2008 advances in dna genotyping and sequencing genotyping and sequencing tad s. sonstegard usda,...
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2008
Advances in DNAAdvances in DNA genotyping and sequencinggenotyping and sequencing
Tad S. SonstegardUSDA, ARS, Bovine Functional Genomics Laboratory
BARC-East Beltsville, MD [email protected]
SBTE XXIIAug. 22, 2008Guaraja, Brazil
2008
Past Genotyping and Sequencing - Livestock
80 90 00 1070
RFLPmicrosatellite
SNP
Sanger Next-Gen
Gene Sequence EST Microarrays DGE
2008
Advances in genotyping
2008
Where to advance from the 50K chip?
• Get Smaller– Low density multiplexing platform– Cost effective use of concentrated
genotypic information• Get Bigger
– Higher density (300-600 K) Infinium II or Affymetrix type platform
– More expensive platform for finer resolution mapping
• Linkage disequilibrium• monogenic traits
– Capture larger proportion of genetic diversity across cattle species in one platform
2008
Goals of BovineSNP50 development
• develop an assay to surpass the minimum number of markers that would span the bovine genome at an average marker density of 100 kb
• achieve an average marker minor allele frequency (MAF) of at least 0.15 among common cattle breeds
2008
MAF taurus>composite>African>indicusParentage SNP had significantly higher average
MAF values
Average MAF by Source/Breed Type
20087
Phylogeny of Bovini
2008
Conclusions from High-Res Hap Study
• Historic geographic ancestry better explains genotypic variation (and haplotype block structure) in cattle than does their more recent selection into breeds
• Estimated that it would be necessary to successfully assay >28,700 SNPs to construct an LD map for association studies
• >574,000 SNPs are needed to characterize the haplotype block structure across the entire bovine genome
2008
Resources in Native Breeds
Phenotypes Massive Limited Very LimitedGenome Draft7X – Sanger None None50K SNP chip 51K Informative SNP >40K SNP >2.5 K SNP.4K Assay In development None None300K chip Not enough SNP
2008
SNP DiscoveryPool ~15-35 gDNA samples for each library
Cut with restriction enzyme and select 70-130 bp
Sequence fragment ends 10-30X and cluster reads
Identify clusters with SNPs (at least 2 alleles)
Map to genomic coordinates and estimate MAF
Design detection assays from genome sequence
3 librariesHolsteinAngus
Other beef
10
3 librariesNelore
BrahmanNative Dairy
2008
Where are we going?• Testing of alternative breeds for WGS
– Test use of 50K chip -> ~30,000 informative markers in Nelore– Genotyping for congenic line selection
• Evaluate and maintain genetic diversity– Pursue SNP discovery for indicine cattle using “next-gen”– Building a bigger chip?
• Chasing of IP/Biological knowledge– Genome partitioning technology for use on “next gen”
• Sequence 5-10 Mbp regions of genome– Sequencing Genomes of other breeds/ruminants– Defining genome activity– Single molecule sequencing “next next-gen”
• Defining true haplotypes• Characterizing copy number variation• Finding rare mutations• Replacing genotyping - $100 Genome
2008
The gap between genetic improvement and biology…
2008
Systems & temporal framework: digestive tract
255 d Juvenile 180 d Fetus
liver
abomasumduodenum
jejunum
colon
ileum
cecum
reticulum
rumen
spleen
liver
omasumabomasumduodenum
jejunum
ileum
reticulum
rumen
omasum
2008
Library Prep Process
AAAAA
cDNA synthesis
TTTTT___Biotin
Ligation of Adaptor
NNNNAdaptor GATC
CTAG
Ligation of Adaptor
AAAAATTTTT___BiotinAdaptor GATC
CTAG
MmeI
Mme I Digestion
Adaptor GATCCTAG
MmeI
NN
Restriction Enzyme Digestion
AAAAATTTTT__Biotin
GATC
AAAAAmRNA
Ready for clustering on Illumina GA flow cells
2008
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2008
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Differentially Expressed GeneDifferentially Expressed Gene
http://animalgenomics.missouri.edu
19
2008
Differentially Expressed UTRDifferentially Expressed UTR
http://animalgenomics.missouri.edu
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2008
Future of functional genomics
Wold - Nat. Genetics 2008
2008
200823
USDA/CSREES/NRIUSDA/CSREES/NRI2006-35616-166972006-35616-166972006-35205-168882006-35205-168882006-35205-167012006-35205-16701
USDA/ARSUSDA/ARS1265-31000-081D1265-31000-081D1265-31000-090-00D1265-31000-090-00D5438-31000-073D5438-31000-073DAIPL LaboratoriesAIPL Laboratories
Paul Van RadenPaul Van RadenGeorge WiggansGeorge Wiggans
Merial, LtdMerial, LtdStewart BauckStewart Bauck
Bull Studs/NAAB/Breed Bull Studs/NAAB/Breed AssociationsAssociationsABS GlobalABS GlobalAccelerated GeneticsAccelerated GeneticsAlta GeneticsAlta GeneticsCRI/GenexCRI/GenexSelect SiresSelect SiresSemex AllianceSemex AllianceTaurus ServiceTaurus ServiceHAUSAHAUSAAJCAAJCA
Funding Agencies/CollaboratorsFunding Agencies/Collaborators
BARCBARCCurt Van TassellCurt Van TassellSteven SchroederSteven SchroederLakshmi MatukumalliLakshmi MatukumalliJeff O’ConnellJeff O’ConnellLarry ShadeLarry ShadeAlicia BertlesAlicia Bertles
MissouriMissouriJerry TaylorJerry TaylorRobert SchnabelRobert Schnabel
Jared DeckerJared Decker
MARCMARCTim SmithTim SmithMarc AllenMarc Allen
Support PersonnelSupport Personnel
Acknowledgements