Data from: Substantial differences in bias between single-digest and double-digest RAD-seq libraries: a case study

The trade‐offs of using single‐digest vs. double‐digest restriction
site‐associated DNA sequencing (RAD‐seq) protocols have been widely
discussed. However, no direct empirical comparisons of the two methods
have been conducted. Here, we sampled a single population of Gulf pipefish
(Syngnathus scovelli) and genotyped 444 individuals using RAD‐seq. Sixty
individuals were subjected to single‐digest RAD‐seq (sdRAD‐seq), and the
remaining 384 individuals were genotyped using a double‐digest RAD‐seq
(ddRAD‐seq) protocol. We analysed the resulting Illumina sequencing data
and compared the two genotyping methods when reads were analysed either
together or separately. Coverage statistics, observed heterozygosity, and
allele frequencies differed significantly between the two protocols, as
did the results of selection components analysis. We also performed an in
silico digestion of the Gulf pipefish genome and modelled five major
sources of bias: PCR duplicates, polymorphic restriction sites, shearing
bias, asymmetric sampling (i.e., genotyping fewer individuals with
sdRAD‐seq than with ddRAD‐seq) and higher major allele frequencies. This
combination of approaches allowed us to determine that polymorphic
restriction sites, an asymmetric sampling scheme, mean allele frequencies
and to some extent PCR duplicates all contribute to different estimates of
allele frequencies between samples genotyped using sdRAD‐seq versus
ddRAD‐seq. Our finding that sdRAD‐seq and ddRAD‐seq can result in
different allele frequencies has implications for comparisons across
studies and techniques that endeavour to identify genomewide signatures of
evolutionary processes in natural populations.