Expressed Exome Capture Sequencing (EecSeq): a method for cost-effective exome sequencing for all organisms with or without genomic resources

Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require a priori knowledge of exon sequences from previous genome and/or transcriptome sequencing and annotation. Here, we present a new method that uses cDNA directly to enrich and capture genomic DNA for expressed exon sequences, eliminating the need for previous bioinformatic analysis and costly probe synthesis. To empirically test the efficiency and accuracy of EecSeq, four adult eastern oysters (Crassostrea virginica) were heat shocked at 36° C for 1 hour along with four control oysters kept at ambient temperature (20° C). Stranded mRNA libraries were prepared for two exposed and two control individuals; half of the libraries were sequenced and the other half used for EecSeq probe synthesis. Genomic DNA was extracted from all eight individuals, enriched via captured probes, and sequenced directly. EecSeq has an average capture sensitivity of 86.8% across all known exons and over 99.4% of exons with detectable levels of expression. Over 47.9% of all mapped reads mapped to exons with 37.0% of reads on average mapping to expressed targets. Unlike other exome capture techniques, EecSeq captures displayed relatively even coverage within exons (i.e. little to no "edge effects") and coverage uniformly distributed across exon GC content. Across six replicate pools of eight individuals, 5,951 SNPs with a minumum average coverage of 80X were discovered, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.