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. 2014 Jul 3;15(1):550.
doi: 10.1186/1471-2164-15-550.

Design and development of exome capture sequencing for the domestic pig (Sus scrofa)

Christelle Robert, Pablo Fuentes-Utrilla, Karen Troup, Julia Loecherbach, Frances Turner, Richard Talbot, Alan L Archibald, Alan Mileham, Nader Deeb, David A Hume, Mick Watson  1
Affiliations

Design and development of exome capture sequencing for the domestic pig (Sus scrofa)

Christelle Robert et al. BMC Genomics. .

Abstract

Background: The domestic pig (Sus scrofa) is both an important livestock species and a model for biomedical research. Exome sequencing has accelerated identification of protein-coding variants underlying phenotypic traits in human and mouse. We aimed to develop and validate a similar resource for the pig.

Results: We developed probe sets to capture pig exonic sequences based upon the current Ensembl pig gene annotation supplemented with mapped expressed sequence tags (ESTs) and demonstrated proof-of-principle capture and sequencing of the pig exome in 96 pigs, encompassing 24 capture experiments. For most of the samples at least 10x sequence coverage was achieved for more than 90% of the target bases. Bioinformatic analysis of the data revealed over 236,000 high confidence predicted SNPs and over 28,000 predicted indels.

Conclusions: We have achieved coverage statistics similar to those seen with commercially available human and mouse exome kits. Exome capture in pigs provides a tool to identify coding region variation associated with production traits, including loss of function mutations which may explain embryonic and neonatal losses, and to improve genomic assemblies in the vicinity of protein coding genes in the pig.

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Figures

Figure 1
Figure 1
Comparison of exome regions. The size of the core Ensembl protein-coding exome in a range of species, including human, important agricultural species (chicken, cattle, pig); important rodent model species (rat, mouse); and important primate (gorilla, chimp) species.
Figure 2
Figure 2
Additional gene: putative SMG1 phosphatidylinositol 3-kinase. Genomic region displayed in Ensembl with good alignment to a UniGene mRNA sequence representing an 11 kb putative SMG1 phosphatidylinositol 3-kinase-related sequence which is homologous to the human SMG1 gene. Alignments to several cDNA and EST sequences are annotated in Ensembl in this region (22,400-100,300 bp) on unplaced scaffold GL894597.1 but no Ensembl gene model has been built or core gene defined. The red regions show the pig exome capture regions described in this paper, derived from alignments of UniGene sequences against the genome (see “Methods”). NCBI annotation of the pig genome includes a gene model for SMG1 at this location.
Figure 3
Figure 3
Additional gene: putative GPCR98. Genomic region displayed in Ensembl with good alignment to a UniGene mRNA sequence representing an 8.7 kb putative GPCR98 mRNA. Alignments to cDNA and EST sequences are annotated in Ensembl in this region (100,918,831-100,958,358 bp) on chromosome 2 but no Ensembl gene model has been built or core gene defined. The red regions show the pig exome capture regions described in this paper, derived from alignments of UniGene sequences against the genome (see “Methods”).
Figure 4
Figure 4
Dot plot comparison of Ensembl and EMBL/GenBank IGF2 mRNA sequences. A dot plot comparison of Ensembl transcript ENSSSCT00000022466 and sequence accession X56094, both of which represent IGF2. Clearly, ENSSSCT00000022466 represents only a fragment of the full length mRNA.
Figure 5
Figure 5
Heatmap showing percentage of bases at a range of coverage levels for all 96 samples and 16 capture pools.
See this image and copyright information in PMC

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