articles

Genome

sequence

of

the

Brown

Norway

rat

yields

insights

into

mammalian

evolution

Rat

Genome

Sequencing

Project

Consortium

Lists

of

participants

and

affiliations

appear

at

the

end

of

the

paper

The

laboratory

rat

-LRB-

Rattus

norvegicus

-RRB-

is

an

indispensable

tool

in

experimental

medicine

and

drug

development

having

made

inestimable

contributions

to

human

health

We

report

here

the

genome

sequence

of

the

Brown

Norway

-LRB-

BN

-RRB-

rat

strain

The

sequence

represents

a

high-quality

draft

covering

over

of

the

genome

The

BN

rat

sequence

is

the

third

complete

mammalian

genome

to

be

deciphered

and

three-way

comparisons

with

the

human

and

mouse

genomes

resolve

details

of

mammalian

evolution

This

first

comprehensive

analysis

includes

genes

and

proteins

and

their

relation

to

human

disease

repeated

sequences

comparative

genome-wide

studies

of

mammalian

orthologous

chromosomal

regions

and

rearrangement

breakpoints

reconstruction

of

ancestral

karyotypes

and

the

events

leading

to

existing

species

rates

of

variation

and

lineage-specific

and

lineageindependent

evolutionary

events

such

as

expansion

of

gene

families

orthology

relations

and

protein

evolution

Darwin

believed

that

natural

selection

will

always

act

very

slowly

often

only

at

long

intervals

of

time

The

consequences

of

evolution

over

timescales

of

approximately

millions

of

years

-LRB-

Myr

-RRB-

and

Myr

were

investigated

in

publications

comparing

the

human

with

invertebrate

and

mouse

genomes

Here

we

describe

changes

in

mammalian

genomes

that

occurred

in

a

shorter

time

interval

approximately

Myr

-LRB-

refs

-RRB-

since

the

common

ancestor

of

rat

and

mouse

The

comparison

of

these

genomes

has

produced

a

number

of

insights

The

rat

genome

-LRB-

gigabases

Gb

-RRB-

is

smaller

than

the

human

-LRB-

Gb

-RRB-

but

appears

larger

than

the

mouse

-LRB-

initially

Gb

-LRB-

ref

-RRB-

but

given

as

Gb

in

NCBI

build

see

-RRB-

The

rat

mouse

and

human

genomes

encode

similar

numbers

of

genes

The

majority

have

persisted

without

deletion

or

duplication

since

the

last

common

ancestor

Intronic

structures

are

well

conserved

Some

genes

found

in

rat

but

not

mouse

arose

through

expansion

of

gene

families

These

include

genes

producing

pheromones

or

involved

in

immunity

chemosensation

detoxification

or

proteolysis

Almost

all

human

genes

known

to

be

associated

with

disease

have

orthologues

in

the

rat

genome

but

their

rates

of

synonymous

substitution

are

significantly

different

from

the

remaining

genes

About

of

the

rat

genome

is

in

large

segmental

duplications

a

fraction

intermediate

between

mouse

-LRB-

-RRB-

and

human

-LRB-

-RRB-

These

occur

predominantly

in

pericentromeric

regions

Recent

expansions

of

major

gene

families

are

due

to

these

genomic

duplications

The

eutherian

core

of

the

rat

genome

that

is

bases

that

align

orthologously

to

mouse

and

human

comprises

a

billion

nucleotides

-LRB-

of

the

euchromatic

rat

genome

-RRB-

and

contains

the

vast

majority

of

exons

and

known

regulatory

elements

-LRB-

of

the

genome

-RRB-

A

portion

of

this

core

constituting

of

the

genome

appears

to

be

under

selective

constraint

in

rodents

and

primates

while

the

remainder

appears

to

be

evolving

neutrally

Approximately

of

the

rat

genome

aligns

only

with

mouse

a

considerable

portion

of

which

is

rodent-specific

repeats

Of

the

non-aligning

portion

at

least

half

is

rat-specific

repeats

More

genomic

changes

occurred

in

the

rodent

lineages

than

the

primate

-LRB-

-RRB-

These

rodent

genomic

changes

include

approximately

large

rearrangements

between

a

hypothetical

murid

ancestor

and

human

approximately

from

the

murid

ancestor

to

rat

and

about

the

same

from

the

murid

ancestor

to

mouse

-LRB-

-RRB-

A

threefoldhigher

rate

of

base

substitution

in

neutral

DNA

is

found

along

the

rodent

lineage

when

compared

with

the

human

lineage

with

the

rate

on

the

rat

branch

higher

than

along

the

mouse

branch

-LRB-

-RRB-

Microdeletions

occur

at

an

approximately

twofold-higher

rate

than

microinsertions

in

both

rat

and

mouse

branches

A

strong

correlation

exists

between

local

rates

of

microinsertions

and

microdeletions

transposable

element

insertion

and

nucleotide

substitutions

since

divergence

of

rat

and

mouse

even

though

these

events

occurred

independently

in

the

two

lineages

Background

History

of

the

rat

The

rat

hated

and

loved

at

once

is

both

scourge

and

servant

to

mankind

The

Devil

's

Lapdog

is

the

first

sign

in

the

Chinese

zodiac

and

traditionally

carries

the

Hindu

god

Rats

are

a

reservoir

of

pathogens

known

to

carry

over

diseases

They

are

involved

in

the

transmission

of

infectious

diseases

to

man

including

cholera

bubonic

plague

typhus

leptospirosis

cowpox

and

hantavirus

infections

The

rat

remains

a

major

pest

contributing

to

famine

with

other

rodents

by

eating

around

one-fifth

of

the

world

's

food

harvest

Paradoxically

the

rat

's

contribution

to

human

health

can

not

be

overestimated

from

testing

new

drugs

to

understanding

essential

nutrients

to

increasing

knowledge

of

the

pathobiology

of

human

disease

In

many

parts

of

the

world

the

rat

remains

a

source

of

meat

The

laboratory

rat

-LRB-

R.

norvegicus

-RRB-

originated

in

central

Asia

and

its

success

at

spreading

throughout

the

world

can

be

directly

attributed

to

its

relationship

with

J.

Berkenhout

in

his

treatise

Outline

of

the

Natural

History

of

Great

Britain

mistakenly

took

it

to

be

from

Norway

and

used

R.

norvegicus

Berkenhout

in

the

first

formal

Linnaean

description

of

the

species

Whereas

the

black

rat

-LRB-

Rattus

rattus

-RRB-

was

part

of

the

European

landscape

from

at

least

the

third

century

AD

and

is

the

species

associated

with

the

spread

of

bubonic

plague

R.

norvegicus

probably

originated

in

northern

China

and

migrated

to

Europe

somewhere

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

around

the

eighteenth

They

may

have

entered

Europe

after

an

earthquake

in

by

swimming

the

Volga

river

The

rat

in

research

BCM-HGSC

analysis

was

performed

by

an

international

team

representing

over

groups

in

six

countries

and

relying

largely

on

gene

and

protein

predictions

produced

by

Ensembl

R.

norvegicus

was

the

first

mammalian

species

to

be

domesticated

for

scientific

research

with

work

dating

to

before

-LRB-

ref

-RRB-

The

first

recorded

breeding

colony

for

rats

was

established

in

-LRB-

ref

-RRB-

Rat

genetics

had

a

surprisingly

early

start

The

first

studies

by

Crampe

from

to

focused

on

the

inheritance

of

coat

Following

the

rediscovery

of

Mendel

's

laws

at

the

turn

of

the

century

Bateson

used

these

concepts

in

to

demonstrate

that

rat

coat

colour

is

a

mendelian

The

first

inbred

rat

strain

PA

was

established

by

King

in

the

same

year

that

systematic

inbreeding

began

for

the

Despite

this

the

mouse

became

the

dominant

model

for

mammalian

geneticists

while

the

rat

became

the

model

of

choice

for

physiologists

nutritionists

and

other

biomedical

researchers

Nevertheless

there

are

over

inbred

strains

of

R.

norvegicus

developed

by

selective

breeding

which

fixes

natural

disease

alleles

in

particular

strains

or

Over

the

past

century

the

role

of

the

rat

in

medicine

has

transformed

from

carrier

of

contagious

diseases

to

indispensable

tool

in

experimental

medicine

and

drug

development

Current

examples

of

use

of

the

rat

in

human

medical

research

include

psychiatric

including

behavioural

and

neural

and

bone

space

motion

and

cardiovascular

In

drug

development

the

rat

is

routinely

employed

both

to

demonstrate

therapeutic

and

to

assess

toxicity

of

novel

therapeutic

compounds

before

human

clinical

The

Rat

Genome

Project

Determination

of

the

genome

sequence

Atlas

and

the

combined

sequencing

strategy

Over

the

past

decade

investigators

and

funding

agencies

have

participated

in

rat

genomics

to

develop

valuable

resources

Before

the

launch

of

the

Rat

Genome

Sequencing

Project

-LRB-

RGSP

-RRB-

there

was

much

debate

about

the

overall

value

of

the

rat

genome

sequence

and

its

contribution

to

the

utility

of

the

rat

as

a

model

organism

The

debate

was

fuelled

by

the

naive

belief

that

the

rat

and

mouse

were

so

similar

morphologically

and

evolutionarily

that

the

rat

sequence

would

be

redundant

Nevertheless

an

effort

spearheaded

by

two

NIH

agencies

-LRB-

NHGRI

and

NHLBI

-RRB-

culminated

in

the

formation

of

the

RGSP

Consortium

-LRB-

RGSPC

-RRB-

The

RGSP

was

to

generate

a

draft

sequence

of

the

rat

genome

and

unlike

the

comparable

human

and

mouse

projects

errors

would

not

ultimately

be

corrected

in

a

finished

Consequently

the

draft

quality

was

critical

Although

it

was

expected

to

have

gaps

and

areas

of

inaccuracy

the

overall

sequence

quality

had

to

be

high

enough

to

support

detailed

analyses

The

BN

rat

was

selected

as

a

sequencing

target

by

the

research

community

An

inbred

animal

-LRB-

BN/SsNHsd

-RRB-

was

obtained

by

the

Medical

College

of

Wisconsin

-LRB-

MCW

-RRB-

from

Harlan

Sprague

Dawley

Microsatellite

studies

indicated

heterozygosity

so

over

generations

of

additional

inbreeding

were

performed

at

the

MCW

resulting

in

BN/SsNHsd/Mcwi

animals

Most

of

the

sequence

data

were

from

two

females

with

a

small

amount

of

whole

genome

shotgun

-LRB-

WGS

-RRB-

and

flow-sorted

Y

chromosome

sequencing

from

a

male

The

Y

chromosome

is

not

included

in

the

current

assembly

A

network

of

centres

generated

data

and

resources

led

by

the

Baylor

College

of

Medicine

Human

Genome

Sequencing

Center

-LRB-

BCM-HGSC

-RRB-

and

including

Celera

Genomics

the

Genome

Therapeutics

Corporation

the

British

Columbia

Cancer

Agency

Genome

Sciences

Centre

The

Institute

for

Genomic

Research

the

University

of

Utah

the

Medical

College

of

Wisconsin

The

Children

's

Hospital

of

Oakland

Research

Institute

and

the

Max

Delbruck

Center

for

Molecular

Medicine

Berlin

After

assembly

of

the

genome

at

the

Despite

progress

in

assembling

draft

the

question

of

which

method

produces

the

highest-quality

products

is

unresolved

A

significant

issue

is

the

choice

between

logistically

simpler

WGS

approaches

versus

more

complex

strategies

employing

bacterial

artificial

chromosome

-LRB-

BAC

-RRB-

In

the

Public

Human

Genome

a

BAC

by

BAC

hierarchical

approach

was

used

and

provided

advantages

in

assembling

difficult

parts

of

the

genome

The

draft

mouse

sequence

was

a

pure

WGS

approach

using

the

ARACHNE

but

underrepresented

duplicated

regions

owing

to

collapses

in

the

This

limitation

of

the

mouse

draft

sequence

was

tolerable

owing

to

the

planned

full

use

of

BAC

clones

in

constructing

the

final

finished

sequence

The

RGSPC

opted

to

develop

a

combined

approach

using

both

WGS

and

BAC

sequencing

-LRB-

Fig.

-RRB-

In

the

combined

approach

WGS

data

are

progressively

melded

with

light

sequence

coverage

of

individual

BACs

-LRB-

BAC

skims

-RRB-

to

yield

intermediate

products

called

enriched

BACs

-LRB-

eBACs

-RRB-

eBACs

covering

the

whole

genome

are

then

joined

into

longer

structures

-LRB-

bactigs

-RRB-

Bactigs

are

joined

to

form

larger

structures

superbactigs

then

ultrabactigs

During

this

process

other

data

are

introduced

including

BAC

end

sequences

DNA

fingerprints

and

other

long-range

information

-LRB-

genetic

markers

syntenic

information

-RRB-

but

the

process

is

constrained

by

eBAC

structures

To

execute

the

combined

strategy

we

developed

the

Atlas

software

-LRB-

Fig.

-RRB-

The

Atlas

suite

includes

a

BAC-Fisher

component

that

performs

the

functions

needed

to

generate

eBACs

WGS

genome

coverage

was

generated

ahead

of

complete

BAC

coverage

so

a

BAC-Fisher

web

server

was

established

at

the

BCM-HGSC

to

enable

users

to

access

the

combined

BAC

and

WGS

reads

as

each

BAC

was

processed

-LRB-

see

Methods

for

data

access

-RRB-

Each

eBAC

is

assembled

with

high

stringency

to

represent

the

local

sequence

accurately

and

so

provide

a

valuable

intermediate

product

that

assists

all

users

of

the

genome

data

Additional

Atlas

modules

joined

eBACs

and

linked

bactigs

to

give

the

complete

assembly

-LRB-

Fig.

-RRB-

Overall

the

combined

approach

takes

advantage

of

the

strengths

of

both

previous

methods

with

few

of

the

disadvantages

Sequence

and

genome

data

Over

million

DNA

sequence

reads

were

generated

-LRB-

Table

Methods

-RRB-

Following

removal

of

low-quality

reads

and

vector

contaminants

million

reads

were

used

for

Atlas

assembly

which

retained

million

reads

This

was

sequence

coverage

with

provided

by

WGS

and

from

BACs

Slightly

different

estimates

came

from

considering

the

entire

trimmed

length

of

the

sequence

data

-LRB-

-RRB-

or

only

the

portion

of

quality

or

higher

-LRB-

-RRB-

The

sequence

data

were

end-reads

from

clones

either

derived

directly

from

the

genome

-LRB-

insert

sizes

of

kb

kb

kb

and

kb

-RRB-

or

from

small

insert

plasmids

subcloned

from

BACs

Overall

these

provided

clone

coverage

with

coverage

having

both

paired

ends

represented

Approximately

equal

contributions

of

clone

coverage

were

from

the

different

categories

Over

BACs

were

used

for

BAC

skims

-LRB-

coverage

-RRB-

with

an

average

sequence

depth

of

giving

an

overall

genomic

sequence

coverage

from

BACs

This

was

slightly

more

than

the

most

efficient

procedure

would

require

-LRB-

each

-RRB-

because

the

genome

size

was

not

known

at

the

project

start

Simultaneous

with

sequencing

clones

from

the

BAC

were

fingerprinted

by

restriction

enzyme

NATURE

VOL

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Nature

Publishing

Group

articles

a

b

Figure

The

new

combined

sequence

strategy

and

Atlas

software

a

Formation

of

eBACs

The

RGSP

strategy

combined

the

advantages

of

both

BAC

and

WGS

sequence

Modest

sequence

coverage

-LRB-

-RRB-

from

a

BAC

is

used

as

bait

to

catch

WGS

reads

from

the

same

region

of

the

genome

These

reads

and

their

mate

pairs

are

assembled

using

Phrap

to

form

an

eBAC

This

stringent

local

assembly

retains

of

the

catch

b

Creation

of

higher-order

structures

Multiple

eBACs

are

assembled

into

bactigs

based

on

sequence

overlaps

The

bactigs

are

joined

into

superbactigs

by

large

clone

mate-pair

information

-LRB-

at

least

two

links

-RRB-

extended

into

ultrabactigs

using

additional

information

-LRB-

single

links

FPC

contigs

synteny

markers

-RRB-

and

ultimately

aligned

to

genome

mapping

data

-LRB-

radiation

hybrid

and

physical

maps

-RRB-

to

form

the

complete

assembly

digestion

representing

genomic

-LRB-

Methods

-RRB-

These

were

assembled

into

a

fingerprint

contig

-LRB-

FPC

-RRB-

map

-LRB-

a

contig

is

a

set

of

overlapping

segments

of

DNA

-RRB-

containing

FPCs

BAC

selection

for

sequence

skimming

was

based

on

overlaps

between

BACs

using

FPC

-LRB-

M.K.

and

C.F.

unpublished

work

-RRB-

ongoing

BAC

end

sequencing

-LRB-

S.Z.

unpublished

work

-RRB-

and

BAC

sequence

This

strategy

led

to

the

sequence

of

a

tiling

path

of

BAC

clones

covering

the

whole

genome

In

addition

to

the

FPC

map

a

yeast

artificial

chromosome

-LRB-

YAC

-RRB-

based

physical

map

was

constructed

BAC

and

artificial

chromosome

-LRB-

PAC

-RRB-

clones

from

and

respectively

were

anchored

to

YAC

clones

originating

from

two

tenfold-coverage

YAC

assembled

into

This

map

was

subsequently

integrated

with

the

FPC

map

and

the

sequence

assembly

reducing

the

total

number

of

map

contigs

to

-LRB-

minimum

length

of

contig

containing

the

typical

nucleotide

N

clones

Mb

anchored

to

the

sequence

assembly

Supplementary

Information

-RRB-

The

combined

strategy

enabled

development

of

resources

such

as

the

FPC

map

BAC

end

sequences

and

BAC

skim

sequences

in

parallel

rather

than

sequentially

In

addition

to

allowing

ongoing

quality

checking

this

permitted

the

data-gathering

phase

of

the

project

to

be

completed

in

less

than

two

years

Atlas

assembly

Statistics

for

the

assembly

are

in

Table

Contigs

within

eBACs

were

ordered

and

oriented

using

read-pair

information

Read-pair

information

was

also

used

to

add

WGS

reads

to

eBACs

even

when

sequence

overlaps

could

not

be

reliably

detected

owing

to

repeated

sequences

BAC

skim

reads

with

repeats

were

included

in

the

assembly

of

eBACs

because

they

clearly

originated

within

BAC

insert

sequences

Over

eBACs

were

eventually

generated

More

than

of

eBACs

were

successfully

merged

to

form

bactigs

-LRB-

Fig.

-RRB-

Bactigs

were

subsequently

reassembled

to

process

all

reads

from

overlapping

BACs

simultaneously

and

then

ordered

and

oriented

with

respect

to

each

other

using

FPC

map

and

BAC

end

sequence

read-pair

information

These

superbactig

and

ultrabactig

structures

-LRB-

see

below

-RRB-

were

aligned

with

chromosomes

using

external

information

such

as

positions

of

genetic

markers

Ultrabactigs

represented

the

largest

sequence

units

used

to

build

chromosomes

The

current

release

of

the

rat

genome

assembly

version

Table

Clones

and

reads

used

in

the

RGSP

Insert

size

-LRB-

kb

-RRB-

Source

or

vector

All

Total

WGS

Total

Plasmid

Plasmid

Plasmid

Plasmid

BAC

BAC

skims

Reads

-LRB-

millions

-RRB-

Used

Paired

Assembled

Bases

-LRB-

billions

-RRB-

Trimmed

Sequence

coverage

Trimmed

k

Clone

coverage

Grouped

in

ranges

of

sizes

for

individual

libraries

tracked

to

specific

multiples

of

kb

Total

bases

in

used

reads

divided

by

sampled

genome

size

including

all

cloned

and

sequenced

euchromatic

or

heterochromatic

regions

Estimated

as

sum

of

insert

sizes

divided

by

sampled

genome

size

WGS

reads

available

on

the

NCBI

Trace

Archive

as

of

March

BAC

skim

reads

attempted

at

BCM-HGSC

as

of

May

BAC

end

reads

obtained

directly

from

TIGR

k

Refers

to

coverage

from

kb

subclones

from

BACs

The

BACs

that

were

skimmed

amounted

to

clone

coverage

NATURE

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Nature

Publishing

Group

articles

Table

Statistics

of

the

RGSP

draft

sequence

assembly

Features

Number

length

-LRB-

kb

-RRB-

Bases

-LRB-

Gb

-RRB-

Bases

plus

gaps

-LRB-

Gb

-RRB-

Percentage

of

genome

Sampled

-LRB-

Gb

-RRB-

Bases

Bases

gaps

Assembled

-LRB-

Gb

-RRB-

Bases

Bases

gaps

Anchored

sequences

are

those

that

can

be

placed

on

chromosomes

because

they

contain

known

markers

The

main

scaffold

for

each

superbactig

is

the

largest

set

of

contigs

-LRB-

in

terms

of

total

contig

sequence

-RRB-

that

can

be

ordered

and

oriented

using

mate-pair

links

and

ordering

of

BACs

Scaffolds

that

can

not

be

ordered

and

oriented

with

respect

to

the

main

scaffold

are

termed

minor

scaffolds

Ambiguous

bases

-LRB-

N

-RRB-

are

counted

in

the

gap

sizes

and

excluded

in

the

base

counts

Computed

as

bases

plus

gaps

divided

by

estimated

genome

size

Sampled

genome

size

is

based

on

oligonucleotide

frequency

statistics

of

unassembled

WGS

reads

Assembled

genome

size

is

based

on

cumulative

contig

sequence

following

assembly

Anchored

contigs

Anchored

superbactig

scaffolds

Anchored

ultrabactigs

Unanchored

superbactigs

main

scaffolds

Unanchored

ultrabactigs

All

superbactigs

main

scaffolds

Minor

scaffolds

783 291 134 128

was

generated

using

the

data

in

Table

Earlier

releases

-LRB-

Methods

-RRB-

were

used

for

a

substantial

part

of

the

annotation

and

analysis

of

genes

and

proteins

whereas

the

current

release

provided

the

genome

description

has

contigs

with

N

length

kb

larger

than

the

expected

genomic

extent

of

a

mammalian

gene

These

sequence

contigs

were

linked

into

superbactigs

that

were

anchored

to

the

radiation

hybrid

These

larger

units

had

N

length

Mb

Another

smaller

superbactigs

-LRB-

N

length

Mb

-RRB-

could

not

be

anchored

presumably

because

they

fell

into

gaps

between

markers

or

because

they

were

in

repeated

regions

that

could

not

be

unambiguously

placed

From

placement

on

the

radiation

hybrid

map

adjacent

superbactigs

were

further

linked

to

maximize

continuity

of

sequence

if

appropriate

read-pair

mates

existed

or

FPC

suggested

links

This

reduced

linked

superbactigs

to

pieces

with

singletons

ultrabactigs

with

N

length

of

nearly

Mb

were

placed

on

chromosomes

Orthology

information

with

mouse

and

human

sequences

was

also

used

to

resolve

conflicts

and

suggest

placement

of

sequence

units

Most

of

the

unplaced

units

were

either

singletons

or

small

superbactigs

that

consisted

of

few

clones

Thus

nearly

the

entire

genome

was

represented

in

less

than

large

sequence

units

Quality

assessment

somes

and

chromosome

sizes

in

are

in

Supplementary

Table

Landscape

and

evolution

of

the

rat

genome

Genome

size

Thirteen

megabases

of

high-quality

finished

rat

sequence

from

BACs

were

available

for

comparison

with

-LRB-

Methods

-RRB-

This

analysis

showed

that

the

majority

of

draft

bases

from

within

contigs

were

high

quality

-LRB-

mismatches

per

kb

-RRB-

This

is

essentially

the

accepted

accuracy

standard

for

finished

sequence

-LRB-

errors

per

kb

-RRB-

so

the

overwhelming

majority

of

contig

bases

are

highly

accurate

The

highest

frequency

of

mismatches

occurred

at

the

ends

of

contigs

We

calculate

the

average

size

of

these

loweraccuracy

regions

to

be

base

pairs

-LRB-

bp

-RRB-

and

they

amount

to

less

than

of

the

genome

These

regions

arise

from

misassembly

of

terminal

reads

due

to

repeated

sequences

Few

mismatches

were

found

within

contigs

Six

were

found

within

contigs

when

compared

with

the

Mb

of

finished

sequence

or

one

case

per

Mb

All

were

insertions

or

deletions

and

may

represent

polymorphisms

Thus

at

the

fine

structure

level

the

bulk

of

sequences

that

make

up

contigs

is

nearly

the

quality

of

finished

sequence

We

judged

accuracy

of

assembly

at

the

chromosomal

level

by

alignment

with

linkage

and

radiation

hybrid

-LRB-

Fig.

-RRB-

Thirteen

markers

out

of

from

the

SHRSP

BN

genetic

map

were

placed

on

different

chromosomes

in

the

assembly

and

in

the

genetic

map

Similarly

of

the

sequence

tagged

sites

placed

on

both

the

assembly

and

radiation

hybrid

-LRB-

-RRB-

map

had

consistent

chromosome

Initial

alignments

identified

regions

of

misassembly

and

these

were

corrected

so

that

in

the

maps

are

congruent

except

for

possible

mismapped

markers

The

distribution

of

assembled

sequence

among

the

Genomic

assemblies

are

usually

smaller

than

the

actual

genome

size

owing

to

under-representation

of

sequences

affected

by

cloning

bias

and

sequencing

and

assembly

difficulties

Simply

equating

the

assembled

genome

size

with

the

euchromatic

cloneable

portion

does

not

take

into

account

heterochromatin

that

may

be

We

therefore

estimated

both

an

assembled

genome

size

scaled

by

the

inverse

of

the

fraction

of

features

-LRB-

genetic

markers

expressed

sequence

tags

-LRB-

ESTs

-RRB-

and

so

on

-RRB-

found

in

the

assembly

and

a

cloneable

-LRB-

or

sampled

-RRB-

genome

size

which

was

the

part

of

the

genome

present

in

the

WGS

reads

before

assembly

as

measured

by

analysing

the

distribution

of

short

The

former

may

be

an

underestimate

because

non-repetitive

easily

assembled

regions

can

be

enriched

for

known

features

The

latter

should

be

an

overestimate

because

there

are

likely

to

be

regions

-LRB-

such

as

repeats

-RRB-

that

can

be

cloned

and

sequenced

but

not

assembled

For

the

rat

genome

the

assembled

and

cloneable

genome

sizes

are

very

close

Considering

the

fraction

of

the

marker

set

successfully

mapped

to

-LRB-

-RRB-

or

the

fraction

of

sequence

finished

outside

the

BCM-HGSC

-LRB-

to

reduce

bias

-RRB-

present

in

-LRB-

-RRB-

together

with

the

assembled

bases

in

main

scaffolds

-LRB-

Gb

Table

-RRB-

we

suggest

a

genome

size

of

Gb

Alternatively

analysis

of

the

WGS

oligomers

of

length

to

predicted

a

genome

size

of

between

and

billion

bases

We

have

used

the

more

conservative

value

of

Gb

for

the

rat

genome

size

but

this

is

Figure

Map

correspondence

Correspondence

between

positions

of

markers

on

two

genetic

maps

of

the

rat

-LRB-

SHRSP

BN

intercross

and

FHH

ACI

-RRB-

on

the

rat

radiation

hybrid

and

their

position

on

the

rat

genome

assembly

-LRB-

-RRB-

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

still

considerably

higher

-LRB-

Mb

-RRB-

than

the

Gb

currently

reported

for

the

mouse

draft

genome

sequence

A

fraction

of

the

size

differences

in

these

rodent

genomes

results

from

the

different

repeat

content

-LRB-

see

below

-RRB-

however

it

is

also

recognized

that

segmental

duplications

may

be

under-represented

in

the

mouse

WGS

draft

sequence

for

technical

Telomeres

centromeres

and

mitochondrial

sequence

The

rat

has

both

metacentric

and

telocentric

chromosomes

in

contrast

to

the

wholly

telocentric

mouse

chromosomes

As

expected

from

previous

draft

sequences

the

rat

draft

does

not

contain

complete

telomeres

or

centromeres

Their

physical

location

relative

to

the

rat

draft

sequence

can

however

be

approximated

the

centromeres

of

the

telocentric

rat

chromosomes

-LRB-

and

X

-RRB-

must

be

positioned

before

nucleotide

of

these

assemblies

and

those

for

the

remaining

chromosomes

are

estimated

as

indicated

in

Fig.

Several

of

these

putative

centromere

positions

coincide

with

both

segmental

duplication

blocks

-LRB-

see

below

-RRB-

and

classical

satellite

clusters

consistent

with

enrichment

of

both

of

these

sequence

features

in

rat

pericentromeric

DNA

Human

subtelomere

regions

are

characterized

by

both

an

abundance

of

segmentally

duplicated

DNA

and

an

enrichment

of

internal

-LRB-

TTAGGG

-RRB-

n-like

sequence

Approximately

one-third

of

the

euchromatic

rat

subtelomeric

regions

are

similarly

enriched

suggesting

that

might

extend

very

close

to

the

chromosome

ends

Fragments

of

the

rat

mitochondrial

genome

were

also

propagated

within

the

WGS

libraries

and

subsequently

sequenced

allowing

the

assembly

of

the

complete

bp

mitochondrial

genome

-LRB-

Supplementary

Information

-RRB-

Comparison

with

existing

mitochondrial

sequences

in

the

public

databases

revealed

variable

positions

totalling

bp

-LRB-

-RRB-

between

this

strain

and

the

wild

brown

rat

Considerably

more

variation

-LRB-

-RRB-

was

found

when

compared

with

the

Wistar

strain

bp

differences

over

the

whole

genome

including

positions

that

are

conserved

in

the

other

mammalian

sequences

Such

variation

has

also

been

reported

in

mouse

mitochondrial

sequences

and

attributed

to

errors

in

previously

Figure

Distribution

of

segmental

duplications

in

the

rat

genome

Interchromosomal

duplications

-LRB-

red

-RRB-

and

intrachromosomal

duplications

-LRB-

blue

-RRB-

are

depicted

for

all

duplications

with

sequence

identity

and

kb

length

The

intrachromosomal

duplications

are

drawn

with

connecting

blue

line

segments

those

with

no

apparent

connectors

are

local

duplications

-LRB-

spaced

below

the

figure

resolution

limit

-RRB-

p

arms

are

on

the

left

and

the

q

arms

on

the

right

Chromosomes

and

X

are

telocentric

the

assemblies

begin

with

pericentric

sequences

of

the

q

arms

and

no

centromeres

are

indicated

For

the

remaining

chromosomes

the

approximate

centromere

positions

were

NATURE

VOL

APRIL

estimated

from

the

most

proximal

STS/gene

marker

to

the

p

and

q

arm

as

determined

by

fluorescent

in

situ

hybridization

-LRB-

FISH

-RRB-

-LRB-

cyan

vertical

lines

no

chromosome

data

-RRB-

The

Chr

Un

sequence

consists

of

contigs

not

incorporated

into

any

chromosomes

Green

arrows

indicate

Mb

intervals

with

more

than

tenfold

enrichment

of

classic

rat

satellite

repeats

within

the

assembly

Orange

diamonds

indicate

Mb

intervals

with

more

than

tenfold

enrichment

of

internal

-LRB-

TTAGGG

-RRB-

n-like

sequences

For

more

detail

see

http

ratparalogy.cwru.edu

Nature

Publishing

Group

articles

sequenced

The

current

sequence

is

very

accurate

and

we

therefore

favour

the

BN

sequence

as

a

reference

for

the

rat

mitochondrial

genome

Orthologous

chromosomal

segments

and

large-scale

rearrangements

Multi-megabase

segments

of

the

chromosomes

of

the

primate

rodent

ancestor

have

been

passed

on

to

human

and

murid

rodent

descendants

with

minimal

rearrangements

of

gene

These

intact

regions

which

are

bounded

by

the

breaks

that

occurred

during

ancient

large-scale

chromosomal

rearrangements

are

referred

to

as

orthologous

chromosomal

segments

The

same

phenomenon

has

occurred

in

the

descent

of

the

rat

and

mouse

from

the

genome

of

their

common

murid

ancestor

and

we

were

able

to

use

the

human

genome

and

in

some

cases

other

outgroup

data

to

tentatively

reconstruct

the

sequence

of

many

of

these

rearrangements

in

these

lineages

To

visualize

the

extent

of

orthologous

chromosomal

segments

each

genome

was

painted

with

the

orthologous

segments

of

the

other

two

species

-LRB-

Fig.

-RRB-

using

the

Virtual

Genome

Painting

method

-LRB-

M.L.G.-G

et

al.

unpublished

work

-RRB-

Inspection

shows

the

interleaving

of

events

that

both

preceded

and

occurred

subsequently

to

the

rat

mouse

divergence

Comparing

the

three

species

at

Mb

resolution

PatternHunter/Grimm-Synteny

and

associated

merging

produce

virtually

indistinguishable

sets

of

orthologous

chromosomal

segments

PatternHunter

and

the

GRIMMSynteny

detect

orthologous

segments

between

human

and

rat

and

between

human

and

mouse

The

mouse

rat

comparison

reveals

a

smaller

number

of

segments

-LRB-

-RRB-

of

larger

average

size

The

larger

number

of

breaks

in

orthologous

segments

between

the

human

to

the

rodent

pair

is

expected

because

of

the

latter

's

closer

evolutionary

relationship

Understanding

the

number

and

timing

of

rearrangement

events

that

have

occurred

in

each

of

the

three

individual

lineages

-LRB-

see

tree

in

Fig.

-RRB-

since

the

common

primate

rodent

ancestor

required

a

more

detailed

analysis

We

initially

focused

on

the

X

chromosome

because

rearrangements

between

the

X

and

the

autosomes

are

and

its

history

is

somewhat

easier

to

trace

completely

The

X

chromosome

consists

of

human

mouse

rat

orthologous

segments

of

at

least

kb

in

-LRB-

Fig.

-RRB-

In

the

most

parsimonious

scenario

-LRB-

found

with

MGR

and

-RRB-

these

were

created

by

inversions

in

the

descent

from

the

primate

rodent

ancestor

-LRB-

Fig.

-RRB-

Outgroup

data

from

cat

and

resolved

the

timing

of

these

rearrangements

more

precisely

Most

of

these

events

occurred

in

the

rodent

lineage

five

-LRB-

or

four

-RRB-

before

the

divergence

of

rat

and

mouse

five

in

the

rat

lineage

and

five

in

the

mouse

lineage

At

most

one

rearrangement

occurred

in

the

human

lineage

since

divergence

from

the

common

ancestor

with

rodents

The

timing

of

this

one

event

was

ambiguous

owing

to

the

limited

resolution

of

the

outgroup

data

Even

given

this

uncertainty

it

is

clear

that

the

large-scale

architecture

of

the

X

chromosome

in

humans

is

largely

unchanged

since

the

primate

rodent

whereas

there

has

been

considerable

activity

in

the

rodents

The

assignment

of

the

accelerated

activity

to

the

rodent

branch

following

the

primate

rodent

divergence

is

consistent

with

previous

studies

at

significantly

lower

resolution

-LRB-

these

showed

complete

conservation

of

marker

order

between

the

X

chromosomes

of

human

and

human

and

and

human

and

as

well

as

similar

karyotypes

of

the

X

chromosomes

in

human

chimpanzees

gorillas

and

-RRB-

Large-scale

reconstruction

of

the

entire

ancestral

murid

genome

suggests

that

it

retained

many

previously

postulated

chromosome

associations

of

the

placental

The

most

parsimonious

scenario

we

found

requires

a

total

of

rearrangements

between

the

murid

ancestor

and

human

from

the

murid

ancestor

to

mouse

and

from

the

murid

ancestor

to

rat

A

recent

implies

that

most

of

the

rearrangements

between

the

murid

ancestor

and

human

occurred

on

the

evolutionary

subpath

from

the

squirrel

mouse

rat

ancestor

to

the

murid

ancestor

Our

Figure

Map

of

conserved

synteny

between

the

human

mouse

and

rat

genomes

For

each

species

each

chromosome

-LRB-

x

axis

-RRB-

is

a

two-column

boxed

pane

-LRB-

p

arm

at

the

bottom

-RRB-

coloured

according

to

conserved

synteny

to

chromosomes

of

the

other

two

species

The

same

chromosome

colour

code

is

used

for

all

species

-LRB-

indicated

below

-RRB-

For

example

the

first

Mb

of

mouse

chromosome

is

shown

to

be

similar

to

part

of

human

chromosome

-LRB-

by

the

red

in

left

column

-RRB-

and

part

of

rat

chromosome

-LRB-

by

the

olive

in

right

column

-RRB-

An

interactive

version

is

accessible

-LRB-

-RRB-

Figure

Substitutions

and

microindels

-LRB-

bp

-RRB-

in

the

evolution

of

the

human

mouse

and

rat

genomes

a

The

lengths

of

the

labelled

branches

in

the

tree

are

proportional

to

the

number

of

substitutions

per

site

inferred

using

the

REV

from

all

sites

with

aligned

bases

in

all

three

genomes

b

The

table

shows

the

midpoint

and

variation

in

these

branch-length

estimates

when

estimated

from

different

sequence

alignment

programs

and

different

neutral

sites

including

sites

from

ancestral

fourfold

degenerate

sites

in

codons

and

rodent-specific

sites

-LRB-

in

neutral

sites

only

row

Supplementary

Information

-RRB-

Other

rows

give

midpoints

and

variation

for

micro-indels

on

each

branch

of

the

tree

in

a.

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

analyses

confirm

that

the

rate

of

rearrangements

in

murid

rodents

is

much

higher

than

in

the

human

Segmental

duplications

Segmental

duplications

are

defined

here

as

regions

of

the

genome

that

are

repeated

over

at

least

kb

of

length

and

identity

The

rat

has

approximately

of

its

bases

in

these

duplicated

regions

-LRB-

Fig.

-RRB-

whereas

the

human

genome

has

In

contrast

to

the

greater

rate

of

large-scale

rearrangement

the

mouse

genome

shows

substantially

fewer

of

these

with

only

of

its

sequenced

bases

in

duplicated

regions

These

duplicated

structures

are

particularly

challenging

to

assemble

and

we

attribute

at

least

some

of

the

mouse

rat

differences

to

the

BAC-based

approach

we

used

for

compared

with

the

WGS

mouse

approach

The

vast

majority

of

these

sequences

-LRB-

of

Mb

-RRB-

were

regions

with

identity

and

thus

were

not

simply

overlapping

sequences

that

had

not

been

joined

by

the

assembly

program

Phrap

The

unplaced

chromosome

in

showed

a

marked

enrichment

for

blocks

of

segmental

duplication

-LRB-

nearly

of

the

total

-RRB-

which

indicates

problems

with

anchoring

these

elements

to

the

genome

Intrachromosomal

duplications

are

represented

at

a

three-to-one

excess

when

compared

with

interchromosomal

duplications

and

are

significantly

enriched

near

the

telomeres

and

in

centromeric

regions

-LRB-

Fig.

-RRB-

The

pericentromeric

accumulation

of

segmental

duplications

in

the

rat

is

reminiscent

of

that

observed

in

human

and

and

seems

to

be

a

general

property

of

mammalian

chromosome

architecture

We

observed

considerable

clustering

of

including

discrete

genomic

regions

larger

than

Mb

in

size

in

which

duplications

appear

to

be

organized

into

groups

with

kb

between

duplicated

segments

For

many

of

these

clusters

the

underlying

sequence

alignments

showed

a

wide

range

in

the

degree

of

sequence

identity

suggesting

that

these

areas

have

been

subject

to

duplication

events

more

or

less

continuously

over

millions

of

years

In

contrast

an

analysis

of

the

evolutionary

distance

between

all

duplicated

regions

showed

an

unusual

bimodal

distribution

particularly

for

intrachromosomal

segmental

duplications

Two

peaks

were

observed

at

substitutions

per

site

and

substitutions

per

site

Given

that

the

rat

genome

has

accumulated

substitutions

-LRB-

see

below

-RRB-

since

the

speciation

from

mouse

Myr

ago

this

bimodal

distribution

may

correspond

to

bursts

of

segmental

duplication

that

occurred

approximately

and

Myr

ago

respectively

The

segmental

duplications

in

the

rat

genome

were

of

considerable

interest

because

they

represent

an

important

mechanism

for

the

generation

of

new

genes

We

found

that

NCBI

reference

-LRB-

RefSeq

see

-RRB-

genes

were

located

completely

or

partially

within

rat

duplicated

regions

out

of

a

genome

total

of

rat

RefSeq

genes

As

discussed

below

many

of

these

genes

are

present

in

multiple

copies

and

belong

to

gene

familes

that

have

been

recently

duplicated

and

contribute

to

distinctive

elements

of

rat

biology

Gains

and

losses

of

DNA

In

addition

to

large

rearrangements

and

segmental

duplications

genome

architecture

is

strongly

influenced

by

insertion

and

deletion

events

that

add

and

remove

DNA

over

evolutionary

time

To

characterize

the

origins

and

losses

of

sequence

elements

in

the

human

mouse

and

rat

genomes

we

categorized

all

the

nucleotides

in

each

of

the

three

genomes

using

our

alignment

data

and

Figure

X

chromosome

in

each

pair

of

species

a

computes

three-way

orthologous

segments

-LRB-

kb

-RRB-

on

the

X

chromosome

of

human

mouse

and

rat

shown

for

each

pair

of

species

using

consistent

colours

b

The

arrangement

-LRB-

order

and

orientation

-RRB-

of

the

blocks

implies

that

at

least

rearrangement

events

occurred

during

X

chromosome

evolution

of

these

species

The

program

MGR

-LRB-

http

-RRB-

determined

that

evolutionary

scenarios

NATURE

VOL

APRIL

with

events

are

achievable

and

all

have

the

same

median

ancestor

-LRB-

located

at

the

last

common

mouse

rat

ancestor

-RRB-

Shown

is

a

possible

-LRB-

not

unique

-RRB-

most

parsimonious

inversion

scenario

from

each

species

to

that

ancestor

We

note

that

the

last

common

ancestor

of

human

mouse

and

rat

should

be

on

the

evolutionary

path

between

this

median

ancestor

and

human

Nature

Publishing

Group

articles

RepeatMasker

annotations

of

the

insertions

of

repetitive

elements

-LRB-

Fig.

-RRB-

The

rodent

repeat

database

used

by

RepeatMasker

was

greatly

expanded

by

analysing

the

rat

and

mouse

but

it

is

clear

that

not

all

repeats

are

being

recognized

especially

the

older

ones

Thus

these

estimates

of

the

amount

of

rodent

repeats

represent

lower

bounds

About

a

billion

nucleotides

-LRB-

of

the

euchromatic

rat

genome

-RRB-

align

in

all

three

species

constituting

an

ancestral

core

that

is

retained

in

these

genomes

This

ancestral

core

contains

of

the

known

coding

exons

and

regulatory

regions

Comparisons

between

the

human

and

mouse

genomes

using

transposon

relics

retained

in

both

species

-LRB-

mammalian

ancestral

repeats

-RRB-

to

model

neutral

evolution

have

been

used

to

estimate

the

fraction

of

the

human

genome

that

is

accumulating

substitutions

more

slowly

than

the

neutral

rate

in

both

lineages

since

their

divergence

and

hence

may

be

under

some

level

of

purifying

Depending

on

details

of

methodology

such

estimates

have

ranged

between

about

and

The

levels

of

three-way

conservation

observed

here

between

the

human

mouse

and

rat

genomes

in

the

ancestral

core

lend

further

support

to

these

earlier

estimates

giving

values

in

the

range

of

when

measured

by

two

quite

different

methods

-LRB-

see

Methods

and

ref

-RRB-

In

this

constrained

fraction

non-coding

regions

outnumber

coding

regions

regardless

of

the

strength

of

an

observation

that

supports

recent

comparative

analyses

limited

to

subsets

of

the

The

preponderance

of

non-coding

elements

in

the

most

constrained

fraction

of

the

genome

underscores

the

likelihood

that

they

play

critical

roles

in

mammalian

biology

About

Mb

-LRB-

-RRB-

of

the

rat

euchromatic

genome

aligns

only

with

the

mouse

At

least

of

this

comprises

of

rodent-specific

repeats

inserted

on

the

branch

from

the

primate

rodent

ancestor

to

the

murid

ancestor

and

some

of

the

remainder

can

be

recognized

as

mammalian

ancestral

repeats

whose

orthologues

were

deleted

in

the

human

lineage

-LRB-

Fig.

-RRB-

Another

part

is

likely

to

consist

of

singlecopy

ancestral

DNA

deleted

in

the

human

lineage

but

retained

in

rodents

Although

this

Mb

of

rodent-specific

DNA

is

primarily

neutral

it

may

also

contain

some

functional

elements

lost

in

the

human

lineage

in

addition

to

sequences

representing

gains

of

rodent-specific

functions

including

some

coding

The

remainder

of

the

euchromatic

rat

genome

-LRB-

Mb

-RRB-

aligns

with

neither

mouse

nor

human

-LRB-

Fig.

-RRB-

At

least

half

of

this

-LRB-

of

the

rat

genome

-RRB-

consists

of

rat-specific

repeats

and

another

large

fraction

-LRB-

of

the

rat

genome

-RRB-

consists

of

rodent-specific

repeats

whose

orthologues

are

deleted

in

the

mouse

Substitution

rates

Figure

Aligning

portions

and

origins

of

sequences

in

rat

mouse

and

human

genomes

Each

outlined

ellipse

is

a

genome

and

the

overlapping

areas

indicate

the

amount

of

sequence

that

aligns

in

all

three

species

-LRB-

rat

mouse

and

human

-RRB-

or

in

only

two

species

Non-overlapping

regions

represent

sequence

that

does

not

align

Types

of

repeats

classified

by

ancestry

those

that

predate

the

human

rodent

divergence

-LRB-

grey

-RRB-

those

that

arose

on

the

rodent

lineage

before

the

rat

mouse

divergence

-LRB-

lavender

-RRB-

species-specific

-LRB-

orange

for

rat

green

for

mouse

blue

for

human

-RRB-

and

simple

-LRB-

yellow

-RRB-

placed

to

illustrate

the

approximate

amount

of

each

type

in

each

alignment

category

Uncoloured

areas

are

non-repetitive

DNA

the

bulk

is

assumed

to

be

ancestral

to

the

human

rodent

divergence

Numbers

of

nucleotides

-LRB-

in

Mb

-RRB-

are

given

for

each

sector

-LRB-

type

of

sequence

and

alignment

category

-RRB-

Detailed

results

are

tabulated

-LRB-

Supplementary

Table

-RRB-

The

alignment

data

allow

relatively

precise

estimates

of

the

rates

of

neutral

substitutions

and

microindel

events

-LRB-

bp

-RRB-

Both

synonymous

fourfold

degenerate

-LRB-

-RRB-

sites

in

protein-coding

regions

and

sites

in

mammalian

ancestral

repeats

were

used

in

this

analysis

as

in

previous

studies

comparing

human

and

We

additionally

used

a

class

of

primarily

neutral

sites

whose

identification

is

made

uniquely

possible

by

the

addition

of

the

rat

genome

sequence

namely

the

rodent-specific

sites

discussed

above

identified

by

their

failure

to

align

to

human

sequence

Our

estimates

for

the

neutral

substitution

level

between

the

two

rodents

range

from

to

substitutions

per

site

while

estimates

for

the

entire

tree

of

human

mouse

and

rat

range

from

to

substitutions

per

site

-LRB-

Fig.

-RRB-

This

difference

was

predictable

because

of

the

evolutionary

closeness

of

the

two

rodents

For

all

classes

of

neutral

sites

analysed

however

the

branch

connecting

the

rat

to

the

common

rodent

ancestor

is

longer

than

the

mouse

branch

-LRB-

Fig.

-RRB-

Thus

for

as

yet

unknown

reasons

the

rat

lineage

has

accumulated

substantially

more

point

substitutions

than

the

mouse

lineage

since

their

last

common

ancestor

We

also

analysed

four-way

alignments

including

sequence

from

orthologous

ancestral

repeats

in

human

mouse

and

rat

along

with

the

repeat

consensus

sequences

which

approximate

the

sequence

of

the

progenitor

of

the

corresponding

repeat

family

-LRB-

Methods

-RRB-

These

alignments

allow

us

to

distinguish

substitutions

on

the

branch

from

the

primate

rodent

ancestor

to

the

rodent

ancestor

from

substitutions

on

the

branch

descending

to

This

revealed

an

overall

speed-up

in

rodent

substitution

rates

relative

to

human

of

about

three-to-one

larger

than

estimated

but

consistent

with

other

more

recent

studies

which

also

use

multiple

sequence

Estimates

for

rates

of

microdeletion

events

are

for

all

branches

approximately

twofold

higher

than

rates

of

microinsertion

-LRB-

Fig.

-RRB-

suggesting

a

fundamental

difference

in

the

mechanisms

that

generate

these

mutations

Furthermore

there

are

substantial

rate

differences

for

each

class

of

event

between

the

various

lineages

In

particular

the

rat

lineage

has

accumulated

microdeletions

more

rapidly

than

the

mouse

while

the

opposite

holds

true

for

microinsertions

As

with

substitutions

both

microinsertion

and

microdeletion

rates

are

substantially

slower

in

the

human

lineage

The

size

distribution

of

microindels

-LRB-

bp

-RRB-

on

the

rat

branch

was

heavily

weighted

towards

the

smallest

indels

of

indels

are

single

bases

are

bp

are

bp

are

bp

and

so

on

monotonically

decreasing

Separate

distributions

for

insertions

and

for

deletions

were

similar

as

were

distributions

of

indel

sizes

on

the

mouse

branch

NATURE

VOL

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Nature

Publishing

Group

articles

Male

mutation

bias

As

mouse

and

rat

are

similar

in

generation

time

and

number

of

germline

cell

we

investigated

a

potential

sex

bias

in

different

types

of

observed

genome

changes

We

compared

substitution

and

indel

rates

between

the

X

chromosome

and

autosomes

in

ancestral

repeat

sites

-LRB-

Mb

and

Mb

in

total

for

X

and

autosomes

-RRB-

We

discovered

that

in

rodents

small

indels

-LRB-

bp

-RRB-

are

male-biased

with

a

male-to-female

rate

ratio

of

This

is

in

contrast

to

a

recent

study

in

primates

based

on

a

substantially

smaller

data

set

that

indicates

no

sex

bias

in

small

Our

male-to-female

nucleotide

substitution

rate

ratio

in

rodents

is

confirming

earlier

When

substitution

rates

are

compared

for

all

sites

aligned

between

mouse

and

rat

-LRB-

Mb

and

Mb

respectively

-RRB-

we

again

observe

an

approximately

twofold

excess

of

small

indels

and

nucleotide

substitutions

originating

in

males

compared

with

Interestingly

the

ratio

in

the

number

of

cell

divisions

between

the

male

and

female

germlines

is

also

about

suggesting

that

these

substitutions

may

arise

from

mutations

that

occur

primarily

during

DNA

replication

content

and

CpG

islands

The

G

C

content

of

the

rat

varies

significantly

across

the

genome

-LRB-

Fig.

-RRB-

and

the

distribution

more

closely

resembles

that

of

mouse

than

human

The

variation

in

G

C

content

is

coupled

with

differences

in

the

distribution

of

CpG

islands

short

regions

that

are

associated

with

the

ends

of

genes

and

gene

and

that

escape

the

depletion

of

CpG

dinucleotides

that

occurs

from

deamination

of

methylated

The

Gb

rat

genome

assembly

-LRB-

including

unmapped

sequences

-RRB-

contains

CpG

islands

in

non-repetitive

sequences

of

the

genome

This

is

similar

to

the

CpG

islands

reported

in

the

Gb

mouse

but

far

fewer

than

the

reported

in

the

human

A

summary

of

the

CpG

island

distributions

by

chromosome

is

given

in

Fig.

Chromosome

X

with

a

low

G

C

content

of

has

the

fewest

islands

-LRB-

-RRB-

and

the

lowest

density

of

islands

-LRB-

per

Mb

-RRB-

Chromosome

is

at

the

other

end

of

the

range

with

a

G

C

content

of

and

the

highest

density

of

CpG

islands

-LRB-

islands

per

Mb

-RRB-

This

is

similar

to

chromosome

with

islands

per

Mb

The

average

density

of

CpG

islands

is

islands

per

Mb

over

the

whole

genome

and

CpG

islands

per

Mb

averaged

by

chromosome

which

is

similar

to

the

distribution

in

Neither

rodent

genome

shows

the

extreme

outliers

in

CpG

island

density

that

are

seen

for

human

chromosome

-LRB-

ref

-RRB-

The

density

of

CpG

islands

in

the

rat

genome

correlates

positively

with

the

density

of

predicted

genes

-LRB-

R

of

-RRB-

-LRB-

Fig.

-RRB-

These

data

show

that

the

overall

changes

in

CpG

island

content

predate

the

rat

mouse

split

and

are

consistent

with

the

accelerated

loss

of

CpG

dinucleotides

in

rodents

compared

with

It

remains

possible

however

that

occurrences

such

as

the

greater

number

of

human

regions

with

extremely

high

G

C

content

are

due

to

distributional

changes

mostly

in

the

primate

rather

than

in

the

rodent

lineage

Shift

in

substitution

spectra

between

mouse

and

rat

The

non-repetitive

fraction

of

the

rat

genome

is

enriched

for

G

C

content

relative

to

the

mouse

genome

by

over

billion

nucleotides

This

is

a

subtle

but

substantial

difference

that

may

be

explained

at

least

in

part

by

differences

in

the

spectra

of

mutation

events

that

have

accumulated

in

the

mouse

and

rat

lineages

We

analysed

all

alignment

columns

in

which

substitution

events

can

be

assigned

to

either

the

mouse

or

the

rat

lineage

by

virtue

of

a

nucleotide

match

between

human

and

only

one

note

that

this

is

a

small

minority

of

substitutions

Of

the

million

alignment

columns

meeting

this

criteria

million

involve

a

change

in

the

rat

lineage

versus

million

in

the

mouse

reflecting

the

increase

in

rates

of

point

substitution

in

the

rat

lineage

-LRB-

Fig.

-RRB-

While

of

these

changes

in

rat

involve

a

substitution

from

an

A/T

to

a

G/C

these

events

constitute

only

of

all

mouse

changes

The

complementary

change

G/C

to

A/T

exhibits

relative

excess

in

the

mouse

versus

the

rat

lineage

-LRB-

versus

respectively

-RRB-

No

substantial

difference

between

changes

that

do

not

alter

G

C

content

is

observed

In

addition

this

bias

is

not

confined

to

particular

transition

or

transversion

events

nor

can

it

be

explained

simply

as

a

result

of

divergent

substitution

rates

of

CpG

dinucleotides

-LRB-

data

not

shown

-RRB-

Thus

this

shift

appears

to

be

a

general

change

that

results

in

an

increase

in

G

C

content

in

the

rat

genome

Biochemical

changes

in

repair

or

replication

enzymes

might

be

responsible

and

the

observation

that

recombination

rates

are

slightly

higher

in

rat

than

in

may

suggest

a

role

for

G

C-biased

mismatch

However

population

genetic

factors

such

as

selection

can

not

be

ruled

out

Evolutionary

hotspots

Figure

Base

composition

distribution

analysis

a

The

fraction

of

kb

non-overlapping

with

a

given

G

C

content

is

shown

for

human

mouse

and

rat

b

The

number

of

Ensembl-predicted

genes

per

chromosome

and

the

number

of

CpG

islands

per

chromosome

The

density

of

CpG

islands

averages

islands

per

Mb

across

chromosomes

and

islands

per

Mb

across

the

genome

Chromosome

has

more

CpG

islands

than

other

chromosomes

yet

neither

the

island

density

nor

ratio

to

predicted

genes

exceeds

the

normal

distribution

The

number

of

CpG

islands

per

chromosome

and

the

number

of

predicted

genes

are

correlated

-LRB-

R

-RRB-

NATURE

VOL

APRIL

Comparison

of

the

two

rodent

genomes

using

human

as

outgroup

reveals

regions

that

are

conserved

yet

under

different

levels

of

constraint

in

mouse

and

rat

These

regions

may

have

distinct

functional

roles

and

contribute

to

species-specific

differences

Analysis

of

the

MAVID

revealed

regions

bp

in

which

there

was

at

least

a

tenfold

difference

in

the

estimated

number

of

substitutions

per

site

on

the

mouse

and

rat

branches

To

avoid

alignment

problems

and

fast-evolving

regions

the

analysis

was

restricted

to

regions

where

the

human

branch

had

substitutions

per

These

regions

are

enriched

twofold

in

transcribed

regions

of

mouse

hotspots

were

found

in

the

Nature

Publishing

Group

articles

of

the

mouse

genome

covered

by

RefSeq

genes

and

of

the

rat

hotspots

were

found

in

the

of

the

rat

genome

covered

by

RefSeq

genes

Similar

numbers

are

observed

when

examining

coding

exon

and

EST

regions

-LRB-

not

shown

-RRB-

Half

of

all

hotspots

in

the

mouse

genome

lie

totally

in

non-coding

regions

Many

hotspots

are

several

hundred

bases

long

with

average

length

bp

Future

work

aimed

at

identifying

the

genomic

differences

that

contribute

to

phenotypic

evolution

may

benefit

from

analyses

such

as

these

which

will

become

more

powerful

as

the

repertoire

of

mammalian

genome

sequences

expands

Covariation

of

evolutionary

and

genomic

features

To

illustrate

the

genomic

and

evolutionary

landscape

of

a

single

rat

chromosome

in

depth

we

characterized

features

for

rat

chromosome

at

Mb

resolution

-LRB-

Fig.

-RRB-

This

high-resolution

analysis

uncovered

strong

correlations

between

certain

microevolutionary

Particularly

strongly

correlated

are

the

local

rates

of

microdeletion

-LRB-

R

Fig.

-RRB-

microinsertion

-LRB-

R

Fig.

-RRB-

and

point

substitution

-LRB-

R

Fig.

-RRB-

between

the

two

independent

lineages

of

mouse

and

rat

In

addition

microinsertion

rates

are

correlated

with

microdeletion

rates

-LRB-

R

Fig.

-RRB-

These

strong

correlations

are

also

observed

in

an

independent

genome-wide

analysis

both

on

the

original

data

and

after

factoring

out

the

effects

of

G

C

content

-LRB-

not

shown

see

Supplementary

Information

-RRB-

Perhaps

surprisingly

substantially

less

correlation

is

seen

between

microindel

and

point

substitution

rates

-LRB-

compare

Fig.

and

b

-RRB-

The

amount

of

correlation

varies

among

chromosomes

-LRB-

not

shown

-RRB-

but

is

generally

weaker

than

the

relationships

mentioned

above

Further

studies

will

be

required

to

determine

whether

local

evolutionary

pressures

which

must

have

remained

stable

since

the

separation

of

the

mouse

and

rat

lineages

differentially

drive

microindel

and

point

substitution

rates

We

also

find

that

the

local

point

substitution

rate

in

sites

common

to

human

mouse

and

rat

strongly

correlates

with

that

in

rodentspecific

sites

-LRB-

R

Fig.

blue

line

versus

red/green

-RRB-

These

two

classes

of

sites

while

interdigitated

at

the

level

of

tens

to

thousands

of

bases

constitute

sites

that

are

otherwise

evolutionarily

independent

This

result

confirms

that

local

rate

variation

is

not

solely

determined

by

stochastic

effects

and

extends

at

high

resolution

the

previously

documented

regional

correlation

in

rate

between

sites

and

ancestral

repeat

Evolution

of

genes

A

substantial

motivation

for

sequencing

the

rat

genome

was

to

study

protein-coding

genes

Besides

being

the

first

step

in

accurately

defining

the

rat

proteome

this

fundamental

data

set

yields

insights

into

differences

between

the

rat

and

other

mammalian

species

with

a

complete

genome

sequence

Estimation

of

the

rat

gene

content

is

possible

because

of

relatively

mature

gene-prediction

programs

and

rodent

transcript

data

Mouse

and

human

genome

sequences

also

allow

characterization

of

mutational

events

in

proteins

such

as

amino

acid

repeats

and

codon

insertions

and

deletions

The

quality

of

the

rat

sequence

also

allows

us

to

distinguish

between

functional

genes

and

pseudogenes

We

estimate

-LRB-

on

the

basis

of

a

subset

-RRB-

that

of

rat

genes

Figure

Variability

of

several

evolutionary

and

genomic

features

along

rat

chromosome

a

Rates

of

microdeletion

and

microinsertion

events

-LRB-

less

than

bp

-RRB-

in

the

mouse

and

rat

lineages

since

their

last

common

ancestor

revealing

regional

correlations

b

Rates

of

point

substitution

in

the

mouse

and

rat

lineages

Red

and

green

lines

represent

rates

of

substitution

within

each

lineage

estimated

from

sites

common

to

human

mouse

and

rat

Blue

represents

the

neutral

distance

separating

the

rodents

as

estimated

from

rodent-specific

sites

Note

the

regional

correlation

among

all

three

plots

despite

being

estimated

in

different

lineages

-LRB-

mouse

and

rat

-RRB-

and

from

different

sites

-LRB-

mammalian

versus

rodent-specific

-RRB-

c

Density

of

SINEs

inserted

independently

into

the

rat

or

mouse

genomes

after

their

last

common

ancestor

d

A

T

content

of

the

rat

and

density

in

the

rat

genome

of

LINEs

and

SINEs

that

originated

since

the

last

common

ancestor

of

human

mouse

and

rat

Pink

boxes

highlight

regions

of

the

chromosome

in

which

substitution

rates

A

T

content

and

LINE

density

are

correlated

Blue

boxes

highlight

regions

in

which

SINE

density

is

high

but

LINE

density

is

low

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

possess

strict

orthologues

in

both

mouse

and

human

genomes

Our

studies

also

identified

genes

arising

from

recent

duplication

events

occurring

only

in

rat

and

not

in

mouse

or

human

These

genes

contribute

characteristic

features

of

rat-specific

biology

including

aspects

of

reproduction

immunity

and

toxin

metabolism

By

contrast

almost

all

human

disease

genes

have

rat

orthologues

This

emphasizes

the

importance

of

the

rat

as

a

model

organism

in

experimental

science

Construction

of

gene

set

and

determination

of

orthology

The

Ensembl

gene

prediction

predicted

genes

with

transcripts

and

exons

-LRB-

Methods

-RRB-

These

genes

contain

an

average

of

exons

with

a

median

exon

number

of

At

least

of

the

genes

are

alternatively

spliced

with

an

average

of

transcripts

predicted

per

gene

Of

the

single

exon

transcripts

contain

frameshifts

relative

to

the

predicted

protein

and

are

probably

processed

pseudogenes

Of

the

transcripts

have

both

and

untranslated

regions

-LRB-

UTRs

-RRB-

predicted

and

have

at

least

one

UTR

predicted

These

gene

predictions

considered

homology

to

other

sequences

including

rodent

proteins

non-rodent

vertebrate

proteins

rat

complementary

DNAs

from

RefSeq

and

EMBL

and

mouse

cDNAs

from

Riken

RefSeq

and

EMBL

The

majority

-LRB-

-RRB-

of

transcripts

are

supported

by

rodent

transcript

evidence

When

combined

with

additional

private

EST

data

the

fraction

of

genes

supported

by

transcript

evidence

could

be

increased

to

A

number

of

other

ab

initio

-LRB-

-RRB-

similarity-based

-LRB-

FGENESH

ref

-RRB-

and

comparative

-LRB-

-RRB-

gene-prediction

programs

were

used

to

analyse

the

rat

genome

The

number

of

genes

predicted

by

these

programs

ranged

from

to

suggesting

coding

densities

ranging

from

to

The

coding

fraction

of

RefSeq

genes

covered

by

these

predictions

ranged

from

to

Such

comparative

ab

initio

programs

using

the

rat

genome

were

successfully

used

to

identify

and

experimentally

verify

genes

missed

by

other

methods

in

and

The

predictions

of

these

programs

can

be

accessed

through

the

UCSC

genome

browser

and

Ensembl

websites

RefSeq

genes

-LRB-

human

mouse

and

rat

-RRB-

mapped

onto

genome

assemblies

with

and

the

UCSC

browser

revealed

that

the

number

of

coding

exons

per

gene

and

average

exon

length

were

similar

in

the

three

species

Differences

were

observed

in

intron

length

with

an

average

of

bp

in

human

bp

in

mouse

and

bp

in

rat

These

differences

were

also

found

in

a

smaller

collection

of

confidently

mapped

orthologous

intron

triads

-LRB-

see

Conservation

of

intronic

splice

signals

section

below

-RRB-

average

intron

lengths

in

this

collection

were

bp

in

human

bp

in

mouse

and

bp

in

rat

Properties

of

orthologous

genes

Orthology

relationships

were

predicted

on

the

basis

of

BLASTp

reciprocal

best-hits

between

proteins

of

genome

pairs

-LRB-

human

rat

rat

mouse

and

mouse

human

-RRB-

-LRB-

Supplementary

Information

-RRB-

Using

these

methods

and

the

ENSEMBL

prediction

sets

rat

genes

showed

clear

unambiguous

correspondence

with

a

gene

in

the

mouse

genome

This

is

an

underestimate

because

random

sampling

of

different

classes

of

rat

genes

with

less

stringent

criteria

for

comparison

to

mouse

always

identified

additional

gene

pairs

Errors

arose

from

pseudogene

misclassification

sequence

loss

duplication

or

fragmentation

in

assemblies

and

missing

or

inappropriate

gene

predictions

including

coding-gene

predictions

from

non-coding

RNAs

Taking

these

errors

into

account

we

estimate

the

true

proportion

of

orthologues

in

rat

and

mouse

genomes

to

lie

between

and

-LRB-

Methods

-RRB-

The

remaining

genes

were

associated

with

lineage-specific

gene

family

expansions

or

contractions

These

overall

observations

are

consistent

with

a

careful

analysis

of

rat

proteases

showing

that

of

these

genes

have

orthologues

in

Surprisingly

a

similar

proportion

-LRB-

to

-RRB-

of

rat

genes

possessed

a

single

orthologue

in

the

human

genome

Because

human

represents

an

outgroup

to

the

two

rodents

it

was

expected

that

mouse

and

rat

would

share

a

higher

fraction

of

orthologues

A

close

inspection

of

gene

relationships

indicates

that

these

findings

may

suffer

from

incompleteness

of

rodent

genome

sequences

together

with

problems

of

misassembly

and

gene

prediction

within

clusters

of

gene

paralogues

Further

analysis

of

orthologous

pairs

considered

the

occurrence

of

nucleotide

changes

within

protein-coding

regions

that

reflected

synonymous

or

non-synonymous

substitutions

The

majority

of

these

studies

measured

evolutionary

rates

by

determination

of

K

A

-LRB-

number

of

non-synonymous

substitutions

per

non-synonymous

site

-RRB-

and

K

S

-LRB-

number

of

synonymous

substitutions

per

synonymous

site

-RRB-

K

A/K

S

ratios

of

less

than

indicate

purifying

selection

values

of

suggest

neutral

evolution

and

values

greater

than

indicate

positive

Evolutionary

rates

were

first

calculated

from

a

reduced

set

of

orthologue

pairs

that

are

embedded

in

orthologous

genomic

segments

and

are

related

by

conservative

values

of

K

S

-LRB-

Table

-RRB-

-LRB-

Methods

-RRB-

A

slight

increase

in

median

K

S

values

for

rat

human

as

compared

with

mouse

human

was

found

indicating

that

the

rat

lineage

has

more

neutral

substitutions

in

gene

coding

regions

than

the

mouse

lineage

Sequence

conservation

values

were

similar

to

those

previously

found

using

smaller

data

and

the

overall

trend

is

consistent

with

results

of

other

evolutionary

rate

analyses

discussed

above

-LRB-

Fig.

-RRB-

Next

we

investigated

examples

of

rat

genes

shared

with

mouse

but

with

no

counterparts

in

human

Such

genes

might

be

rapidly

evolving

so

that

homologues

are

not

discernible

in

human

or

they

might

have

arisen

from

non-coding

DNA

or

their

orthologues

in

the

human

lineage

might

have

formed

pseudogenes

Thirty-one

Ensembl

rat

genes

were

collected

that

have

no

non-rodent

homologues

in

current

databases

-LRB-

Methods

-RRB-

These

are

twofold

overrepresented

among

genes

in

paralogous

gene

clusters

and

threefold

over-represented

among

genes

whose

proteins

are

likely

to

be

secreted

This

is

consistent

with

that

clusters

of

paralogous

genes

and

secreted

proteins

evolve

relatively

rapidly

Detailed

examination

of

the

genes

using

PSI-BLAST

determined

that

ten

genes

can

not

be

assigned

homology

relationships

to

experimentally

described

mammalian

genes

These

ten

rodent

Table

One-to-one

orthologous

genes

in

human

mouse

and

rat

genomes

Human

mouse

orthologue

relationships

Median

K

S

values

Median

K

A/K

S

values

Median

amino

acid

identity

Median

nucleotide

identity

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Human

rat

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mouse

rat

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Data

obtained

from

Ensembl

Homo

sapiens

version

-LRB-

genes

-RRB-

Mus

musculus

version

-LRB-

genes

-RRB-

Rattus

norvegicus

version

-LRB-

genes

-RRB-

Numbers

in

parentheses

represent

the

and

percentiles

This

value

is

consistent

with

previous

findings

-LRB-

in

ref

-RRB-

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

specific

genes

may

have

evolved

particularly

rapidly

or

have

noncoding

DNA

homologues

or

be

erroneous

predictions

The

paucity

of

rodent-specific

genes

indicates

that

de

novo

invention

of

complete

genes

in

rodents

is

rare

This

is

not

unexpected

because

the

majority

of

eukaryotic

protein-coding

genes

are

modular

structures

containing

coding

and

non-coding

exons

splicing

signals

and

regulatory

sequences

and

the

chances

of

independent

evolution

and

successful

assembly

of

these

elements

into

a

functional

gene

are

small

given

the

relatively

short

evolutionary

time

available

since

the

mouse

rat

split

However

individual

rodent-specific

exons

may

arise

more

frequently

particularly

if

the

exon

is

alternatively

Applying

a

K

A

K

S

ratio

to

sequences

that

align

only

between

rat

and

mouse

we

identified

potential

novel

rodent-specific

exons

with

EST

support

in

BLASTZ

alignments

of

rat

and

mouse

sequences

of

these

individual

exons

matched

human

transcripts

but

approximately

half

-LRB-

-RRB-

appear

to

be

present

in

alternative

splice

forms

found

in

rodents

We

speculate

that

these

exons

contain

the

few

successful

lineage-specific

survivors

of

the

constant

process

of

gene

evolution

by

birth

and

death

of

individual

exons

Indels

and

repeats

in

protein-coding

sequences

In

contrast

to

small

indels

occurring

in

the

bulk

of

the

genome

-LRB-

above

-RRB-

indels

within

protein-coding

regions

are

probably

lethal

or

deleterious

and

so

are

rapidly

removed

from

the

population

by

purifying

selection

Indel

rates

within

rat

coding

sequences

were

lower

than

in

bulk

genomic

The

whole

genome

excess

of

deletions

compared

with

insertions

-LRB-

Fig.

-RRB-

was

also

evident

in

coding

sequences

The

magnitude

was

less

with

a

genome-wide

deletion-to-insertion

ratio

of

reducing

to

in

the

rat

In

mouse

this

value

reduced

from

to

-LRB-

ref

-RRB-

These

data

suggest

that

deletions

are

more

likely

than

insertions

to

be

removed

from

coding

sequences

by

selection

Owing

to

the

triplet

nature

of

the

genetic

code

indels

of

multiples

of

three

nucleotides

in

length

-LRB-

indels

-RRB-

are

less

likely

to

be

deleterious

Direct

comparison

of

indel

rates

between

bulk

DNA

-LRB-

indels

per

kb

for

mouse

indels

per

kb

for

rat

-RRB-

and

coding

sequence

-LRB-

indels

per

kb

for

mouse

and

indel

per

kb

for

rat

-RRB-

showed

that

indels

were

ninefold

underrepresented

in

coding

sequences

At

least

of

indels

were

duplicative

insertion

or

deletion

of

a

tandemly

duplicated

sequence

collectively

termed

sequence

Sequence

slippage

contributed

approximately

equally

to

observed

insertions

and

deletions

The

overall

excess

of

deletions

could

be

attributed

specifically

to

an

excess

of

non-slippage

deletion

over

non-slippage

insertion

in

both

mouse

and

rat

Of

the

slippage

indels

were

in

the

context

of

trinucleotide

repeats

-LRB-

n

excluding

the

inserted

or

deleted

sequence

-RRB-

which

are

known

to

be

particularly

prone

to

sequence

slippage

and

encode

homopolymeric

amino

acid

To

gain

better

understanding

of

dynamic

changes

in

the

length

of

homopolymeric

amino

acid

tracts

on

gene

evolution

and

disease

susceptibility

we

searched

for

other

characteristics

of

amino

acid

repeat

variation

by

analysing

all

size-five

or

longer

amino

acid

repeats

in

a

data

set

of

rat

mouse

and

human

orthologous

protein

Most

species-specific

amino

acid

repeats

-LRB-

-RRB-

were

found

in

indel

regions

and

regions

encoding

speciesspecific

repeats

were

more

likely

to

contain

tandem

trinucleotide

repeats

than

those

encoding

conserved

repeats

This

was

consistent

with

the

involvement

of

slippage

in

the

generation

of

novel

repeats

in

proteins

and

extended

previous

observations

for

glutamine

repeats

in

a

more

limited

human

mouse

data

The

percentage

of

proteins

containing

amino

acid

repeats

was

in

rat

in

mouse

and

in

The

most

frequently

occurring

tandem

amino

acid

repeats

were

glutamic

acid

proline

alanine

leucine

serine

glycine

glutamine

and

lysine

Using

the

same

threshold

size

cut-off

tandem

trinucleotide

repeats

were

significantly

more

abundant

in

human

than

in

rodent

coding

sequences

in

striking

contrast

to

the

frequencies

observed

in

bulk

genomic

sequences

-LRB-

trinucleotide

repeats

per

Mb

in

rat

repeats

per

Mb

in

mouse

and

repeats

per

Mb

in

human

see

discussion

of

the

general

simple

repeat

structure

below

-RRB-

The

conservation

of

human

repeats

was

higher

in

mouse

-LRB-

-RRB-

than

in

rat

-LRB-

-RRB-

suggesting

a

higher

rate

of

repeat

loss

in

the

rat

lineage

than

the

mouse

lineage

Functional

consequences

of

these

in-frame

changes

in

rat

mouse

and

human

were

through

clustering

of

proteins

based

on

annotation

of

function

and

cellular

and

mapping

indels

onto

protein

structural

and

sequence

features

The

rate

that

indels

accumulated

in

secreted

-LRB-

indels

per

amino

acid

-RRB-

and

nuclear

-LRB-

-RRB-

proteins

is

approximately

twice

that

of

cytoplasmic

-LRB-

-RRB-

and

mitochondrial

-LRB-

-RRB-

proteins

Likewise

ligand-binding

proteins

acquire

indels

-LRB-

-RRB-

at

a

higher

rate

than

enzymes

-LRB-

-RRB-

These

trends

exactly

mirror

those

observed

for

amino

acid

substitution

suggesting

tight

coupling

of

selective

constraints

between

indels

and

substitutions

Transcription

regulators

showed

the

highest

rate

of

indels

-LRB-

-RRB-

a

finding

that

may

relate

to

the

over-representation

of

homopolymorphic

amino

acid

tracts

in

these

Known

protein

domains

exhibited

fewer

indels

than

expected

by

chance

again

paralleling

nucleotide

substitution

rate

differences

between

domains

and

non-domain

Of

the

protein-sequence

and

structural

categories

considered

-LRB-

transmembrane

protein

domain

signal

peptide

coiled

coil

and

low

complexity

-RRB-

the

transmembrane

regions

were

the

most

refractory

to

accumulating

indels

exhibiting

a

sixfold

reduction

compared

with

that

expected

by

chance

Low-complexity

regions

were

enriched

reflecting

their

relatively

unstructured

nature

and

enrichment

in

indel-prone

trinucleotide

repeats

Mapping

of

indels

onto

groups

of

known

structures

revealed

that

indels

are

more

likely

to

be

tolerated

in

loop

regions

than

the

structural

core

of

the

We

observed

that

indel

frequency

and

amino

acid

repeat

occurrence

both

correlated

positively

with

the

G

C

coding

sequence

content

of

the

local

sequence

This

may

be

explained

in

part

by

the

correlation

of

polymerase

slippage-prone

trinucleotide

repeat

sequences

and

G

C

There

is

also

a

positive

correlation

between

CpG

dinucleotide

frequency

and

coding

sequence

insertions

but

not

deletions

This

effect

diminishes

rapidly

with

increasing

distance

from

the

site

of

the

Transcription-associated

substitution

strand

asymmetry

A

recent

study

reported

a

significant

strand

asymmetry

for

neutral

substitutions

in

transcribed

Within

introns

of

nine

genes

the

higher

rate

of

A!G

substitutions

over

that

of

T!C

substitutions

together

with

a

smaller

excess

of

G!A

over

C!T

substitutions

leads

to

an

excess

of

G

T

over

C

A

on

the

coding

strand

-LRB-

also

verified

on

human

chromosome

-RRB-

The

hypothesized

that

the

asymmetries

are

a

byproduct

of

transcription

Table

Strand

asymmetry

of

substitutions

in

introns

of

rat

genes

Base

frequencies

on

coding

strand

-LRB-

G

T

-RRB-

-LRB-

C

A

-RRB-

Ratio

of

purine

transitions

to

pyrimidine

transitions

Rate

-LRB-

A$

G

-RRB-

Rate

-LRB-

C$

T

-RRB-

Rate

of

transitions

Rate

-LRB-

A!G

-RRB-

Rate

-LRB-

T!C

-RRB-

Rate

-LRB-

G!A

-RRB-

Rate

-LRB-

C!T

-RRB-

Rat

genome

Rat

mouse

Rat

Rat

human

Mouse

Computed

from

the

rat

genome

Computed

from

pairwise

alignments

Computed

from

three-way

alignments

Nature

Publishing

Group

NATURE

VOL

APRIL

articles

coupled

repair

in

germline

cells

Examining

the

three-way

alignments

of

rat

mouse

and

human

we

verified

that

the

strand

asymmetries

for

neutral

substitutions

exist

in

introns

across

the

genome

-LRB-

Table

-RRB-

Under

the

assumption

of

independence

of

sequence

positions

large

sample

normal

approximations

to

the

binomial

distribution

allow

us

to

test

whether

the

fraction

of

G

T

exceeds

and

whether

the

rate

at

the

numerator

exceeds

the

rate

at

the

denominator

for

each

of

the

ratios

in

Table

With

the

large

amount

of

data

provided

by

pooling

introns

genome-wide

the

tests

are

all

highly

significant

-LRB-

P

values

-RRB-

except

for

the

rate

of

G!A

in

mouse

which

does

not

significantly

exceed

that

of

C!T

-LRB-

P

value

-RRB-

These

asymmetries

are

also

seen

if

the

study

is

limited

to

ancestral

repeat

sites

excludes

ancestral

repeat

sites

excludes

CpG

dinucleotides

is

limited

to

positions

flanked

by

sites

that

are

identical

in

the

aligned

sequences

-LRB-

in

the

case

of

observations

and

in

Table

-RRB-

or

considers

introns

of

RefSeq

genes

for

human

or

mouse

Thus

it

appears

that

strand

asymmetry

of

substitution

events

within

transcribed

regions

of

the

genome

is

a

robust

genome-wide

phenomenon

Conservation

of

intronic

splice

signals

Using

human

mouse

rat

orthologous

introns

from

genes

-LRB-

Methods

-RRB-

we

examined

the

dynamics

of

evolution

of

consensus

splice

signals

in

mammalian

genes

We

found

that

intron

is

extremely

well

conserved

we

did

not

observe

any

to

intron

conversion

or

vice

versa

nor

within

introns

did

we

find

any

switching

between

the

major

AT

AC

and

GT

AG

subtypes

although

such

events

are

documented

at

larger

evolutionary

In

contrast

conversions

between

canonical

GT

AG

and

non-canonical

GC

AG

subtypes

of

introns

are

not

uncommon

Only

of

GC

AG

introns

are

conserved

between

human

and

mouse/rat

and

only

are

conserved

between

mouse

and

rat

Using

human

as

the

outgroup

we

detected

nine

GT

to

GC

conversions

after

divergence

of

mouse

and

rat

-LRB-

from

introns

that

were

likely

to

have

been

GT

AG

before

human

and

rodents

split

-RRB-

and

two

GC

to

GT

conversions

-LRB-

from

GC

AG

introns

that

probably

predated

the

human

and

rodent

split

-RRB-

These

results

give

some

indication

of

the

degree

to

which

mutation

from

T

to

C

is

tolerated

in

donor

sites

The

GC

donor

site

appears

to

be

better

tolerated

in

introns

with

very

strong

donor

sites

because

in

these

introns

the

proportion

of

GC

donor

sites

is

much

higher

than

the

overall

frequency

of

GC

donor

sites

in

introns

Although

we

found

a

variety

of

other

non-canonical

configurations

in

introns

very

few

are

conserved

which

suggests

that

most

correspond

to

transient

evolutionarily

unstable

states

pseudogenes

or

mis-annotations

Gene

duplications

synonymous

site

-LRB-

K

S

-RRB-

between

all

pairs

of

homologous

genes

constructed

K

S-derived

phylogenetic

trees

and

predicted

orthology

or

paralogy

gene

duplication

events

automatically

from

their

topologies

-LRB-

Supplementary

Information

-RRB-

The

results

showed

that

the

neutral

substitution

rate

varies

among

orthologues

by

approximately

twofold

-LRB-

Fig.

-RRB-

This

is

similar

to

chromosomal

variation

shown

previously

by

a

study

of

mouse

and

human

ancestral

Rates

of

change

among

ancestral

gene

duplications

-LRB-

those

that

predate

the

mouse

rat

split

-RRB-

were

relatively

constant

Mouse-specific

and

rat-specific

duplications

occurred

at

similar

rates

except

for

those

with

K

S

which

are

reduced

in

mousespecific

duplications

-LRB-

Fig.

-RRB-

More

data

are

required

to

determine

whether

this

reduction

is

a

biological

effect

as

it

might

be

accounted

for

by

different

protocols

for

assembling

mouse

and

rat

genomes

which

differentially

collapse

areas

of

nearly

identical

sequence

The

rat

paralogue

pairs

that

probably

arose

after

the

rat

mouse

split

-LRB-

Myr

ago

-RRB-

have

K

S

values

of

-LRB-

Table

-RRB-

We

found

K

S

gene

duplication

events

in

rat

a

lower

number

than

is

found

in

mouse

-LRB-

-RRB-

For

both

rodents

this

represents

a

likelihood

of

a

gene

duplicating

of

between

and

every

Myr

These

are

necessarily

estimates

because

gene

deletions

conversions

and

pseudogene

formation

are

not

considered

Interestingly

the

data

are

consistent

with

a

previous

estimate

for

Drosophila

genes

but

are

an

order

of

magnitude

lower

than

an

estimate

for

Caenorhabditis

elegans

A

subset

of

clusters

have

at

least

three

gene

duplications

with

K

S

-LRB-

Table

-RRB-

These

are

expected

to

be

enriched

in

genes

whose

duplications

persist

as

a

consequence

of

positive

selection

The

group

is

dominated

by

genes

involved

in

adaptive

immune

response

and

Inspection

of

the

K

S-derived

trees

allowed

us

to

infer

the

gene

numbers

in

these

clusters

for

the

common

ancestor

of

rat

and

mouse

-LRB-

that

is

at

K

S

-RRB-

assuming

no

gene

deletions

or

pseudogene

generation

-LRB-

Table

-RRB-

Immunoglobulin

T-cell

receptor

a-chain

and

genes

appear

to

be

duplicating

at

the

fastest

rates

in

the

rat

genome

-LRB-

Table

-RRB-

Since

divergence

with

mouse

these

rat

clusters

have

increased

gene

content

several-fold

This

recapitulates

previous

observations

that

rapidly

evolving

and

duplicating

genes

are

over-represented

in

olfaction

and

odorant

detection

antigen

recognition

and

An

examination

of

duplicated

genomic

segments

showed

this

enrichment

for

most

of

the

same

genes

and

also

elements

involved

in

foreign

compound

detoxification

-LRB-

cytochrome

and

carboxylesterase

genes

-RRB-

Together

these

are

exciting

findings

because

each

of

these

categories

can

easily

be

associated

with

a

Duplication

of

genomic

segments

represents

a

frequent

and

robust

mechanism

for

generating

new

Because

there

were

no

compelling

data

showing

rat-specific

genes

arising

directly

from

non-coding

sequences

we

examined

gene

duplications

to

measure

their

potential

contribution

to

rat-specific

biology

A

previous

study

showed

that

gene

clusters

in

mouse

without

counterparts

in

human

are

subject

to

rapid

adaptive

We

used

two

methods

to

identify

recent

gene

duplications

methods

that

directly

identified

paralogous

clusters

and

methods

that

analysed

genomic

segmental

duplications

-LRB-

see

above

-RRB-

Using

the

first

approach

we

found

rat

paralogue

clusters

containing

genes

-LRB-

Methods

-RRB-

This

was

lower

than

in

mouse

-LRB-

genes

-RRB-

but

the

difference

probably

reflects

the

larger

number

of

gene

predictions

from

the

mouse

assembly

To

investigate

the

timing

of

expansion

of

these

individual

families

we

measured

rates

of

local

gene

duplication

and

retention

within

clusters

BLAST

is

not

suited

to

and

so

we

instead

calculated

the

number

of

synonymous

substitutions

per

NATURE

VOL

APRIL

Figure

Variation

in

the

frequency

of

gene

duplications

during

the

evolutionary

histories

of

the

rat

and

mouse

The

sequence

of

gene

duplication

events

was

inferred

from

phylogenetic

trees

determined

from

pairwise

estimates

of

genetic

divergence

under

neutral

selection

-LRB-

K

S

Methods

-RRB-

The

median

K

S

value

for

mouse

rat

orthologues

is

This

value

corresponds

to

the

divergence

time

of

mouse

and

rat

lineages

Nature

Publishing

Group

articles

familiar

feature

of

rat-specific

biology

and

further

investigation

could

explain

some

differences

between

rats

and

their

evolutionary

neighbours

Conservation

of

gene

regulatory

regions

As

the

third

mammal

to

be

fully

sequenced

the

rat

can

add

significantly

to

the

utility

of

nucleotide

alignments

for

identifying

conserved

non-coding

This

power

increases

roughly

as

a

function

of

the

total

amount

of

neutral

substitution

represented

in

the

and

rat

adds

about

to

the

human

mouse

comparison

-LRB-

Fig.

-RRB-

Many

conserved

mammalian

non-coding

sequences

are

expected

to

have

regulatory

function

and

can

be

predicted

using

further

analyses

based

upon

these

We

applied

such

methods

for

detecting

significantly

conserved

and

scoring

regulatory

to

the

genomewide

human

mouse

rat

alignments

Typical

results

show

strong

conservation

for

a

coding

exon

as

well

as

for

several

non-coding

regions

-LRB-

Fig.

-RRB-

For

example

the

intronic

region

in

Fig.

contains

bp

that

are

highly

conserved

in

human

mouse

and

rat

The

last

bp

of

this

alignment

block

are

identical

in

all

three

species

Peaks

in

regulatory

potential

score

are

correlated

with

conservation

score

and

in

the

highly

conserved

intronic

segment

they

are

higher

for

the

three-way

regulatory

potential

score

than

for

the

two-way

scores

using

human

and

just

one

These

data

are

illustrative

but

form

the

foundation

of

ongoing

efforts

to

identify

genome

sequences

involved

in

gene

regulation

Requiring

conservation

among

mammalian

genomes

greatly

increases

the

specificity

of

predictions

of

transcription

factor

binding

sites

Transcription

factor

databases

such

as

contain

known

transcription

factor

binding

sites

and

some

knowledge

of

their

distribution

but

simply

searching

a

sequence

with

these

motifs

provides

little

discriminatory

power

For

example

all

of

the

known

regulatory

and

functional

have

TRANSFAC

matches

but

so

do

of

the

mammalian

ancestral

repeats

most

representing

false-positive

predictions

The

introduction

of

conservation

as

a

criterion

for

regulatory

element

identification

greatly

increases

specificity

with

only

a

modest

cost

in

sensitivity

If

we

insist

that

the

TRANSFAC

matches

be

present

and

orthologously

aligned

in

all

three

species

human

mouse

and

rat

then

only

matches

are

recorded

in

ancestral

repeats

-LRB-

-RRB-

while

-LRB-

-RRB-

of

the

above

matches

in

known

regulatory

elements

and

-LRB-

-RRB-

in

functional

promoters

are

retained

Overall

using

a

set

of

weight

matrices

for

transcription

factors

extracted

from

we

find

matches

in

the

April

reference

human

genome

sequence

but

this

was

reduced

to

only

by

demanding

conservation

in

the

human

mouse

rat

three-way

alignments

This

is

a

increase

in

specificity

We

examined

one

region

in

more

detail

a

complex

cis-regulatory

region

consisting

of

a

bp

segment

containing

two

regulatory

modules

hypersensitive

sites

and

from

the

locus

control

region

of

the

HBB

Considerable

experimental

work

has

identified

six

functional

binding

sites

for

the

transcription

factor

in

this

segment

Requiring

that

matches

to

binding

sites

be

conserved

in

all

three

species

and

occur

within

regions

of

strong

regulatory

potential

is

sufficient

to

find

these

six

functional

binding

sites

and

only

these

six

in

the

bp

segment

Thus

in

this

example

we

observed

complete

sensitivity

and

specificity

by

requiring

this

level

of

conservation

Pseudogenes

and

gene

loss

To

complement

the

identification

and

analysis

of

protein-coding

regions

we

sought

to

examine

rat

pseudogenes

Using

a

previously

described

we

found

pseudogenes

in

intergenic

regions

Pseudogenes

are

normally

not

subjected

to

selective

con

Table

Recent

gene

duplications

-LRB-

K

S

-RRB-

in

the

rat

lineage

Cluster

ID

249 640 346 190

578 400 743

500 51 256 488

248 393 522 550

79 88 109 294

310 353 399 638

690 239 253 274

297 523 746

Recent

duplication

events

Numbers

of

genes

involved

Extant

cluster

size

Ancestral

cluster

size

Chromosome

Annotation

Process

Duplications

involving

retroviral

genes

fragmented

genes

with

internal

repeats

and

likely

pseudogene

clusters

were

removed

from

this

list

Only

gene

clusters

exhibiting

at

least

three

duplications

are

shown

Immunoglobulin

k-chain

V

TCR

a-chain

V

Immunoglobulin

heavy

chain

V

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Glandular

kallikrein

Vomeronasal

receptor

Olfactory

receptor

Granzyme

serine

protease

Trace

amine

receptor

GPCR

Vomeronasal

receptor

Olfactory

receptor

Keratin-associated

protein

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

Olfactory

receptor

urinary

protein

RNase

A

Prolactin

paralogue

Prolactin-induced

protein

Camello-like

N-acetyltransferase

lectin

natural

killer

cell

protein

Interferon-a

Keratin-associated

protein

MHC

class

-LRB-

-RRB-

Immunity

Immunity

Immunity

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Reproduction

Chemosensation

Chemosensation

Immunity

Neuropeptide

receptors

Chemosensation

Chemosensation

Epithelial

cell

function

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Chemosensation

Immunity

Reproduction

Reproduction

Developmental

regulator

Immunity

Immunity

Epithelial

cell

function

Chemosensation

Nature

Publishing

Group

NATURE

VOL

APRIL

articles

straint

and

therefore

accumulate

sequence

modifications

neutrally

Indeed

nearly

all

of

our

identified

pseudogenes

-LRB-

-RRB-

evolved

under

neutrality

according

to

a

K

A/K

S

test

and

therefore

are

consistent

with

being

pseudogenic

We

classified

these

pseudogenes

according

to

whether

they

arose

from

retrotransposition

in

which

case

they

integrated

into

the

genome

randomly

or

whether

they

arose

from

tandem

duplication

and

neutral

sequence

substitution

Using

human

rat

synteny

we

found

that

of

pseudogenes

exhibited

no

significant

similarity

to

the

corresponding

human

orthologous

region

and

therefore

were

considered

retrotransposed

processed

pseudogenes

The

total

pseudogene

count

and

processed

pseudogene

proportion

are

consistent

with

those

found

for

These

numbers

are

greater

than

those

previously

reported

for

However

reanalysis

using

the

method

employed

here

detects

a

similar

pseudogene

number

-LRB-

-RRB-

to

that

found

for

human

and

rat

This

suggests

that

the

rate

of

pseudogene

creation

is

similar

among

these

mammals

As

with

the

human

the

largest

group

of

rat

pseudogenes

-LRB-

totalling

-RRB-

according

to

consists

of

ribosomal

protein

genes

Other

large

rat

pseudogene

families

arose

from

olfactory

receptors

-LRB-

see

below

-RRB-

dehydrogenase

-LRB-

GAPDH

-RRB-

-LRB-

-RRB-

protein

kinases

-LRB-

-RRB-

and

RNA

binding

proteins

-LRB-

-RRB-

Pseudogenes

homologous

to

a

meiotic

spindle-associated

protein

are

particularly

numerous

in

rat

-LRB-

at

least

copies

-RRB-

compared

with

mouse

-LRB-

approximately

three

copies

-RRB-

This

suggests

that

spindlin

pseudogenes

may

have

distributed

rapidly

by

a

recently

active

transposable

element

We

investigated

the

much-studied

metabolic

enzyme

and

observed

that

-LRB-

-RRB-

the

GAPDS

gene

arose

from

a

duplication

of

the

GAPDH

gene

-LRB-

-RRB-

biogenesis

of

the

GAPDH

pseudogenes

has

been

occurring

steadily

over

time

both

before

and

after

rodent

human

and

mouse

rat

divergence

and

-LRB-

-RRB-

the

GAPDS

gene

has

undergone

little

retrotransposition

in

all

three

genomes

compared

with

its

relative

the

GAPDH

gene

-LRB-

consistent

with

respective

gene-expression

levels

in

the

germ

line

-RRB-

In

situ

loss

of

rat

genes

As

an

organism

evolves

its

need

for

certain

genes

may

be

reduced

or

lost

owing

to

changes

in

its

ecological

niche

Loss

of

selective

constraints

leads

to

accumulation

of

nonsense

and/or

frameshift

mutations

without

retrotransposition

or

duplication

These

nonprocessed

pseudogenes

are

interesting

because

they

link

environmental

changes

to

genomic

mutation

events

However

predicted

pseudogenes

with

disrupted

reading

frames

might

also

be

indicative

of

errors

in

genome

sequence

or

assembly

By

constraining

the

search

to

orthologous

genomic

regions

we

identified

rat

putative

non-processed

pseudogenes

-LRB-

Table

-RRB-

with

apparently

functional

single

human

and

mouse

orthologues

Half

of

these

contain

one

inframe

stop

or

frameshift

whereas

the

remainder

contain

more

We

expect

this

number

of

identified

pseudogenic

orthologues

to

be

conservative

because

the

methods

employed

required

high

fidelity

of

both

gene

prediction

and

orthologue

identification

in

all

three

species

-LRB-

Methods

-RRB-

Nevertheless

as

only

recently

evolved

pseudogene

candidates

were

identified

this

indicates

that

the

genome

sequence

and

assembly

-LRB-

-RRB-

is

of

high

quality

The

improved

quality

of

the

most

recent

assembly

is

underscored

by

additional

candidate

pseudogenes

predicted

from

rat

assembly

that

are

apparently

functional

full-length

genes

in

Consequently

some

of

the

current

candidates

in

particular

those

that

are

involved

in

fundamental

processes

of

eukaryotic

biology

may

yet

be

repaired

by

sequence

changes

in

future

assemblies

and

thus

be

recognized

as

genic

However

genes

associated

with

innate

immunity

-LRB-

which

is

particularly

susceptible

to

change

via

adaptive

evolution

-RRB-

such

as

Forssman

glycolipid

synthetase

and

complement

factor

I

may

yet

be

found

to

survive

as

true

pseudogenes

in

the

rat

Non-coding

RNA

genes

Figure

Close-up

of

-LRB-

peroxisomal

membrane

protein

-RRB-

locus

on

human

chromosome

-LRB-

with

homologous

mouse

chromosome

and

rat

chromosome

-RRB-

Conservation

score

computed

on

three-way

human

mouse

rat

alignments

-LRB-

parsimony

P

-RRB-

presents

a

clear

coding

exon

peak

-LRB-

grey

bar

-RRB-

and

very

high

values

in

a

bp

non-coding

intronic

segment

-LRB-

right

last

bp

of

alignment

are

identical

in

all

three

organisms

-RRB-

The

latter

segment

showed

a

striking

difference

between

the

inferred

mouse

and

rat

branch

the

grey

bracket

corresponds

to

a

phylogenetic

tree

where

the

logarithm

of

mouse

to

rat

branch-length

ratio

is

Regulatory

potential

that

discriminate

between

conserved

regulatory

elements

and

neutrally

evolving

DNA

are

calculated

from

three-way

-LRB-

human

mouse

rat

-RRB-

and

two-way

-LRB-

human

rodent

-RRB-

alignments

Here

the

three-way

regulatory

potential

scores

are

enhanced

over

the

two-way

scores

NATURE

VOL

APRIL

We

investigated

the

abundance

and

distribution

of

non-coding

-LRB-

nc

-RRB-

RNAs

in

rat

Cytoplasmic

transfer

-LRB-

t

-RRB-

RNA

gene

identification

in

rodents

is

complicated

by

tRNA-derived

identifier

-LRB-

ID

-RRB-

short

interspersed

nucleotide

-LRB-

SINEs

-RRB-

-LRB-

and

ID

-RRB-

tRNAscan-SE

predicted

tRNAs

-LRB-

genes

and

pseudogenes

-RRB-

however

the

majority

-LRB-

-RRB-

were

SINEs

identified

by

RepeatMasker

This

is

far

greater

than

the

number

found

in

mouse

-LRB-

-RRB-

or

human

-LRB-

-RRB-

Of

the

remaining

predictions

were

annotated

as

tRNA

pseudogenes

and

four

were

annotated

as

undetermined

by

tRNAscan-SE

An

additional

predictions

were

removed

because

their

best

database

match

in

either

human

mouse

or

rat

tRNA

databases

matched

tRNAs

with

either

a

different

amino

acid

or

anticodon

-LRB-

violating

the

wobble

rules

that

specify

the

distinct

anticodons

expected

-RRB-

The

total

of

tRNAs

-LRB-

including

a

single

selenocysteine

tRNA

-RRB-

identified

in

the

rat

genome

is

comparable

to

that

for

mouse

tRNAs

-LRB-

version

from

the

UCSC

genome

browser

-RRB-

and

human

tRNAs

-LRB-

from

the

genomic

tRNA

database

-RRB-

These

three

species

share

a

core

set

of

approximately

tRNAs

using

a

cutoff

of

sequence

identity

and

sequence

length

A

total

of

ncRNAs

-LRB-

other

than

tRNAs

-RRB-

were

identified

by

sequence

comparison

to

known

ncRNAs

-LRB-

Supplementary

Information

-RRB-

These

include

micro

-LRB-

mi

-RRB-

RNAs

five

ribosomal

RNAs

small

nucleolar

-LRB-

sno

-RRB-

RNAs

and

small

nuclear

-LRB-

sn

-RRB-

RNAs

various

other

ncRNAs

such

as

signal

recognition

particle

-LRB-

SRP

-RRB-

RNA

RNA

telomerase

RNA

RNase

P

RNA

brain-specific

repetitive

-LRB-

bsr

-RRB-

RNA

non-coding

transcript

abundantly

expressed

in

brain

-LRB-

ntab

-RRB-

RNA

small

cytoplasmic

-LRB-

sc

-RRB-

RNA

and

pseudogenes

Complete

and

rRNA

genes

and

more

rRNAs

were

not

identified

presumably

owing

to

assembly

issues

Nature

Publishing

Group

articles

Table

Candidate

rat

pseudogenes

orthologous

to

mouse

and

human

functional

genes

Mouse

gene

Human

gene

Strand

Rat

genome

coordinates

Frameshifts/stops

Annotation

Sorting

nexin

Dual-specificity

phosphatase

Diacylglycerol

kinase

d

Protein

kinase

PKNb

Forssman

glycolipid

synthetase

Zinc

finger

protein

Complement

factor

I

Ubiquitin-protein

ligase

-LRB-

-RRB-

Copine

-LRB-

membrane

trafficking

-RRB-

Cardiac

troponin

I

Zinc

finger

protein

Dysferlin-like

protein

Similar

to

yeast

RNA-binding

protein

Brush

border

kDa-like

protein

Coordinates

from

rat

Mouse

genes

were

used

as

templates

for

predicting

rat

pseudogenes

Evolution

of

transposable

elements

Most

interspersed

repeats

are

immobilized

copies

of

transposable

elements

that

have

accrued

substitutions

in

proportion

to

their

time

spent

fixed

in

the

genome

-LRB-

for

-RRB-

About

of

the

rat

genome

draft

is

identified

as

interspersed

repetitive

DNA

derived

from

transposable

elements

similar

to

that

for

the

-LRB-

Table

-RRB-

and

lower

than

for

the

human

-LRB-

almost

-RRB-

The

latter

difference

is

mainly

due

to

the

lower

substitution

rate

in

the

human

lineage

which

allows

us

to

recognize

much

older

-LRB-

Mesozoic

-RRB-

sequences

as

interspersed

repeats

Almost

all

repeats

are

derived

from

retroposons

elements

that

procreate

via

reverse

transcription

of

their

transcripts

As

in

mouse

there

is

no

evidence

for

activity

of

DNA

transposons

since

the

rat

mouse

split

Many

aspects

of

the

rat

and

the

mouse

genomes

repeat

structure

are

shared

here

we

focus

on

the

differences

activity

in

the

rat

lineage

The

long

interspersed

nucleotide

element

-LRB-

LINE

-RRB-

-LRB-

-RRB-

is

an

autonomous

retroelement

containing

an

internal

RNA

polymerase

II

promoter

and

two

open

reading

frames

-LRB-

ORFs

-RRB-

The

product

is

an

RNA

binding

protein

with

chaperone-like

activity

suggesting

a

role

in

mediating

nucleic

acid

strand

transfer

steps

during

reverse

whereas

encodes

a

protein

with

both

reverse

transcriptase

and

DNA

endonuclease

activity

LINEs

are

characteristically

truncated

so

that

only

a

small

subset

extends

to

include

the

promoter

region

and

can

function

as

a

source

for

more

copies

Many

classes

of

LINE-like

elements

exist

but

only

has

been

active

in

rodents

Over

half

a

million

copies

in

variable

stages

of

decay

comprise

of

the

rat

genome

Although

of

the

human

genome

is

comprised

of

copies

introduced

before

the

rodent

primate

split

owing

to

the

fast

substitution

rate

in

the

rodent

lineage

only

of

the

rat

genome

could

be

recognized

as

such

Thus

probably

well

over

one-quarter

of

all

rat

DNA

is

derived

directly

from

the

gene

Following

the

mouse

rat

split

activity

appears

to

have

increased

in

rat

The

UTR

sequences

defined

six

rat-specific

subfamilies

represented

by

copies

that

cover

of

the

rat

genome

copies

accumulated

over

the

same

period

in

mouse

cover

only

of

the

genome

-LRB-

Table

-RRB-

This

higher

accumulation

of

copies

could

explain

some

of

the

size

difference

of

the

rat

and

mouse

genome

In

addition

to

the

traditional

elements

there

are

copies

Table

Composition

of

interspersed

repeats

in

the

rat

genome

Rat

Copies

-LRB-

-RRB-

LINEs

SINEs

-LRB-

Alu

-RRB-

-LRB-

-RRB-

ID

MIR

LTR

elements

I

II

ERVL

-LRB-

III

-RRB-

MaLRs

DNA

elements

Charlie

-LRB-

hAT

-RRB-

Tigger

-LRB-

-RRB-

Unclassified

Total

Small

RNAs

Satellites

Simple

repeats

384 359 225 328

141 74 302 108

Mouse

Lineage-specific

-LRB-

-RRB-

Fraction

of

genome

-LRB-

-RRB-

Lineage-specific

-LRB-

-RRB-

Total

length

-LRB-

Mb

-RRB-

Fraction

of

genome

-LRB-

-RRB-

Data

for

and

October

mouse

-LRB-

-RRB-

excluding

Y

chromosome

using

the

December

version

of

RepeatMasker

To

highlight

the

differences

between

rat

and

mouse

repeat

content

columns

and

show

the

fractions

of

the

genomes

comprising

lineage-specific

repeats

The

numbers

include

all

copies

whereas

all

scRNA

and

diverged

tRNA-Ala

matches

far

more

common

than

other

small

RNA

pseudogenes

and

closely

related

to

ID

have

been

counted

as

ID

matches

Nature

Publishing

Group

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articles

-LRB-

Mb

-RRB-

of

a

non-autonomous

element

that

is

derived

from

by

deletion

of

most

of

its

A

similar

element

active

in

Mesozoic

times

has

been

called

-LRB-

for

Half-a-LINE

-RRB-

Given

their

low

divergence

we

conclude

that

the

currently

identified

elements

operated

only

a

few

million

years

ago

in

the

mouse

lineage

-LRB-

-RRB-

and

still

propagate

in

the

rat

genome

-LRB-

-RRB-

contains

only

an

whereas

encoded

an

endonuclease

as

well

although

no

reverse

transcriptase

The

bases

of

are

identical

to

the

currently

active

in

rat

-LRB-

or

-RRB-

Unlike

ancient

elements

which

shared

the

UTR

with

a

contemporary

the

end

of

is

unrelated

to

other

repeats

The

repeated

origin

and

high

copy

number

of

suggest

that

the

product

which

binds

strongly

to

its

messenger

may

render

this

transcript

a

superior

target

for

reverse

transcription

In

this

way

resembles

the

non-autonomous

endogenous

retrovirusderived

MaLR

elements

-LRB-

below

-RRB-

which

for

over

million

years

retained

only

the

retroviral

gag

ORF

that

encodes

an

RNA

binding

protein

A

potential

advantage

of

over

is

its

shorter

length

which

considering

the

usual

truncation

of

copies

increases

the

chance

that

a

copy

may

include

the

internal

promoter

elements

and

become

a

source

gene

Different

activity

of

SINEs

in

the

rat

and

mouse

lineage

Co-localization

of

SINEs

in

rat

and

mouse

The

most

successful

usurpers

of

the

retrotransposition

machinery

however

are

SINEs

These

are

small

RNA-derived

sequences

with

an

internal

RNA

polymerase

III

promoter

Recently

the

human

Alu

SINE

has

been

experimentally

proven

to

be

transposed

by

Most

SINEs

share

the

end

with

their

associated

LINE

elements

like

the

Mesozoic

mammalian

-LRB-

-RRB-

and

MIR

pair

increasing

the

efficiency

with

which

a

LINE

reverse

transcriptase

recognizes

the

end

of

a

dependent

SINE

However

does

not

show

sequence

specificity

and

rodent

and

primate

SINE

sequences

are

unrelated

to

Although

any

transcript

can

be

retroposed

as

can

be

seen

from

the

numerous

processed

pseudogenes

in

mammalian

genomes

SINEs

probably

have

features

that

make

them

especially

efficient

targets

of

the

reverse

transcriptase

Although

before

the

radiation

of

most

mammalian

orders

was

at

least

as

active

as

the

MIR

was

the

only

known

-LRB-

and

very

abundant

-RRB-

SINE

of

that

time

All

of

the

currently

active

SINEs

in

different

mammalian

orders

appear

to

have

arisen

after

the

demise

of

-LRB-

and

consequently

MIR

-RRB-

as

though

an

opportunity

-LRB-

or

necessity

-RRB-

arose

for

the

creation

and

expansion

of

other

SINEs

Four

different

SINEs

are

distinguished

in

rat

and

mouse

The

element

seems

to

share

its

origin

from

a

RNA

gene

with

the

primate

This

probably

happened

just

before

the

rodent

primate

split

and

after

the

speciation

from

most

other

eutherians

where

elements

are

not

known

The

other

SINEs

are

rodentspecific

and

have

tRNA-like

internal

promoter

regions

ID

elements

consist

only

of

this

tRNA-like

region

which

in

older

ID

copies

closely

match

an

Ala-tRNA

from

which

it

may

have

been

derived

resembles

a

fusion

of

an

ID

and

SINE

Finally

has

a

tRNA-like

region

of

unknown

affiliation

followed

by

a

unique

bp

region

The

fortunes

of

these

SINEs

during

mouse

and

rat

evolution

have

been

different

-LRB-

Fig.

-RRB-

probably

became

extinct

before

the

mouse

rat

speciation

while

has

remained

productive

in

both

lineages

scattering

copies

in

each

genome

after

this

time

Interestingly

the

fate

of

the

and

ID

SINEs

has

been

opposite

in

rat

and

mouse

While

is

still

active

in

mouse

having

left

over

mouse-specific

copies

in

its

trail

the

youngest

of

the

rat-specific

copies

are

diverged

from

their

source

indicating

a

relatively

early

extinction

in

the

rat

lineage

On

the

other

hand

after

the

mouse

rat

split

only

a

few

hundred

ID

copies

may

have

inserted

in

mouse

whereas

this

previously

minor

SINE

-LRB-

copies

predate

the

speciation

-RRB-

increased

its

activity

in

rat

to

produce

ID

copies

NATURE

VOL

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Despite

the

different

fates

of

SINE

families

the

number

of

SINEs

inserted

after

speciation

in

each

lineage

is

remarkably

similar

copies

Reminiscent

of

the

replacement

of

MIR

by

driven

SINEs

it

seems

that

the

demise

of

in

rat

allowed

the

expansion

of

IDs

Moreover

these

independently

inserted

and

unrelated

SINEs

-LRB-

ID

and

share

only

a

mechanism

of

retroposition

-RRB-

accumulated

at

orthologous

sites

the

density

of

ratspecific

SINEs

in

kb

windows

in

the

rat

genome

is

highly

correlated

-LRB-

R

-RRB-

with

the

density

of

mouse-specific

SINEs

in

orthologous

regions

in

mouse

To

avoid

including

elements

fixed

before

the

speciation

only

SINEs

labelled

lineagespecific

on

the

basis

of

subfamily

assignment

-LRB-

-RRB-

were

tallied

with

a

divergence

from

the

consensus

that

was

well

below

the

average

for

neutral

sites

-LRB-

Fig.

-RRB-

These

data

corroborate

and

refine

the

observation

of

a

strong

correlation

between

the

location

of

primate

and

rodent-specific

SINEs

in

Mb

At

kb

no

correlation

is

seen

for

interspersed

repeats

other

than

SINEs

Insertions

of

SINEs

at

the

same

location

in

different

species

have

been

and

the

correlation

could

reflect

the

existence

of

conserved

hotspots

for

SINE

insertions

However

only

five

of

human

specific

Alu

elements

have

an

Alu

inserted

within

bp

in

any

of

six

other

primate

Likewise

gene

conversions

of

shared

Alus

into

lineage-specific

copies

were

observed

five

times

in

the

same

set

too

low

a

level

to

contribute

significantly

to

the

observed

Figure

displays

the

lineage-specific

SINE

densities

on

rat

chromosome

and

in

the

mouse

orthologous

blocks

showing

a

stronger

correlation

than

any

other

feature

The

cause

of

the

unusual

distribution

patterns

of

SINEs

accumulating

in

gene-rich

regions

where

other

interspersed

repeats

are

scarce

is

apparently

a

conserved

feature

independent

of

the

primary

sequence

of

the

SINE

and

effective

over

regions

smaller

than

isochores

In

the

human

genome

the

most

recent

-LRB-

unfixed

-RRB-

Alus

are

distributed

similarly

to

whereas

older

copies

gradually

take

on

the

opposite

distribution

of

This

suggested

that

SINEs

insert

in

the

same

places

as

LINEs

and

that

the

typical

SINE

pattern

is

due

to

selection

-LRB-

or

deletion

bias

-RRB-

rather

than

a

mechanistic

insertion

bias

shared

by

all

-LRB-

unrelated

-RRB-

SINEs

but

not

by

LINEs

that

use

the

same

insertion

process

This

led

to

a

proposal

that

SINEs

are

preferentially

maintained

in

regions

where

they

can

easily

be

if

so

this

could

be

the

local

feature

conserved

between

mammalian

genomes

that

leads

to

the

strong

correlation

of

local

SINE

densities

in

different

mammals

However

we

did

not

observe

this

temporal

shift

in

SINE

distribution

pattern

in

mouse

nor

currently

in

the

rat

genome

despite

a

considerable

effort

to

define

the

potentially

unfixed

SINEs

in

both

species

-LRB-

see

ref

for

details

-RRB-

The

observations

in

human

could

reflect

a

recent

change

in

Alu

behaviour

which

would

necessitate

another

explanation

for

the

contrary

insertion-preference

of

older

Alus

and

all

other

SINEs

Some

regions

of

high

LINE

content

coincide

with

regions

that

exhibit

both

higher

AT

content

and

an

increased

rate

of

point

substitution

-LRB-

Fig.

pink

rectangles

-RRB-

In

a

genome-wide

analysis

LINE

content

correlates

strongly

with

substitution

rates

and

about

of

this

correlation

is

explained

by

higher

rates

in

AT-rich

SINE

density

shows

the

opposite

correlation

both

on

chromosome

-LRB-

Fig.

-RRB-

and

These

phenomena

in

conjunction

with

an

overall

trend

in

substitution

rates

towards

AT-richness

suggest

a

model

in

which

quickly

evolving

regions

accumulate

a

higher-than-average

AT

content

which

attracts

LINE

elements

Although

distinct

cause

effect

relationships

such

as

this

remain

largely

speculative

these

results

reinforce

the

idea

that

local

genomic

context

strongly

shapes

local

genomic

features

and

rates

of

evolution

Endogenous

retroviruses

and

derivatives

The

other

major

contributors

to

interspersed

repeats

in

the

rodent

Nature

Publishing

Group

articles

genome

are

retrovirus-like

elements

These

have

several

bp

long

terminal

repeats

-LRB-

LTRs

-RRB-

with

transcriptional

regulatory

sequences

that

flank

an

internal

sequence

that

in

autonomous

elements

encodes

all

proteins

necessary

for

retrotransposition

All

mammalian

LTR

elements

are

endogenous

retroviruses

-LRB-

ERVs

-RRB-

or

their

nonautonomous

derivatives

They

fall

into

three

groups

of

which

representatives

in

mouse

are

murine

leukaemia

virus

-LRB-

MuLV

-RRB-

-LRB-

class

I

-RRB-

intracisternal

A-particle

-LRB-

IAP

-RRB-

and

MMTV

-LRB-

class

II

-RRB-

and

MERVL

-LRB-

class

III

-RRB-

The

most

productive

retrovirus

in

mammals

has

been

the

class

III

element

ERV-L

primarily

through

its

ancient

non-autonomous

derivatives

called

MaLRs

with

copies

occupying

of

the

rat

genome

-LRB-

Table

-RRB-

Human

ERV-L

and

MaLR

copies

are

diverged

from

their

reconstructed

source

genes

and

must

have

died

out

around

the

time

of

human

speciation

from

New

World

monkeys

In

mouse

several

thousand

almost

identical

MaLR

and

ERV-L

copies

suggest

sustained

In

contrast

rat

ERV-L

activity

must

have

been

silenced

a

few

million

years

ago

given

that

the

least

diverged

MaLR

and

ERV-L

-LRB-

and

-RRB-

copies

differ

by

from

each

other

Other

class

III

ERVs

were

active

earlier

in

rodent

evolution

before

the

mouse

rat

speciation

In

contrast

to

class

III

ERVs

class

I

and

class

II

elements

still

thrive

in

rat

We

reconstructed

four

rat-specific

autonomous

class

I

ERVs

of

which

two

appear

still

active

and

nine

class

II

ERVs

of

which

four

may

still

be

active

The

non-autonomous

NICER

and

RAL

elements

represent

over

of

all

rat-specific

class

I

elements

The

autonomous

drivers

of

this

group

and

with

several

intact

copies

are

closely

related

to

the

mouse-specific

MuLV

Among

the

potentially

active

autonomous

class

II

ERVs

are

related

to

the

Mys

element

in

Peromyscus

and

several

IAP

elements

one

with

a

full-length

envelope

gene

The

most

prolific

still-active

class

II

ERV

is

distantly

related

to

the

simian

retroviruses

and

like

ERV-L

and

NICER

has

spawned

abundant

non-autonomous

elements

characterized

by

closely

related

LTRs

Simple

repeats

over

Remarkable

differences

were

noted

between

the

SSR

contents

of

the

human

and

mouse

Three

times

as

many

base

pairs

are

contained

in

near

-LRB-

-RRB-

perfect

SSRs

in

mouse

than

in

human

and

a

excess

was

revealed

when

excluding

SSRs

contained

in

or

seeded

by

interspersed

repeats

-LRB-

primarily

SSRs

derived

from

the

poly

A

or

simple

repeat

tails

of

SINEs

and

LINEs

-RRB-

SSRs

are

both

more

frequent

and

on

average

longer

in

mouse

Polypurine

-LRB-

or

polypyrimidine

-RRB-

repeats

are

especially

-LRB-

tenfold

-RRB-

over-represented

in

the

mouse

genome

As

discussed

above

this

contrasts

sharply

with

the

greater

frequency

of

triplet

repeats

coding

for

amino

acids

in

human

than

in

the

rodents

Rat

and

mouse

SSR

contents

show

perhaps

not

surprisingly

much

smaller

differences

They

represent

almost

the

same

amount

of

the

rat

and

mouse

genomes

-LRB-

for

perfect

elements

compared

with

in

human

-RRB-

and

are

of

similar

average

length

for

example

the

average

perfect

-LRB-

CA

-RRB-

n

repeat

the

most

common

SSR

in

mammals

is

bp

long

in

mouse

and

bp

in

rat

Some

potentially

significant

differences

are

that

polypurine

SSRs

are

of

similar

average

length

but

are

more

common

in

mouse

whereas

the

rare

SSRs

containing

CG

dimers

are

more

frequently

observed

in

rat

Whereas

the

above

interspersed

repeats

derive

from

transposed

sequences

mammalian

genomes

also

contain

interspersed

simple

sequence

repeats

-LRB-

SSRs

-RRB-

regions

of

tandemly

repeated

short

-LRB-

bp

-RRB-

units

that

probably

arise

from

slippage

during

DNA

replication

and

can

expand

and

compress

by

unequal

crossing

Figure

Historical

view

of

rodent

repeated

sequences

Relationships

of

the

major

families

of

interspersed

repeats

-LRB-

Table

-RRB-

are

shown

for

the

rat

and

mouse

genomes

indicating

losses

and

gains

of

repeat

families

after

speciation

The

lines

indicate

activity

as

a

function

of

time

Note

that

elements

appear

to

have

arisen

in

both

the

mouse

and

rat

lineages

Figure

Adaptive

remodelling

of

genomes

and

genes

a

Orthologous

regions

of

rat

human

and

mouse

genomes

encoding

pheromone-carrier

proteins

of

the

lipocalin

family

-LRB-

in

rat

and

major

urinary

proteins

in

mouse

-RRB-

shown

in

brown

zinc

finger

genes

are

shown

in

blue

Filled

arrows

represent

likely

genes

whereas

striped

arrows

represent

likely

pseudogenes

Gene

expansions

are

bracketed

Arrowhead

orientation

represents

transcriptional

direction

Flanking

genes

and

are

TSCOT

and

respectively

b

Site-specific

K

A/K

S

analysis

of

rat

Shown

in

red

are

side-chains

from

codons

subject

to

positive

selection

These

have

been

mapped

to

a

ribbon

representation

of

the

crystal

structure

of

rat

chain

A.

NATURE

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Nature

Publishing

Group

articles

b

Figure

Evolution

of

cytochrome

-LRB-

CYP

-RRB-

protein

families

in

rat

mouse

and

human

a

Dendrogram

topology

from

full-length

sequences

sequences

of

amino

acids

subfamily

names

and

chromosome

numbers

are

shown

Black

branches

have

bootstrap

support

Incomplete

sequences

-LRB-

they

contain

Ns

-RRB-

are

included

in

counts

of

functional

genes

-LRB-

rat

mouse

and

human

-RRB-

and

pseudogenes

-LRB-

including

fragments

not

shown

rat

mouse

and

human

-RRB-

rat

genes

and

pseudogenes

were

in

predicted

gene

sets

Human

is

a

allele

owing

to

an

in-frame

STOP

codon

in

the

genome

although

a

full-length

translation

exists

-LRB-

SwissProt

-RRB-

Rat

missing

in

the

genome

is

incomplete

because

there

is

a

RefSeq

entry

-LRB-

-RRB-

Grouped

subfamilies

and

occur

in

gene

clusters

thus

nine

loci

contain

multiple

functional

genes

in

a

species

One

-LRB-

-RRB-

has

fewer

rat

genes

than

human

seven

have

more

rodent

than

human

and

all

NATURE

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nine

differ

in

rodent

copy

numbers

is

a

rat-specific

subfamily

-LRB-

orthologues

are

pseudogenes

-RRB-

has

no

rodent

counterpart

Rodent-specific

expansion

rat

is

illustrated

below

b

The

neighbour-joining

with

the

single

human

gene

contains

clear

mouse

-LRB-

Mm

-RRB-

and

rat

-LRB-

Rn

-RRB-

orthologous

pairs

-LRB-

bootstrap

values

trials

shown

-RRB-

Bar

indicates

substitutions

per

site

c

All

rat

genes

have

a

single

mouse

counterpart

except

for

which

has

further

expanded

in

mouse

-LRB-

mouse

and

-RRB-

by

two

consecutive

single

duplications

The

genes

flanking

the

orthologous

regions

-LRB-

rat

chromosome

Mb

mouse

chromosome

Mb

human

chromosome

Mb

-RRB-

are

-LRB-

pink

-RRB-

and

nuclear

factor

I/A

-LRB-

NFIA

cyan

-RRB-

Genes

-LRB-

solid

-RRB-

and

gene

fragments

-LRB-

dashed

boxes

-RRB-

are

shown

above

-LRB-

forward

strand

-RRB-

and

below

-LRB-

reverse

strand

-RRB-

the

horizontal

line

No

orthology

relation

could

be

concluded

for

most

of

these

cases

Nature

Publishing

Group

articles

Figure

Comparative

analysis

of

rat

mouse

and

human

proteases

The

complete

nonredundant

set

of

proteases

and

protease

homologues

from

each

species

is

distributed

in

five

catalytic

classes

and

families

Each

square

represents

a

single

protease

and

is

coloured

according

to

its

presence

or

absence

in

rat

mouse

and

human

as

indicated

in

the

inset

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

Prevalent

medium-length

duplications

in

rodents

In

addition

to

the

transpositionally

derived

interspersed

repeats

and

simple

repeats

detected

by

RepeatMasker

and

Tandem

Repeat

Finder

the

rat

and

mouse

genomes

contain

a

substantial

amount

of

medium-length

unclassified

duplications

-LRB-

typically

bp

-RRB-

These

are

readily

seen

in

self-comparisons

and

in

intrarodent

comparisons

after

masking

the

known

repeats

but

they

are

substantially

less

prevalent

in

comparisons

with

the

human

genome

-LRB-

Supplementary

Information

-RRB-

Clearly

a

substantial

fraction

of

the

rodent

genomes

consists

of

currently

unexplained

repeats

and

a

full

characterization

awaits

further

studies

The

unclassified

duplications

may

include

-LRB-

-RRB-

novel

families

of

low-copy

rodent

interspersed

repeats

-LRB-

-RRB-

extensions

of

known

but

not

fully

characterized

rodent

repeats

and

-LRB-

-RRB-

duplications

generated

by

a

mechanism

different

from

transposition

Rat-specific

biology

A

principal

ambition

of

the

RGSP

was

to

reveal

genetic

differences

between

rats

and

mice

that

might

specify

their

differences

in

physiology

and

behaviour

This

view

was

well

supported

by

the

current

draft

sequence

and

predicted

gene

set

In

particular

recently

duplicated

genes

are

enriched

in

elements

involved

in

chemosensation

and

functional

aspects

of

reproduction

-LRB-

Table

-RRB-

Here

we

illustrate

the

differences

in

the

gene

complements

of

rat

and

mouse

by

in-depth

analyses

of

olfactory

receptors

-LRB-

ORs

-RRB-

pheromones

cytochromes

proteases

and

protease

inhibitors

Chemosensation

The

ability

to

emit

and

sense

specific

smells

is

a

key

feature

of

survival

for

most

animals

in

the

wild

Another

describes

the

evolution

of

rat

and

mouse

pheromones

vomeronasal

receptors

and

ORs

whose

genes

were

duplicated

frequently

during

the

time

since

the

common

ancestor

of

rats

and

mice

-LRB-

Table

-RRB-

Their

study

yielded

over

aligned

codons

predicted

to

have

been

subject

to

adaptive

evolution

They

attribute

the

rapid

evolution

of

these

genes

to

conspecific

competition

in

particular

sexual

selection

Using

a

homology-based

identification

procedure

with

manual

we

found

ORs

in

locations

in

the

rat

genome

multi-gene

clusters

and

single

genes

After

adjusting

for

missing

sequences

-LRB-

the

assembly

covers

of

the

genome

-RRB-

we

extrapolate

that

there

are

OR

genes

and

pseudogenes

The

rat

therefore

has

more

OR

genes

and

pseudogenes

than

the

ORs

of

the

assuming

similar

representation

of

recently

duplicated

sequences

in

the

two

genome

assemblies

used

Of

the

OR

sequences

that

are

not

interrupted

by

assembly

gaps

-LRB-

-RRB-

encode

intact

proteins

while

the

remaining

-LRB-

-RRB-

sequences

are

probably

pseudogenes

with

in-frame

stop

codons

frameshifts

and/or

interspersed

repeat

elements

Fewer

mouse

OR

homologues

are

pseudogenes

-LRB-

-RRB-

but

the

larger

family

size

in

rat

still

leaves

it

with

substantially

more

intact

ORs

than

the

mouse

-LRB-

versus

-RRB-

Striking

rat-specific

expansions

of

two

ancestral

clusters

account

for

much

of

the

difference

in

OR

family

size

and

pseudogene

content

between

rat

and

mouse

although

many

other

clusters

exhibit

more

subtle

changes

-LRB-

not

shown

-RRB-

Significant

differences

between

human

and

mouse

OR

families

have

also

been

but

the

functional

implications

of

OR

repertoire

size

on

the

ability

of

different

species

to

detect

and

discriminate

odorants

are

not

yet

known

pheromones

The

genes

are

odorant-binding

proteins

that

also

contribute

to

essential

survival

functions

in

animals

homologues

are

likely

to

be

highly

heterogeneous

among

murid

species

Several

homologues

-LRB-

major

urinary

proteins

-RRB-

sequenced

from

the

BALB/c

mouse

are

distinct

from

their

mouse

counterparts

and

these

also

appear

to

be

arranged

differently

along

its

Moreover

two

full-length

genes

from

other

mouse

NATURE

VOL

APRIL

differ

from

their

orthologues

either

lacking

two

of

the

bases

or

retaining

of

the

bases

that

render

the

sequences

likely

to

be

pseudogenes

-LRB-

not

shown

-RRB-

The

evolution

of

genes

on

rat

chromosome

has

clearly

driven

a

significant

remodelling

of

this

genomic

region

-LRB-

Fig.

-RRB-

The

orthologous

human

genomic

region

contains

a

single

homologue

suggesting

that

the

common

ancestor

of

rodents

and

human

possessed

one

gene

The

genome

of

mice

contains

four

homologous

genes

and

seven

pseudogenes

whereas

the

rat

genome

contains

ten

genes

and

pseudogenes

in

a

single

region

-LRB-

Fig.

-RRB-

Phylogenetic

trees

constructed

using

amino

acid

and

non-coding

DNA

sequences

show

that

surprisingly

the

rat

gene

clusters

appear

to

have

arisen

recently

via

a

rapid

burst

of

gene

duplication

since

the

rat

mouse

split

-LRB-

Table

data

not

shown

-RRB-

This

is

consistent

with

the

zinc-finger-like

pseudogene

having

uniquely

hitchhiked

for

virtually

all

of

the

rat-specific

gene

duplications

-LRB-

Fig.

-RRB-

The

sequences

of

these

genes

are

also

evolving

rapidly

with

median

K

A/K

S

values

of

and

for

rat

and

mouse

genes

respectively

Amino

acid

sites

that

appear

to

have

been

subject

to

adaptive

evolution

are

situated

both

within

the

ligand-binding

cavity

and

on

the

solvent-exposed

periphery

of

the

-LRB-

Fig.

-RRB-

This

demonstrates

how

genome

analysis

can

reveal

the

imprint

of

adaptive

evolution

from

megabase

to

single-base

levels

The

rapid

evolution

of

these

genes

and

the

remodelling

of

their

genomic

regions

can

be

attributed

to

the

known

roles

of

rat

and

mouse

major

urinary

proteins

in

conspecific

competition

and

sexual

selection

These

proteins

are

pheromones

and

pheromone

carriers

that

are

present

in

large

quantities

in

rodent

urine

and

act

as

scent

markers

indicating

dominance

and

subspecies

Detoxification

Cytochrome

is

a

well-recognized

participant

in

metabolic

detoxification

and

we

also

observe

rapid

evolution

within

this

family

These

enzymes

metabolize

a

large

number

of

toxic

and

endogenous

and

thus

are

particularly

relevant

to

clinical

and

pharmacological

studies

in

humans

As

rodents

are

important

model

organisms

for

understanding

human

drug

metabolism

it

is

important

to

identify

orthologues

and

species-specific

expansions

and

Compared

with

human

genes

there

are

clear

expansions

of

several

rodent

subfamilies

but

there

are

also

significant

differences

between

rat

and

mouse

subfamilies

-LRB-

Fig.

-RRB-

The

fastest-evolving

subfamily

seems

to

be

containing

a

single

gene

in

human

but

at

least

four

in

rat

and

eight

in

mouse

-LRB-

Fig.

c

-RRB-

enzymes

catalyse

the

NADPH-dependent

oxidation

of

arachidonic

acid

to

various

eicosanoids

which

in

turn

possess

numerous

biological

activities

including

modulation

of

ion

transport

control

of

bronchial

and

vascular

smooth

muscle

tone

and

stimulation

of

peptide

hormone

The

genomic

ordering

of

genes

and

their

phylogenetic

tree

indicate

an

ongoing

expansion

in

the

rodents

-LRB-

Fig.

c

-RRB-

This

suggests

that

adaptive

evolution

has

been

involved

in

diversifying

their

functions

Moreover

detailed

study

of

the

nuclear

receptors

a

highly

conserved

family

of

transcription

factors

revealed

that

PXR

and

CAR

two

nuclear

receptors

regulating

CYP

genes

involved

with

have

the

two

highest

nucleotide

substitution

rates

in

their

ligand

binding

domains

whereas

the

nuclear

receptor

regulating

-LRB-

ref

-RRB-

which

has

not

undergone

expansion

is

more

conserved

like

other

nuclear

Proteolysis

Protease

and

protease

inhibitor

genes

also

represent

an

example

of

rapid

evolution

in

the

rat

genome

Proteases

are

a

structurally

and

functionally

heterogeneous

group

of

enzymes

involved

in

multiple

biological

and

pathological

The

rat

contains

Nature

Publishing

Group

articles

protease

genes

of

the

rat

gene

more

than

human

-LRB-

-RRB-

but

similar

to

mouse

-LRB-

-RRB-

Of

the

rat

protease

genes

are

absent

from

human

and

are

absent

from

mouse

-LRB-

Fig.

-RRB-

Several

rat

gene

families

have

expanded

including

placental

cathepsins

testases

kallikreins

and

haematopoietic

serine

proteases

others

appear

to

have

formed

pseudogenes

in

humans

-LRB-

Table

-RRB-

These

protease

families

are

mainly

involved

in

reproductive

or

immunological

functions

and

have

evolved

independently

in

the

rat

and

mouse

lineages

The

rat

protease

inhibitor

complement

contains

members

similar

to

mouse

-LRB-

-RRB-

but

larger

than

human

-LRB-

-RRB-

As

with

the

protease

genes

the

rapid

evolution

in

protease

inhibitors

derives

from

differential

expansions

of

specific

families

such

as

serpins

and

cystatins

The

concomitant

expansions

in

rat

and

mouse

proteases

and

their

inhibitors

appear

to

reflect

homeostasis

of

protein

turnover

These

gene

family

expansions

dramatically

illustrate

how

largescale

genomic

changes

have

accompanied

species-specific

innovation

Positive

selection

of

duplicated

genes

has

afforded

the

rat

an

enhanced

repertoire

of

precisely

those

genes

that

allow

reproductive

success

despite

severe

competition

from

both

within

its

own

and

with

other

species

This

serves

as

a

general

illustration

of

the

importance

of

chemosensation

detoxification

and

proteolysis

in

innovation

and

adaptation

the

rat

human

orthologues

predicted

by

Ensembl

were

found

in

orthologous

genomic

regions

We

asked

if

these

disease

orthologue

pairs

were

distinguishable

from

other

rat

human

orthologues

Ensembl

automatically

predicts

that

human

genes

have

rat

orthologues

-LRB-

corresponding

to

of

all

Ensembl

predicted

human

genes

-RRB-

By

contrast

a

much

higher

proportion

-LRB-

-RRB-

of

human

disease

genes

have

Ensembl-predicted

rat

orthologues

Careful

analysis

of

the

remaining

human

genes

that

were

not

predicted

by

Ensembl

to

show

orthology

indicated

that

only

six

of

the

human

disease

genes

lack

likely

rat

orthologues

among

genome

cDNA

EST

and

protein

Thus

it

appears

that

in

general

genes

involved

in

human

disease

are

unlikely

to

have

diverged

or

to

have

become

duplicated

deleted

or

lost

as

pseudogenes

between

rat

and

human

-LRB-

conservation

of

orthologues

discussed

above

-RRB-

We

next

compared

K

S

K

A

and

the

K

A/K

S

ratio

values

of

disease

orthologues

with

those

of

all

remaining

orthologue

pairs

Only

the

K

S

distributions

differed

suggesting

that

coding

regions

of

human

disease

genes

and

their

rat

counterparts

have

mutated

more

rapidly

than

the

non-disease

genes

This

might

result

from

factors

influencing

the

specific

loci

or

the

disease

genes

may

characteristically

reside

in

genomic

regions

that

exhibit

higher

mutation

rates

The

disease

gene

set

was

next

grouped

into

disease-system

categories

and

analysed

using

a

non-parametric

test

for

K

A/K

S

-LRB-

human/rat

-RRB-

-LRB-

Fig.

-RRB-

Only

five

disease

systems

exhibited

significant

K

A/K

S

differences

with

respect

to

the

remaining

samples

-LRB-

P

-RRB-

Neurological

and

malformation-syndrome

disease

categories

manifested

the

lowest

median

K

A/K

S

ratios

that

are

consistent

with

purifying

selection

acting

on

these

gene

sets

With

a

comparison

of

the

mean

to

the

mean

and

standard

deviation

of

the

hypothesis

-LSB-

-LRB-

Mean

-RRB-

-RSB-

of

-LRB-

P

-RRB-

the

neurological

disease

gene

set

revealed

the

most

evidence

for

purifying

selection

of

the

disease

gene

categories

examined

In

contrast

the

pulmonary

haematological

and

immune

categories

manifested

the

highest

median

K

A/K

S

ratios

and

the

genes

of

the

immune

system

disease

category

with

a

value

for

-LRB-

Mean

-RRB-

of

-LRB-

P

-RRB-

show

the

highest

K

A

K

S

ratios

These

results

are

consistent

with

a

role

for

more

positive

selection

or

reduced

selective

constraints

among

these

genes

Where

possible

we

further

considered

conservation

of

these

pathophysiology-based

gene

sets

among

orthologues

of

more

diverse

phyla

including

mouse

fish

fly

nematode

worm

and

Human

disease

gene

orthologues

in

the

rat

genome

A

further

strong

motivation

for

sequencing

the

rat

genome

was

to

enhance

its

utility

in

biomedical

research

Although

the

rat

is

already

recognized

as

the

premier

model

for

studying

the

physiological

aspects

of

many

human

diseases

it

has

not

had

as

prominent

a

role

in

the

study

of

simple

genetic

disease

traits

As

more

than

human

mendelian

disorders

now

have

associated

loci

and

alleles

there

is

now

a

tremendous

opportunity

to

link

the

new

knowledge

of

the

rat

genome

with

data

from

the

human

disease

examples

The

precise

identification

of

the

rat

orthologues

of

human

genes

that

are

mutated

in

disease

creates

further

opportunities

to

discover

and

develop

rat

models

Predicted

rat

genes

were

compared

with

well-characterized

human

disease

that

were

verified

and

classified

on

the

basis

of

pathophysiology

-LRB-

H.H.

E.E.W.

H.W.

K.G.W.

H.X.

L.G.

P.D.S.

D.N.C.

D.S.

M.M.A.

C.P.P.

and

K.F.

unpublished

work

-RRB-

As

predicted

by

Ensembl

-LRB-

-RRB-

have

orthologues

in

the

rat

These

predictions

are

likely

to

be

of

high

quality

because

of

Table

Protease-expanded

gene

families

and

pseudogenes

in

rat

mouse

and

human

genomes

Protease

Absent

genes

in

assembly

Expanded

families

Placental

cathepsins

Testins

Glandular

kallikreins

Mast

cell

chymases/granzymes

Human

pseudogenes

Chymosin

Distal

intestinal

serine

proteases

Pancreatic

elastase

Fertilins

and

reproductive

ADAMs

Testases

Testis

serine

proteases

Implantation

serine

proteases

Airway

trypsin-like

proteases

Rat

pseudogenes

Calpain

Pyroglutamyl-peptidase

II

transamidase

Aminopeptidase

MAMS/L-RAP

Carboxypeptidase

O

Procollagen

III

N-endopeptidase

and

Testis-specific

protein

Rat

gene

locus

from

-LRB-

-RRB-

genes

genes

genes

genes

gene

genes

gene

genes

various

loci

genes

genes

various

loci

genes

genes

ps

ps

ps

ps

ps

ps

ps

ps

Human

gene

locus

from

-LRB-

-RRB-

Absent

Absent

Absent

genes

ps

ps

ps

ps

various

loci

ps

ps

various

loci

ps

ps

gene

gene

gene

gene

gene

gene

genes

gene

Mouse

gene

locus

from

-LRB-

-RRB-

genes

genes

genes

genes

gene

genes

gene

genes

various

loci

genes

genes

various

loci

genes

genes

gene

gene

ps

ps

ps

ps

ps

gene

Reproduction

Reproduction

Reproduction

Host

defence

Digestion

Digestion

Digestion

Reproduction

Reproduction

Reproduction

Reproduction

Host

defence

Reproduction

Metabolism

Metabolism

Host

defence

Unknown

Metabolism

Reproduction

Reproduction

Function

Nature

Publishing

Group

NATURE

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articles

yeast

orthologues

Overall

we

obtained

results

consistent

with

those

reported

here

for

these

rat

human

orthologous

gene

disease

These

results

demonstrate

that

the

individual

genes

that

constitute

various

disease

systems

exhibit

significantly

different

average

evolutionary

rates

The

higher

evolutionary

rates

noted

for

the

immune

system

disease

genes

are

consistent

with

a

previous

finding

that

lymphocyte-specific

genes

evolve

relatively

and

may

indicate

rapid

diversification

of

the

functions

of

the

immune

systems

of

rodents

and

humans

This

is

expected

for

genes

involved

in

controlling

species-restricted

infectious

agents

if

strong

adaptive

pressure

acts

during

host

pathogen

co-evolution

Thus

the

results

of

studies

of

these

rodent

genes

may

be

less

directly

relevant

to

our

understanding

of

human

immune

system

diseases

than

results

obtained

for

other

pathophysiology

disease

systems

where

conservation

is

greater

and

purifying

selection

is

stronger

We

have

also

specifically

examined

a

number

of

genes

that

harbour

triplet

nucleotide

repeats

and

are

involved

in

human

neurological

disorders

such

as

Huntington

's

disease

a

condition

known

to

be

caused

by

CAG

triplet

repeat

expansion

producing

abnormally

long

polyglutamine

tracts

in

an

otherwise

normal

Analysis

of

the

rat

human

orthologues

of

these

disease

genes

indicated

that

repeat-expansion

disease

genes

exhibit

a

repeat

length

that

is

substantially

shorter

in

the

rat

than

that

found

in

the

normal

human

gene

-LRB-

Fig.

-RRB-

In

all

cases

human

disease

genes

localize

below

the

line

demarcating

length

correlation

showing

that

rat

orthologues

uniformly

bear

shorter

repeats

At

present

there

are

no

naturally

occurring

rat

strains

described

that

exhibit

neurological

disease

associated

with

repeat-expansion

mechanisms

The

shorter

repeat

length

of

these

orthologues

in

the

rat

would

be

consistent

with

either

the

lack

of

repeat-expansion

mutational

mechanisms

in

the

rat

or

the

failure

of

these

orthologues

to

achieve

a

critical

repeat

length

susceptible

to

such

mutational

mechanisms

Other

human

genes

not

at

present

known

to

be

associated

with

disease

also

contain

glutamine

repeats

that

are

much

shorter

in

the

rat

orthologues

and

thus

could

be

investigated

as

potential

disease

These

triplet-repeat-bearing

genes

may

be

susceptible

to

mutations

that

arise

through

repeat-expansion

mechanisms

In

Fig.

it

may

also

be

observed

that

a

relatively

high

proportion

of

repeats

are

significantly

longer

in

the

rat

than

in

their

corresponding

human

orthologue

In

addition

to

enabling

the

direct

comparison

of

rat

human

disease

orthologues

the

rat

genome

sequence

itself

is

an

invaluable

aid

for

the

discovery

of

additional

rat

genes

that

can

be

studied

as

disease

models

Two

general

modes

can

now

be

pursued

First

genes

underlying

disease

phenotypes

with

simple

inheritance

that

have

been

mapped

to

chromosomal

regions

can

be

more

easily

pursued

in

both

species

Indeed

the

rearrangements

of

conserved

segments

between

the

two

species

in

this

map

were

found

to

have

significant

value

because

they

tighten

the

boundaries

of

the

mapped

disease

regions

and

thus

reduce

the

number

of

genes

that

could

potentially

be

associated

with

a

given

disease

Second

the

identification

of

multiple

alleles

contributing

to

quantitative

and

complex

trait

differences

that

are

involved

in

disease

processes

can

be

pursued

with

more

accuracy

both

in

the

initial

association

phases

and

in

subsequent

efforts

to

detect

causative

alleles

Rat

single

nucleotide

polymorphisms

The

discovery

and

cataloguing

of

the

natural

DNA

variation

that

persists

between

individual

rat

strains

will

allow

further

research

using

rat

model

systems

Although

many

rat

microsatellites

have

been

characterized

and

studied

single

nucleotide

polymorphisms

-LRB-

SNPs

-RRB-

are

of

more

general

interest

because

of

their

probable

ubiquity

and

the

ease

with

which

they

can

be

assayed

SNP

data

have

three

broad

applications

-LRB-

-RRB-

the

individual

markers

can

be

used

in

ongoing

efforts

to

associate

phenotypes

that

have

complex

underlying

genetic

components

with

specific

sites

in

the

genome

-LRB-

-RRB-

A

panel

of

such

markers

can

be

used

in

conjunction

with

selective

breeding

and

chromosome

mechanics

to

generate

rat

strains

that

are

amenable

to

the

kinds

of

manipulations

that

will

hasten

the

discovery

of

important

alleles

-LRB-

-RRB-

A

set

of

such

markers

can

be

used

to

detail

the

history

of

the

different

genomic

events

that

have

led

to

the

structure

of

the

genomes

of

contemporary

rat

strains

A

detailed

map

of

these

events

has

a

utility

analogous

to

the

current

human

haplotype

-LRB-

HapMap

-RRB-

mapping

and

will

probably

Figure

Selective

constraints

differ

for

human

disease

systems

in

the

rat

genome

Human

disease

system

categories

showing

significant

differences

-LRB-

P

-RRB-

in

a

nonparametric

test

-LRB-

Mann

Whitney

Wilcoxon

-RRB-

comparing

K

A

K

S

-LRB-

human

rat

-RRB-

ratios

P

values

from

two-level

tests

between

genes

from

one

disease

system

and

the

remaining

genes

-LRB-

Mean

-RRB-

values

from

multi-level

tests

from

categorized

disease

systems

Negative

values

-LRB-

shown

in

yellow

and

orange

-RRB-

for

neurological

-LRB-

-RRB-

and

malformationsyndrome

-LRB-

-RRB-

categories

were

observed

to

be

consistent

with

K

A

K

S

ranges

in

which

purifying

selection

predominates

Immune

haematological

and

pulmonary

categories

show

positive

values

of

and

respectively

-LRB-

for

complete

data

set

and

details

see

ref

-RRB-

NATURE

VOL

APRIL

Figure

Polyglutamine

repeat

length

comparison

between

human

and

rat

Points

represent

protein

poly-Q

length

for

rat

and

human

Red

points

correspond

to

repeats

in

genes

associated

with

human

disease

spinocerebellar

ataxia

protein

or

spinocerebellar

ataxia

protein

MJD

Machado

Joseph

disease

protein

spinocerebellar

ataxia

protein

or

calcium

channel

alpha

subunit

isoform

DRPLA

dentatorubral

pallidoluysian

atrophy

protein

HD

Huntington

's

disease

protein

or

huntingtin

TBP

TATA

binding

protein

or

spinocerebellar

ataxia

protein

Repeat

lengths

over

ten

were

examined

green

shading

delineates

the

range

not

included

in

our

analysis

Also

noted

are

a

set

that

are

expanded

in

rat

and

human

-LRB-

black

circle

-RRB-

and

a

set

where

repeats

are

expanded

in

the

rat

Nature

Publishing

Group

articles

aid

disease

gene

identification

as

recently

suggested

for

the

The

draft

sequence

was

generated

primarily

from

DNA

of

a

single

inbred

rat

line

This

maximized

the

likelihood

of

deriving

an

accurate

sequence

assembly

but

reduced

any

likely

discovery

of

natural

variation

in

this

phase

of

the

project

As

a

consequence

there

has

been

no

large-scale

public

SNP

discovery

from

rat

genomic

sequencing

A

pilot

project

based

on

coding

-LRB-

c

-RRB-

SNP

discovery

has

been

initiated

as

these

cSNPs

represent

a

particularly

important

subset

of

variants

that

may

have

direct

functional

These

data

have

illustrated

both

immediate

applications

and

the

long-term

potential

for

an

effort

aimed

at

comprehensive

SNP

discovery

online

as

well

as

by

the

informally

assessed

increases

in

overall

genomic

research

of

this

model

The

expected

benefit

of

a

third

mammalian

sequence

providing

an

outgroup

by

which

to

discriminate

the

timing

of

events

that

had

already

been

noted

between

mouse

and

human

was

fully

realized

Using

the

three

sequences

and

other

partial

data

sets

from

additional

organisms

it

was

possible

to

measure

some

of

the

overall

faster

rate

of

evolutionary

change

in

the

rodent

lineage

shared

by

mice

and

rats

as

well

as

the

peculiar

acceleration

of

some

aspects

of

rat-specific

evolution

The

observation

of

specific

expanded

gene

families

in

the

rat

should

provide

material

for

targeted

studies

for

some

time

At

this

time

there

is

no

plan

to

further

upgrade

or

finish

the

rat

genome

sequence

This

programme

decision

is

a

consequence

of

the

high

cost

of

converting

draft

sequence

to

finished

data

and

the

pressing

need

to

analyse

new

genomes

However

as

the

distant

objective

of

very-low-cost

sequencing

or

other

advances

that

can

improve

draft

sequences

inexpensively

are

realized

it

might

be

envisioned

that

a

rat

sequence

that

approaches

the

quality

of

the

current

human

data

will

be

produced

A

finished

rat

genome

may

answer

many

questions

as

specific

clues

already

show

that

areas

of

the

genome

that

are

most

difficult

to

resolve

in

a

random

sequencing

project

are

also

those

areas

that

are

most

dynamic

and

therefore

of

high

potential

interest

in

an

evolutionary

context

Despite

the

advances

represented

here

we

are

clearly

still

at

the

beginning

of

the

full

analysis

of

the

mammalian

genome

and

its

complex

evolutionary

history

Much

of

the

additional

data

that

are

required

to

complete

this

story

will

be

from

other

genomes

distantly

related

to

rat

Nevertheless

a

considerable

body

of

data

remains

to

be

developed

from

this

species

In

addition

to

the

distant

prospect

of

a

finished

rat

genome

analysis

of

other

rat

strains

may

yield

genome-wide

polymorphism

data

while

targeted

efforts

to

generate

cDNA

clone

collections

will

provide

rat-specific

reagents

for

routine

use

in

research

Together

with

the

ongoing

efforts

to

fully

develop

methods

to

genetically

manipulate

whole

rats

and

provide

effective

gene

knockouts

the

current

and

future

rat

genome

resources

will

ensure

a

place

for

this

organism

in

genomic

and

biomedical

research

for

some

time

A

Conclusions

As

the

third

mammalian

genome

to

be

sequenced

the

rat

genome

has

provided

both

predictable

and

surprising

information

about

mammalian

species

Although

it

was

clear

at

the

outset

of

this

programme

that

ongoing

rat

research

would

benefit

from

the

resource

of

a

genome

sequence

there

was

uncertainty

about

how

many

new

insights

would

be

found

especially

considering

the

superficial

similarities

between

the

rat

and

the

already

sequenced

mouse

Instead

the

results

of

the

sequencing

and

analysis

have

generated

some

deep

insights

into

the

evolutionary

processes

that

have

given

rise

to

these

different

species

In

addition

the

project

has

been

invaluable

in

further

developing

the

methods

for

the

generation

and

analysis

of

large

genome

sequence

data

sets

The

generation

of

the

rat

draft

tested

the

new

combined

approach

for

large

genome

sequencing

As

the

overall

assembly

is

of

high

quality

there

is

no

doubt

that

this

overall

strategy

and

the

supporting

software

we

have

developed

provides

a

suitable

approach

for

this

problem

Because

we

included

a

BAC

skimming

component

in

the

underlying

data

set

the

assembly

recovered

a

fraction

of

the

genome

that

was

expected

by

analogy

to

the

mouse

project

to

be

difficult

to

assemble

from

pure

WGS

data

In

addition

the

BAC

skimming

component

allowed

progressive

generation

of

high-quality

local

assemblies

that

were

of

use

to

the

rat

research

community

as

the

project

developed

On

the

other

hand

although

the

BAC

component

used

here

was

far

less

expensive

than

the

fully

ordered

and

highly

redundant

set

used

in

the

hierarchical

approach

to

sequencing

the

human

genome

it

nevertheless

increased

the

overall

cost

of

data

production

relative

to

a

WGS

approach

The

issue

of

efficacy

of

WGS

versus

other

approaches

to

the

sequencing

of

large

genomes

remains

a

matter

of

earnest

scientific

debate

In

ongoing

projects

at

different

centres

that

participated

in

the

RGSP

consortium

different

approaches

are

being

used

to

tackle

new

genomes

These

include

pure

WGS

methods

the

combined

approach

and

variations

on

that

methodology

The

future

application

of

the

different

procedures

depends

on

the

target

genome

sizes

the

expected

degree

of

heterogeneity

-LRB-

that

is

polymorphism

-RRB-

in

the

organism

to

be

sequenced

and

the

preferences

of

the

individual

centre

So

far

all

the

genomes

that

have

been

analysed

by

RGSP

consortium

members

have

been

of

high

quality

and

we

anticipate

that

this

will

continue

as

the

benefits

and

disadvantages

of

different

approaches

are

further

studied

and

analysed

The

rat

genome

data

have

improved

the

utility

of

the

rat

model

enormously

Now

that

near-complete

knowledge

of

the

rat

gene

content

is

realizable

individual

researchers

have

a

data

source

for

the

rat

parts

list

that

can

be

explored

with

the

high

degree

of

confidence

and

precision

that

is

appropriate

for

biomedical

research

A

similar

improvement

has

been

made

in

the

resources

for

physical

and

genetic

mapping

because

the

relative

position

of

individual

markers

is

now

known

with

high

confidence

and

there

are

now

computational

resources

to

bridge

the

process

of

genetic

association

with

gene

modelling

and

experimental

investigation

These

advances

have

been

reflected

by

measured

increases

in

the

use

of

all

the

rat-specific

public

genome

data

sets

that

can

be

accessed

Methods

DNA

sequencing

and

data

access

Paired-end

reads

from

BAC

and

WGS

libraries

were

produced

as

previously

Unprocessed

sequence

reads

are

available

from

the

NCBI

Trace

Archive

-LRB-

ftp

ftp.ncbi.nih.gov

pub/TraceDB/rattus

norvegicus

-RRB-

raw

eBAC

assembly

data

are

available

from

the

BCM-HGSC

-LRB-

-RRB-

and

the

released

assembly

is

available

from

the

BCM-HGSC

-LRB-

-RRB-

the

NCBI

-LRB-

-RRB-

and

the

UCSC

-LRB-

http

genome.ucsc.edu/downloads.html

-RRB-

Genome

assembly

Assembly

of

the

rat

genome

by

the

Atlas

system

is

described

in

detail

Earlier

assemblies

-LRB-

-RRB-

of

the

initial

data

set

were

based

on

million

total

reads

and

BAC

skims

These

assemblies

spanned

Gb

and

comprised

over

ultrabactigs

with

N

of

over

Mb

They

differed

only

in

the

removal

of

short

artefactual

duplications

from

includes

another

BACs

selected

to

fill

gaps

in

Because

of

the

comprehensive

coverage

of

the

genome

by

it

was

used

for

the

initial

predictions

of

genes

and

proteins

BAC

fingerprints

An

agarose-gel-based

fingerprinting

was

employed

to

generate

HindIII

fingerprints

from

clones

in

the

BAC

library

The

contig

assembly

was

subjected

to

manual

review

and

editing

to

refine

clone

order

within

contigs

and

to

make

merges

between

contigs

using

tools

provided

in

the

FPC

Fingerprints

for

and

BACs

were

subsequently

added

to

allow

correlation

between

the

fingerprint

map

and

a

developing

YAC

map

of

the

rat

genome

BAC

and

PAC

clones

are

available

through

BACPAC

Resources

at

CHORI

-LRB-

bacpacorders@chori.org

-RRB-

BAC

PAC

and

YAC

maps

Markers

generated

from

BAC

and

PAC

clones

were

hybridized

against

-LRB-

R.D.

Pmatch

unpublished

software

-RRB-

and

radiation

hybrid

to

produce

independent

maps

that

were

subsequently

combined

Genetic

markers

from

two

rat

NATURE

VOL

APRIL

Nature

Publishing

Group

articles

genetic

and

the

radiation

hybrid

were

aligned

to

the

assembly

using

-LRB-

when

sequence

was

available

-RRB-

or

electronic

polymerase

chain

reaction

-LRB-

EPCR

-RRB-

Kuntz

C.

et

al.

Comparison

of

laparoscopic

versus

conventional

technique

in

colonic

and

liver

resection

in

a

tumor-bearing

small

animal

model

Surg

Endosc

-LRB-

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Kitagawa

K.

Hamada

Y.

Nakai

K.

Kato

Y.

Okumura

T.

Comparison

of

one

and

two-step

procedures

in

a

rat

model

of

small

bowel

transplantation

Transplant

Proc

-LRB-

-RRB-

Sauve

Y.

Girman

S.

V.

Wang

S.

Keegan

D.

J.

Lund

R.

D.

Preservation

of

visual

responsiveness

in

the

superior

colliculus

of

RCS

rats

after

retinal

pigment

epithelium

cell

transplantation

Neuroscience

-LRB-

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Wang

H.

et

al.

Attenuation

of

acute

xenograft

rejection

by

short-term

treatment

with

and

monoclonal

antibody

against

in

a

rat-to-mouse

cardiac

transplantation

model

Transplantation

-LRB-

-RRB-

Alves

A.

et

al.

Total

vascular

exclusion

of

the

liver

enhances

the

efficacy

of

retroviral-mediated

associated

thymidine

kinase

and

genes

transfer

against

multiple

hepatic

tumors

in

rats

Surgery

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Liu

M.

Y.

Poellinger

L.

Walker

C.

L.

Up-regulation

of

hypoxia-inducible

factor

in

renal

cell

carcinoma

associated

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loss

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tumor

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Cancer

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Jin

X.

et

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Effects

of

leptin

on

endothelial

function

with

OB-Rb

gene

transfer

in

Zucker

fatty

rats

Atherosclerosis

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Ravingerova

T.

Neckar

J.

Kolar

F.

Ischemic

tolerance

of

rat

hearts

in

acute

and

chronic

phases

of

experimental

diabetes

Mol

Cell

Biochem

-LRB-

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Taylor

J.

R.

et

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An

animal

model

of

Tourette

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syndrome

Am

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Psychiatry

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Smyth

M.

D.

Barbaro

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A.

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Surg

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P.

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healing

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T.

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Space

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et

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progression

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K.

et

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gene

expression

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Drugs

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M.

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improves

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Kostrubsky

V.

E.

et

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of

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of

bile

acid

transport

in

cultured

primary

human

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and

intact

rats

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Sci

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J.

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et

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the

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of

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and

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the

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Appl

Pharmacol

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Tam

R.

C.

et

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ribavirin

analog

ICN

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with

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of

both

immunomodulatory

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and

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of

hepatitis-induced

serum

alanine

aminotransferase

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Agents

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P.

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F.

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and

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of

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lansoprazole

in

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rats

exposed

from

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through

sexual

maturity

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Toxicol

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National

Institutes

of

Health

Network

for

Large-Scale

Sequencing

of

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Rat

Genome

khttp

grants/guide/rfa-files

htmll

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J.

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A

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of

the

rice

genome

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Oryza

sativa

L.

ssp

indica

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Science

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Goff

S.

A.

et

al.

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draft

sequence

of

the

rice

genome

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Oryza

sativa

L.

ssp

japonica

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Science

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M.

D.

et

al.

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genome

sequence

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melanogaster

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et

al.

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draft

genome

of

Ciona

intestinalis

insights

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and

vertebrate

origins

Science

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E.

W.

et

al.

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of

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E.

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G.

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H.

O.

Adams

M.

D.

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J.

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and

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of

the

human

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Natl

Acad

Sci

USA

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H.

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J.

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the

sequencing

of

the

human

genome

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Natl

Acad

Sci

USA

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H.

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E.

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Sulston

J.

E

More

on

the

sequencing

of

the

human

genome

Proc

Natl

Acad

Sci

USA

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author

reply

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P.

Whole-genome

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Proc

Natl

Acad

Sci

USA

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S.

et

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a

whole-genome

shotgun

assembler

Genome

Res

-LRB-

-RRB-

Finished

sequence

used

for

quality

assessment

of

the

assembly

To

assess

the

accuracy

of

the

Atlas

assembly

the

sequence

was

compared

to

Mb

of

sequences

that

had

been

finished

to

high

quality

Large-scale

rearrangements

We

compared

these

assemblies

Human

-LRB-

April

NCBI

build

-RRB-

Mouse

-LRB-

February

NCBI

build

-RRB-

and

Rat

-LRB-

June

-RRB-

Repeats

were

masked

using

RepeatMasker

-LRB-

A.S.

P.

Green

unpublished

work

see

-RRB-

and

Local

alignments

were

produced

using

-LRB-

Supplementary

Information

-RRB-

Repeat

contamination

was

removed

and

the

remaining

similarities

combined

into

two

and

threeway

and

synteny

blocks

produced

at

various

resolutions

using

Genome-wide

visualization

of

conserved

synteny

Pairwise

comparisons

of

the

genomes

of

human

mouse

and

rat

using

and

were

merged

into

blocks

of

conserved

and

the

images

were

displayed

using

the

Virtual

Genome

Painting

method

-LRB-

M.L.G.-G

et

al.

unpublished

work

http

-RRB-

Rat

segmental

duplications

Segmental

duplications

kb

were

identified

extracted

and

aligned

as

and

paralogous

sequence

relationships

were

assessed

using

PARASIGHT

visualization

software

-LRB-

J.A.B.

unpublished

work

Supplementary

Information

-RRB-

Venn

diagram

Pairwise

and

three-way

alignments

generated

using

and

or

were

analysed

to

classify

each

nucleotide

in

the

three

genomes

by

the

species

with

which

it

aligns

in

all

three

species

aligning

between

human

and

rat

-LRB-

but

not

mouse

-RRB-

between

human

and

mouse

-LRB-

but

not

rat

-RRB-

or

between

mouse

and

rat

-LRB-

but

not

human

-RRB-

Other

nucleotides

are

species-specific

unassigned

nucleotides

occupying

gaps

in

the

genome

assemblies

were

excluded

On

the

basis

of

output

from

and

nucleotides

were

assigned

to

categories

-LRB-

of

non-repetitive

repetitive

with

a

certain

ancestry

or

repetitive

but

unassigned

-RRB-

and

counted

See

Supplementary

Table

for

details

Gene

prediction

ENSEMBL

transcript

models

were

built

from

rodent

proteins

that

were

aligned

to

the

genome

using

a

combination

of

Pmatch

-LRB-

R.D.

unpublished

software

-RRB-

and

Models

based

on

vertebrate

proteins

were

added

in

regions

without

rodent-protein-based

models

UTRs

were

added

using

transcripts

built

from

different

rat

cDNAs

aligned

to

the

genome

using

BLAT

with

coverage

and

identity

This

procedure

-LRB-

as

but

without

GENSCAN

predictions

-RRB-

gave

rise

to

genes

and

transcripts

This

is

the

protein-based

gene

set

Rat

and

mouse

cDNA

and

rat

EST-based

gene

sets

were

also

built

See

Supplementary

Information

for

details

Non-processed

pseudogene

identification

Human

and

mouse

genes

related

by

orthology

and

lacking

an

apparent

rat

orthologue

were

considered

See

Supplementary

Information

for

details

High-resolution

analyses

of

chromosome

These

were

performed

predominantly

on

the

whole

genome

Plots

in

Fig.

were

generated

by

sliding

windows

of

width

Mb

and

a

step

size

of

kb

-LRB-

total

windows

-RRB-

See

Supplementary

Information

for

details

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December

accepted

February

doi

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C.

On

The

Origin

of

Species

by

Means

of

Natural

Selection

edn

Ch

-LRB-

John

Murray

London

-RRB-

International

Human

Genome

Sequencing

Consortium

Initial

sequencing

and

analysis

of

the

human

genome

Nature

-LRB-

-RRB-

Mouse

Genome

Sequencing

Consortium

Initial

sequencing

and

comparative

analysis

of

the

mouse

genome

Nature

-LRB-

-RRB-

Adkins

R.

M.

Gelke

E.

L.

Rowe

D.

Honeycutt

R.

L.

Molecular

phylogeny

and

divergence

time

estimates

for

major

rodent

groups

evidence

from

multiple

genes

Mol

Biol

Evol

-LRB-

-RRB-

Springer

M.

S.

Murphy

W.

J.

Eizirik

E.

O'Brien

S.

J.

Placental

mammal

diversification

and

the

Cretaceous-Tertiary

boundary

Proc

Natl

Acad

Sci

USA

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Canby

T.

Y

The

rat

lapdog

of

the

devil

Nat

Geogr

July

-LRB-

-RRB-

Robinson

R.

Genetics

of

the

Norway

Rat

-LRB-

Pergamon

Oxford

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Barnett

S.

A

The

Story

of

Rats

Their

Impact

on

Us

and

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on

Them

Ch

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and

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Crows

Nest

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Hedrich

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in

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Biol

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Mouchiroud

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rates

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not

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rate

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Biol

Evol

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Reddy

P.

S.

Housman

D.

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complex

pathology

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International

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Nature

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C.

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Nature

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J.

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H.

C

When

the

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goes

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disease-producing

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thaliana

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Nature

Genet

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M.

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throughput

fingerprint

analysis

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clones

Genome

Res

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Human

Genome

Mapping

Consortium

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physical

map

of

the

human

genome

Nature

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E.

A.

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a

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Genome

Res

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contigs

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Bioinformatics

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and

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rat

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VOL

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G.

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by

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PCR

Genome

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Benson

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repeats

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to

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sequences

Nucleic

Acids

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Blanchette

M.

et

al.

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multiple

genomic

sequences

with

the

threaded

blockset

aligner

Genome

Res

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Brudno

M.

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and

Multi-LAGAN

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tools

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of

genomic

DNA

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Brudno

M.

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whole-genome

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mouse

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J.

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Massa

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E.

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Birney

E.

Durbin

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Genome

Res

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Yang

Z.

Goldman

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Friday

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Pachter

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K-BROWSER

Genome

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N.

Nei

M

The

neighbor-joining

method

a

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trees

Mol

Biol

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Haldi

M.

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rat

genome

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Genome

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Acknowledgements

Work

at

Baylor

College

of

Medicine

was

supported

by

a

grant

from

the

NHGRI

and

NHLBI

to

R.A.G.

Work

at

Genome

Therapeutics

was

supported

by

grants

from

the

NHGRI

to

D.S.

A.S.

acknowledges

support

from

the

NIGMS

M.B.

acknowledges

support

from

the

NIH

N.H.

was

supported

by

the

NGFN/BMBF

-LRB-

German

Ministry

for

Research

and

Education

-RRB-

B.J.T.

and

J.M.Y.

are

supported

by

an

NIH

grant

from

the

NIDCD

K.M.R.

and

G.M.C.

are

Howard

Hughes

Medical

Institute

Predoctoral

Fellows

L.M.D

'S

K.M.

and

K.J.K.

are

supported

by

training

fellowships

from

the

W.

M.

Keck

Foundation

to

the

Gulf

Coast

Consortia

through

the

Keck

Center

for

Computational

and

Structural

Biology

Work

at

Case

Western

Reserve

was

supported

in

part

by

NIH

grants

to

E.E.E.

Work

at

IMIM

was

supported

by

a

grant

from

Plan

Nacional

de

I

D

-LRB-

Spain

-RRB-

M.M.A.

acknowledges

support

from

programme

Ramon

y

Cajal

and

a

grant

from

the

Spanish

Ministry

of

Science

and

Technology

Work

at

Universidad

de

Oviedo

was

supported

by

grants

from

the

European

Union

Obra

Social

Cajastur

and

Gobierno

del

Principado

de

Asturias

Work

at

Penn

State

University

was

supported

by

NHGRI

grants

Work

at

the

University

of

California

Berkeley

was

supported

by

a

grant

from

the

NIH

Work

at

the

Washington

University

School

of

Medicine

Genome

Sequencing

Center

and

the

British

Columbia

Cancer

Agency

Genome

Sciences

Centre

was

supported

by

an

NIH

grant

Work

at

UCSC

and

CHORI

was

supported

by

the

NHGRI

Competing

interests

statement

The

authors

declare

that

they

have

no

competing

financial

interests

Correspondence

and

requests

for

materials

should

be

addressed

to

R.A.G.

-LRB-

agibbs@bcm.tmc.edu

-RRB-

The

genomic

sequence

is

available

under

accession

numbers

to

in

the

international

sequence

databases

-LRB-

GenBank

DDBJ

and

EMBL

-RRB-

Supplementary

Information

accompanies

the

paper

on

Rat

Genome

Sequencing

Project

Consortium

-LRB-

Participants

are

arranged

under

area

of

contribution

and

then

by

institution

-RRB-

DNA

sequencing

Baylor

College

of

Medicine

Richard

A.

Gibbs

-LRB-

Principal

Investigator

-RRB-

George

M.

Weinstock

-LRB-

Co-principal

Investigator

-RRB-

Michael

L.

Donna

M.

Erica

J.

Steven

Graham

David

Kim

C.

Paula

E.

Geoffrey

Sandra

Lora

Christine

Oliver

Shannon

George

Margaret

Alicia

Rachel

Celera

Robert

A.

Holt

-LRB-

Principal

Investigator

-RRB-

Mark

D.

Peter

G.

Holly

Mary

Soo

Cheryl

A.

Steve

Carl

Anna

Zhiping

Don

Cheryl

L.

Trixie

Cynthia

M.

Cynthia

Granger

G.

J.

Craig

Trevor

Genome

Therapeutics

Douglas

Smith

-LRB-

Principal

Investigator

-RRB-

Hong-Mei

Erik

Patrick

Arnold

Lynn

Keith

Kim

University

of

Utah

Robert

B.

Weiss

-LRB-

Principal

Investigator

-RRB-

Diane

M.

NISC

Comparative

Sequencing

Program

NHGRI

Eric

D.

Robert

W.

Gerard

G.

BAC

library

production

Children

's

Hospital

Oakland

Research

Institute

Pieter

J.

de

Jong

-LRB-

Principal

Investigator

-RRB-

Kazutoyo

Baoli

BAC

fingerprinting

British

Columbia

Cancer

Agency

Canada

's

Michael

Smith

Genome

Sciences

Centre

Marco

Marra

-LRB-

Principal

Investigator

-RRB-

Jacqueline

Schein

-LRB-

Principal

Investigator

-RRB-

Ian

Chris

Steven

Martin

Carrie

Asim

Natasja

Genome

Sequencing

Center

Washington

University

School

of

Medicine

John

BAC

end

sequencing

TIGR

Shaying

Zhao

-LRB-

Principal

Investigator

-RRB-

Claire

M.

Jyoti

Sofiya

Keita

Yixin

Sofyia

William

C.

Sequence

assembly

Baylor

College

of

Medicine

Richard

A.

Gibbs

-LRB-

Principal

Investigator

-RRB-

George

M.

Weinstock

-LRB-

Principal

Investigator

-RRB-

Paul

H.

Rui

K.

James

Amy

Yanru

Xing-Zhi

Bingshan

Yue

Xiang

Analysis

and

annotation

Affymetrix

Simon

Baylor

College

of

Medicine

George

M.

Weinstock

-LRB-

Coordinator

-RRB-

Kim

C.

Worley

-LRB-

Overall

Coordinator

-RRB-

A.

J.

Richard

A.

Lisa

M.

Kirt

Jia

Qian

Manuel

L.

Andrew

R.

Kenneth

J.

Michael

P.

Aleksandar

Davinder

Andrei

David

A.

Zhengdong

Case

Western

Reserve

University

Jeffrey

A.

Evan

E.

Eray

EBI

Wellcome

Trust

Genome

Campus

Ewan

Emmanuel

Abel

Cara

EMBL

Heidelberg

Evgeny

Peer

Mikita

David

Fraunhofer-Chalmers

Research

Centre

for

Industrial

Mathematics

Gothenburg

Marina

Fred

Hutchinson

Cancer

Research

Center

Barbara

J.

Janet

M.

Genome

Therapeutics

Douglas

Smith

-LRB-

Principal

Investigator

-RRB-

Hui

Kim

Huajun

Heming

Keith

Incyte

Corporation

Sue

Darryl

Jeanette

Kristian

Ursula

Jim

Institut

Municipal

d'Investigacio

Medica

Barcelona

Francisco

M.

Mar

Josep

F.

Roderic

The

Institute

for

Systems

Biology

Arian

Lawrence

Berkeley

National

Laboratory

Inna

Edward

M.

Olivier

Alexander

Max

Delbruck

Center

for

Molecular

Medicine

Norbert

Detlev

Claudia

Oliver

Thomas

Young-Ae

Jan

Herbert

Heike

Nature

Publishing

Group

NATURE

VOL

APRIL

articles

Max

Planck

Institute

for

Molecular

Genetics

Berlin

Heinz

Hans

Medical

College

of

Wisconsin

Howard

J.

Jacob

-LRB-

Principal

Investigator

-RRB-

Susan

Jo

Michael

I.

Anne

E.

Jozef

Dean

Peter

J.

Simon

MRC

Functional

Genetics

Unit

University

of

Oxford

Chris

P.

Ponting

-LRB-

Leader

Genes

and

Proteins

Analysis

Group

-RRB-

Jose

M.

Stephen

Leo

Scott

A.

Richard

D.

Eitan

E.

Caleb

MWG-Biotech

Petra

Gerald

Pennsylvania

State

University

Margaret

Francesca

Laura

Pallavi

Ross

C.

Minmei

Diana

Kateryna

Webb

Anton

Cathy

Scott

James

Shan

Yi

Roche

Genetics

and

Roche

Center

for

Medical

Genomics

Klaus

Sanger

Institute

T.

Dan

Mario

Michele

Laura

Valerie

Richard

Eduardo

Stephen

M.

Stanford

University

Gregory

M.

Cooper

-LRB-

Co-Leader

Evolutionary

Analysis

Group

-RRB-

Serafim

Michael

Arend

Eric

A.

The

Center

for

the

Advancement

of

Genomics

J.

Craig

University

of

Arizona

Bret

A.

Universite

de

Montreal

Guillaume

Universidad

de

Oviedo

Carlos

Xose

S.

University

of

California

Berkeley

Kushal

Sourav

Colin

Lior

Nicolas

Von

Bing

Anat

University

of

California

San

Diego

Glenn

Pavel

A.

University

of

California

Santa

Cruz

David

Haussler

-LRB-

Co-Leader

Evolutionary

Analysis

Group

-RRB-

Krishna

M.

Robert

Hiram

Terrence

S.

Angie

S.

Donna

William

J.

Kate

R.

Heather

Matt

University

of

Wales

College

of

Medicine

David

N.

Peter

D.

University

of

Western

Ontario

Bin

Washington

University

Michael

Manimozhiyan

David

Wellcome

Trust

Centre

for

Human

Genetics

University

of

Oxford

Richard

R.

Martin

S.

The

Wistar

Institute

Harold

Uma

Scientific

management

Jane

Mark

Adam

Susan

Stephen

Francis

Affiliations

for

participants

Human

Genome

Sequencing

Center

Department

of

Molecular

and

Human

Genetics

Baylor

College

of

Medicine

MS

One

Baylor

Plaza

Houston

Texas

USA

khttp

British

Columbia

Cancer

Agency

Canada

's

Michael

Smith

Genome

Sciences

Centre

W

Avenue

Vancouver

British

Columbia

Canada

khttp

Celera

West

Gude

Drive

Rockville

Maryland

USA

Department

of

Genetics

and

the

Center

for

Computational

Genomics

Case

Western

Reserve

University

School

of

Medicine

Euclid

Avenue

Cleveland

Ohio

USA

DSM

Pharmaceuticals

Inc.

NW

Greenville

Blvd

Greenville

North

Carolina

USA

The

Institute

for

Biological

Energy

Alternatives

-LRB-

IBEA

-RRB-

Research

Blvd

Rockville

Maryland

USA

Intronn

Inc.

Clopper

Road

South

Building

Gaithersburg

Maryland

USA

The

Center

for

the

Advancement

of

Genomics

-LRB-

TCAG

-RRB-

Research

Blvd

Suite

Rockville

Maryland

USA

Avalon

Pharmaceuticals

SenecaMeadows

Parkway

Germantown

Maryland

USA

Basic

Immunology

Branch

Division

of

Allergy

Immunology

and

Transplantation

National

Institute

of

Allergy

and

Infectious

Diseases

-LRB-

NIAID

-RRB-

NIH

DHHS

Rockledge

Blvd

Room

Bethesda

Maryland

USA

DynPort

Vaccine

Company

LLC

Thomas

Jefferson

Drive

Frederick

Maryland

USA

Genome

Therapeutics

Corporation

Beaver

Street

Waltham

Massachusetts

USA

Agencourt

Bioscience

Corporation

Cummings

Center

Beverly

Massachusetts

USA

Department

of

Human

Genetics

University

of

Utah

Salt

Lake

City

Utah

USA

NIH

Intramural

Sequencing

Center

-LRB-

NISC

-RRB-

and

Genome

Technology

Branch

National

Human

Genome

Research

Institute

-LRB-

NHGRI

-RRB-

National

Institutes

of

Health

Bethesda

Maryland

USA

BACPAC

Resources

Children

's

Hospital

Oakland

Research

Institute

Street

Oakland

California

USA

khttp

bacpac.chori.orgl

Genome

Sequencing

Centre

Washington

University

School

of

Medicine

Forest

Park

Blvd

St

Louis

Missouri

USA

khttp

genome.wustl.edul

The

Institute

for

Genomic

Research

Medical

Center

Dr.

Rockville

Maryland

USA

khttp

Affymetrix

Vallejo

St

Suite

Emeryville

California

USA

Department

of

Cell

Biology

Baylor

College

of

Medicine

One

Baylor

Plaza

Houston

Texas

USA

EBI

Wellcome

Trust

Genome

Campus

Hinxton

Cambridgeshire

UK

EMBL

Meyerhofstrasse

Heidelberg

Germany

Max

Delbruck

Center

for

Molecular

Medicine

-LRB-

MDC

-RRB-

Experimental

Genetics

of

Cardiovascular

Disease

Robert-Rossle-Strasse

Berlin

Germany

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Fraunhofer-Chalmers

Research

Centre

for

Industrial

Mathematics

Chalmers

Science

Park

Gothenburg

Sweden

Division

of

Human

Biology

Fred

Hutchinson

Cancer

Research

Center

Fairview

Avenue

N.

Seattle

Washington

USA

khttp

labs/trask/l

Incyte

Corporation

Porter

Drive

Palo

Alto

California

USA

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Grup

de

Recerca

en

Informatica

Biomedia

Institut

Municipal

d'Investigacio

Medica

Universitat

Pompeu

Fabra

and

Programa

de

Bioinfomatica

i

Genomica

Centre

de

Regulacio

Genomica

C

Dr.

Aiguader

Barcelona

Catalonia

Spain

Computational

Biology

Group

The

Institute

for

Systems

Biology

North

Street

Seattle

Washington

USA

Genomics

Division

Lawrence

Berkeley

National

Laboratory

Cyclotron

Rd.

Berkeley

California

USA

khttp

US

Department

of

Energy

Joint

Genome

Institute

Mitchell

Drive

Walnut

Creek

California

USA

khttp

jgi.doe.govl

Max

Planck

Institute

for

Molecular

Genetics

Ihnestrasse

Berlin

Germany

Human

and

Molecular

Genetics

Center

Bioinformatics

Research

Center

and

Department

of

Physiology

Medical

College

of

Wisconsin

Milwaukee

Wisconsin

USA

Rat

Genome

Database

Bioinformatics

Research

Center

Medical

College

of

Wisconsin

Milwaukee

Wisconsin

USA

MRC

Functional

Genetics

Unit

University

of

Oxford

Department

of

Human

Anatomy

and

Genetics

South

Parks

Road

Oxford

UK

MWG-Biotech

Anzinger

Strasse

Ebersberg

Germany

Center

for

Comparative

Genomics

and

Bioinformatics

Huck

Institutes

of

Life

Sciences

Departments

of

Biology

Statistics

Biochemistry

and

Molecular

Biology

Computer

Science

and

Engineering

and

Health

Evaluation

Sciences

The

Pennsylvania

State

University

University

Park

Pennsylvania

USA

Roche

Genetics

and

Roche

Center

for

Medical

Genomics

F.

Hoffmann-La

Roche

Ltd

Basel

Switzerland

Sanger

Institute

The

Wellcome

Trust

Genome

Campus

Hinxton

Cambridgeshire

UK

Departments

of

Pathology

and

Genetics

Stanford

University

Stanford

California

USA

James

H.

Clark

Center

Department

of

Computer

Science

Stanford

University

Stanford

California

USA

Department

of

Ecology

and

Evolutionary

Biology

University

of

Arizona

Tucson

Arizona

USA

Centre

de

Recherches

Mathematiques

Universite

de

Montreal

Chemin

de

la

tour

Montreal

Quebec

Canada

khttp

Departamento

de

Bioquimica

y

Biologia

Molecular

Instituto

Universitario

de

Oncologia

Universidad

de

Oviedo

Oviedo

Spain

khttp

web.uniovi.es

degradomel

Department

of

Electrical

Engineering

and

Computer

Science

University

of

California

Berkeley

Berkeley

California

USA

Department

of

Mathematics

University

of

California

Berkeley

Berkeley

California

USA

Bioengineering

Graduate

Group

University

of

California

Berkeley

Berkeley

California

USA

University

of

California

San

Diego

Department

of

Mathematics

Gilman

Drive

San

Diego

California

USA

khttp

University

of

California

San

Diego

Department

of

Computer

Science

and

Engineering

Gilman

Drive

San

Diego

California

USA

khttp

Howard

Hughes

Medical

Institute

Center

for

Biomolecular

Science

Engineering

Mailstop

SOE

Baskin

School

of

Engineering

University

of

California

Santa

Cruz

High

Street

Santa

Cruz

California

USA

UCSC

Genome

Bioinformatics

Group

Center

for

Biomolecular

Science

and

Engineering

Mailstop

SOE

Baskin

School

of

Engineering

University

of

California

Santa

Cruz

High

Street

Santa

Cruz

California

USA

Institute

of

Medical

Genetics

University

of

Wales

College

of

Medicine

Heath

Park

Cardiff

UK

Department

of

Computer

Science

University

of

Western

Ontario

London

Ontario

Canada

Laboratory

for

Computational

Genomics

Campus

Box

Washington

University

St

Louis

Missouri

USA

khttp

genes.cse.wustl.edul

Wellcome

Trust

Centre

for

Human

Genetics

University

of

Oxford

Roosevelt

Drive

Oxford

UK

The

Wistar

Institute

Spruce

Street

Philadelphia

Pennsyvania

USA

US

National

Institutes

of

Health

National

Human

Genome

Research

Institute

Center

Drive

Bethesda

Maryland

USA

US

National

Institutes

of

Health

National

Heart

Lung

and

Blood

Institute

Bethesda

Maryland

USA

Program

in

Structural

and

Computational

Biology

and

Molecular

Biophysics

Baylor

College

of

Medicine

One

Baylor

Plaza

Houston

Texas

USA

NATURE

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APRIL

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