Data from: Evaluating hybridization capture with RAD probes as a tool for museum genomics with historical bird specimens

Laboratory techniques for high-throughput sequencing have enhanced our ability to generate DNA sequence data from millions of natural history specimens collected prior to the molecular era, but remain poorly tested at shallower evolutionary time scales. Hybridization capture using restriction site-associated DNA probes (hyRAD) is a recently developed method for population genomics with museum specimens. The hyRAD method employs fragments produced in a restriction site-associated double digestion as the basis for probes that capture orthologous loci in samples of interest. While promising in that it does not require a reference genome, hyRAD has yet to be applied across study systems in independent laboratories. Here, we provide an independent assessment of the effectiveness of hyRAD on both fresh avian tissue and dried tissue from museum specimens up to 140 years old and investigate how variable quantities of input DNA affect sequencing, assembly, and population genetic inference. We present a modified bench protocol and bioinformatics pipeline, including three steps for detection and removal of microbial and mitochondrial DNA contaminants. We confirm that hyRAD is an effective tool for sampling thousands of orthologous SNPs from historic museum specimens to describe phylogeographic patterns. We find that modern DNA performs significantly better than historical DNA better during sequencing but that assembly performance is largely equivalent. We also find that the quantity of input DNA predicts %GC content of assembled contiguous sequences, suggesting PCR bias. We caution against sampling schemes that include taxonomic or geographic autocorrelation across modern and historic samples.高通量测序实验技术提升了我们从分子生物学时代前采集的数百万件自然历史标本中获取DNA序列数据的能力，但该技术在较晚近的进化时间尺度下的应用效果仍未得到充分验证。基于限制性酶切位点相关DNA探针的杂交捕获技术（hyRAD）是一种新近开发的适用于博物馆标本的群体基因组学研究方法。该hyRAD技术以限制性酶切位点相关的双酶切产物片段作为探针设计基础，用于捕获目标样本中的直系同源基因座（orthologous loci）。尽管该技术无需参考基因组，颇具应用前景，但目前尚未在独立实验室的不同研究体系中得到验证。本研究对hyRAD技术在新鲜鸟类组织以及最长可达140年的博物馆干燥标本组织中的应用效果进行了独立评估，并探究了不同投入量的模板DNA对测序、序列组装以及群体遗传推断的影响。本研究提出了一套优化后的实验室操作流程与生物信息学分析流程，其中包含三步用于检测并去除微生物与线粒体DNA污染物的流程。本研究证实，hyRAD技术可作为一种高效工具，从历史博物馆标本中获取数千个直系同源单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点，以解析系统地理格局。研究发现，现代DNA在测序过程中的表现显著优于历史DNA，但序列组装的表现则基本相当。同时我们发现，模板DNA的投入量可预测组装得到的连续序列的GC含量占比，这提示存在聚合酶链式反应（Polymerase Chain Reaction, PCR）偏倚。本研究提醒研究者应避免在现代与历史样本的采样方案中引入分类学或地理学上的自相关性。