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. 2009 Aug 5;4(8):e6524.
doi: 10.1371/journal.pone.0006524.

Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology

Antonio M Ramos  1 , Richard P M A Crooijmans, Nabeel A Affara, Andreia J Amaral, Alan L Archibald, Jonathan E Beever, Christian Bendixen, Carol Churcher, Richard Clark, Patrick Dehais, Mark S Hansen, Jakob Hedegaard, Zhi-Liang Hu, Hindrik H Kerstens, Andy S Law, Hendrik-Jan Megens, Denis Milan, Danny J Nonneman, Gary A Rohrer, Max F Rothschild, Tim P L Smith, Robert D Schnabel, Curt P Van Tassell, Jeremy F Taylor, Ralph T Wiedmann, Lawrence B Schook, Martien A M Groenen
Affiliations

Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology

Antonio M Ramos et al. PLoS One. .

Abstract

Background: The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay.

Methodology/principal findings: A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina's Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274.

Conclusions/significance: Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.

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Conflict of interest statement

Competing Interests: The author Mark S. Hansen is employed by Illumina Inc.

Figures

Figure 1
Figure 1. SNP distribution on each of the GA read positions.
The distribution represents all de novo identified SNPs, from the RRLs generated. The number of transitions and transversions identified is also illustrated.
Figure 2
Figure 2. Distribution of the low minor allele count SNPs on each of the GA read positions.
For these SNPs two reads were identified for the minor allele. The number of transitions and transversions identified is also illustrated.
Figure 3
Figure 3. Distances between the SNPs included on the 60K+porcine Beadchip.
The distances (x axis) were calculated using builds 7 (blue) and 8 (red) of the pig genome sequence assembly.
Figure 4
Figure 4. Correlation between sequence-derived and genotype-derived allele frequencies.
The scatter plot was determined using the frequencies of the PorcineSNP60 SNPs derived from the RRLs generated.
Figure 5
Figure 5. Relationship between sequence depth and the correlation between sequence-derived and genotype-derived allele frequencies.
This relationship was determined for the PorcineSNP60 SNPs derived from the RRLs generated.
See this image and copyright information in PMC

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