A simple strategy for recovering ultraconserved elements, exons, and introns from low coverage shotgun sequencing of museum specimens: placement of the partridge genus Tropicoperdix within the Galliformes

Next-generation DNA sequencing (NGS) offers a promising way to obtain massive numbers of orthologous loci to understand phylogenetic relationships among organisms. Of particular interest are old museum specimens and other samples with degraded DNA, where traditional sequencing methods have proven to be challenging. Low coverage shotgun sequencing and sequence capture are two widely used NGS approaches for degraded DNA. Sequence capture can yield sequence data for large numbers of orthologous loci, but it can only be used to sequence genomic regions near conserved sequences that can be used as probes. Low coverage shotgun sequencing has the potential to yield different data types throughout the genome. However, many studies using this method have often generated mitochondrial sequences, and few nuclear sequences, suggesting orthologous nuclear sequences are likely harder to recover. To determine the phylogenetic position of the galliform genus Tropicoperdix, whose phylogenetic position is currently uncertain, we explored two strategies to maximize data extraction from low coverage shotgun sequencing from approximately 100-year-old museum specimens from two species of Tropicoperdix. One approach, a simple read mapping strategy, outperformed the other (a reduced complexity assembly approach), and allowed us to obtain a large number of ultraconserved element (UCE) loci, relatively conserved exons, more variable introns, as well as mitochondrial genomes. Additionally, we demonstrated some simple approaches to explore possible artifacts that may result from the use of degraded DNA. Our data placed Tropicoperdix within a clade that includes many taxa characterized with ornamental eyespots (peafowl, argus pheasants, and peacock pheasants), and established relationships among species within the genus. Therefore, our study demonstrated that low coverage shotgun sequencing can easily be leveraged to yield substantial amounts and varying types of data, which opens the door for many research questions that might require information from different data types from museum specimens.

下一代DNA测序（Next-generation DNA sequencing，NGS）可为获取海量直系同源位点（orthologous loci）以解析生物间的系统发育关系提供极具前景的途径。其中，老旧博物馆标本及其他DNA降解样本尤为受关注——传统测序方法在这类样本上的应用已被证实颇具挑战。低覆盖度鸟枪法测序（low coverage shotgun sequencing）与序列捕获（sequence capture）是两类常用于降解DNA样本的NGS策略。序列捕获可获取大量直系同源位点的序列数据，但仅能对可作为探针的保守序列附近的基因组区域进行测序。而低覆盖度鸟枪法测序则有望获取全基因组范围内不同类型的数据。然而，采用该方法的诸多研究往往仅能得到线粒体序列，核序列产出极少，这表明直系同源核序列的获取难度可能更高。为明确目前系统发育位置尚存争议的鸡形目（Galliformes）热带鹑属（Tropicoperdix）的分类地位，我们针对两个热带鹑属物种的约百年历史博物馆标本，探索了两种可从低覆盖度鸟枪法测序数据中最大化提取信息的策略。其中一种简单的读段比对策略表现优于另一种（即简化复杂度组装策略），使我们得以获取大量超保守元件（ultraconserved element，UCE）位点、相对保守的外显子、多态性更高的内含子以及完整的线粒体基因组。此外，我们还提出了若干简易方法，用于排查因使用降解DNA样本而可能产生的测序伪影。我们的测序数据将热带鹑属归入了一个包含诸多具装饰性眼斑类群的演化支——包括孔雀、眼斑雉以及孔雀雉，并明确了该属内物种间的系统发育关系。综上，本研究证实低覆盖度鸟枪法测序可便捷地获取大量且类型多样的数据，这为诸多需要从博物馆标本中获取不同类型数据的研究问题开辟了新的研究路径。