Double-digest RAD-sequencing: do pre- and post-sequencing protocol parameters impact biological results?

Next-generation sequencing technologies have opened a new era of research in population genetics. Following these opportunities, the use restriction enzyme-based genotyping techniques, such as site-associated DNA (RAD-seq) or double-digest RAD-sequencing (ddRAD-seq), has dramatically increased last decade. From sampling to SNP calling, laboratory and bioinformatic parameters techniques been investigated literature. However, impact those on downstream analyses biological results remains less documented. In this study, we effects sevral pre- post-sequencing settings ddRAD-seq for two systems: complex butterfly species (Coenonympha sp.) several populations common beech (Fagus sylvatica). Our suggest that pre-sequencing (i.e., quantity, number PCR cycles during library preparation) significant recovered reads SNPs, unique alleles individual heterozygosity. same way, found clustering minimum coverage thresholds) influenced loci reconstruction (e.g., loci, mean coverage) calling SNPs; heterozygosity) but had only marginal measure genetic differentiation, estimation admixture, demographic inferences). addition, replication confirmed reproducibility procedure. Overall, study assesses degree sensitivity data protocols, illustrates its robustness when studying