Accuracy of allele frequency estimation using pooled RNA-Seq

For non-model organisms, genome-wide information that describes functionally relevant variation may be obtained by RNA-Seq following de novo transcriptome assembly. While sequencing has become relatively inexpensive, the preparation of a large number of sequencing libraries remains prohibitively expensive for population genetic analyses of non-model species. Pooling samples may be then an attractive alternative. To test whether pooled RNA-Seq accurately predicts true allele frequencies, we analyzed the liver transcriptomes of 10 bank voles. Each sample was sequenced both as an individually barcoded library and as a part of a pool. Equal amounts of total RNA from each vole were pooled prior to mRNA selection and library construction. Reads were mapped onto the de novo assembled reference transcriptome. High-quality genotypes for individual voles, determined for 23,682 SNPs, provided information on “true” allele frequencies; allele frequencies estimated from the pool were then compared to...