Data from: Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

古DNA与档案DNA样本是研究各类历史进程的宝贵资源。其中，博物馆标本可获取时间与空间跨度均极大的生物群资源，有助于解析长期的生态与演化变化。然而，档案标本的处理难度颇高：它们通常脆弱且不可替代，且往往仅含有少量变性DNA的短片段。本研究提供一套可用于此类样本的前沿遗传分析处理工具。首先，我们报道了一种针对昆虫博物馆标本的低破坏性DNA提取方案，所有受测样本均通过该方案获得了可用于测序的DNA。本次分析的11份标本的DNA均已碎片化，长度极少超过100碱基对（bp），且无法通过针对线粒体细胞色素氧化酶I基因的常规聚合酶链式反应（PCR）完成扩增。本方案可令此类样本适配于主流的下一代测序分子分析工具，包括RAD标签技术（RAD-tagging）与鸟枪法基因组重测序（shotgun genome re-sequencing）。首先，我们使用三种各带有参考基因组的蚂蚁博物馆标本开展RAD标签定位分析。我们可通过对降解DNA序列进行全测序，识别重复测序读段并在碱基识别前完成过滤。其次，我们对6个夏威夷果蝇属（Drosophila）物种开展了重测序：这些物种间的分化时长可达数百万年，但仅存在一套可用的参考基因组。尽管每碱基的测序深度仅为0.37±0.42，我们仍可获取足够数量的重叠单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点，得以完整解析物种树；该物种树与早期的核型研究及既往分子研究结果一致，至少在这些研究可覆盖的树分支区域内如此。尽管本套技术专为降解DNA样本开发，但所有方法均可轻松适配于较新鲜的组织样本，且兼容液体处理自动化流程。