fungal

transcriptional

regulatory

protein

N

terminus

-LSB-

-RSB-

-LRB-

-RRB-

domain

of

unknown

function

-LSB-

-RSB-

Macmillan

Magazines

Ltd

articles

a

Br

A

Y

Br

Ba

Znf

Ba

hmg

W

b

Y

PHD

PHD

F

H

PHD

PHD

PHD

PHD

PHD

Znf

Br

Br

BMB

Znf

W

F

V

Br

Br

Br

Br

Br

Br

W

PHD

Ch

Ch

PHD

Sa

hmg

F

H

Ch

Ch

Sa

PHD

peregrin

Y

F

V

E

-LRB-

Pc

-RRB-

like

F

PHD

PHD

PHD

PHD

c

H

PHD

PHD

PHD

sja

C

CC

SET

C

Br

Trx

W

F

H

A

A

A

Me

Br

PHD

sja

C

CC

SET

C

Hrx

PHD

PHD

Common

ancestor

hmg

sja

C

CC

ALR

SET

C

Figure

Examples

of

domain

accretion

in

chromatin

proteins

Domain

accretion

in

various

lineages

before

the

animal

divergence

in

the

apparent

coelomate

lineage

and

the

vertebrate

lineage

are

shown

using

schematic

representations

of

domain

architectures

-LRB-

not

to

scale

-RRB-

Asterisks

mobile

domains

that

have

participated

in

the

accretion

Species

in

which

a

domain

architecture

has

been

identi

ed

are

indicated

above

the

diagram

-LRB-

Y

yeast

W

worm

F

y

V

vertebrate

-RRB-

Protein

names

are

below

the

diagrams

The

domains

are

SET

a

chromatin

protein

methyltransferase

domain

a

superfamily

II

helicase/ATPase

domain

Sa

sant

domain

Br

bromo

domain

Ch

chromodomain

C

a

cysteine

triad

motif

associated

with

the

and

SET

domains

A

AT

hook

motif

enhancer

of

polycomb

domains

and

Znf

zinc

nger

sja

SET-JOR-associated

domain

-LRB-

L.

Aravind

unpublished

-RRB-

Me

DNA

methylase/Hrx-associated

DNA

binding

zinc

nger

Ba

bromo-associated

homology

motif

a

c

Different

examples

of

accretion

vertebrate

innovation

lies

in

the

expansion

of

protein

families

Table

shows

the

most

prevalent

protein

domains

and

protein

families

in

humans

together

with

their

relative

ranks

in

the

other

species

About

of

families

are

more

numerous

in

the

human

than

in

any

of

the

other

four

organisms

This

shows

that

gene

duplication

has

been

a

major

evolutionary

force

during

vertebrate

evolution

A

a

Conserved

domain

architectures

in

chromatin

proteins

Human

and

fly

Human

and

worm

Worm

and

fly

All

three

b

Conserved

domain

architectures

in

apoptotic

proteins

Human

and

fly

Human

and

worm

Worm

and

fly

All

three

Figure

Conservation

of

architectures

between

animal

species

The

pie

charts

illustrate

the

shared

domain

architectures

of

apparent

orthologues

that

are

conserved

in

at

least

two

of

the

three

sequenced

animal

genomes

If

an

architecture

was

detected

in

fungi

or

plants

as

well

as

two

of

the

animal

lineages

it

was

omitted

as

ancient

and

its

absence

in

the

third

animal

lineage

attributed

to

gene

loss

a

Chromatin-associated

proteins

b

Components

of

the

programmed

cell

death

system

comparison

of

relative

expansions

in

human

versus

y

is

shown

in

Fig.

Many

of

the

families

that

are

expanded

in

human

relative

to

y

and

worm

are

involved

in

distinctive

aspects

of

vertebrate

physiology

An

example

is

the

family

of

immunoglobulin

-LRB-

IG

-RRB-

domains

rst

identi

ed

in

antibodies

thirty

years

ago

Classic

-LRB-

as

opposed

to

divergent

-RRB-

IG

domains

are

completely

absent

from

the

yeast

and

mustard

weed

proteomes

and

although

prokaryotic

homologues

exist

they

have

probably

been

transferred

horizontally

from

Most

IG

superfamily

proteins

in

invertebrates

are

cell-surface

proteins

In

vertebrates

the

IG

repertoire

includes

immune

functions

such

as

those

of

antibodies

MHC

proteins

antibody

receptors

and

many

lymphocyte

cell-surface

proteins

The

large

expansion

of

IG

domains

in

vertebrates

shows

the

versatility

of

a

single

family

in

evoking

rapid

and

effective

response

to

infection

Two

prominent

families

are

involved

in

the

control

of

development

The

human

genome

contains

broblast

growth

factors

-LRB-

FGFs

-RRB-

as

opposed

to

two

FGFs

each

in

the

y

and

worm

It

contains

transforming

growth

factor-bs

-LRB-

TGFbs

-RRB-

compared

with

nine

and

six

in

the

y

and

worm

respectively

These

growth

factors

are

involved

in

organogenesis

such

as

that

of

the

liver

and

the

lung

A

y

FGF

protein

branchless

is

involved

in

developing

respiratory

organs

-LRB-

tracheae

-RRB-

in

Thus

developmental

triggers

of

morphogenesis

in

vertebrates

have

evolved

from

related

but

simpler

systems

in

Another

example

is

the

family

of

intermediate

lament

proteins

with

family

members

This

expansion

is

almost

entirely

due

to

keratins

which

are

chordate-speci

c

intermediate

lament

proteins

that

form

laments

in

epithelia

The

large

number

of

human

keratins

suggests

multiple

cellular

structural

support

roles

for

the

many

specialized

epithelia

of

vertebrates

Finally

the

olfactory

receptor

genes

comprise

a

huge

gene

family

of

about

genes

and

The

number

of

olfactory

receptors

testi

es

to

the

importance

of

the

sense

of

smell

in

vertebrates

A

total

of

olfactory

receptor

genes

and

pseudogenes

could

be

identi

ed

in

the

draft

genome

sequence

two-thirds

of

which

were

not

previously

annotated

About

are

found

in

about

two

dozen

clusters

ranging

from

to

genes

and

encompassing

about

Mb

-LRB-

-RRB-

of

the

human

genome

Despite

the

importance

of

smell

among

our

vertebrate

ancestors

hominids

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

appear

to

have

considerably

less

interest

in

this

sense

About

of

the

olfactory

receptors

in

the

draft

genome

sequence

have

disrupted

ORFs

and

appear

to

be

pseudogenes

consistent

with

recent

suggesting

massive

functional

gene

loss

in

the

last

Interestingly

there

appears

to

be

a

much

higher

proportion

of

intact

genes

among

class

I

than

class

II

olfactory

receptors

suggesting

functional

importance

Vertebrates

are

not

unique

in

employing

gene

family

expansion

For

many

domain

types

expansions

appear

to

have

occurred

independently

in

each

of

the

major

eukaryotic

lineages

A

good

example

is

the

classical

family

of

zinc

nger

domains

which

have

expanded

independently

in

the

yeast

worm

y

and

human

lineages

-LRB-

Fig.

-RRB-

These

independent

expansions

have

resulted

in

numerous

zinc

nger

domain-containing

proteins

that

are

speci

c

to

each

lineage

In

ies

the

important

components

of

the

zinc

nger

expansion

are

architectures

in

which

it

is

combined

with

the

POZ

domain

and

the

domain

-LRB-

a

metalbinding

domain

found

only

in

y

-RRB-

In

humans

the

most

prevalent

expansions

are

combinations

of

the

zinc

nger

with

POZ

-LRB-

independent

of

the

one

in

insects

-RRB-

and

the

vertebrate-speci

c

KRAB

and

SCAN

domains

The

homeodomain

is

similarly

expanded

in

all

animals

and

is

present

in

both

architectures

that

are

conserved

and

lineage-speci

c

architectures

-LRB-

Fig.

-RRB-

This

indicates

that

the

ancestral

animal

probably

encoded

a

signi

cant

number

of

homeodomain

proteins

but

subsequent

evolution

involved

multiple

independent

expansions

and

domain

shuf

ing

after

lineages

diverged

Thus

the

most

prevalent

transcription

factor

families

are

different

in

worm

y

and

human

-LRB-

Fig.

-RRB-

This

has

major

biological

implications

because

transcription

factors

are

critical

in

animal

development

and

differentiation

The

emergence

of

major

variations

in

the

developmental

body

plans

that

accompanied

the

early

radiation

of

the

could

have

been

driven

by

lineage-speci

c

proliferation

of

such

transcription

factors

Beyond

these

large

expansions

of

protein

families

protein

components

of

particular

functional

systems

such

as

the

cell

death

signalling

system

show

a

general

increase

in

diversity

and

numbers

in

the

vertebrates

relative

to

other

animals

For

example

there

are

greater

numbers

of

and

more

novel

architectures

in

cell

death

regulatory

proteins

such

as

TNFR

and

NFkB

from

vertebrates

Conclusion

Five

lines

of

evidence

point

to

an

increase

in

the

complexity

of

the

proteome

from

the

single-celled

yeast

to

the

multicellular

invertebrates

and

to

vertebrates

such

as

the

human

Speci

cally

the

human

contains

greater

numbers

of

genes

domain

and

protein

families

paralogues

multidomain

proteins

with

multiple

functions

and

domain

architectures

According

to

these

measures

the

relatively

greater

complexity

of

the

human

proteome

is

a

consequence

not

simply

of

its

larger

size

but

also

of

large-scale

protein

innovation

An

important

question

is

the

extent

to

which

the

greater

phenotypic

complexity

of

vertebrates

can

be

explained

simply

by

two

or

threefold

increases

in

proteome

complexity

The

real

explanation

may

lie

in

combinatorial

ampli

cation

of

these

modest

differences

by

mechanisms

that

include

alternative

splicing

post-translational

modi

cation

and

cellular

regulatory

networks

The

potential

numbers

of

different

proteins

and

protein

protein

interactions

are

vast

and

their

actual

numbers

can

not

readily

be

discerned

from

the

genome

sequence

Elucidating

such

systemlevel

properties

presents

one

of

the

great

challenges

for

modern

biology

Table

The

most

populous

InterPro

families

in

the

human

proteome

and

other

species

Human

InterPro

ID

No

of

genes

765 706 575 569

433 350 300 277

276 267 252 242

222 215 210 188

171 165 162 155

150 133 131 123

119 118 114 113

Rank

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

No

of

genes

140 357 319

198 10 157 162

81 72 114 115

Fly

Rank

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Worm

No

of

genes

64 151 437 358

50 96 102 107

12 16 117 108

Rank

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Yeast

No

of

genes

Rank

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mustard

weed

No

of

genes

331 80 255 210

15 148 125

Rank

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Immunoglobulin

domain

zinc

nger

Eukaryotic

protein

kinase

Rhodopsin-like

GPCR

superfamily

P-loop

motif

Reverse

transcriptase

-LRB-

RNA-dependent

DNA

polymerase

-RRB-

rrm

domain

G-protein

b

repeats

Ankyrin

repeat

Homeobox

domain

PH

domain

EF-hand

family

EGF-like

domain

domain

RING

nger

Leucine-rich

repeat

KRAB

box

Fibronectin

type

III

domain

PDZ

domain

Helicase

C-terminal

domain

TPR

repeat

Ion

transport

protein

Helix

loop

helix

DNA-binding

domain

domain

domain

Serine

protease

trypsin

family

Cadherin

domain

BTB/POZ

domain

Tyrosine-speci

c

protein

phosphatase

and

dual

speci

city

protein

phosphatase

family

Collagen

triple

helix

repeat

Esterase/lipase/thioesterase

-LRB-

high

mobility

group

-RRB-

box

Neutral

zinc

metallopeptidase

PHD

nger

Cation

channels

-LRB-

non-ligand

gated

-RRB-

LIM

domain

VWA

domain

Calponin

homology

domain

RhoGAP

domain

Forty

most

populous

Interpro

families

found

in

the

human

proteome

compared

with

equivalent

numbers

from

other

species

not

applicable

-LRB-

used

when

there

are

no

proteins

in

an

organism

in

that

family

-RRB-

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

Segmental

history

of

the

human

genome

In

bacteria

genomic

segments

often

convey

important

information

about

function

genes

located

close

to

one

another

often

encode

proteins

in

a

common

pathway

and

are

regulated

in

a

common

operon

In

mammals

genes

found

close

to

each

other

only

rarely

have

common

functions

but

they

are

still

interesting

because

they

have

a

common

history

In

fact

the

study

of

genomic

segments

can

shed

light

on

biological

events

as

long

as

Myr

ago

and

as

recently

as

years

ago

Conserved

segments

between

human

and

mouse

Humans

and

mice

shared

a

common

ancestor

about

Myr

ago

Despite

the

Myr

of

evolutionary

distance

between

the

species

a

signi

cant

fraction

of

genes

show

synteny

between

the

two

being

preserved

within

conserved

segments

Genes

tightly

linked

in

one

mammalian

species

tend

to

be

linked

in

others

In

fact

conserved

segments

have

been

observed

in

even

more

distant

species

humans

show

conserved

segments

with

and

even

with

invertebrates

such

as

y

and

In

general

the

likelihood

that

a

syntenic

relationship

will

be

disrupted

correlates

with

the

physical

distance

between

the

loci

and

the

evolutionary

distance

between

the

species

Studying

conserved

segments

between

human

and

mouse

has

several

uses

First

conservation

of

gene

order

has

been

used

to

identify

likely

orthologues

between

the

species

particularly

when

investigating

disease

phenotypes

Second

the

study

of

conserved

segments

among

genomes

helps

us

to

deduce

evolutionary

ancestry

300 250 200 150

Human

-LRB-

number

of

genes

-RRB-

Figure

Relative

expansions

of

protein

families

between

human

and

y

These

data

have

not

been

normalized

for

proteomic

size

differences

Blue

line

equality

between

normalized

family

sizes

in

the

two

organisms

Green

line

equality

between

unnormalized

family

sizes

Numbered

InterPro

entries

-LRB-

-RRB-

immunoglobulin

domain

-LSB-

-RSB-

-LRB-

-RRB-

zinc

nger

type

-LSB-

-RSB-

-LRB-

-RRB-

eukaryotic

protein

kinase

-LSB-

-RSB-

-LRB-

-RRB-

rhodopsinlike

GPCR

superfamily

-LSB-

-RSB-

-LRB-

-RRB-

ATP/GTP-binding

site

motif

A

-LRB-

P-loop

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

reverse

transcriptase

-LRB-

RNA-dependent

DNA

polymerase

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

RNA-binding

region

-LRB-

RNA

recognition

motif

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

G-proteinb

repeats

-LSB-

-RSB-

-LRB-

-RRB-

ankyrin

repeat

-LSB-

-RSB-

-LRB-

-RRB-

homeobox

domain

-LSB-

-RSB-

-LRB-

-RRB-

PH

domain

-LSB-

-RSB-

-LRB-

-RRB-

EF-hand

family

-LSB-

-RSB-

-LRB-

-RRB-

EGFlike

domain

-LSB-

-RSB-

-LRB-

-RRB-

Src

homology

-LRB-

-RRB-

domain

-LSB-

-RSB-

-LRB-

-RRB-

RING

nger

-LSB-

-RSB-

-LRB-

-RRB-

KRAB

box

-LSB-

-RSB-

-LRB-

-RRB-

leucine-rich

repeat

-LSB-

-RSB-

-LRB-

-RRB-

bronectin

type

III

domain

-LSB-

-RSB-

-LRB-

-RRB-

PDZ

domain

-LRB-

also

known

as

DHR

or

GLGF

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

TPR

repeat

-LSB-

-RSB-

-LRB-

-RRB-

helicase

C-terminal

domain

-LSB-

-RSB-

-LRB-

-RRB-

ion

transport

protein

-LSB-

-RSB-

-LRB-

-RRB-

helix

loop

helix

DNA-binding

domain

-LSB-

-RSB-

-LRB-

-RRB-

cadherin

domain

-LSB-

-RSB-

-LRB-

-RRB-

intermediate

lament

proteins

-LSB-

-RSB-

-LRB-

-RRB-

domain

-LSB-

-RSB-

-LRB-

-RRB-

Src

homology

-LRB-

-RRB-

domain

-LSB-

-RSB-

-LRB-

-RRB-

serine

proteases

trypsin

family

-LSB-

-RSB-

-LRB-

-RRB-

BTB/POZ

domain

-LSB-

-RSB-

-LRB-

-RRB-

tyrosine-speci

c

protein

phosphatase

and

dual

speci

city

protein

phosphatase

family

-LSB-

-RSB-

-LRB-

-RRB-

collagen

triple

helix

repeat

-LSB-

-RSB-

-LRB-

-RRB-

esterase/lipase/thioesterase

-LSB-

-RSB-

-LRB-

-RRB-

neutral

zinc

metallopeptidases

zincbinding

region

-LSB-

-RSB-

-LRB-

-RRB-

ATP-binding

transport

protein

P-loop

motif

-LSB-

-RSB-

-LRB-

-RRB-

ABC

transporters

family

-LSB-

-RSB-

-LRB-

-RRB-

cytochrome

enzyme

-LSB-

-RSB-

-LRB-

-RRB-

insect

cuticle

protein

-LSB-

-RSB-

And

third

detailed

comparative

maps

may

assist

in

the

assembly

of

the

mouse

sequence

using

the

human

sequence

as

a

scaffold

Two

types

of

linkage

conservation

are

commonly

Conserved

synteny

indicates

that

at

least

two

genes

that

reside

on

a

common

chromosome

in

one

species

are

also

located

on

a

common

chromosome

in

the

other

species

Syntenic

loci

are

said

to

lie

in

a

conserved

segment

when

not

only

the

chromosomal

position

but

the

linear

order

of

the

loci

has

been

preserved

without

interruption

by

other

chromosomal

rearrangements

An

initial

survey

of

homologous

loci

in

human

and

suggested

that

the

total

number

of

conserved

segments

would

be

about

Subsequent

estimates

based

on

increasingly

detailed

comparative

maps

have

remained

close

to

this

-LRB-

-RRB-

The

distribution

of

segment

lengths

has

corresponded

reasonably

well

to

the

truncated

negative

exponential

curve

predicted

by

the

random

breakage

The

availability

of

a

draft

human

genome

sequence

allows

the

rst

global

human

mouse

comparison

in

which

human

physical

distances

can

be

measured

in

Mb

rather

than

cM

or

orthologous

gene

counts

We

identi

ed

likely

orthologues

by

reciprocal

comparison

of

the

human

and

mouse

mRNAs

in

the

LocusLink

database

using

megaBLAST

For

each

orthologous

pair

we

mapped

the

location

of

the

human

gene

in

the

draft

genome

sequence

and

then

checked

the

location

of

the

mouse

gene

in

the

Mouse

Genome

Informatics

database

-LRB-

-RRB-

Using

a

conservative

threshold

we

identi

ed

orthologous

pairs

in

which

the

human

gene

could

be

mapped

on

the

draft

genome

sequence

with

high

con

dence

Of

these

corresponding

mouse

genes

had

a

known

position

in

the

mouse

genome

We

then

searched

for

de

nitive

conserved

segments

de

ned

as

human

regions

containing

orthologues

of

at

least

two

genes

from

the

same

mouse

chromosome

region

-LRB-

cM

-RRB-

without

interruption

by

segments

from

other

chromosomes

We

identi

ed

de

nitive

conserved

segments

-LRB-

Fig.

-RRB-

The

average

segment

length

was

Mb

with

the

largest

segment

being

Mb

and

the

smallest

kb

There

were

also

singletons

segments

that

contained

only

a

single

locus

these

are

not

counted

in

the

statistics

Although

some

of

these

could

be

short

conserved

segments

they

could

also

re

ect

incorrect

choices

of

orthologues

or

problems

with

the

human

or

mouse

maps

Because

of

this

conservative

approach

the

observed

number

of

de

nitive

segments

is

likely

be

lower

than

the

correct

total

One

piece

of

evidence

for

this

conclusion

comes

from

a

more

detailed

analysis

on

human

chromosome

-LRB-

ref

-RRB-

which

identi

ed

conserved

segments

of

which

three

were

singletons

Our

analysis

revealed

only

de

nitive

segments

on

this

chromosome

with

nine

singletons

The

frequency

of

observing

a

particular

gene

count

in

a

conserved

segment

is

plotted

on

a

logarithmic

scale

in

Fig.

If

chromosomal

breaks

occur

in

a

random

fashion

-LRB-

as

has

been

proposed

-RRB-

and

differences

in

gene

density

are

ignored

a

roughly

straight

line

should

result

There

is

a

clear

excess

for

n

suggesting

that

or

more

of

the

singletons

are

indeed

artefactual

Thus

we

estimate

that

true

number

of

conserved

segments

is

around

in

good

agreement

with

the

original

Nadeau

Taylor

Figure

shows

a

plot

of

the

frequency

of

lengths

of

conserved

segments

where

the

x-axis

scale

is

shown

in

Mb

As

before

there

is

a

fair

amount

of

scatter

in

the

data

for

the

larger

segments

-LRB-

where

the

numbers

are

small

-RRB-

but

the

trend

appears

to

be

consistent

with

a

random

breakage

model

We

attempted

to

ascertain

whether

the

breakpoint

regions

have

any

special

characteristics

This

analysis

was

complicated

by

imprecision

in

the

positioning

of

these

breaks

which

will

tend

to

blur

any

relationships

With

orthologues

the

average

interval

within

which

a

break

is

known

to

have

occurred

is

about

Mb

We

compared

the

aggregate

features

of

these

breakpoint

intervals

with

the

genome

as

a

whole

The

mean

gene

density

was

lower

in

breakpoint

regions

than

in

the

conserved

segments

-LRB-

versus

NATURE

VOL

FEBRUARY

Fly

-LRB-

number

of

genes

-RRB-

Macmillan

Magazines

Ltd

articles

per

Mb

-RRB-

This

suggests

that

breakpoints

may

be

more

likely

to

occur

or

to

undergo

xation

in

gene-poor

intervals

than

in

generich

intervals

The

occurrence

of

breakpoints

may

be

promoted

by

homologous

recombination

among

repeated

When

the

sequence

of

the

mouse

genome

is

nished

this

analysis

can

be

revisited

more

precisely

A

number

of

examples

of

extended

conserved

segments

and

syntenies

are

apparent

in

Fig.

As

has

been

noted

almost

all

human

genes

on

chromosome

are

found

on

mouse

chromosome

with

two

members

of

the

placental

lactogen

family

from

mouse

inserted

Apart

from

two

singleton

loci

human

chromosome

appears

to

be

entirely

orthologous

to

mouse

chromosome

apparently

in

a

single

segment

The

largest

apparently

contiguous

conserved

segment

in

the

human

genome

is

on

chromosome

including

roughly

Mb

of

human

DNA

that

is

orthologous

to

mouse

chromosome

This

analysis

also

allows

us

to

infer

the

likely

location

of

thousands

of

mouse

genes

for

which

the

human

orthologue

has

been

located

in

the

draft

genome

sequence

but

the

mouse

locus

has

not

yet

been

mapped

With

about

conserved

segments

between

mouse

and

human

and

about

Myr

of

evolution

from

their

common

we

obtain

an

estimated

rate

of

about

chromosomal

rearrangement

being

xed

per

Myr

However

there

is

good

evidence

that

the

rate

of

chromosomal

rearrangement

-LRB-

like

the

rate

of

nucleotide

substitutions

see

above

-RRB-

differs

between

the

two

species

Among

mammals

rodents

may

show

unusually

rapid

chromosome

alteration

By

comparison

very

few

rearrangements

have

been

observed

among

primates

and

studies

of

a

broader

array

of

mammalian

orders

including

cats

cows

sheep

and

pigs

suggest

an

average

rate

of

chromosome

alteration

of

only

about

rearrangements

per

Myr

in

these

Additional

evidence

that

rodents

are

outliers

comes

from

a

recent

analysis

of

synteny

between

the

human

and

zebra

sh

genomes

From

a

study

of

orthologues

it

was

possible

to

project

conserved

Assuming

Myr

since

a

common

vertebrate

ancestor

of

zebra

sh

and

we

obtain

an

estimate

of

rearrangements

per

Myr

Recent

estimates

of

rearrangement

rates

in

plants

have

suggested

bimodality

with

some

pairs

showing

rates

of

rearrangements

per

Myr

and

others

showing

higher

rates

of

rearrangements

per

With

additional

detailed

genome

maps

of

multiple

species

it

should

be

possible

to

determine

whether

this

particular

molecular

clock

is

truly

operating

at

a

different

rate

in

various

branches

of

the

evolutionary

tree

and

whether

variations

in

that

rate

are

bimodal

or

continuous

It

should

also

be

possible

to

reconstruct

the

karyotypes

of

common

ancestors

Ancient

duplicated

segments

in

the

human

genome

Another

approach

to

genomic

history

is

to

study

segmental

duplications

within

the

human

genome

Earlier

we

discussed

examples

of

recent

duplications

of

genomic

segments

to

pericentromeric

and

subtelomeric

regions

Most

of

these

events

appear

to

be

evolutionary

dead-ends

resulting

in

nonfunctional

pseudogenes

however

segmental

duplication

is

also

an

important

mode

of

evolutionary

innovation

a

duplication

permits

one

copy

of

each

gene

to

drift

and

potentially

to

acquire

a

new

function

Segmental

duplications

can

occur

through

unequal

crossing

over

to

create

gene

families

in

speci

c

chromosomal

regions

This

mechanism

can

create

both

small

families

such

as

the

ve

related

genes

of

the

b-globin

cluster

on

chromosome

and

large

ones

such

as

the

olfactory

receptor

gene

clusters

which

together

contain

nearly

genes

and

pseudogenes

Worm

POZ

MATH

Fly

POZ

Human

POZ

Homeodomain

Paired

Homeodomain

Paired

Homeodomain

Paired

POZ

K

HD

HD

HD

Lim

HD

POZ

POZ

HD

HD

HD

Lim

HD

POZ

POZ

HD

HD

HD

Lim

HD

POZ

POZ

Pou

A

Pou

bZip

Pou

Zn

LB

SAZ

SAZ

SAZ

Krab

Scan

bHLH

bHIH

bHIH

PAS

PAS

bHLH

bHIH

bHIH

PAS

PAS

Unique

and

shared

domain

organizations

in

animals

Tig

Tig

ank

ank

Tig

Fly

ATX

HMG

NFAT

NFKB

ank

ank

ank

ank

ank

Tig

wd

wd

wd

wd

wd

wd

wd

wd

Hf

POZ

A

A

A

A

A

A

A

A

A

A

PHD

MeF

YCS

HMG

Histone

fold

S

SPRy

ATPase

SAF-A

STAT

SSRP

HMG

SAZ

HMG

HMG

HMG

HMG

HMG

CGBP

kdwk

my

HD

HD

HD

HD

HD

A

HMG

MTF

SSRP

Worm

Unique

to

fly

or

worm

Ancient

architectures

conserved

in

all

animals

Shared

by

fly

and

human

Unique

to

human

Figure

Lineage-speci

c

expansions

of

domains

and

architectures

of

transcription

factors

Top

speci

c

families

of

transcription

factors

that

have

been

expanded

in

each

of

the

proteomes

Approximate

numbers

of

domains

identi

ed

in

each

of

the

-LRB-

nearly

-RRB-

complete

proteomes

representing

the

lineages

are

shown

next

to

the

domains

and

some

of

the

most

common

architectures

are

shown

Some

are

shared

by

different

animal

lineages

others

are

lineage-speci

c.

Bottom

samples

of

architectures

from

transcription

factors

that

are

shared

by

all

animals

-LRB-

ancient

architectures

-RRB-

shared

by

y

and

human

and

unique

to

each

lineage

Domains

K

kelch

HD

homeodomain

Zn

zinc-binding

domain

LB

ligand-binding

domain

novel

Zn

cluster

with

four

cysteines

probably

involved

in

protein

protein

interactions

-LRB-

L.

Aravind

unpublished

-RRB-

MATH

meprin-associated

TRAF

NATURE

VOL

FEBRUARY

domain

novel

domain

in

transcription

factors

-LRB-

L.

Aravind

unpublished

-RRB-

MTF

myelin

transcription

factor

domain

SAZ

specialized

Myb-like

helixturn-helix

-LRB-

HTH

-RRB-

domain

found

in

Stonewall

and

Zeste

-LRB-

L.

Aravind

unpublished

-RRB-

A

AT-hook

motif

winged

HTH

DNA-binding

domain

GHL

gyraseB-histidine

kinaseMutL

ATPase

domain

ATX

ATaXin

domain

RFX

RFX

winged

HTH

DNA

binding

domain

My

MYND

domain

KDWK

KDWK

DNA-binding

domain

POZ

Pox

zinc

nger

domain

S

SAP

domain

fold

domain

HF

histone

fold

ANK

ankyrin

repeat

TIG

transcription

factor

Ig

domain

SSRP

structure-speci

c

recognition

protein

domain

metal

binding

domain

classic

zinc

nger

domain

WD

repeats

NHR

SAZ

-LRB-

MYB-like

-RRB-

Krab

Scan

GHL

HMG

RFX

A

Macmillan

Magazines

Ltd

articles

The

most

extreme

mechanism

is

whole-genome

duplication

-LRB-

WGD

-RRB-

through

a

polyploidization

event

in

which

a

diploid

organism

becomes

tetraploid

Such

events

are

classi

ed

as

autopolyploidy

or

allopolyploidy

depending

on

whether

they

involve

hybridization

between

members

of

the

same

species

or

different

species

Polyploidization

is

common

in

the

plant

kingdom

with

many

known

examples

among

wild

and

domesticated

crop

species

Alfalfa

-LRB-

Medicago

sativa

-RRB-

is

a

naturally

occurring

and

Nicotiana

tabacum

some

species

of

cotton

-LRB-

Gossypium

-RRB-

and

several

of

the

common

brassicas

are

allotetraploids

containing

pairs

of

homeologous

chromosome

pairs

In

principle

WGD

provides

the

raw

material

for

great

bursts

of

innovation

by

allowing

the

duplication

and

divergence

of

entire

pathways

suggested

that

WGD

has

played

a

key

role

in

evolution

There

is

evidence

for

an

ancient

WGD

event

in

the

ancestry

of

yeast

and

several

independent

such

events

in

the

ancestry

of

mustard

Such

ancient

WGD

events

can

be

hard

to

detect

because

only

a

minority

of

the

duplicated

loci

may

be

retained

with

the

result

that

the

genes

in

duplicated

segments

can

not

be

aligned

in

a

one-to-one

correspondence

but

rather

require

many

gaps

In

addition

duplicated

segments

may

be

subsequently

rearranged

For

example

the

ancient

duplication

in

the

yeast

genome

appears

to

have

been

followed

by

loss

of

more

than

of

the

newly

duplicated

One

of

the

most

controversial

hypotheses

about

vertebrate

evolution

is

the

proposal

that

two

WGD

events

occurred

early

in

the

vertebrate

lineage

around

the

time

of

jawed

shes

some

Myr

ago

Some

have

seen

support

for

this

theory

in

the

fact

that

many

human

genes

occur

in

sets

of

four

homologues

most

notably

the

four

extensive

HOX

gene

clusters

on

chromosomes

and

whose

duplication

dates

to

around

the

correct

time

However

other

authors

have

disputed

this

suggesting

that

these

cases

may

re

ect

unrelated

duplications

of

speci

c

regions

rather

than

successive

WGD

We

analysed

the

draft

genome

sequence

for

evidence

that

might

bear

on

this

question

The

analysis

provides

many

interesting

observations

but

no

convincing

evidence

of

ancient

WGD

We

looked

for

evidence

of

pairs

of

chromosomal

regions

containing

many

homologous

genes

Although

we

found

many

pairs

containing

a

few

homologous

genes

the

human

genome

does

not

appear

to

contain

any

pairs

of

regions

where

the

density

of

duplicated

genes

approaches

the

densities

seen

in

yeast

or

mustard

We

also

examined

human

proteins

in

the

IPI

for

which

the

orthologues

among

y

or

worm

proteins

occur

in

the

ratios

and

so

on

-LRB-

Fig.

-RRB-

The

number

of

such

families

falls

smoothly

with

no

peak

at

four

and

some

instances

of

ve

or

more

homologues

Although

this

does

not

rule

out

two

rounds

of

WGD

followed

by

extensive

gene

loss

and

some

unrelated

gene

duplication

it

provides

no

support

for

the

theory

More

probatively

if

two

successive

rounds

of

genome

duplication

occurred

phylogenetic

analysis

of

the

proteins

having

ratios

between

human

y

and

worm

would

be

expected

to

show

more

trees

with

the

topology

-LRB-

A

B

-RRB-

-LRB-

C

D

-RRB-

for

the

human

sequences

than

-LRB-

A

-LRB-

B

-LRB-

C

D

-RRB-

-RRB-

-RRB-

However

of

sets

studied

carefully

only

of

the

trees

constructed

from

the

set

have

the

former

topology

this

is

not

signi

cantly

different

from

what

would

be

expected

under

the

hypothesis

of

random

sequential

duplication

of

individual

loci

Occurrences

Genes

per

conserved

segment

Figure

Distribution

of

number

of

genes

per

conserved

segment

between

human

and

mouse

genomes

X

Y

Occurrences

X

Y

Figure

Conserved

segments

in

the

human

and

mouse

genome

Human

chromosomes

with

segments

containing

at

least

two

genes

whose

order

is

conserved

in

the

mouse

genome

as

colour

blocks

Each

colour

corresponds

to

a

particular

mouse

chromosome

Centromeres

subcentromeric

heterochromatin

of

chromosomes

and

and

the

repetitive

short

arms

of

and

are

in

black

Conserved

segment

length

-LRB-

Mb

-RRB-

Figure

Distribution

of

lengths

-LRB-

in

bins

-RRB-

of

conserved

segments

between

human

and

mouse

genomes

omitting

singletons

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

We

also

searched

for

sets

of

four

chromosomes

where

there

are

multiple

genes

with

homologues

on

each

of

the

four

The

strongest

example

was

chromosomes

and

containing

the

HOX

clusters

as

well

as

additional

genes

These

four

chromosomes

appear

to

have

an

excess

of

quadruplicated

genes

The

genes

are

not

all

clustered

in

a

single

region

this

may

re

ect

intrachromosomal

rearrangement

since

the

duplication

of

these

genes

or

it

may

indicate

that

they

result

from

several

independent

events

Of

the

genes

with

homologues

on

chromosomes

and

many

of

those

missing

on

chromosome

are

clustered

on

chromosome

suggesting

a

translocation

Several

additional

examples

of

groups

of

four

chromosomes

were

found

although

they

were

connected

by

fewer

homologous

genes

Although

the

analyses

are

sensitive

to

the

imperfect

quality

of

the

gene

predictions

our

results

so

far

are

insuf

cient

to

settle

whether

two

rounds

of

WGD

occurred

around

Myr

ago

It

may

be

possible

to

resolve

the

issue

by

systematically

estimating

the

time

of

each

of

the

many

gene

duplication

events

on

the

basis

of

sequence

divergence

although

this

is

beyond

the

scope

of

this

report

Another

approach

to

determining

whether

a

widespread

duplication

occurred

at

a

particular

time

in

vertebrate

evolution

would

be

to

sequence

the

genomes

of

organisms

whose

lineages

diverged

from

vertebrates

at

appropriate

times

such

as

amphioxus

and

sheds

light

on

the

unique

properties

and

history

of

each

genomic

region

The

average

heterozygosity

at

a

locus

will

tend

to

increase

in

proportion

to

the

local

mutation

rate

and

the

age

of

the

locus

-LRB-

which

can

be

de

ned

as

the

average

number

of

generations

since

the

most

recent

common

ancestor

of

two

randomly

chosen

copies

in

the

population

-RRB-

For

example

positive

selection

can

cause

a

locus

to

be

unusually

young

and

balancing

selection

can

cause

it

to

be

unusually

old

An

extreme

example

is

the

HLA

region

in

which

a

high

SNP

density

is

observed

re

ecting

the

fact

that

diverse

HLA

haplotypes

have

been

maintained

for

many

millions

of

years

by

balancing

selection

and

greatly

predate

the

origin

of

the

human

species

SNPs

can

also

be

used

to

study

linkage

disequilibrium

in

the

human

Linkage

disequilibrium

refers

to

the

persistence

of

ancestral

haplotypes

that

is

genomic

segments

carrying

particular

combinations

of

alleles

descended

from

a

common

ancestor

It

can

provide

a

powerful

tool

for

mapping

disease

and

for

probing

population

There

has

been

considerably

controversy

concerning

the

typical

distance

over

which

linkage

disequilibrium

extends

in

the

human

With

the

collection

of

SNPs

now

available

it

should

be

possible

to

resolve

this

important

issue

Recent

history

from

human

polymorphism

The

recent

history

of

genomic

segments

can

be

probed

by

studying

the

properties

of

SNPs

segregating

in

the

current

human

population

The

sequence

information

generated

in

the

course

of

this

project

has

yielded

a

huge

collection

of

SNPs

These

SNPs

were

extracted

in

two

ways

by

comparing

overlapping

large-insert

clones

derived

from

distinct

haplotypes

-LRB-

either

different

individuals

or

different

chromosomes

within

an

individual

-RRB-

and

by

comparing

random

reads

from

whole-genome

shotgun

libraries

derived

from

multiple

individuals

The

analysis

con

rms

an

average

heterozygosity

rate

in

the

human

population

of

about

in

bp

-LRB-

ref

-RRB-

More

than

million

SNPs

have

been

assembled

into

a

genome-wide

map

and

are

analysed

in

detail

in

an

accompanying

SNP

density

is

also

displayed

across

the

genome

in

Fig.

The

SNPs

have

an

average

spacing

of

kb

and

of

intervals

contain

a

SNP

These

polymorphisms

are

of

immediate

utility

for

medical

genetic

studies

Whereas

investigators

studying

a

gene

previously

had

to

expend

considerable

effort

to

discover

polymorphisms

across

the

region

of

interest

the

current

collection

now

provides

then

with

about

SNPs

for

gene

loci

of

average

size

The

density

of

SNPs

-LRB-

adjusted

for

ascertainment

that

is

polymorphisms

per

base

screened

-RRB-

varies

considerably

across

the

Applications

to

medicine

and

biology

In

most

research

papers

the

authors

can

only

speculate

about

future

applications

of

the

work

Because

the

genome

sequence

has

been

released

on

a

daily

basis

over

the

past

four

years

however

we

can

already

cite

many

direct

applications

We

focus

on

a

handful

of

applications

chosen

primarily

from

medical

research

Disease

genes

Human

-LRB-

x

-RRB-

Fly

-LRB-

-RRB-

Worm

-LRB-

-RRB-

Frequency

800 600 400 200

No

of

human

paralogues

Figure

Number

of

human

paralogues

of

genes

having

single

orthologues

in

worm

and

y.

NATURE

VOL

FEBRUARY

A

key

application

of

human

genome

research

has

been

the

ability

to

nd

disease

genes

of

unknown

biochemical

function

by

positional

This

method

involves

mapping

the

chromosomal

region

containing

the

gene

by

linkage

analysis

in

affected

families

and

then

scouring

the

region

to

nd

the

gene

itself

Positional

cloning

is

powerful

but

it

has

also

been

extremely

tedious

When

the

approach

was

rst

proposed

in

the

early

a

researcher

wishing

to

perform

positional

cloning

had

to

generate

genetic

markers

to

trace

inheritance

perform

chromosomal

walking

to

obtain

genomic

DNA

covering

the

region

and

analyse

a

region

of

around

Mb

by

either

direct

sequencing

or

indirect

gene

identi

cation

methods

The

rst

two

barriers

were

eliminated

with

the

development

in

the

of

comprehensive

genetic

and

physical

maps

of

the

human

chromosomes

under

the

auspices

of

the

Human

Genome

Project

The

remaining

barrier

however

has

continued

to

be

formidable

All

that

is

changing

with

the

availability

of

the

human

draft

genome

sequence

The

human

genomic

sequence

in

public

databases

allows

rapid

identi

cation

in

silico

of

candidate

genes

followed

by

mutation

screening

of

relevant

candidates

aided

by

information

on

gene

structure

For

a

mendelian

disorder

a

gene

search

can

now

often

be

carried

out

in

a

matter

of

months

with

only

a

modestly

sized

team

At

least

disease

-LRB-

Table

-RRB-

have

been

positionally

cloned

in

research

efforts

that

depended

directly

on

the

publicly

available

genome

sequence

As

most

of

the

human

sequence

has

only

arrived

in

the

past

twelve

months

it

is

likely

that

many

similar

discoveries

are

not

yet

published

In

addition

there

are

many

cases

in

which

the

genome

sequence

played

a

supporting

role

such

as

providing

candidate

microsatellite

markers

for

ner

genetic

linkage

analysis

The

genome

sequence

has

also

helped

to

reveal

the

mechanisms

leading

to

some

common

chromosomal

deletion

syndromes

In

several

instances

recurrent

deletions

have

been

found

to

result

from

homologous

recombination

and

unequal

crossing

over

between

large

nearly

identical

intrachromosomal

duplications

Examples

Macmillan

Magazines

Ltd

articles

include

the

DiGeorge/velocardiofacial

syndrome

region

on

chromosome

-LRB-

ref

-RRB-

and

the

Williams

Beuren

syndrome

recurrent

deletion

on

chromosome

-LRB-

ref

-RRB-

The

availability

of

the

genome

sequence

also

allows

rapid

identi

cation

of

paralogues

of

disease

genes

which

is

valuable

for

two

reasons

First

mutations

in

a

paralogous

gene

may

give

rise

to

a

related

genetic

disease

A

good

example

discovered

through

use

of

the

genome

sequence

is

achromatopsia

-LRB-

complete

colour

blindness

-RRB-

The

gene

encoding

the

a-subunit

of

the

cone

photoreceptor

cyclic

GMP-gated

channel

had

been

shown

to

harbour

mutations

in

some

families

with

achromatopsia

Computational

searching

of

the

genome

sequences

revealed

the

paralogous

gene

encoding

the

corresponding

b-subunit

-LRB-

which

had

not

been

apparent

from

EST

databases

-RRB-

The

gene

was

rapidly

shown

to

be

the

cause

of

achromatopsia

in

other

Another

example

is

provided

by

the

and

genes

in

which

mutations

can

cause

early-onset

Alzheimer

's

Second

the

paralogue

may

provide

an

opportunity

for

therapeutic

intervention

as

exempli

ed

by

attempts

to

reactivate

the

fetally

expressed

haemoglobin

genes

in

individuals

with

sickle

cell

disease

or

b-thalassaemia

caused

by

mutations

in

the

b-globin

We

undertook

a

systematic

search

for

paralogues

of

known

human

disease

genes

with

entries

in

both

the

Online

Mendelian

Inheritance

in

Man

-LRB-

OMIM

-RRB-

database

-LRB-

gov/Omim

-RRB-

and

either

the

SwissProt

or

TrEMBL

protein

databases

We

identi

ed

potential

paralogues

-LRB-

with

the

requirement

of

a

match

of

at

least

amino

acids

with

identity

greater

than

but

less

than

if

on

the

same

chromosome

and

less

than

if

on

a

different

chromosome

-RRB-

Although

this

analysis

may

have

identi

ed

some

pseudogenes

of

the

matches

showed

homology

over

more

than

one

exon

in

the

new

target

sequence

suggesting

that

many

are

functional

This

analysis

shows

the

potential

for

rapid

identi

cation

of

disease

gene

paralogues

in

silico

Drug

targets

Over

the

past

century

the

pharmaceutical

industry

has

largely

depended

upon

a

limited

set

of

drug

targets

to

develop

new

therapies

A

recent

lists

drug

targets

as

accounting

for

virtually

all

drugs

on

the

market

Knowing

the

complete

set

of

human

genes

and

proteins

will

greatly

expand

the

search

for

suitable

drug

targets

Although

only

a

minority

of

human

genes

may

be

drug

targets

it

has

been

predicted

that

the

number

will

exceed

several

thousand

and

this

prospect

has

led

to

a

massive

expansion

of

genomic

research

in

pharmaceutical

research

and

development

A

few

examples

will

illustrate

the

point

-LRB-

-RRB-

The

neurotransmitter

serotonin

-LRB-

-RRB-

mediates

rapid

excitatory

responses

through

ligand-gated

channels

The

previously

identi

ed

receptor

gene

produces

functional

receptors

but

with

a

much

smaller

conductance

than

observed

in

vivo

Cross-hybridization

experiments

and

analysis

of

ESTs

failed

to

reveal

any

other

homologues

of

the

known

receptor

Recently

however

by

searching

the

human

draft

genome

sequence

at

low

stringency

a

putative

homologue

was

identi

ed

within

a

PAC

clone

from

the

long

arm

of

chromosome

-LRB-

ref

-RRB-

The

homologue

was

shown

to

be

expressed

in

the

amygdala

caudate

and

hippocampus

and

a

full-length

cDNA

was

subsequently

obtained

The

gene

which

codes

for

a

serotonin

receptor

was

named

When

assembled

in

a

heterodimer

with

it

was

shown

to

account

for

the

large-conductance

neuronal

serotonin

channel

Given

the

central

role

of

the

serotonin

pathway

in

mood

disorders

and

schizophrenia

the

discovery

of

a

major

new

therapeutic

target

is

of

considerable

interest

-LRB-

-RRB-

The

contractile

and

in

ammatory

actions

of

the

cysteinyl

leukotrienes

formerly

known

as

the

slow

reacting

substance

of

anaphylaxis

-LRB-

SRS-A

-RRB-

are

mediated

through

speci

c

receptors

The

second

such

receptor

was

identi

ed

using

the

combination

of

a

rat

EST

and

the

human

genome

sequence

This

led

to

the

cloning

of

a

gene

with

amino-acid

identity

to

the

only

other

receptor

that

had

previously

been

identi

This

new

receptor

which

shows

high-af

nity

binding

to

several

leukotrienes

maps

to

a

region

of

chromosome

that

is

linked

to

atopic

asthma

The

gene

is

expressed

in

airway

smooth

muscles

and

in

the

heart

As

the

leukotriene

pathway

has

been

a

signi

cant

target

for

the

development

of

drugs

against

asthma

the

discovery

of

a

new

receptor

has

obvious

and

important

consequences

-LRB-

-RRB-

Abundant

deposition

of

b-amyloid

in

senile

plaques

is

the

hallmark

of

Alzheimer

's

disease

b-Amyloid

is

generated

by

proteolytic

processing

of

the

amyloid

precursor

protein

-LRB-

APP

-RRB-

One

of

the

enzymes

involved

is

the

b-site

APP-cleaving

enzyme

-LRB-

BACE

-RRB-

which

is

a

transmembrane

aspartyl

protease

Computational

searching

of

the

public

human

draft

genome

sequence

recently

identi

ed

a

new

sequence

homologous

to

BACE

encoding

a

protein

now

named

which

has

amino-acid

sequence

identity

to

BACE

contains

two

active

protease

sites

and

maps

to

the

obligatory

Down

's

syndrome

region

of

chromosome

as

does

APP

This

raises

the

question

of

whether

the

extra

copies

of

both

and

APP

may

contribute

to

accelerated

deposition

of

b-amyloid

in

the

brains

of

Down

's

syndrome

patients

The

development

of

antagonists

to

BACE

and

represents

a

promising

approach

to

preventing

Alzheimer

's

disease

Given

these

examples

we

undertook

a

systematic

effort

to

identify

paralogues

of

the

classic

drug

target

proteins

in

the

draft

genome

sequence

The

target

was

used

to

identify

entries

in

the

SwissProt

database

with

unique

accession

numbers

These

were

then

searched

against

the

current

genome

sequence

database

using

the

requirement

that

a

match

should

have

identity

to

at

least

amino

acids

Matches

to

named

proteins

were

ignored

as

we

assumed

that

these

represented

known

homologues

We

found

putative

novel

paralogues

-LRB-

Table

-RRB-

including

apparent

dopamine

receptors

purinergic

receptors

and

insulin-like

growth

factor

receptors

In

six

cases

the

novel

paralogue

matches

at

least

one

EST

adding

con

dence

that

this

search

process

can

identify

novel

functional

genes

For

the

remaining

putative

paralogues

without

an

EST

match

all

have

long

ORFs

and

all

but

NATURE

VOL

FEBRUARY

Table

Disease

genes

positionally

cloned

using

the

draft

genome

sequence

Locus

AIRE

PDS

XLP

SEDL

LGMD

EVC

AASS

MUL

XLCSNB

GAN

AAAS

Disorder

Breast

cancer

susceptibility

Autoimmune

polyglandular

syndrome

type

-LRB-

or

APECED

-RRB-

Peroxisome

biogenesis

disorder

Pendred

syndrome

X-linked

lymphoproliferative

disease

Nonsyndromic

deafness

Darier

's

disease

X-linked

spondyloepiphyseal

dysplasia

tarda

Progressive

pseudorheumatoid

dysplasia

Cerebral

cavernous

malformations

Nonsyndromic

deafness

Limb-girdle

muscular

dystrophy

Ellis-Van

Creveld

syndrome

Weyer

's

acrodental

dysostosis

Familial

focal

segmental

glomerulosclerosis

Generalized

epilepsy

with

febrile

seizures

plus

type

Familial

hyperlysinaemia

Hereditary

motor

and

sensory

neuropathy-Lom

Total

colour-blindness

Mulibrey

nanism

Usher

type

May-Hegglin

anomaly

Carney

's

complex

Nonsyndromic

hereditary

deafness

Spinocerebellar

ataxia

type

Optic

atrophy

X-linked

congenital

stationary

night

blindness

Hypophosphataemic

rickets

Giant

axonal

neuropathy

Triple-A

syndrome

Schwartz-Jampel

syndrome

Reference

-LRB-

s

-RRB-

55 389 390

391 392 393 394

395 396 397 398

399 400 401 402

403 404 405 406

408 409 410

413 414 415 416

417 418 419 420

Macmillan

Magazines

Ltd

articles

one

show

similarity

spanning

multiple

exons

separated

by

introns

so

these

are

not

processed

pseudogenes

They

are

likely

to

represent

interesting

new

candidate

drug

targets

Basic

biology

Although

the

examples

above

re

ect

medical

applications

there

are

also

many

similar

applications

to

basic

physiology

and

cell

biology

To

cite

one

satisfying

example

the

publicly

available

sequence

was

used

to

solve

a

mystery

that

had

vexed

investigators

for

several

decades

the

molecular

basis

of

bitter

Humans

and

other

animals

are

polymorphic

for

response

to

certain

bitter

tastes

Recently

investigators

mapped

this

trait

in

both

humans

and

mice

and

then

searched

the

relevant

region

of

the

human

draft

genome

sequence

for

G-protein

coupled

receptors

These

studies

led

in

quick

succession

to

the

discovery

of

a

new

family

of

such

proteins

the

demonstration

that

they

are

expressed

almost

exclusively

in

taste

buds

and

the

experimental

con

rmation

that

the

receptors

in

cultured

cells

respond

to

speci

c

bitter

months

All

chromosomes

should

be

essentially

completed

by

if

not

sooner

Finally

techniques

must

be

developed

to

close

recalcitrant

gaps

Several

hundred

such

gaps

in

the

euchromatic

sequence

will

probably

remain

in

the

genome

after

exhaustive

screening

of

existing

large-insert

libraries

New

methodologies

will

be

needed

to

recover

sequence

from

these

segments

and

to

de

ne

biological

reasons

for

their

lack

of

representation

in

standard

libraries

Ideally

it

would

be

desirable

to

obtain

complete

sequence

from

all

heterochromatic

regions

such

as

centromeres

and

ribosomal

gene

clusters

although

most

of

this

sequence

will

consist

of

highly

polymorphic

tandem

repeats

containing

few

protein-coding

genes

Developing

the

IGI

and

IPI

The

draft

genome

sequence

has

provided

an

initial

look

at

the

human

gene

content

but

many

ambiguities

remain

A

high

priority

will

be

to

re

ne

the

IGI

and

IPI

to

the

point

where

they

accurately

re

ect

every

gene

and

every

alternatively

spliced

form

Several

steps

are

needed

to

reach

this

ambitious

goal

Finishing

the

human

sequence

will

assist

in

this

effort

but

the

experiences

gained

on

chromosomes

and

show

that

sequence

alone

is

not

enough

to

allow

complete

gene

identi

cation

One

powerful

approach

is

cross-species

sequence

comparison

with

related

organisms

at

suitable

evolutionary

distances

The

sequence

coverage

from

the

puffer

sh

T.

nigroviridis

has

already

proven

valuable

in

identifying

potential

this

work

is

expected

to

continue

from

its

current

state

of

onefold

coverage

to

reach

at

least

vefold

coverage

later

this

year

The

genome

sequence

of

the

laboratory

mouse

will

provide

a

particularly

powerful

tool

for

exon

identi

cation

as

sequence

similarity

is

expected

to

identify

of

the

exons

as

well

as

a

signi

cant

number

of

regulatory

A

public-private

consortium

is

speeding

this

effort

by

producing

freely

accessible

whole-genome

shotgun

coverage

that

can

be

readily

used

for

cross-species

More

than

onefold

coverage

from

the

strain

has

already

been

completed

and

threefold

is

expected

within

the

next

few

months

In

the

slightly

longer

term

a

program

is

under

way

to

produce

a

nished

sequence

of

the

laboratory

mouse

Another

important

step

is

to

obtain

a

comprehensive

collection

of

full-length

human

cDNAs

both

as

sequences

and

as

actual

clones

The

Mammalian

Gene

Collection

project

has

been

underway

for

a

and

expects

to

produce

human

full-length

cDNAs

over

the

coming

year

which

will

be

available

without

restrictions

on

use

The

Genome

Exploration

Group

of

the

RIKEN

Genomic

Sciences

Center

is

similarly

developing

a

collection

of

cDNA

clones

from

which

is

a

valuable

complement

The

next

steps

Considerable

progress

has

been

made

in

human

sequencing

but

much

remains

to

be

done

to

produce

a

nished

sequence

Even

more

work

will

be

required

to

extract

the

full

information

contained

in

the

sequence

Many

of

the

key

next

steps

are

already

underway

Finishing

the

human

sequence

The

human

sequence

will

serve

as

a

foundation

for

biomedical

research

in

the

years

ahead

and

it

is

thus

crucial

that

the

remaining

gaps

be

lled

and

ambiguities

be

resolved

as

quickly

as

possible

This

will

involve

a

three-step

program

The

rst

stage

involves

producing

nished

sequence

from

clones

spanning

the

current

physical

map

which

covers

more

than

of

the

euchromatic

regions

of

the

genome

About

Gb

of

nished

sequence

is

already

completed

Almost

all

of

the

remaining

clones

are

already

sequenced

to

at

least

draft

coverage

and

the

rest

have

been

selected

for

sequencing

All

clones

are

expected

to

reach

full

shotgun

coverage

-LRB-

redundancy

-RRB-

by

about

and

nished

form

-LRB-

accuracy

-RRB-

not

long

thereafter

using

established

and

increasingly

automated

protocols

The

next

stage

will

be

to

screen

additional

libraries

to

close

gaps

between

clone

contigs

Directed

probing

of

additional

large-insert

clone

libraries

should

close

many

of

the

remaining

gaps

Unclosed

gaps

will

be

sized

by

FISH

techniques

or

other

methods

Two

chromosomes

and

have

already

been

assembled

in

this

essentially

complete

form

in

this

and

chromosomes

Y

and

are

likely

to

reach

this

status

in

the

next

few

Table

New

paralogues

of

common

drug

targets

identi

ed

by

searching

the

draft

human

genome

sequence

Drug

target

HGM

symbol

Aquaporin

Arachidonate

Calcitonin

Calcium

channel

voltage-dependent

g-subunit

DNA

polymerase-d

small

subunit

Dopamine

receptor

Dopamine

receptor

Eukaryotic

translation

elongation

factor

FKBP

tacrolimus

binding

protein

binding

protein

Glutamic

acid

decarboxylase

Glycine

receptor

Heparan

N-deacetylase/N-sulphotransferase

Insulin-like

growth

receptor

Na,K-ATPase

a-subunit

Purinergic

receptor

-LRB-

-RRB-

ligand-gated

ion

channel

Tubulin

e-chain

Tubulin

x-chain

Voltage-gated

potassium

channel

CALCA

TUBE

Drug

target

SwissProt

accession

Chromosome

Novel

match

IGI

number

Chromosome

containing

paralogue

Per

cent

identity

dbEST

GenBank

accession

HGM

symbol

unknown

X

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

because

of

the

availability

of

tissues

from

all

developmental

time

points

A

challenge

will

be

to

de

ne

the

gene-speci

c

patterns

of

alternative

splicing

which

may

affect

half

of

human

genes

Existing

collections

of

ESTs

and

cDNAs

may

allow

identi

cation

of

the

most

abundant

of

these

isoforms

but

systematic

exploration

of

this

problem

may

require

exhaustive

analysis

of

cDNA

libraries

from

multiple

tissues

or

perhaps

high-throughput

reverse

transcription

PCR

studies

Deep

understanding

of

gene

function

will

probably

require

knowledge

of

the

structure

tissue

distribution

and

abundance

of

these

alternative

forms

Large-scale

identi

cation

of

regulatory

regions

The

one-dimensional

script

of

the

human

genome

shared

by

essentially

all

cells

in

all

tissues

contains

suf

cient

information

to

provide

for

differentiation

of

hundreds

of

different

cell

types

and

the

ability

to

respond

to

a

vast

array

of

internal

and

external

in

uences

Much

of

this

plasticity

results

from

the

carefully

orchestrated

symphony

of

transcriptional

regulation

Although

much

has

been

learned

about

the

cis-acting

regulatory

motifs

of

some

speci

c

genes

the

regulatory

signals

for

most

genes

remain

uncharacterized

Comparative

genomics

of

multiple

vertebrates

offers

the

best

hope

for

large-scale

identi

cation

of

such

regulatory

Previous

studies

of

sequence

alignment

of

regulatory

domains

of

orthologous

genes

in

multiple

species

has

shown

a

remarkable

correlation

between

sequence

conservation

dubbed

phylogenetic

footprints

and

the

presence

of

binding

motifs

for

transcription

factors

This

approach

could

be

particularly

powerful

if

combined

with

expression

array

technologies

that

identify

cohorts

of

genes

that

are

coordinately

regulated

implicating

a

common

set

of

cisacting

regulatory

It

will

also

be

of

considerable

interest

to

study

epigenetic

modi

cations

such

as

cytosine

methylation

on

a

genome-wide

scale

and

to

determine

their

biological

Towards

this

end

a

pilot

Human

Epigenome

Project

has

been

Sequencing

of

additional

large

genomes

More

generally

comparative

genomics

allows

biologists

to

peruse

evolution

's

laboratory

notebook

to

identify

conserved

functional

features

and

recognize

new

innovations

in

speci

c

lineages

Determination

of

the

genome

sequence

of

many

organisms

is

very

desirable

Already

projects

are

underway

to

sequence

the

genomes

of

the

mouse

rat

zebra

sh

and

the

puffer

shes

T.

nigroviridis

and

Takifugu

rubripes

Plans

are

also

under

consideration

for

sequencing

additional

primates

and

other

organisms

that

will

help

de

ne

key

developments

along

the

vertebrate

and

nonvertebrate

lineages

To

realize

the

full

promise

of

comparative

genomics

however

it

needs

to

become

simple

and

inexpensive

to

sequence

the

genome

of

any

organism

Sequencing

costs

have

dropped

over

the

last

years

corresponding

to

a

roughly

twofold

decrease

every

months

This

rate

is

similar

to

Moore

's

law

concerning

improvements

in

semiconductor

manufacture

In

both

sequencing

and

semiconductors

such

improvement

does

not

happen

automatically

but

requires

aggressive

technological

innovation

fuelled

by

major

investment

Improvements

are

needed

to

move

current

dideoxy

sequencing

to

smaller

volumes

and

more

rapid

sequencing

times

based

upon

advances

such

as

microchannel

technology

More

revolutionary

methods

such

as

mass

spectrometry

singlemolecule

sequencing

and

nanopore

have

not

yet

been

fully

developed

but

hold

great

promise

and

deserve

strong

encouragement

Completing

the

catalogue

of

human

variation

The

human

draft

genome

sequence

has

already

allowed

the

identi

cation

of

more

than

million

SNPs

comprising

a

substantial

proportion

of

all

common

human

variation

This

program

should

be

extended

to

obtain

a

nearly

complete

catalogue

of

common

variants

and

to

identify

the

common

ancestral

haplotypes

present

in

the

population

In

principle

these

genetic

tools

should

make

it

possible

to

perform

association

studies

and

linkage

disequilibrium

to

identify

the

genes

that

confer

even

relatively

modest

risk

for

common

diseases

Launching

such

an

intense

era

of

human

molecular

epidemiology

will

also

require

major

advances

in

the

cost

ef

ciency

of

genotyping

technology

in

the

collection

of

carefully

phenotyped

patient

cohorts

and

in

statistical

methods

for

relating

large-scale

SNP

data

to

disease

phenotype

From

sequence

to

function

The

scienti

c

program

outlined

above

focuses

on

how

the

genome

sequence

can

be

mined

for

biological

information

In

addition

the

sequence

will

serve

as

a

foundation

for

a

broad

range

of

functional

genomic

tools

to

help

biologists

to

probe

function

in

a

more

systematic

manner

These

will

need

to

include

improved

techniques

and

databases

for

the

global

analysis

of

RNA

and

protein

expression

protein

localization

protein

protein

interactions

and

chemical

inhibition

of

pathways

New

computational

techniques

will

be

needed

to

use

such

information

to

model

cellular

circuitry

A

full

discussion

of

these

important

directions

is

beyond

the

scope

of

this

paper

Concluding

thoughts

The

Human

Genome

Project

is

but

the

latest

increment

in

a

remarkable

scienti

c

program

whose

origins

stretch

back

a

hundred

years

to

the

rediscovery

of

Mendel

's

laws

and

whose

end

is

nowhere

in

sight

In

a

sense

it

provides

a

capstone

for

efforts

in

the

past

century

to

discover

genetic

information

and

a

foundation

for

efforts

in

the

coming

century

to

understand

it

We

nd

it

humbling

to

gaze

upon

the

human

sequence

now

coming

into

focus

In

principle

the

string

of

genetic

bits

holds

longsought

secrets

of

human

development

physiology

and

medicine

In

practice

our

ability

to

transform

such

information

into

understanding

remains

woefully

inadequate

This

paper

simply

records

some

initial

observations

and

attempts

to

frame

issues

for

future

study

Ful

lling

the

true

promise

of

the

Human

Genome

Project

will

be

the

work

of

tens

of

thousands

of

scientists

around

the

world

in

both

academia

and

industry

It

is

for

this

reason

that

our

highest

priority

has

been

to

ensure

that

genome

data

are

available

rapidly

freely

and

without

restriction

The

scienti

c

work

will

have

profound

long-term

consequences

for

medicine

leading

to

the

elucidation

of

the

underlying

molecular

mechanisms

of

disease

and

thereby

facilitating

the

design

in

many

cases

of

rational

diagnostics

and

therapeutics

targeted

at

those

mechanisms

But

the

science

is

only

part

of

the

challenge

We

must

also

involve

society

at

large

in

the

work

ahead

We

must

set

realistic

expectations

that

the

most

important

bene

ts

will

not

be

reaped

overnight

Moreover

understanding

and

wisdom

will

be

required

to

ensure

that

these

bene

ts

are

implemented

broadly

and

equitably

To

that

end

serious

attention

must

be

paid

to

the

many

ethical

legal

and

social

implications

-LRB-

ELSI

-RRB-

raised

by

the

accelerated

pace

of

genetic

discovery

This

paper

has

focused

on

the

scienti

c

achievements

of

the

human

genome

sequencing

efforts

This

is

not

the

place

to

engage

in

a

lengthy

discussion

of

the

ELSI

issues

which

have

also

been

a

major

research

focus

of

the

Human

Genome

Project

but

these

issues

are

of

comparable

importance

and

could

appropriately

ll

a

paper

of

equal

length

Finally

it

is

has

not

escaped

our

notice

that

the

more

we

learn

about

the

human

genome

the

more

there

is

to

explore

We

shall

not

cease

from

exploration

And

the

end

of

all

our

exploring

will

be

to

arrive

where

we

started

and

know

the

place

for

the

rst

time

T.

S.

M

Received

December

accepted

January

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C.

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Supplementary

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is

available

on

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's

World-Wide

Web

site

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-RRB-

or

as

paper

copy

from

the

London

editorial

of

ce

of

Nature

Acknowledgements

Beyond

the

authors

many

people

contributed

to

the

success

of

this

work

E.

Jordan

provided

helpful

advice

throughout

the

sequencing

effort

We

thank

D.

Leja

and

J.

Shehadeh

for

their

expert

assistance

on

the

artwork

in

this

paper

especially

the

foldout

gure

K.

Jegalian

for

editorial

assistance

J.

Schloss

E.

Green

and

M.

Seldin

for

comments

on

an

earlier

version

of

the

manuscript

P.

Green

and

F.

Ouelette

for

critiques

of

the

submitted

version

C.

Caulcott

A.

Iglesias

S.

Renfrey

B.

Skene

and

J.

Stewart

of

the

Wellcome

Trust

P.

Whittington

and

T.

Dougans

of

NHGRI

and

M.

Meugnier

of

Genoscope

for

staff

support

for

meetings

of

the

international

consortium

and

the

University

of

Pennsylvania

for

facilities

for

a

meeting

of

the

genome

analysis

group

We

thank

Compaq

Computer

Corporations

's

High

Performance

Technical

Computing

Group

for

providing

a

Compaq

Biocluster

-LRB-

a

node

con

guration

of

AlphaServer

containing

CPUs

serving

as

compute

nodes

and

a

le

server

with

one

terabyte

of

secondary

storage

-RRB-

to

assist

in

the

annotation

and

analysis

Compaq

provided

the

systems

and

implementation

services

to

set

up

and

manage

the

cluster

for

continuous

use

by

members

of

the

sequencing

consortium

Platform

Computing

Ltd.

provided

its

LSF

scheduling

and

loadsharing

software

without

license

fee

In

addition

to

the

data

produced

by

the

members

of

the

International

Human

Genome

Sequencing

Consortium

the

draft

genome

sequence

includes

published

and

unpublished

human

genomic

sequence

data

from

many

other

groups

all

of

whom

gave

permission

to

include

their

unpublished

data

Four

of

the

groups

that

contributed

particularly

signi

cant

amounts

of

data

were

M.

Adams

et

al.

of

the

Institute

for

Genomic

Research

E.

Chen

et

al.

of

the

Center

for

Genetic

Medicine

and

Applied

Biosystems

S.-F

Tsai

of

National

Yang-Ming

University

Institute

of

Genetics

Taipei

Taiwan

Republic

of

China

and

Y.

Nakamura

K.

Koyama

et

al.

of

the

Institute

of

Medical

Science

University

of

Tokyo

Human

Genome

Center

Laboratory

of

Molecular

Medicine

Minato-ku

Tokyo

Japan

Many

other

groups

provided

smaller

numbers

of

database

entries

We

thank

them

all

a

full

list

of

the

contributors

of

unpublished

sequence

is

available

as

Supplementary

Information

This

work

was

supported

in

part

by

the

National

Human

Genome

Research

Institute

of

the

US

NIH

The

Wellcome

Trust

the

US

Department

of

Energy

Of

ce

of

Biological

and

Environmental

Research

Human

Genome

Program

the

UK

MRC

the

Human

Genome

Sequencing

Project

from

the

Science

and

Technology

Agency

-LRB-

STA

-RRB-

Japan

the

Ministry

of

Education

Science

Sport

and

Culture

Japan

the

French

Ministry

of

Research

the

Federal

German

Ministry

of

Education

Research

and

Technology

-LRB-

BMBF

-RRB-

through

Projekttrager

DLR

in

the

framework

of

the

German

Human

Genome

Project

BEO

Projekttrager

Biologie

Energie

Umwelt

des

BMBF

und

BMWT

the

Max-Planck-Society

DFG

Deutsche

Forschungsgemeinschaft

TMWFK

Thuringer

Ministerium

fur

Wissenschaft

Forschung

und

Kunst

EC

European

Commission

Directorate

Science

Research

and

Development

Chinese

Academy

of

Sciences

-LRB-

CAS

-RRB-

Ministry

of

Science

and

Technology

-LRB-

MOST

-RRB-

National

Natural

Science

Foundation

of

China

-LRB-

NSFC

-RRB-

US

National

Science

Foundation

EPSCoR

and

The

SNP

Consortium

Ltd.

Additional

support

for

members

of

the

Genome

Analysis

group

came

in

part

from

an

ARCS

Foundation

Scholarship

to

T.S.F.

a

Burroughs

Wellcome

Foundation

grant

to

C.B.B.

and

P.A.S.

a

DFG

grant

to

P.B.

DOE

grants

to

D.H.

E.E.E.

and

T.S.F.

an

EU

grant

to

P.B.

a

Marie-Curie

Fellowship

to

L.C.

an

NIH-NHGRI

grant

to

S.R.E.

an

NIH

grant

to

E.E.E.

an

NIH

SBIR

to

D.K.

an

NSF

grant

to

D.H.

a

Swiss

National

Science

Foundation

grant

to

L.C.

the

David

and

Lucille

Packard

Foundation

the

Howard

Hughes

Medical

Institute

the

University

of

California

at

Santa

Cruz

and

the

W.

M.

Keck

Foundation

Correspondence

and

requests

for

materials

should

be

addressed

to

E.

S.

Lander

-LRB-

e-mail

lander@genome.wi.mit.edu

-RRB-

R.

H.

Waterston

-LRB-

e-mail

bwaterst@watson.wustl.edu

-RRB-

J.

Sulston

-LRB-

e-mail

jes@sanger.ac.uk

-RRB-

or

F.

S.

Collins

-LRB-

e-mail

-RRB-

Af

liations

for

authors

Whitehead

Institute

for

Biomedical

Research

Center

for

Genome

Research

Nine

Cambridge

Center

Cambridge

Massachusetts

USA

The

Sanger

Centre

The

Wellcome

Trust

Genome

Campus

Hinxton

Cambridgeshire

United

Kingdom

Washington

University

Genome

Sequencing

Center

Box

Forest

Park

Avenue

St.

Louis

Missouri

USA

US

DOE

Joint

Genome

Institute

Mitchell

Drive

Walnut

Creek

California

USA

Baylor

College

of

Medicine

Human

Genome

Sequencing

Center

Department

of

Molecular

and

Human

Genetics

One

Baylor

Plaza

Houston

Texas

USA

Department

of

Cellular

and

Structural

Biology

The

University

of

Texas

Health

Science

Center

at

San

Antonio

Floyd

Curl

Drive

San

Antonio

Texas

USA

Department

of

Molecular

Genetics

Albert

Einstein

College

of

Medicine

Poplar

Street

Bronx

New

York

USA

Baylor

College

of

Medicine

Human

Genome

Sequencing

Center

and

the

Department

of

Microbiology

Molecular

Genetics

University

of

Texas

Medical

School

PO

Box

Houston

Texas

USA

RIKEN

Genomic

Sciences

Center

Suehiro-cho

Tsurumi-ku

Yokohama-city

Kanagawa

Japan

Genoscope

and

CNRS

Rue

Gaston

Cremieux

CP

Evry

Cedex

France

GTC

Sequencing

Center

Genome

Therapeutics

Corporation

Beaver

Street

Waltham

Massachusetts

USA

Department

of

Genome

Analysis

Institute

of

Molecular

Biotechnology

Beutenbergstrasse

Jena

Germany

Beijing

Genomics

Institute/Human

Genome

Center

Institute

of

Genetics

Chinese

Academy

of

Sciences

Beijing

China

Southern

China

National

Human

Genome

Research

Center

Shanghai

China

Northern

China

National

Human

Genome

Research

Center

Beijing

China

Multimegabase

Sequencing

Center

The

Institute

for

Systems

Biology

Roosevelt

Way

NE

Suite

Seattle

Washington

USA

Stanford

Genome

Technology

Center

California

Avenue

Palo

Alto

California

USA

Stanford

Human

Genome

Center

and

Department

of

Genetics

Stanford

University

School

of

Medicine

Stanford

California

USA

University

of

Washington

Genome

Center

Fluke

Hall

on

Mason

Road

Seattle

Washington

USA

Department

of

Molecular

Biology

Keio

University

School

of

Medicine

Shinanomachi

Shinjuku-ku

Tokyo

Japan

University

of

Texas

Southwestern

Medical

Center

at

Dallas

Harry

Hines

Blvd.

Dallas

Texas

USA

University

of

Oklahoma

's

Advanced

Center

for

Genome

Technology

Dept.

of

Chemistry

and

Biochemistry

University

of

Oklahoma

Parrington

Oval

Rm

Norman

Oklahoma

USA

Max

Planck

Institute

for

Molecular

Genetics

Ihnestrasse

Berlin

Germany

Cold

Spring

Harbor

Laboratory

Lita

Annenberg

Hazen

Genome

Center

Bungtown

Road

Cold

Spring

Harbor

New

York

USA

GBF

German

Research

Centre

for

Biotechnology

Mascheroder

Weg

Braunschweig

Germany

National

Center

for

Biotechnology

Information

National

Library

of

Medicine

National

Institutes

of

Health

Bldg.

Rockville

Pike

Bethesda

Maryland

USA

Department

of

Genetics

Case

Western

Reserve

School

of

Medicine

and

University

Hospitals

of

Cleveland

BRB

Euclid

Ave.

Cleveland

Ohio

USA

EMBL

European

Bioinformatics

Institute

Wellcome

Trust

Genome

Campus

Hinxton

Cambridge

United

Kingdom

Max

Delbruck

Center

for

Molecular

Medicine

Robert-Rossle-Strasse

Berlin-Buch

Germany

EMBL

Meyerhofstrasse

Heidelberg

Germany

Dept.

of

Biology

Massachusetts

Institute

of

Technology

Massachusetts

Ave.

Cambridge

Massachusetts

USA

Howard

Hughes

Medical

Institute

Dept.

of

Genetics

Washington

University

School

of

Medicine

Saint

Louis

Missouri

USA

Dept.

of

Computer

Science

University

of

California

at

Santa

Cruz

Santa

Cruz

California

USA

Affymetrix

Inc.

St

Berkeley

California

USA

Genome

Exploration

Research

Group

Genomic

Sciences

Center

RIKEN

Yokohama

Institute

Suehiro-cho

Tsurumi-ku

Yokohama

Kanagawa

Japan

Howard

Hughes

Medical

Institute

Department

of

Computer

Science

University

of

California

at

Santa

Cruz

California

USA

University

of

Dublin

Trinity

College

Department

of

Genetics

Smur

t

Institute

Dublin

Ireland

Cambridge

Research

Laboratory

Compaq

Computer

Corporation

and

MIT

Genome

Center

Cambridge

Center

Cambridge

Massachusetts

USA

Dept.

of

Mathematics

University

of

California

at

Santa

Cruz

Santa

Cruz

California

USA

Dept.

of

Biology

University

of

California

at

Santa

Cruz

Santa

Cruz

California

USA

Crown

Human

Genetics

Center

and

Department

of

Molecular

Genetics

The

Weizmann

Institute

of

Science

Rehovot

Israel

Dept.

of

Genetics

Stanford

University

School

of

Medicine

Stanford

California

USA

The

University

of

Michigan

Medical

School

Departments

of

Human

Genetics

and

Internal

Medicine

Ann

Arbor

Michigan

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd

articles

USA

MRC

Functional

Genetics

Unit

Department

of

Human

Anatomy

and

Genetics

University

of

Oxford

South

Parks

Road

Oxford

UK

Institute

for

Systems

Biology

Roosevelt

Way

NE

Seattle

WA

USA

National

Human

Genome

Research

Institute

US

National

Institutes

of

Health

Center

Drive

Bethesda

Maryland

USA

Of

ce

of

Science

US

Department

of

Energy

Germantown

Road

Germantown

Maryland

USA

The

Wellcome

Trust

Euston

Road

London

UK

Present

addresses

Genome

Sequencing

Project

Egea

Biosciences

Inc.

Sorrento

Valley

Blvd.

Suite

F

San

Diego

CA

USA

-LRB-

G.A.E.

-RRB-

INRA

Station

d'Amelioration

des

Plantes

Clermont-Ferrand

Cedex

France

-LRB-

L.C.

-RRB-

DNA

sequence

databases

GenBank

National

Center

for

Biotechnology

Information

National

Library

of

Medicine

National

Institutes

of

Health

Bldg.

Rockville

Pike

Bethesda

Maryland

USA

EMBL

European

Bioinformatics

Institute

Wellcome

Trust

Genome

Campus

Hinxton

Cambridge

UK

DNA

Data

Bank

of

Japan

Center

for

Information

Biology

National

Institute

of

Genetics

Yata

Mishima-shi

Shizuoka-ken

Japan

NATURE

VOL

FEBRUARY

Macmillan

Magazines

Ltd