Sequence capture and phylogenetic utility of genomic ultraconserved elements obtained from pinned insect specimens more than 100 years-old. Xylocopa

Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a recently developed target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with ages ranging from 2–121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and our DNA yield and sequence capture variables. Additionally, we reconstructed a phylogeny in order to confirm the validity of our sequence data. We recovered 6–972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06–9.8 ng/µL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts than specimens older than 20 years. We found breakpoints in our data between 21–39 years for pre- and post-library DNA concentration and UCE contig length beyond the negative effect of decreased specimen age. Our phylogenetic results confirmed the integrity of our sequences as specimens of the same species grouped together, and give preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss our phylogenetic results with regard to the promise of this UCE approach for large scale projects in insect phylogenomics using museum specimens.

从馆藏历史标本中获取序列数据已成为日益增长的研究热点，而支持对高度降解DNA进行上机测序的新一代测序技术为该领域注入了新的动力。本研究采用新近开发的靶向富集与新一代测序实验方案，从51件木蜂属（Xylocopa）大木蜂标本中获取了超保守元件（ultraconserved elements, UCEs）数据，这些标本涵盖25个物种，保存时长介于2至121年之间。我们通过线性回归模型分析了标本保存时长与DNA产出量（建库前与建库后的DNA浓度）以及多项UCE序列捕获统计指标（原始读段数、靶区域UCE读段数、UCE平均重叠群长度以及UCE位点数量）之间的相关性，并采用分段回归模型检验了标本保存时长与DNA产出量、序列捕获指标之间关系的特定拐点。此外，我们构建了系统发育树以验证所获序列数据的有效性。在建库前DNA浓度介于0.06至9.8 ng/µL的样本中，我们成功获取了6至972个UCE位点。所有被测的DNA产出量与序列捕获指标均与标本保存时长呈显著但仅为中度的负相关关系。保存时长不超过20年的标本，其建库前、后DNA浓度、UCE重叠群长度以及UCE位点数量均显著高于保存时长超过20年的标本。我们发现，在建库前、后DNA浓度以及UCE重叠群长度的相关关系中，其与标本保存时长的负相关拐点出现在21至39年的区间内，在此拐点之后，标本保存时长对上述指标的负向影响进一步加剧。我们的系统发育分析结果证实了序列数据的可靠性：同一物种的标本聚类为单系群，同时该结果也为木蜂属内部的系统发育关系提供了初步的研究见解。我们探讨了本研究未纳入测量的其他因素（如DNA碎片化程度与标本保存方式）对与标本保存时长相关的序列捕获结果的影响，并结合该UCE方法在利用馆藏标本开展昆虫系统发育基因组学大规模项目中的应用前景，对本次系统发育分析结果进行了讨论。