Accelerating positional cloning in miceusing ancestral haplotype patterns

Mark Daly

WhiteheadInstitute for Biomedical Research

Kerstin Lindblad-TohWhitehead/MIT Center for Genome Research

Mouse sequence reveals great similarity with the human genome

Extremely high conservation: 560,000 “anchors”

Mouse-Human Comparisonboth genomes 2.5-3 billion bp long> 99% of genes have homologs> 95% of genome “syntenic”

Mouse history

Mouse Genetics, L. Silver

Recent mouse history

W.E. Castle C.C. Little

Fancy mouse breeding - Asia, Europe(last few centuries)

Retired schoolteacher Abbie Lathropcollects and breeds these mice

Granby, MA – 1900

Castle, Little and others form most commonly usedinbred strains

from Lathrop stock(1908 on)

Critical components of inbred strain diversity

• Asian musculus and European domesticus mice dominate the world but have evolved separately over ~ 1 Million years

• Thousands of years of fancy mouse breeding resulted in highly homogeneous versions of these wild mice being traded and ending up in Lathrop’s schoolhouse

Structure of variation in the laboratory mouse

• Study 1: compare finished BACs from strain 129 to recent C57BL/6J genome assembly

• Study 2: extrapolate general observations utilizing WGS reads from 129, C3H, Balb/c done as part of the MGSC

Distribution of variation rates 70 unlinked 50 kb segments (129 vs. B6)

{

<1 SNP/10 kb

{~40 SNP/10 kb

Distribution of variation rates 70 unlinked 50 kb segments

{

Only 1/3 validate

{~99% validate

Low and High SNP rate suggest recent and distant

ancestry

SNP discovery analysis summary

• Comparisons of 129 and C57BL/6 show alternating regions of high SNP density (~1 per 200-250 bp) and low SNP density (~1 per 20,000 bp)

• Genome-wide shotgun suggests these segments average 1 Mb

• C3H and Balb/c comparisons to C57BL/6 give identical picture with regions of divergence and identity varying

Genetic Background of the inbred lab mice

musc musc

musc

musc

domest

domestdomest

domestdomest

C57BL/6

C3H

DBA

Avg segment size ~ 1-2 Mb{cast

Positional Cloning

C3H (susceptible)

B6 (resistant)

20 Mb

Traditionally: positional cloning is painful(e.g., generating thousands of mice for fine mapping, breeding congenics) –

As a result, countless significant QTLs have been identified in mappingcrosses but only a handful of those have been successfully cloned

Using ancestral haplotypes to localize QTLs

C3H (susc.)

B6 (res.)

Critical Region

20 Mb

Using ancestral haplotypes to localize QTLs

C3H (susc.)

B6 (res.)

DBA (susc.)

Critical Region

20 Mb

Using ancestral haplotypes to localize QTLs

C3H (susc.)

B6 (res.)

DBA (susc.)

Critical Region

20 Mb

One can then also use the map to: - examine correlation of genotype to phenotype of other strains in the critical segments - choosing optimal additional strains for crossing

Pilot Haplotype Map

• ~150 SNPs across 25 Mb of chromosome 4

• Typed in 37 inbred lines and 10 wild-derived isolates of potential founder strains

• Roughly 3-fold less dense than projected to give a finished picture

Strains proposedWild derived ancestral strains Inbred strains

CAST/Ei M.m.castaneus

129S1/SvImJ C57BL/6J DDK NZW/LacJ

WSB/Ei M.m.domesticus

129X1/SvJ C57BLKS/J FVB/NJ PL/J

PERA/Ei M.m.domesticus

A/J C57BR/cdJ I/LnJ RIIIS/J

MOLF/Ei M.m.molossinus

AKR/J C57L/J KK/HlJ RF/J

MAI/PasM.m.musculus

BALB/cByJ C58/J LG/J SEA/GnJ

CZECHII/Ei M.m.musculus

BTBR+Ttf/tf CBA/J LP/J SJL/J

SPRET/Ei M.spretus

BUB/BnJ CE/J MA/MyJ SM/J

SEG/Pas M.spretus

C3H/HeJ DBA/2 NOD/LtJ ST/bJ

BACT/Bon

M.m.bactrianusC57BL/10 020 NON/LtJ SWR/J

First few Mb…129S1 T A * C C C * C G G T A C G A G G G

AKR A G T T T A A T G G T A C G A G G G

A_J A G T T T A A T G G T A C G A G G G

BALB_c T A * C C C G C G G T A C G A G G G

C3H A G T T T A A T G G T A C G A G G G

C57B6 A G T T T A A T C T A G T A C C C A

CBA A G T T T A A T C T A G T A C C C A

DBA2 A G T T T A A T C T A G T A C C C A

FVB A G T T T A A T C T A G T A C C C A

I A G T T T A A T G G T A C G A G G G

NOD A G T T T A A T G G T A C G A G G G

NZB * A C C C C * C C T * G T A C C C A

SJL A G T T T A A T C T A G T A C C C A

SWR A G T T T A A T C T A G T A C C C A

Chr 4 35.7 37.6 37.9 39.4 (Mb)

Regional Comparison 129/S1 vs. C57BL/6J

Red = ancestrally divergent

{

GGGAGCATGGC*CCC*AT

ACCCATGATCTAATTTGA

{

GGAACCGTGATCCACAAGAC

GGAACCGT*ATCCACAAGAC

Blue = ancestrally identical

QTL identified in two crosses

129xB6

DBAxB6

QTL identified in three crosses• QTL identified by three crosses 1x2, 1x3,

and 1x4

• Across 25 Mb, only 4 segments (each between 300 and 700 kb) are ancestrally consistent with the QTL mapping data

• In total – 1.9 out of the 25 Mb is identified as most likely to contain the responsible mutation

A/J vs C3H

Not a software bug – two strains identical at every SNP typedacross the 25 Mb interval

Genetic Background of the inbred lab mice

musc musc

musc

musc

domest

domestdomest

domestdomest

C57BL/6

C3H

DBA

Avg segment size ~ 1-2 Mb{cast

Genetic Background of the inbred lab mice

musc musc

musc

musc

domest

domestdomest

domestdomest

C57BL/6

C3H

DBA

Avg segment size ~ 1-2 Mb{cast

Colleagues

Claire WadeAndrew Kirby

Whitehead Genome CenterKerstin Lindblad-TohEJ KulbokasElinor KarlssonMike ZodyEric Lander

Mouse Genome Sequencing Consortium