Vol

November

doi

ARTICLES

Global

variation

in

copy

number

in

the

human

genome

Richard

Shumpei

Karen

R.

Lars

George

H.

T.

Daniel

Heike

Michael

H.

Andrew

R.

Wenwei

Eun

Kyung

Stephanie

Jennifer

L.

Juan

R.

Monica

Jing

Dimitrios

Daisuke

Jeffrey

R.

Christian

R.

Rui

Lyndal

Kunihiro

Kohji

Fan

Martin

J.

Joelle

Armand

Cara

Fengtang

Junjun

Tatiana

Jane

Lluis

Donald

F.

Xavier

Chris

Nigel

P.

Hiroyuki

Charles

Keith

W.

Stephen

W.

Matthew

E.

Copy

number

variation

-LRB-

CNV

-RRB-

of

DNA

sequences

is

functionally

significant

but

has

yet

to

be

fully

ascertained

We

have

constructed

a

first-generation

CNV

map

of

the

human

genome

through

the

study

of

individuals

from

four

populations

with

ancestry

in

Europe

Africa

or

Asia

-LRB-

the

HapMap

collection

-RRB-

DNA

from

these

individuals

was

screened

for

CNV

using

two

complementary

technologies

single-nucleotide

polymorphism

-LRB-

SNP

-RRB-

genotyping

arrays

and

clone-based

comparative

genomic

hybridization

A

total

of

copy

number

variable

regions

-LRB-

CNVRs

-RRB-

which

can

encompass

overlapping

or

adjacent

gains

or

losses

covering

megabases

-LRB-

of

the

genome

-RRB-

were

identified

in

these

populations

These

CNVRs

contained

hundreds

of

genes

disease

loci

functional

elements

and

segmental

duplications

Notably

the

CNVRs

encompassed

more

nucleotide

content

per

genome

than

SNPs

underscoring

the

importance

of

CNV

in

genetic

diversity

and

evolution

The

data

obtained

delineate

linkage

disequilibrium

patterns

for

many

CNVs

and

reveal

marked

variation

in

copy

number

among

populations

We

also

demonstrate

the

utility

of

this

resource

for

genetic

disease

studies

Genetic

variation

in

the

human

genome

takes

many

forms

ranging

from

large

microscopically

visible

chromosome

anomalies

to

singlenucleotide

changes

Recently

multiple

studies

have

discovered

an

abundance

of

submicroscopic

copy

number

variation

of

DNA

segments

ranging

from

kilobases

-LRB-

kb

-RRB-

to

megabases

-LRB-

Mb

-RRB-

in

Deletions

insertions

duplications

and

complex

multi-site

collectively

termed

copy

number

variations

-LRB-

CNVs

-RRB-

or

copy

number

polymorphisms

-LRB-

CNPs

-RRB-

are

found

in

all

and

other

mammals

We

defined

a

CNV

as

a

DNA

segment

that

is

kb

or

larger

and

present

at

variable

copy

number

in

comparison

with

a

reference

A

CNV

can

be

simple

in

structure

such

as

tandem

duplication

or

may

involve

complex

gains

or

losses

of

homologous

sequences

at

multiple

sites

in

the

genome

-LRB-

Supplementary

Fig.

-RRB-

An

early

association

of

CNV

with

a

phenotype

was

described

yr

ago

with

the

duplication

of

the

Bar

gene

in

Drosophila

melanogaster

being

shown

to

cause

the

Bar

eye

CNVs

influence

gene

expression

phenotypic

variation

and

adaptation

by

disrupting

genes

and

altering

gene

and

can

cause

disease

as

in

microdeletion

or

microduplication

or

confer

risk

to

complex

disease

traits

such

as

infection

and

CNVs

often

represent

an

appreciable

minority

of

causative

alleles

at

genes

at

which

other

types

of

mutation

are

strongly

associated

with

specific

diseases

CHARGE

and

Parkinson

's

and

Alzheimer

's

Furthermore

CNVs

can

influence

gene

expression

indirectly

through

position

effects

predispose

to

deleterious

genetic

changes

or

provide

substrates

for

chromosomal

change

in

In

this

study

we

investigated

genome-wide

characteristics

of

CNV

in

four

populations

with

different

ancestry

and

classified

CNVs

into

different

types

according

to

their

complexity

and

whether

copies

have

been

gained

or

lost

-LRB-

Supplementary

Fig.

-RRB-

To

maximize

the

utility

of

these

data

and

the

potential

for

integration

of

CNVs

with

SNPs

for

genetic

studies

we

performed

experiments

with

the

International

HapMap

DNA

and

cell-line

derived

from

apparently

healthy

individuals

The

result

is

the

first

comprehensive

map

of

copy

number

variation

in

the

human

genome

which

provides

an

important

resource

for

studies

of

genome

structure

and

human

disease

Two

platforms

for

assessing

genome-wide

CNV

The

HapMap

collection

comprises

four

populations

parent

offspring

trios

of

the

Yoruba

from

Nigeria

-LRB-

YRI

-RRB-

parent

offspring

trios

of

European

descent

from

Utah

USA

-LRB-

CEU

-RRB-

unrelated

The

Wellcome

Trust

Sanger

Institute

Wellcome

Trust

Genome

Campus

Hinxton

Cambridge

UK

Science

and

and

High

Performance

Computing

Research

Center

for

Advanced

Science

and

Technology

University

of

Tokyo

Komaba

Meguro

Tokyo

Japan

Inc.

Santa

Clara

California

USA

The

Centre

for

Applied

Genomics

and

Program

in

Genetics

and

Genomic

Biology

The

Hospital

for

Sick

Children

MaRS

Centre

East

Tower

College

Street

Room

Toronto

Ontario

Canada

of

Molecular

and

Medical

Genetics

Faculty

of

Medicine

University

of

Toronto

Canada

of

Pathology

Brigham

and

Women

's

Hospital

Boston

Massachusetts

USA

and

Disease

Program

Center

for

Genomic

Regulation

Charles

Darwin

s/n

Barcelona

Biomedical

Research

Park

Barcelona

Catalonia

Spain

of

Medical

Genetics

and

Pediatrics

University

of

Alberta

Edmonton

Alberta

Canada

of

Human

Genetics

tim

University

of

Chicago

East

Street

Chicago

Illinois

USA

Fabra

University

Charles

Darwin

s/n

and

National

Genotyping

Centre

-LRB-

CeGen

-RRB-

Passeig

Mari

Barcelona

Biomedical

Research

Park

Barcelona

Catalonia

Spain

Science

and

Technology

Agency

Kawaguchi

Saitama

Japan

Medical

School

Boston

Massachusetts

USA

Nature

Publishing

Group

NATURE

Vol

November

ARTICLES

Japanese

from

Tokyo

Japan

-LRB-

JPT

-RRB-

and

unrelated

Han

Chinese

from

Beijing

China

-LRB-

CHB

-RRB-

Genomic

DNA

from

Epstein

Barrvirus-transformed

lymphoblastoid

cell-lines

was

used

Two

technology

platforms

were

used

to

assess

CNV

-LRB-

Fig.

-RRB-

-LRB-

-RRB-

comparative

analysis

of

hybridization

intensities

on

Affymetrix

GeneChip

Human

Mapping

early

access

arrays

-LRB-

EA

-RRB-

in

which

SNPs

were

analysed

and

-LRB-

-RRB-

comparative

genomic

hybridization

with

a

Whole

Genome

TilePath

-LRB-

WGTP

-RRB-

array

that

comprises

large-insert

clones

representing

of

the

euchromatic

portion

of

the

human

Stringent

quality

control

criteria

were

set

for

each

platform

and

experiments

were

repeated

for

individuals

on

the

WGTP

and

individuals

on

the

EA

platforms

The

quality

of

the

final

data

sets

was

assessed

by

the

standard

deviation

among

ratios

of

autosomal

probes

-LRB-

after

normalization

and

filtering

for

cell-line

artefacts

-RRB-

which

for

the

WGTP

platform

was

-LRB-

Supplementary

Fig.

-RRB-

and

for

the

EA

platform

was

both

of

which

are

improvements

on

published

The

different

nature

of

the

two

data

sets

required

the

development

of

distinct

algorithms

to

identify

CNVs

In

essence

these

algorithms

segment

a

continuous

distribution

of

intensity

ratios

into

discrete

regions

of

CNV

To

train

the

threshold

parameters

we

attempted

to

validate

experimentally

CNVs

that

had

been

defined

with

varying

degrees

of

confidence

in

two

well-characterized

-LRB-

and

-RRB-

By

performing

technical

replicate

experiments

on

both

platforms

we

assessed

the

proportion

of

CNV

calls

that

were

false

positives

for

different

algorithm

parameters

across

a

set

of

experiments

representing

the

spectrum

of

data

quality

The

threshold

parameters

for

both

algorithms

were

set

to

achieve

an

average

false-positive

rate

per

experiment

beneath

-LRB-

Methods

see

also

Supplementary

Methods

Supplementary

Tables

and

refs

-RRB-

Because

all

DNAs

were

derived

from

lymphoblastoid

cell

lines

we

differentiated

somatic

artefacts

-LRB-

such

as

culture-induced

rearrangements

and

aneuploidies

-RRB-

from

germline

CNVs

We

karyotyped

all

available

HapMap

cell

lines

-LRB-

Supplementary

Table

-RRB-

and

sought

evidence

for

chromosomal

abnormalities

in

the

WGTP

and

EA

intensity

data

We

identified

cell

lines

with

unusual

chromosomal

constitutions

-LRB-

Supplementary

Table

and

Supplementary

Fig.

-RRB-

and

removed

the

aberrant

chromosomes

from

further

analyses

Chromosomes

and

X

seemed

to

be

particularly

prone

to

trisomy

For

a

cell

line

with

mosaic

trisomy

of

chromosome

we

confirmed

by

array

comparative

genomic

hybridization

that

this

trisomy

was

not

apparent

in

blood

DNA

from

the

same

individual

-LRB-

Supplementary

Fig.

-RRB-

Furthermore

we

sought

signals

of

somatic

deletions

within

the

SNP

genotypes

of

HapMap

trios

A

somatic

deletion

in

a

parental

genome

manifests

as

a

cluster

of

SNPs

at

which

alleles

present

in

the

offspring

are

not

found

in

either

We

assessed

all

of

our

preliminary

CNV

calls

in

trio

parents

and

found

that

-LRB-

of

-RRB-

fell

in

genomic

regions

that

harbour

highly

significant

clusters

of

HapMap

Phase

II

SNP

genotypes

compatible

with

a

somatic

deletion

in

a

parental

genome

-LRB-

Supplementary

Table

Supplementary

Fig.

and

Supplementary

Note

-RRB-

These

putative

cell-line

artefacts

were

removed

from

further

analyses

Extrapolating

Comparative

genome

hybridization

Whole

Genome

TilePath

array

Reference

DNA

Test

DNA

Reference

DNA

Test

DNA

Test

DNA

Comparative

intensity

analysis

Affymetrix

early

access

SNP

chip

Test

DNA

StyI

Test

DNA

Test

DNA

StyI

NspI

NspI

Combine

chips

Genome

profile

-LRB-

test/reference

-RRB-

16 17 1819 20212

X

Y

Combine

chips

Compare

samples

Combine

dye-swaps

16 17 181920212

X

Chromosome

profile

-LRB-

test/reference

-RRB-

Chromosome

Chromosome

Mb

Mb

window

-LRB-

test/reference

-RRB-

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Mb

Figure

Protocol

outline

for

two

CNV

detection

platforms

The

experimental

procedures

for

comparative

genome

hybridization

on

the

WGTP

array

and

comparative

intensity

analysis

on

the

EA

platform

are

shown

schematically

-LRB-

see

Supplementary

Methods

for

details

-RRB-

for

a

comparison

of

two

male

genomes

-LRB-

and

-RRB-

The

genome

profile

shows

the

ratio

of

copy

number

in

these

two

genomes

chromosome-by-chromosome

The

EA

data

are

smoothed

over

a

fiveprobe

window

Below

the

genome

profiles

are

expanded

plots

of

chromosome

and

a

window

containing

a

large

duplication

in

identified

on

both

platforms

-LRB-

indicated

by

the

red

bracket

-RRB-

Nature

Publishing

Group

ARTICLES

NATURE

Vol

November

this

analysis

to

the

entire

HapMap

collection

suggests

that

less

than

of

the

deletions

we

observed

were

likely

to

have

been

somatic

artefacts

The

quality

of

resultant

CNV

calls

was

assessed

in

additional

Technical

replicate

experiments

-LRB-

triplicates

for

ten

individuals

-RRB-

demonstrated

that

CNV

calls

are

highly

replicable

-LRB-

Supplementary

Table

-RRB-

and

that

noisier

experiments

are

characterized

by

higher

false-negative

rates

rather

than

higher

false-positive

rates

-LRB-

Supplementary

Fig.

-RRB-

Heritability

of

CNVs

within

trios

was

investigated

at

biallelic

CNVs

at

which

CNV

genotypes

could

be

inferred

-LRB-

Fig.

see

also

Supplementary

Table

-RRB-

Of

biallelic

CNV

genotypes

only

exhibited

mendelian

discordance

which

probably

reflects

the

genotyping

error

rate

rather

than

the

rate

of

de

novo

events

at

these

loci

Additional

locus-specific

experimental

validation

was

performed

on

subsets

of

CNVs

-LRB-

Supplementary

Table

-RRB-

CNVs

called

in

only

a

single

individual

-LRB-

singleton

CNVs

-RRB-

are

more

likely

to

be

false

positives

compared

with

CNVs

identified

in

several

individuals

We

attempted

to

validate

singleton

CNVs

called

on

only

one

platform

-LRB-

from

each

platform

-RRB-

and

singleton

CNVs

called

on

both

platforms

All

singleton

CNVs

replicated

by

both

platforms

were

verified

as

true

positives

whereas

out

of

of

CNVs

called

by

only

one

platform

were

similarly

confirmed

-LRB-

false-positive

rate

of

-RRB-

Extrapolating

these

validation

rates

across

the

entire

data

set

suggests

that

only

-LRB-

multiplied

by

the

frequency

of

singleton

CNVs

called

on

only

one

platform

-RRB-

of

the

CNV

regions

we

identify

-LRB-

see

below

-RRB-

are

likely

to

be

false

positives

a

Frequency

ratios

A

genome-wide

map

of

copy

number

variation

The

average

number

of

CNVs

detected

per

experiment

was

and

for

the

WGTP

and

EA

platforms

respectively

-LRB-

Supplementary

Tables

-RRB-

Owing

to

the

nature

of

the

comparative

analysis

each

WGTP

experiment

detects

CNVs

in

both

test

and

reference

genomes

whereas

each

EA

experiment

detects

CNV

in

a

single

genome

The

median

size

of

CNVs

from

the

two

platforms

was

kb

-LRB-

WGTP

-RRB-

and

kb

-LRB-

EA

-RRB-

and

the

mean

size

was

kb

and

kb

respectively

Consequently

the

average

length

of

the

genome

shown

to

be

copy

number

variable

in

a

single

experiment

is

Mb

and

Mb

on

the

WGTP

and

EA

platforms

respectively

The

larger

median

size

of

the

WGTP

CNVs

partially

reflects

inevitable

overestimation

of

CNV

boundaries

on

a

platform

comprising

largeinsert

clones

as

CNV

encompassing

only

a

fraction

of

a

clone

can

be

detected

but

will

be

reported

as

if

the

whole

clone

was

involved

By

merging

overlapping

CNVs

identified

in

each

individual

we

delineated

a

minimal

set

of

discrete

copy

number

variable

regions

-LRB-

CNVRs

-RRB-

among

the

samples

-LRB-

Fig.

see

also

Supplementary

Table

-RRB-

We

identified

CNVRs

on

the

WGTP

platform

and

CNVRs

on

the

EA

platform

and

mapped

their

genomic

distribution

-LRB-

Fig.

-RRB-

Approximately

half

of

these

CNVRs

were

called

in

more

than

one

individual

and

of

all

CNVs

identified

on

one

platform

were

replicated

on

the

other

Combining

the

data

resulted

in

a

total

of

discrete

CNVRs

covering

-LRB-

Mb

-RRB-

of

the

human

genome

Using

locus-specific

quantitative

assays

on

a

subset

of

regions

we

validated

-LRB-

-RRB-

of

these

CNVRs

-LRB-

Supplementary

Tables

and

-RRB-

A

minority

-LRB-

-RRB-

of

these

CNVRs

overlapped

Figure

Heritability

of

five

CNVs

in

four

HapMap

trios

a

The

distribution

of

WGTP

ratios

at

five

CNVs

with

genotype

information

Each

histogram

of

ratios

in

HapMap

individuals

exhibits

three

clusters

each

corresponding

to

a

genotype

of

a

biallelic

CNV

with

the

two

alleles

depicted

by

broken

and

complete

bars

representing

lower

and

higher

copy

number

alleles

respectively

Red

lines

above

each

histogram

denote

ratios

in

the

individuals

represented

in

b.

b

Mendelian

inheritance

of

five

CNVs

in

four

parent

offspring

trios

The

individual

CNVs

were

genotyped

from

WGTP

clones

green

yellow

blue

red

black

b

Frequency

ratios

Frequency

ratios

Frequency

ratios

Frequency

ratios

Nature

Publishing

Group

NATURE

Vol

November

ARTICLES

Both

overlaps

threshold

One

overlap

threshold

One

overlap

threshold

Both

overlaps

threshold

Thresholds

WGTP

of

length

EA

of

SNPs

Individual

A

Individual

B

Individual

C

Individual

D

Individual

E

CNV

regions

-LRB-

CNVR

-RRB-

CNVs

both

overlaps

threshold

CNV

ends

enriched

for

breakpoints

Figure

Defining

CNVRs

CNVs

and

CNV

ends

Overlapping

CNVs

called

in

five

individuals

are

shown

schematically

for

four

loci

-LRB-

in

blue

-RRB-

dashed

lines

indicate

overlap

Copy

number

variable

regions

-LRB-

CNVRs

-RRB-

represent

the

union

of

overlapping

CNVs

-LRB-

in

green

-RRB-

Independent

juxtaposed

CNVs

-LRB-

in

black

-RRB-

are

identified

by

requiring

that

only

individual-specific

CNVs

that

overlap

by

more

than

a

threshold

proportion

be

merged

Intervals

encompassing

CNV

breakpoints

-LRB-

in

red

-RRB-

are

defined

using

platformdependent

criteria

-LRB-

Supplementary

Methods

-RRB-

and

contain

a

significant

paucity

of

recombination

-LRB-

Supplementary

Table

-RRB-

which

results

from

the

enrichment

of

segmental

duplications

within

which

fewer

inferred

recombination

hotspots

reside

those

identified

in

previous

Combining

different

classes

of

experimental

replication

revealed

that

-LRB-

-RRB-

of

the

CNVRs

detected

here

have

been

replicated

on

both

WGTP

and

EA

platforms

or

with

a

locus-specific

assay

or

in

another

individual

or

in

a

previous

study

-LRB-

Supplementary

Table

-RRB-

Wholegenome

views

of

CNV

show

that

although

common

large-scale

CNV

is

distributed

in

a

heterogeneous

manner

throughout

the

genome

-LRB-

Supplementary

Fig.

-RRB-

no

large

stretches

of

the

genome

are

exempt

from

CNV

-LRB-

Fig.

-RRB-

and

the

proportion

of

any

given

chromosome

susceptible

to

CNV

varies

from

to

-LRB-

Supplementary

Fig.

-RRB-

Gaps

within

the

reference

human

genome

assembly

have

an

extremely

high

likelihood

of

being

associated

with

CNVs

out

of

the

gaps

in

the

build

assembly

-LRB-

out

of

-RRB-

are

flanked

or

overlapped

by

CNVRs

This

finding

highlights

the

complexity

in

generating

a

reference

sequence

in

regions

of

structural

dynamism

and

emphasizes

the

need

for

ongoing

characterization

of

these

genomic

regions

Comparing

the

CNVRs

identified

on

the

two

platforms

reveals

that

the

WGTP

and

EA

platforms

largely

complement

one

another

The

EA

platform

is

better

at

detecting

smaller

CNVs

-LRB-

Supplementary

Fig.

-RRB-

whereas

the

WGTP

platform

has

more

power

to

detect

CNVs

in

duplicated

genomic

regions

-LRB-

Supplementary

Table

-RRB-

where

EA

coverage

is

Some

CNVRs

encompass

two

or

more

independent

juxtaposed

CNVs

For

example

a

small

deletion

found

in

one

individual

overlapping

a

much

larger

duplication

in

another

individual

was

merged

into

a

single

CNVR

despite

these

representing

distinct

events

To

delineate

independent

CNVs

-LRB-

CNV

events

-RRB-

we

applied

more

stringent

merging

criteria

to

separate

juxtaposed

CNVs

-LRB-

Fig.

-RRB-

and

identified

and

CNVs

on

the

WGTP

and

EA

X

Y

CNVR

not

associated

with

segmental

duplications

CNVR

associated

with

segmental

duplications

Call

frequency

CNVR

length

kb

kb

Mb

Figure

Genomic

distribution

of

CNVRs

The

chromosomal

locations

of

CNVRs

are

indicated

by

lines

to

either

side

of

ideograms

Green

lines

denote

CNVRs

associated

with

segmental

duplications

blue

lines

denote

CNVRs

not

associated

with

segmental

duplications

The

length

of

righthand

side

lines

represents

the

size

of

each

CNVR

The

length

of

left-hand

side

lines

indicates

the

frequency

that

a

CNVR

is

detected

-LRB-

minor

call

frequency

among

HapMap

samples

-RRB-

When

both

platforms

identify

a

CNVR

the

maximum

call

frequency

of

the

two

is

shown

For

clarity

the

dynamic

range

of

length

and

frequency

are

log

transformed

-LRB-

see

scale

bars

-RRB-

All

data

can

be

viewed

at

the

Database

of

Genomic

Variants

-LRB-

variation

-RRB-

Nature

Publishing

Group

ARTICLES

NATURE

Vol

November

platforms

respectively

-LRB-

Fig.

see

also

Supplementary

Table

-RRB-

We

classified

these

CNVs

into

five

types

-LRB-

-RRB-

deletions

-LRB-

-RRB-

duplications

-LRB-

-RRB-

deletions

and

duplications

at

the

same

locus

-LRB-

-RRB-

multi-allelic

loci

and

-LRB-

-RRB-

complex

loci

whose

precise

nature

was

difficult

to

discern

Owing

to

the

inherently

relative

nature

of

these

comparative

data

it

was

impossible

to

determine

unambiguously

the

ancestral

state

for

most

CNVs

and

hence

whether

they

are

deletions

or

duplications

Here

we

adopted

the

convention

of

assuming

that

the

minor

allele

is

the

derived

thus

deletions

have

a

minor

allele

of

lower

copy

number

and

duplications

have

a

minor

allele

of

higher

copy

number

Approximately

equal

numbers

of

deletions

and

duplications

were

identified

on

the

WGTP

platform

whereas

deletions

outnumbered

duplications

by

approximately

on

the

EA

platform

In

addition

homozygous

deletions

-LRB-

relative

to

the

reference

sequence

-RRB-

identified

on

the

EA

platform

were

experimentally

validated

with

locus-specific

assays

-LRB-

Supplementary

Table

-RRB-

Most

-LRB-

out

of

-RRB-

of

these

have

not

been

observed

in

a

previous

genome-wide

survey

of

To

investigate

mechanisms

of

CNV

formation

we

studied

the

sequence

context

of

sites

of

CNV

Non-allelic

homologous

recombination

can

generate

rearrangements

as

a

result

of

recombination

between

highly

similar

duplicated

Segmental

duplicaWGTP

-LRB-

SegDup

EA

-LRB-

SegDup

associated

-RRB-

-LRB-

-RRB-

Deletion

associated

-RRB-

-LRB-

-RRB-

Duplication

-LRB-

-RRB-

-LRB-

-RRB-

Deletion

duplication

Multi-allelic

Frequency

-LRB-

-RRB-

-LRB-

-RRB-

tions

are

defined

as

sequences

in

the

reference

genome

assembly

sharing

sequence

similarity

over

kb

with

another

genomic

We

found

that

of

the

CNVRs

were

associated

with

segmental

duplications

a

significant

enrichment

-LRB-

P

-RRB-

This

association

results

from

two

factors

-LRB-

-RRB-

rearrangements

generated

by

non-allelic

homologous

recombination

and

-LRB-

-RRB-

not

all

annotated

segmental

duplications

are

fixed

in

humans

but

are

in

fact

CNVs

This

latter

point

highlights

the

essentially

arbitrary

nature

of

defining

segmental

duplications

on

the

basis

of

a

single

genome

sequence

-LRB-

albeit

derived

from

several

individuals

-RRB-

The

likelihood

of

a

CNV

being

associated

with

segmental

duplications

depended

on

its

length

and

its

classification

multi-allelic

CNVs

complex

CNVs

and

loci

at

which

both

deletions

and

duplications

occurred

were

markedly

enriched

for

segmental

duplications

-LRB-

Fig.

see

also

Supplementary

Fig.

-RRB-

This

is

not

surprising

given

the

role

that

non-allelic

homologous

recombination

has

been

shown

to

have

in

generating

complex

structural

arrays

of

tandem

duplications

that

vary

in

and

reciprocal

deletions

and

The

likelihood

of

a

segmental

duplication

being

associated

with

a

CNV

was

greater

for

intrachromosomal

duplications

than

for

interchromosomal

duplications

and

was

highly

correlated

with

increasing

sequence

similarity

to

its

duplicated

copy

-LRB-

Supplementary

Fig.

-RRB-

Non-allelic

homologous

recombination

is

known

to

operate

mainly

on

intrachromosomal

segmental

duplications

and

to

require

sequence

similarity

between

duplicated

This

role

for

non-allelic

homologous

recombination

in

generating

CNVs

in

duplicated

regions

of

the

genome

is

supported

by

the

enrichment

of

segmental

duplications

within

intervals

that

probably

contain

the

breakpoints

of

the

CNV

-LRB-

Fig.

-RRB-

We

identified

CNVs

from

the

EA

platform

and

CNVs

from

the

WGTP

platform

that

contain

a

pair

of

segmental

duplications

one

at

either

end

These

pairs

of

segmental

duplications

were

biased

towards

high

-LRB-

-RRB-

sequence

similarity

and

were

more

frequently

associated

with

the

longest

CNVs

-LRB-

Supplementary

Fig.

-RRB-

In

addition

to

segmental

duplications

there

are

other

types

of

sequence

homologies

that

can

promote

non-allelic

homologous

recombination

for

example

dispersed

repetitive

elements

such

as

Alu

We

performed

an

exhaustive

search

for

sequence

homology

of

all

and

identified

CNVs

from

the

EA

platform

and

on

the

WGTP

platform

that

contain

lengths

of

perfect

sequence

identity

longer

than

bp

between

either

end

of

the

CNV

Genomic

impact

of

CNV

Deletions

are

known

to

be

biased

away

from

as

a

result

of

selection

In

contrast

the

selective

pressures

on

duplications

are

poorly

understood

the

existence

of

gene

families

pays

testament

to

positive

selection

acting

on

some

gene

duplications

over

longer-term

We

identified

the

different

classes

of

functional

sequence

that

fell

within

CNVRs

and

tested

whether

they

were

significantly

enriched

or

impoverished

within

these

CNVRs

compared

to

the

entire

genome

-LRB-

Table

see

also

Supplementary

Table

and

Supplementary

Methods

-RRB-

Table

Functional

sequences

within

CNVRs

Functional

sequence

WGTP

CNVRs

EA

CNVRs

Merged

CNVRs

-LRB-

-RRB-

-LRB-

-RRB-

Complex

-LRB-

-RRB-

ratios

-LRB-

-RRB-

Figure

Classes

of

CNVs

CNVs

identified

from

WGTP

and

EA

platforms

can

be

classified

from

the

population

distribution

of

ratios

-LRB-

exemplified

with

WGTP

data

-RRB-

into

five

different

types

-LRB-

see

text

-RRB-

Biallelic

CNVs

-LRB-

deletions

and

duplications

-RRB-

can

be

genotyped

if

the

clusters

representing

different

genotypes

are

sufficiently

distinct

The

numbers

of

each

class

of

CNV

identified

on

WGTP

and

EA

platforms

are

given

along

with

the

proportion

of

those

CNVs

that

overlap

segmental

duplications

The

overall

proportion

of

CNVRs

overlapping

segmental

duplications

was

and

on

the

EA

and

WGTP

platforms

respectively

RefSeq

genes

OMIM

genes

Ultra-conserved

elements

Conserved

non-coding

elements

Non-coding

RNAs

-LCB-

-LCB-

-LCB-

-LCB-

-LCB-

-LCB-

-LCB-

Statistical

significance

of

the

enrichment

or

paucity

of

functional

sequences

within

CNVRs

was

assessed

by

randomly

permuting

the

genomic

location

of

autosomal

CNVRs

-LRB-

Supplementary

Methods

-RRB-

Significant

observations

are

shown

in

bold

Note

that

both

conserved

non-coding

and

CNVRs

are

biased

away

from

genes

so

an

enrichment

of

conserved

non-coding

elements

in

CNVRs

is

not

unexpected

Significant

-LRB-

P

-RRB-

enrichment

-LCB-

Significant

-LRB-

P

-RRB-

paucity

Nature

Publishing

Group

NATURE

Vol

November

ARTICLES

It

is

not

possible

to

define

precisely

the

breakpoints

of

CNVRs

therefore

some

of

these

functional

sequences

might

flank

rather

than

be

encompassed

by

CNVRs

We

observed

a

significant

paucity

of

all

functional

sequences

-LRB-

with

the

exception

of

conserved

non-coding

-RRB-

in

CNVRs

detected

on

the

EA

platform

which

provided

the

highest

resolution

breakpoint

mapping

-LRB-

Table

-RRB-

Thus

CNVs

are

preferentially

located

outside

of

genes

and

ultra-conserved

elements

in

the

human

We

attempted

to

validate

experimentally

CNVs

containing

ultra-conserved

elements

Although

all

but

two

of

the

CNVs

validated

only

two

ultra-conserved

elements

actually

fell

within

these

CNVs

-LRB-

Supplementary

Table

-RRB-

so

the

selection

against

CNV

at

ultra-conserved

elements

is

likely

to

be

even

stronger

than

this

analysis

would

suggest

Nevertheless

thousands

of

putatively

functional

sequences

including

known

disease-related

genes

flank

or

fall

within

these

CNVs

over

half

-LRB-

-RRB-

of

the

CNVRs

overlap

known

RefSeq

genes

and

more

than

overlap

conserved

non-coding

We

examined

whether

deletions

or

duplications

are

equally

likely

to

encompass

these

different

classes

of

functional

sequences

We

observed

that

a

significantly

lower

proportion

of

deletions

than

duplications

-LRB-

identified

on

the

EA

platform

-RRB-

overlap

with

the

Online

Mendelian

Inheritance

in

Man

-LRB-

OMIM

-RRB-

database

of

disease-related

genes

-LRB-

P

chi-squared

-RRB-

and

RefSeq

genes

-LRB-

P

-RRB-

Thus

deletions

are

biased

away

from

genes

with

respect

to

duplications

The

same

trend

was

observed

with

ultra-conserved

elements

but

their

number

is

too

small

to

provide

statistical

significance

If

deletions

are

under

stronger

purifying

selection

-LRB-

which

removes

deleterious

variants

from

the

population

-RRB-

than

then

deletions

should

on

average

be

both

less

frequent

and

smaller

than

duplications

Although

on

average

deletions

were

almost

threefold

shorter

than

duplications

-LRB-

kb

versus

kb

from

EA

-RRB-

we

detected

no

significant

difference

in

the

frequencies

with

which

deletions

and

duplications

were

called

-LRB-

P

using

G-test

for

on

WGTP

data

-RRB-

We

note

that

our

length

analysis

could

be

confounded

if

long

duplications

arise

more

frequently

than

long

deletions

whereas

our

frequency

analysis

could

be

confounded

if

the

power

to

detect

duplications

was

lower

as

a

result

of

the

smaller

relative

change

in

copy

number

-LRB-

versus

-RRB-

We

identified

functional

categories

of

genes

that

were

enriched

within

CNVs

using

the

Gene

Ontology

-LRB-

GO

-RRB-

database

-LRB-

Supplementary

Table

-RRB-

The

most

enriched

GO

category

among

genes

overlapped

by

the

CNVRs

was

cell

adhesion

Other

highly

enriched

categories

include

sensory

perception

of

smell

and

of

chemical

stimulus

Notably

neurophysiological

processes

were

also

a

highly

enriched

GO

category

The

most

highly

enriched

GO

categories

within

CNVRs

overlapped

appreciably

with

those

identified

in

a

previous

analysis

of

genes

in

Genes

found

in

segmental

duplications

are

known

to

be

biased

in

terms

of

GO

however

an

enrichment

of

cell

adhesion

genes

was

also

observed

within

CNVRs

not

associated

with

segmental

duplications

We

also

investigated

functional

categories

that

are

under-represented

within

CNVRs

as

these

might

reveal

classes

of

genes

that

are

more

likely

to

be

dosage

sensitive

We

noted

an

impoverishment

of

GO

categories

relating

to

cell

signalling

cell

proliferation

and

numerous

kinase

and

phosphorylation-related

categories

The

impoverishment

of

these

gene

functions

within

CNVs

probably

reflects

purifying

selection

acting

both

against

the

altered

copy

number

of

cell-signalling

molecules

vital

for

development

and

of

dosage-sensitive

oncogenes

or

tumour

suppressor

that

could

predispose

to

early-onset

tumorigenesis

Copy

number

variation

of

medical

relevance

In

the

absence

of

phenotypic

information

for

HapMap

donors

our

data

are

most

relevant

for

highlighting

variable

regions

of

the

genome

that

warrant

consideration

in

disease

studies

rather

than

for

immediate

application

to

clinical

diagnostics

We

found

that

out

of

-LRB-

-RRB-

genes

in

the

OMIM

morbid

map

overlapped

with

CNVs

-LRB-

Supplementary

Table

-RRB-

We

observed

numerous

examples

of

possible

relevance

to

both

mendelian

and

complex

diseases

For

example

the

breakpoint

region

-LRB-

s

-RRB-

for

of

loci

involved

in

genomic

disorders

-LRB-

which

cause

different

diseases

-RRB-

such

as

DiGeorge

and

Williams

Beuren

were

found

to

be

highly

polymorphic

-LRB-

Supplementary

Table

-RRB-

CNVs

were

also

identified

within

the

regions

commonly

deleted

in

DiGeorge

Smith

Magenis

Williams

Beuren

Prader

Willi

and

Angelman

syndromes

which

may

be

relevant

for

discerning

uncharacterized

or

atypical

cases

We

also

found

CNVs

at

the

spinal

muscular

atrophy

and

nephronophthisis

loci

as

expected

as

these

diseases

are

recessive

in

nature

with

relatively

high

carrier

Finally

CNVs

were

found

to

reside

within

kb

of

the

ends

of

chromosomal

arms

which

is

relevant

when

assessing

subtelomeric

rearrangements

in

disease

We

found

CNVs

in

genes

already

known

to

be

responsible

for

complex

traits

including

and

Some

new

observations

were

also

documented

Two

CEU

samples

-LRB-

mother

and

offspring

-RRB-

manifested

a

gain

of

involving

the

first

six

exons

of

which

is

disrupted

in

encompassing

the

LPA

apolipoprotein

A

gene

demonstrated

population

variability

which

may

influence

susceptibility

to

atherosclerosis

The

b-crystallin

genes

in

were

observed

as

gains

and

losses

in

copy

number

in

CEU

and

YRI

samples

However

only

gains

were

detected

in

Asians

leading

us

to

speculate

that

variability

in

crystallin

copy

number

may

be

linked

to

population

differences

of

onset

of

age-related

Following

a

similar

rationale

we

highlight

for

possible

involvement

in

sarcoidosis

owing

to

its

proximity

to

the

and

in

psoriasis

susceptibility

because

it

covers

the

-LRB-

-RRB-

susceptibility

We

also

highlight

challenges

in

resolving

genotype

phenotype

correlations

in

complex

CNVRs

and

how

CNV

detection

can

delineate

unstable

genomic

regions

-LRB-

details

in

Supplementary

Note

including

Supplementary

Fig.

-RRB-

We

identified

patients

with

congenital

cardiac

defects

and

lens

that

share

a

deleted

or

duplicated

region

of

containing

pertinent

candidate

genes

Duplication

of

the

same

interval

was

observed

in

other

cases

with

mental

Probands

can

inherit

the

disease-associated

rearrangements

from

unaffected

parents

which

underscores

the

variable

penetrance

of

some

diseases

resulting

from

dosage

We

found

that

this

locus

is

highly

duplicated

polymorphically

inverted

contains

assembly

gaps

and

is

flanked

by

segmental

duplications

of

variable

copy

number

all

features

being

increasingly

observed

in

CNV

regions

of

the

human

genome

Imprint

of

CNV

on

SNP

genotypes

Deletions

perturb

patterns

of

marker

genotypes

within

and

these

patterns

highlight

the

location

of

such

deletions

SNP

genotype

patterns

characteristic

of

deletions

are

enrichment

of

the

following

genotypes

in

homozygous

deletions

mendelian

discrepancies

in

families

and

Hardy

Weinberg

disequilibrium

within

a

Duplications

can

similarly

lead

to

misinterpretation

of

marker

although

their

impact

on

high-density

SNP

maps

is

poorly

understood

We

characterized

the

patterns

of

Phase

I

HapMap

SNP

genotypes

within

deletions

and

duplications

on

a

chromosome-by-chromosome

basis

to

take

account

of

regional

biases

and

found

that

most

classes

of

aberrant

SNP

genotypes

were

significantly

enriched

in

both

deletions

and

duplications

on

most

chromosomes

-LRB-

Supplementary

Fig.

-RRB-

We

replicated

the

patterns

of

SNP

genotypes

within

deletions

-LRB-

described

above

-RRB-

and

demonstrated

that

duplications

also

impact

significantly

on

SNP

genotypes

The

spectrum

of

SNP

failures

enriched

within

duplications

distinguishes

them

from

deletions

-LRB-

Supplementary

Fig.

and

Supplementary

Table

-RRB-

Most

notably

SNPs

exhibiting

mendelian

inconsistencies

are

more

common

within

Nature

Publishing

Group

ARTICLES

NATURE

Vol

November

deletions

than

duplications

whereas

the

opposite

is

true

for

SNPs

in

Hardy

Weinberg

disequilibrium

and

SNPs

with

missing

genotypes

Cell-line

artefacts

also

have

an

impact

on

SNP

genotypes

For

example

the

partial

deletion

of

in

causes

a

distinct

paucity

of

heterozygous

SNP

genotypes

in

that

individual

-LRB-

one-quarter

that

of

the

population

average

-RRB-

over

several

megabases

of

chromosome

-LRB-

HapMap

release

-RRB-

a

Percentage

CNVs

tagged

YRI

CNV

CEU

CNV

CHB+JPT

CNV

YRI

SNP

CEU

SNP

CHB+JPT

SNP

b

Frequency

Frequency

ratio

ratio

Frequency

ratio

Frequency

ratio

Frequency

ratio

Frequency

ratio

Frequency

Frequency

ratio

ratio

Frequency

Frequency

ratio

ratio

Frequency

ratio

Frequency

ratio

Frequency

ratio

Figure

Patterns

of

linkage

disequilibrium

between

CNVs

and

SNPs

a

The

proportion

of

variants

that

are

tagged

by

a

nearby

proxy

SNP

-LRB-

from

Phase

I

HapMap

-RRB-

increases

as

the

pairwise

linkage

disequilibrium

-LRB-

-RRB-

required

for

a

proxy

SNP

is

relaxed

This

cumulative

distribution

is

shown

for

both

Phase

I

HapMap

SNPs

and

for

biallelic

CNVs

b

Histograms

of

the

ratios

among

all

HapMap

individuals

are

shown

for

multi-allelic

CNVs

The

maximal

squared

Pearson

correlation

coefficient

-LRB-

-RRB-

observed

at

a

neighbouring

Phase

I

HapMap

SNP

which

is

highly

correlated

with

pairwise

linkage

disequilibrium

-LRB-

-RRB-

at

biallelic

CNVs

-LRB-

Supplementary

Fig.

-RRB-

is

given

for

each

CNV

Linkage

disequilibrium

around

CNVs

Indirect

methods

to

identify

causative

variants

such

as

co-segregation

of

linked

markers

in

families

and

genetic

association

with

markers

in

linkage

disequilibrium

with

the

causative

variant

are

considered

to

be

blind

to

the

nature

of

the

underlying

This

raises

the

question

of

whether

SNP-based

whole-genome

association

studies

have

the

same

power

to

detect

disease-related

CNVs

as

for

disease-related

SNPs

This

question

can

be

addressed

by

considering

the

maximal

pairwise

linkage

disequilibrium

-LRB-

-RRB-

between

a

particular

variant

-LRB-

CNV

or

SNP

-RRB-

and

any

of

its

neighbouring

polymorphic

markers

If

a

neighbouring

marker

is

in

high

linkage

disequilibrium

-LRB-

close

to

-RRB-

with

the

variant

of

interest

that

variant

is

tagged

by

the

neighbouring

marker

the

genotype

of

the

variant

of

interest

can

be

predicted

with

high

probability

by

knowing

the

genotype

at

the

tagging

marker

Recent

studies

of

linkage

disequilibrium

around

CNVs

have

produced

conflicting

evidence

as

to

the

degree

to

which

CNVs

are

tagged

by

neighbouring

Here

we

performed

a

balanced

comparison

between

the

linkage

disequilibrium

properties

of

biallelic

CNVs

and

Phase

I

HapMap

SNPs

by

considering

CNVs

irrespective

of

their

genomic

location

and

by

analysing

CNV

genotypes

-LRB-

Supplementary

Table

and

Supplementary

Fig.

-RRB-

of

the

same

frequency

and

quality

as

SNP

-LRB-

see

Methods

for

details

-RRB-

We

quantified

pairwise

linkage

disequilibrium

around

biallelic

CNVs

using

the

same

three

analysis

panels

as

for

the

Phase

I

HapMap

-LRB-

CEU

YRI

and

JPT

plus

CHB

-RRB-

Comparing

the

proportion

of

variants

tagged

by

a

neighbouring

SNP

with

an

arbitrary

threshold

of

shows

that

whereas

of

Phase

I

SNPs

in

nonAfrican

populations

were

tagged

only

of

CNVs

were

tagged

in

the

same

populations

-LRB-

Fig.

and

Supplementary

Table

-RRB-

In

the

YRI

both

SNPs

and

CNVs

exhibited

lower

linkage

disequilibrium

with

only

of

CNVs

being

tagged

with

We

considered

three

explanations

for

these

observations

of

lower

apparent

linkage

disequilibrium

around

CNVs

than

SNPs

First

some

duplications

might

represent

transposition

events

that

would

generate

linkage

disequilibrium

around

the

-LRB-

unknown

-RRB-

acceptor

locus

but

not

the

donor

locus

One

of

the

genotyped

CNVs

is

known

to

be

a

duplicative

but

evidence

from

de

novo

pathogenic

duplications

strongly

suggests

a

preference

for

tandem

rather

than

dispersed

duplications

regardless

of

whether

the

duplication

is

caused

by

non-allelic

homologous

Second

some

CNVs

might

undergo

recurrent

mutations

or

reversions

especially

tandem

duplications

which

are

mechanistically

prone

to

unequal

crossing

over

causing

reversions

back

to

a

single

However

duplications

were

not

in

lower

linkage

disequilibrium

with

flanking

SNPs

than

were

deletions

Finally

we

considered

that

CNVs

might

occur

preferentially

in

genomic

regions

with

lower

densities

of

SNP

genotypes

in

HapMap

Phase

I

We

found

that

CNVs

are

enriched

within

segmentally

duplicated

regions

of

the

genome

in

which

there

is

a

paucity

of

genotyped

SNPs

owing

to

technical

Thus

the

strongest

factor

decreasing

apparent

linkage

disequilibrium

around

biallelic

CNVs

is

not

that

linkage

disequilibrium

around

such

CNVs

is

necessarily

lower

but

that

there

is

on

average

lower

coverage

of

these

structurally

dynamic

regions

of

the

genome

by

SNPs

genotyped

in

Phase

I

of

the

HapMap

project

We

investigated

whether

the

copy

number

of

multi-allelic

CNVs

could

be

predicted

reliably

by

nearby

SNPs

We

treated

diploid

genome

copy

number

at

multi-allelic

CNVs

as

a

quantitative

trait

and

asked

which

nearby

SNPs

are

most

predictive

of

this

trait

and

how

strongly

predictive

these

SNPs

are

We

identified

multi-allelic

CNVs

in

which

the

quantitative

WGTP

data

clearly

clustered

into

discrete

diploid

genome

copy

numbers

and

quantified

the

predictive

ability

of

neighbouring

SNPs

using

the

square

of

Pearson

's

correlation

coefficient

-LRB-

-RRB-

-LRB-

Supplementary

Fig.

-RRB-

We

found

that

diploid

copy

number

of

multi-allelic

CNVs

is

poorly

predicted

by

neighbouring

SNPs

-LRB-

Fig.

-RRB-

It

may

be

that

combining

information

from

Nature

Publishing

Group

NATURE

Vol

November

ARTICLES

CEU

CHB

JPT

YRI

several

SNPs

could

provide

greater

power

for

predicting

diploid

genome

copy

number

at

these

loci

Population

genetics

of

copy

number

variation

In

contrast

to

other

classes

of

human

genetic

variation

the

population

genetics

of

copy

number

variation

remains

unexplored

The

distribution

of

copy

number

variation

within

and

among

different

populations

is

shaped

by

mutation

selection

and

demographic

history

A

range

of

polymorphisms

including

and

Alu

insertion

has

been

used

to

investigate

population

structure

To

demonstrate

the

utility

of

copy

number

variation

genotypes

for

population

genetic

inference

we

performed

population

on

genotyped

biallelic

CNVs

We

obtained

the

optimal

clustering

with

the

assumption

of

three

ancestral

populations

with

the

African

European

and

Asian

populations

clearly

differentiated

-LRB-

Fig.

-RRB-

Population

differentiation

of

individual

variants

is

commonly

estimated

by

the

statistic

FST

which

varies

from

-LRB-

undifferentiated

-RRB-

to

-LRB-

population-specific

-RRB-

The

average

FST

for

the

same

autosomal

CNVs

was

very

similar

to

that

observed

for

all

autosomal

Phase

I

HapMap

SNPs

-LRB-

-RRB-

Recent

population-specific

positive

selection

elevates

population

differentiation

To

explore

population

differentiation

at

all

CNVs

we

devised

a

statistic

VST

that

estimates

population

differentiation

based

on

the

quantitative

intensity

data

and

varies

from

to

similar

to

FST

-LRB-

Supplementary

Fig.

-RRB-

Estimating

VST

for

all

clones

on

the

Figure

Population

clustering

from

CNV

genotypes

A

triangle

plot

showing

the

clustering

of

unrelated

HapMap

individuals

assuming

three

ancestral

populations

-LRB-

k

-RRB-

The

proximity

of

an

individual

to

each

apex

of

the

triangle

indicates

the

proportion

of

that

genome

that

is

estimated

to

have

ancestry

in

each

of

the

three

inferred

ancestral

populations

The

clustering

together

of

most

individuals

from

the

same

population

near

a

common

apex

indicates

the

clear

discrimination

between

populations

obtained

through

this

analysis

The

clustering

was

qualitatively

similar

to

that

obtained

previously

with

a

similar

number

of

biallelic

Alu

insertion

polymorphisms

on

different

African

European

and

Asian

population

YRI

JPT+CHB

B

VST

X

Y

CEU

JPT+CHB

D

VST

X

Y

CEU

YRI

C

A

VST

X

Y

A

B

C

D

Near

MAPT

Figure

Population

differentiation

for

copy

number

variation

Population

differentiation

estimated

by

VST

for

each

of

the

three

population

pairwise

comparisons

is

plotted

along

each

chromosome

For

each

pairwise

comparison

the

VST

values

for

all

clones

on

the

WGTP

platform

are

shown

in

the

lighter

colour

with

filled

circles

with

VST

values

of

CNVs

detected

on

the

EA

platform

superimposed

in

a

darker

shade

with

unfilled

circles

Histograms

showing

the

distributions

of

ratios

-LRB-

on

the

WGTP

platform

-RRB-

among

the

unrelated

individuals

in

each

population

are

plotted

for

four

example

CNVs

exhibiting

high

population

differentiation

labelled

A

D.

y

axis

values

indicate

absolute

frequency

Each

example

histogram

is

labelled

with

the

chromosome

coordinates

of

the

WGTP

clone

and

flanking/encompassed

genes

are

given

for

those

CNVs

mentioned

in

the

text

Nature

Publishing

Group

ARTICLES

NATURE

Vol

November

WGTP

array

and

all

CNVs

on

the

EA

array

revealed

a

number

of

outliers

with

levels

of

population

differentiation

suggestive

of

population-specific

selective

pressures

-LRB-

Fig.

see

also

Supplementary

Table

-RRB-

Among

these

outliers

were

two

CNVs

previously

demonstrated

to

have

elevated

population

is

a

gene

encoding

a

UDP-glucuronosyl

transferase

with

roles

in

androgen

metabolism

and

xenobiotic

and

is

a

chemokine-encoding

multi-copy

gene

at

which

greater

copy

numbers

protect

against

Not

all

regions

that

have

been

under

recent

positive

selection

exhibit

elevated

population

To

detect

other

CNVs

that

may

have

recently

been

under

positive

selection

we

identified

CNVRs

that

fell

within

out

of

-LRB-

-RRB-

genomic

locations

previously

to

exhibit

haplotype

patterns

suggestive

of

a

partial

selective

sweep

-LRB-

Supplementary

Table

-RRB-

Two

of

these

overlapping

CNVs

also

fell

within

the

set

exhibiting

highest

population

differentiation

shown

in

Supplementary

Table

One

of

these

selection-associated

CNVs

is

a

duplication

specific

to

the

CEU

-LRB-

Fig.

-RRB-

and

lies

near

to

the

MAPT

gene

which

is

associated

with

a

set

of

neurodegenerative

disorders

known

as

tauopathies

Both

the

MAPT

gene

and

the

duplication

lie

within

a

chromosomal

region

that

has

recently

been

shown

to

have

a

complex

evolutionary

history

characterized

by

a

common

chromosomal

inversion

deep

divergence

between

inverted

haplotypes

and

recent

positive

selection

in

European

We

adapted

methods

used

to

identify

partial

selective

sweeps

at

to

estimate

relative

extended

haplotype

homozygosity

values

-LRB-

REHH

-RRB-

on

either

flank

of

the

CNVs

for

which

we

extracted

genotype

information

-LRB-

Supplementary

Methods

-RRB-

We

identified

no

convincing

signals

-LRB-

P

on

both

flanks

-RRB-

of

positive

selection

on

any

CNV

in

any

one

population

although

there

were

weaker

signals

-LRB-

P

-RRB-

apparent

for

some

CNVs

-LRB-

Supplementary

Fig.

and

Supplementary

Table

-RRB-

Discussion

Our

map

of

copy

number

variation

in

the

human

genome

demonstrates

the

ubiquity

and

complexity

of

this

form

of

genomic

variation

The

abundance

of

functional

sequences

of

all

types

both

within

and

flanking

areas

of

copy

number

variation

suggests

that

the

contribution

of

CNVs

to

phenotypic

variation

is

likely

to

be

appreciable

This

prediction

is

underscored

by

the

impact

of

copy

number

variation

on

variation

in

gene

expression

-LRB-

B.

Stranger

and

E.

Dermitzakis

personal

communication

-RRB-

CNV

assessment

should

now

become

standard

in

the

design

of

all

studies

of

the

genetic

basis

of

phenotypic

variation

including

disease

susceptibility

Similarly

important

will

be

CNV

annotation

in

all

future

genome

assemblies

The

identification

of

CNVs

causing

severe

sporadic

diseases

has

been

hampered

by

an

inability

to

distinguish

between

normal

and

causative

variants

Our

CNV

map

in

tandem

with

the

DECIPHER

-LRB-

decipher

-RRB-

project

's

sharing

of

copy

number

information

on

patients

with

rare

severe

phenotypes

should

advance

progress

in

this

area

For

mendelian

genetic

diseases

our

data

contain

numerous

known

and

candidate

recessive

disease

alleles

and

existing

linkage

data

can

be

used

to

prioritise

CNVs

for

further

investigation

Genetic

association

studies

are

the

predominant

strategy

for

identifying

haplotypes

conferring

risk

for

complex

genetic

diseases

Such

studies

are

typically

based

on

SNP

genotyping

either

within

candidate

loci

or

Our

analysis

of

linkage

disequilibrium

between

CNVs

and

SNPs

gives

us

limited

optimism

that

CNVs

influencing

risk

to

complex

disease

will

be

detected

by

such

approaches

The

tag

SNPs

that

we

have

identified

-LRB-

Supplementary

Table

-RRB-

for

specific

CNVs

can

be

used

as

proxies

for

these

CNVs

Moreover

CNV-specific

genotyping

assays

can

be

developed

for

CNVs

for

which

tag

SNPs

are

not

readily

identifiable

but

whose

proximity

to

candidate

genes

warrants

further

characterization

Finally

we

see

great

merit

in

mining

CNV

information

from

quantitative

SNP

otyping

data

and

in

enriching

future

generations

of

genome-wide

SNP

genotyping

platforms

by

targeting

untaggable

CNVs

The

overall

utility

of

any

map

depends

on

its

coverage

and

its

completeness

Extrapolation

based

on

existing

data

suggests

that

smaller

deletions

-LRB-

kb

-RRB-

are

much

more

frequent

than

larger

deletions

-LRB-

kb

-RRB-

and

the

same

may

be

true

for

duplications

Although

we

have

generated

the

most

complete

CNV

map

yet

described

given

our

lower

power

to

detect

smaller

CNVs

a

substantial

fraction

of

copy

number

variation

kb

in

size

in

these

individuals

remains

to

be

characterized

No

single

available

technology

will

capture

all

variation

Smaller

rearrangements

are

amenable

to

detection

using

technologies

such

as

sequence

assembly

paired-end

sequence

sequence

trace

and

higher-resolution

tiling

Ultimately

it

is

desirable

to

know

the

precise

chromosomal

location

and

sequence

content

of

each

and

every

CNV

At

present

generating

this

information

requires

the

use

of

multiple

experimental

methods

However

in

the

future

the

comparison

of

independently

assembled

whole-genome

sequences

could

provide

a

definitive

solution

Our

study

of

human

genetic

variation

ties

together

cytogenetics

submicroscopic

copy

number

variation

and

single

nucleotide

polymorphisms

providing

a

framework

for

future

genetic

studies

This

framework

will

need

to

be

supported

by

continual

refinement

of

the

reference

genome

sequence

and

robust

nomenclature

and

databasing

for

structural

variation

both

facilitated

through

international

collaboration

to

enable

further

unravelling

of

the

complexity

of

human

genomic

variation

METHODS

CNV

determination

on

WGTP

and

EA

arrays

The

experimental

methods

and

algorithms

used

for

CNV

calling

using

the

WGTP

and

the

EA

platforms

are

described

in

the

Supplementary

Methods

and

EA

and

WGTP

data

quality

assessment

In

order

to

estimate

false-positive

and

false-negative

rates

on

both

platforms

we

used

quantitative

PCR

to

test

experimentally

CNVs

called

from

replicate

experiments

with

-LRB-

ref

Supplementary

Tables

-RRB-

With

EA

the

average

proportion

of

CNV

calls

from

this

sample

that

were

false

positive

was

-LRB-

out

of

CNV

calls

-RRB-

and

the

false-negative

percentage

was

-LRB-

validated

CNVs

not

called

in

any

one

replicate

per

total

validated

CNVs

-RRB-

With

WGTP

we

found

an

average

falsepositive

proportion

of

-LRB-

out

of

-RRB-

and

a

false-negative

percentage

of

-LRB-

non-called

out

of

tested

-RRB-

Reproducibility

was

also

assessed

by

analysing

ten

HapMap

DNAs

in

triplicate

-LRB-

Supplementary

Table

-RRB-

For

the

EA

platform

on

average

of

CNVs

were

called

in

all

three

replicates

were

called

twice

and

were

called

only

once

For

the

WGTP

platform

using

the

replicate

with

the

lowest

number

of

calls

as

the

baseline

on

average

of

CNVs

were

called

in

three

experiments

were

called

twice

and

were

called

Population

genetic

and

statistical

analyses

Sixty-seven

non-redundant

biallelic

CNVs

suitable

for

genotyping

were

identified

on

the

WGTP

and

EA

platforms

Two

procedures

were

used

to

cluster

intensity

ratios

into

discreet

copy

number

genotypes

K-means

and

partitioning

around

medoids

-LRB-

PAM

-RRB-

Pairwise

linkage

disequilibrium

-LRB-

-RRB-

was

estimated

between

biallelic

CNVs

and

all

filtered

non-redundant

Phase

I

HapMap

SNPs

within

kb

of

the

CNV

borders

using

Population

clustering

was

performed

using

and

population-specific

CNVs

were

estimated

using

and

the

new

statistic

VST

-LRB-

Supplementary

Fig.

-RRB-

VST

is

calculated

by

considering

-LRB-

VT

VS

-RRB-

VT

where

VT

is

the

variance

in

ratios

apparent

among

all

unrelated

individuals

and

VS

is

the

average

variance

within

each

population

weighted

for

population

size

For

REHH

we

treated

each

genotyped

CNV

as

a

SNP

located

at

the

CNV

end

and

used

Phase

I

HapMap

SNPs

kb

upstream

and

downstream

of

the

CNV

using

the

program

Sweep

-LRB-

-RRB-

See

Supplementary

Methods

for

more

details

Data

release

The

raw

data

from

the

EA

as

well

as

the

commercial

arrays

are

posted

at

the

Gene

Expression

Omnibus

-LRB-

-RRB-

with

accession

numbers

and

WGTP

data

are

posted

at

ArrayExpress

-LRB-

-RRB-

with

accession

number

and

at

the

Wellcome

Trust

Sanger

Institute

-LRB-

http

-RRB-

CNV

calls

have

been

released

at

the

Database

of

Genomic

Variants

-LRB-

-RRB-

integrated

with

all

other

CNV

data

Nature

Publishing

Group

NATURE

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November

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online

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at

Acknowledgements

We

thank

D.

Altshuler

C.

Bird

Y.

Chen

M.

Daly

C.

Fahey

A.

M.

Joseph-George

Y.

He

K.

Hirose

Z.

Hu

V.

Jayanth

C.

Langford

M.

Li

C.

Lu

G.

Liu

Z.

Liu

H.

Meguro

L.

Pantano

T.

Paton

I.

Pe'er

S.

Pullenayegum

Y.

Qi

S.

Russell

M.

Schachowsky

M.

Shago

K.

Shiina

and

Y.

Xue

for

advice

sharing

data

cell

lines

technical

assistance

or

bioinformatics

support

The

Centre

for

Applied

Genomics

at

The

Hospital

for

Sick

Children

and

the

Microarray

Facility

of

the

Wellcome

Trust

Sanger

Institute

are

acknowledged

for

database

support

and

array

printing

respectively

We

also

thank

J.

R.

Lupski

and

J.

Pritchard

for

comments

on

earlier

versions

of

the

manuscript

The

research

was

supported

by

The

Wellcome

Trust

-LRB-

M.E.H.

N.P.C.

C.T.S.

-RRB-

Canada

Foundation

of

Innovation

and

Ontario

Innovation

Trust

-LRB-

S.W.S.

-RRB-

Canadian

Institutes

of

Health

Research

-LRB-

CIHR

-RRB-

-LRB-

S.W.S.

-RRB-

Genome

Canada/Ontario

Genomics

Institute

-LRB-

S.W.S.

-RRB-

the

McLaughlin

Centre

for

Molecular

Medicine

-LRB-

S.W.S.

-RRB-

Ontario

Ministry

of

Research

and

Innovation

-LRB-

S.W.S.

-RRB-

the

Hospital

for

Sick

Children

Foundation

-LRB-

S.W.S.

-RRB-

The

Department

of

Pathology

Brigham

and

Women

's

Hospital

-LRB-

C.L.

-RRB-

The

Leukemia

and

Lymphoma

Society

-LRB-

C.L.

-RRB-

the

Core

Research

for

Evolutional

Science

and

Technology

-LRB-

CREST

-RRB-

from

the

Japan

Science

and

Technology

Agency

Grant-in-Aid

for

Scientific

Research

-LRB-

H.A.

-RRB-

Grants-in-Aid

for

Young

Scientists

and

Scientific

Research

on

Priority

Areas

Applied

Genomics

from

the

Ministry

of

Education

Culture

Sports

Science

and

Technology

of

Japan

-LRB-

S.I.

-RRB-

the

Departament

d'Universitats

Recerca

i

Societat

de

la

Informacio

-LRB-

X.E.

-RRB-

and

the

Genoma

Espana

and

Genome

Canada

joint

projects

-LRB-

X.E.

and

S.W.S.

-RRB-

the

Generalitat

de

Catalunya

-LRB-

Departament

d'Universitats

and

Departament

de

Salut

-RRB-

the

Fondo

de

Investigaciones

Sanitarias

de

la

Seguridad

Social

Instituto

de

Salud

Carlos

III

and

the

National

Genotyping

Center

Barcelona

Node

-LRB-

CeGen

-RRB-

supported

by

Genoma

Espana

-LRB-

X.E.

-RRB-

and

a

Packard

Fellowship

to

J.

K.

Pritchard

-LRB-

D.C.

-RRB-

L.F.

is

supported

by

a

fellowship

from

CIHR

R.R.

by

a

Sanger

Institute

Postdoctoral

Fellowship

and

A.C.

from

the

Natural

Science

and

Engineering

Research

Council

S.W.S.

is

an

Investigator

of

the

CIHR

and

International

Scholar

of

the

Howard

Hughes

Medical

Institute

Author

Contributions

R.R.

S.I.

K.R.F.

L.F.

and

G.H.P.

are

first

authors

with

equal

contribution

C.T.-S.

N.P.C.

H.A.

C.L.

K.W.J.

S.W.S.

and

M.E.H.

are

principal

investigators

with

equal

contribution

Author

Information

Reprints

and

permissions

information

is

available

at

The

authors

declare

competing

financial

interests

details

accompany

the

paper

on

Correspondence

and

requests

for

materials

should

be

addressed

to

M.E.H.

-LRB-

meh@sanger.ac.uk

-RRB-

or

S.W.S

-LRB-

steve@genet.sickkids.on.ca

-RRB-

Nature

Publishing

Group