1 / 23

Advances in DNA genotyping and sequencing

Explore the evolution of genotyping technologies for cattle breeds, from SNP microsatellites to next-gen sequencing. Learn about the BovineSNP50 chip, SNP discovery methods, and future genomics prospects. This insightful overview focuses on enhancing genetic diversity and discovering rare mutations.

ddukes
Download Presentation

Advances in DNA genotyping and sequencing

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Advances in DNA genotyping and sequencing Tad S. Sonstegard USDA, ARS, Bovine Functional Genomics Laboratory BARC-East Beltsville, MD U.S.A. tad.sonstegard@ars.usda.gov SBTE XXII Aug. 22, 2008 Guaraja, Brazil

  2. Past Genotyping and Sequencing - Livestock SNP microsatellite RFLP 70 80 90 00 10 Sanger Next-Gen Gene Sequence EST Microarrays DGE

  3. Advances in genotyping

  4. 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

  5. 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

  6. Average MAF by Source/Breed Type MAF  taurus>composite>African>indicus Parentage SNP had significantly higher average MAF values

  7. Phylogeny of Bovini 7

  8. 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

  9. Resources in Native Breeds Phenotypes Massive Limited Very Limited Genome Draft 7X – Sanger None None 50K SNP chip 51K Informative SNP >40K SNP >2.5 K SNP .4K Assay In development None None 300K chip Not enough SNP

  10. SNP Discovery • Pool ~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 libraries Nelore Brahman Native Dairy 3 libraries Holstein Angus Other beef 10

  11. 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

  12. The gap between genetic improvement and biology…

  13. Systems & temporal framework: digestive tract spleen rumen rumen reticulum reticulum colon jejunum jejunum liver liver cecum ileum ileum omasum omasum duodenum abomasum duodenum abomasum 180 d Fetus 255 d Juvenile

  14. mRNA AAAAA AAAAA TTTTT___Biotin cDNA synthesis Restriction Enzyme Digestion AAAAA GATC TTTTT__Biotin Ligation of Adaptor MmeI GATC AAAAA Adaptor TTTTT___Biotin CTAG MmeI NN GATC Adaptor CTAG Mme I Digestion Ready for clustering on Illumina GA flow cells Library Prep Process Ligation of Adaptor NN GATC Adaptor NN CTAG

  15. Differentially Expressed Gene http://animalgenomics.missouri.edu 19

  16. Differentially Expressed UTR http://animalgenomics.missouri.edu 20

  17. Future of functional genomics Wold - Nat. Genetics 2008

  18. Acknowledgements Funding Agencies/Collaborators Support Personnel • USDA/CSREES/NRI • 2006-35616-16697 • 2006-35205-16888 • 2006-35205-16701 • USDA/ARS • 1265-31000-081D • 1265-31000-090-00D • 5438-31000-073D • AIPL Laboratories • Paul Van Raden • George Wiggans • Merial, Ltd • Stewart Bauck • Bull Studs/NAAB/Breed Associations • ABS Global • Accelerated Genetics • Alta Genetics • CRI/Genex • Select Sires • Semex Alliance • Taurus Service • HAUSA • AJCA • BARC • Curt Van Tassell • Steven Schroeder • Lakshmi Matukumalli • Jeff O’Connell • Larry Shade • Alicia Bertles • Missouri • Jerry Taylor • Robert Schnabel • Jared Decker • MARC • Tim Smith • Marc Allen 23

More Related