Validation of pooled whole-genome re-sequencing in Arabidopsis lyrata. Validation of Pool-seq

M. Fracassetti, P. C. Griffin and Y. Willi (2015). Validation of pooled whole-genome re-sequencing in Arabidopsis lyrata. Manuscript submitted for publication. Sequencing pooled DNA of multiple individuals of a population instead of individual-specific sequencing has become popular due to cost-effectiveness and simple wet-lab protocol, though the debate on this approach is still open. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq) to prepare Arabidopsis lyrata libraries with low starting DNA (1.6 ng per individual). Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on frequency estimates of single nucleotide polymorphisms (SNPs). The validation was based on comparing population SNP frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS). For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a robust heuristics/statistics approach, while the latter employs a Bayesian approach. Results revealed concordance correlation coefficients well above 0.9, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14) and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual), which increased the concordance correlation coefficient to 0.96. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. Interestingly, we observed that the nucleotide diversity and divergence provide meaningful prior information for SNP calling. We recommend to pool at least 25 individuals and a sequencing depth of 100× to produces satisfactory SNP frequency data.