Abstract
Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called "EecSeq," eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1hr along with four control oysters kept at 14°C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor "edge effects") and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80×, 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.
Highlights
Current address: Department of Biological Sciences, University of Rhode Island, 120 Flagg RD, This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record
For the original probe set synthesis, one microgram of the original cDNA library yielded 2,298 ng of probes, as the biotinylation occurs via a DNA polymerase
Expressed exome capture sequencing (EecSeq) is a novel design for exome capture that uses insitu synthesized biotinylated cDNA probes to enrich for exon sequences, thereby removing the requirement of a priori genomic resources, costly exon probe design, and synthesis
Summary
We present a method for creating cDNA probes from expressed mRNA, which are used to enrich and capture genomic DNA for exon regions. This approach, called “EecSeq”, eliminates the need for costly probe design and synthesis. These data have given insight into classical problems in evolutionary biology, including the repeatability of evolution (e.g., Jones et al 2012), the degree of convergent evolution across distant taxa (e.g., Yeaman et al 2016), and whether selection is driving changes in existing genetic variation or new mutations (e.g., Reid et al 2016) Despite this rapid progress, it is still cost prohibitive to sequence dozens or hundreds of full genomes. This leads to an inherent trade-off between sample size and genomic coverage, leading investigators to make decisions about whether to sequence more individuals (for higher power and precision) versus more of the genome (for making more accurate statements about the genetic basis of adaptation)
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