SNP discovery in swine by reduced representation and high throughput pyrosequencing - PubMed (original) (raw)

SNP discovery in swine by reduced representation and high throughput pyrosequencing

Ralph T Wiedmann et al. BMC Genet. 2008.

Abstract

Background: Relatively little information is available for sequence variation in the pig. We previously used a combination of short read (25 base pair) high-throughput sequencing and reduced genomic representation to discover > 60,000 single nucleotide polymorphisms (SNP) in cattle, but the current lack of complete genome sequence limits this approach in swine. Longer-read pyrosequencing-based technologies have the potential to overcome this limitation by providing sufficient flanking sequence information for assay design. Swine SNP were discovered in the present study using a reduced representation of 450 base pair (bp) porcine genomic fragments (approximately 4% of the swine genome) prepared from a pool of 26 animals relevant to current pork production, and a GS-FLX instrument producing 240 bp reads.

Results: Approximately 5 million sequence reads were collected and assembled into contigs having an overall observed depth of 7.65-fold coverage. The approximate minor allele frequency was estimated from the number of observations of the alternate alleles. The average coverage at the SNPs was 12.6-fold. This approach identified 115,572 SNPs in 47,830 contigs. Comparison to partial swine genome draft sequence indicated 49,879 SNP (43%) and 22,045 contigs (46%) mapped to a position on a sequenced pig chromosome and the distribution was essentially random. A sample of 176 putative SNPs was examined and 168 (95.5%) were confirmed to have segregating alleles; the correlation of the observed minor allele frequency (MAF) to that predicted from the sequence data was 0.58.

Conclusion: The process was an efficient means to identify a large number of porcine SNP having high validation rate to be used in an ongoing international collaboration to produce a highly parallel genotyping assay for swine. By using a conservative approach, a robust group of SNPs were detected with greater confidence and relatively high MAF that should be suitable for genotyping in a wide variety of commercial populations.

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Figures

Figure 1

Figure 1

Distribution of the contig lengths showing that most of the contigs consist of reads from one end of the restriction fragments. About 25% of the contigs span the entire restriction fragment.

Figure 2

Figure 2

The plot shows the distribution of 115,572 SNPs by position in the contigs. The number of SNPs mirrors the profile of contig lengths.

Figure 3

Figure 3

The distribution of contigs by number of SNPs.

Figure 4

Figure 4

Comparison of the distribution of total contigs to contigs containing SNPs as the depth of coverage changes. The average depth of coverage where SNP were detected was 12.6.

Figure 5

Figure 5

Contig spacing along the sequenced pig chromosomes 1, 4, 5, 7, 11, 13 and 14.

Figure 6

Figure 6

Correlation between allele frequency estimated by 454 sequencing and genotyping.

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