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

下一代测序技术开启了群体遗传学研究的新纪元。在这些新的测序机会之后，基于限制性内切酶的基因分型技术（例如与限制性酶切位点相关的DNA测序（RAD-seq）或双消化RAD测序（ddRAD-seq））的使用在过去十年中已大大增加。从DNA采样到SNP调用，已经在文献中研究了基于酶的技术的实验室和生物信息学参数。但是，这些参数对下游分析和生物学结果的影响仍然没有文献记载。在这项研究中，我们调查了两个生物系统中蝶序前和后序设置对ddRAD-seq结果的影响：蝴蝶物种（Coenonympha sp。）和数个普通山毛榉（Fagus sylvatica）。我们的结果表明，预测序参数（即DNA数量，文库制备过程中的PCR循环数）对回收读物和SNP的数量，独特等位基因的数量以及个体杂合性有重大影响。以同样的方式，我们发现测序后的设置（即聚类和最小覆盖阈值）影响了基因座重建（例如，基因座数量，平均覆盖率）和SNP调用（例如，SNP数量；杂合性），但只有一个对下游分析的边际影响（例如，遗传分化的测量，单个混合物的估计以及人口统计学推论）。此外，复制分析证实了ddRAD-seq程序的可重复性。总体而言，这项研究评估了ddRAD-seq数据对测序前后协议的敏感性程度，