Detecting SNPs and estimating allele frequencies in clonal bacterial populations by sequencing pooled DNA

Summary: Here, we present a method for estimating the frequencies of SNP alleles present within pooled samples of DNA using high-throughput short-read sequencing. The method was tested on real data from six strains of the highly monomorphic pathogen Salmonella Paratyphi A, sequenced individually and in a pool. A variety of read mapping and quality-weighting procedures were tested to determine the optimal parameters, which afforded =80% sensitivity of SNP detection and strong correlation with true SNP frequency at poolwide read depth of 40×, declining only slightly at read depths 20–40×.