SNP discovery in swine by reduced representation and high throughput pyrosequencing.
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.


CONCLUSIONS
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.
Wiedmann
Ralph T
RT
USDA, ARS, US Meat Animal Research Center, State Spur 18D, NE 68933-0166, USA. ralph.wiedmann@ars.usda.gov
Smith
Timothy P L
TP
Nonneman
Dan J
DJ
D000818:Animals; D001947:Breeding; D002417:Cattle / Q000235:genetics; D033481:Chromosomes, Mammalian / Q000235:genetics; D020451:Contig Mapping; D004247:DNA / Q000032:analysis; D005787:Gene Frequency; D015723:Gene Library; D005838:Genotype; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D013552:Swine / Q000235:genetics*
D004247:DNA