Location: Reproduction ResearchTitle: Design of a High Density SNP Genotyping Assay in the Pig Using SNPs Identified and Characterized by Next Generation Sequencing Technology) Author
|Nonneman, Danny - Dan|
|Smith, Timothy - Tim|
|Van Tassell, Curtis - Curt|
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/21/2009
Publication Date: 8/5/2009
Publication URL: http://handle.nal.usda.gov/10113/37777
Citation: Ramos, A.M., Crooijmans, R.P.M.A., Affara, N.A., Amaral, A.J., Archibald, A.L., Beever, J.E., Bendixen, C., Churcher, C., Clark, R., Dehais, P., Hansen, M.S., Hedegaard, J., Hu, Z.L., Kerstens, H.H., Law, A.S., Megens, H.J., Milan, D., Nonneman, D.J., Rohrer, G.A., Rothschild, M.F., Smith, T.P.L., Schnabel, R.D., Van Tassell, C.P., Taylor, J.F., Wiedmann, R.T., Schook, L.B., Groenen, M.A.M. 2009. Design of a High Density SNP Genotyping Assay in the Pig Using SNPs Identified and Characterized by Next Generation Sequencing Technology. PLoS One 4(8):E6524. p. 1-13. Interpretive Summary: One of the main limitations to the dissection of economically important traits in livestock has been the lack of a sufficient number of genetic markers for the development of high-density and high-throughput assays for association studies. New “next generation” sequencing technologies have emerged which offer great promise in terms of time and cost to efficiently generate large amounts of sequence data. Our objectives were to perform a large-scale, genome-wide porcine SNP discovery study using next generation sequencing technologies and reduced representation libraries, and to develop and characterize a high-density SNP genotyping chip using these SNPs and others collected from public sources. A combination of Roche 454 and Illumina Genome Analyzer sequencing technologies allowed the construction of reference sequences and simultaneous detection of reliable SNPs in the absence of a sequenced genome. This approach combined both technologies and allowed us to take advantage of the large numbers of genome analyzer sequences for an increased sequence depth, and the longer 454 reads to obtain sufficient sequence information adjacent to each SNP to be able to design primers for the genotyping assay. The breeds used for SNP discovery include the four main breeds used in worldwide pig production (Duroc, Pietrain, Landrace and Large White) as well as the wild boar. Over 375,000 SNP were discovered using this approach and a high-density genotyping array was developed that contains 60,000 SNP of the 500,000 available SNPs dispersed across the genome. It is anticipated that this array will be highly efficient for genomic selection in the pig breeding industry.
Technical Abstract: The dissection of complex traits of economic importance for the pig industry requires the availability of a significant number of genetic markers, such as SNPs. This study was conducted in order to discover thousands of porcine SNPs using next generation sequencing technologies and use those SNPs, as well as others from different sources, to design a high density SNP genotyping assay. A total of 19 reduced representation libraries derived from five swine breeds (Duroc, Landrace, Large White, Pietrain and Wild Boar) and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina’s Genome Analyzer. The SNP discovery effort resulted in the identification of over 375K de novo porcine SNPs. These SNPs, and others from different sources, were used to design the Illumina Porcine 60K+ SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs was included on the beadchip. The results from genotyping 158 individuals with the beadchip showed a high overall call rate for all the SNPs (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic which indicates that the SNP conversion success rate was 94%. The average MAF for all scorable SNPs was 0.274. Overall, the results of this study indicated the utility of using next generation sequencing technologies to identify large number of reliable SNPs. In addition, the first application of the PorcineSNP60 beadchip demonstrated that the beadchip is an excellent tool that will likely be used in a variety of future studies in pigs.