A comparison and evaluation of strategies for bulk sequencing of mitogenomes from museum collections

1. Collecting mitochondrial genome sequences from museum collections has garnered increasing attention in recent years. Currently, several sequencing strategies have been proposed for this purpose, including direct DNA pooling, DNA labeling, and mtDNA enrichment. It is important to understand the advantages and limitations of these methods before embarking on large-scale sequencing efforts. However, there is currently no comprehensive comparison and evaluation of the performance of these sequencing methods. 2. In this study, we evaluated the performance of four different strategies for sequencing mitogenomes by comparing reagent expense, experiment time, bioinformatics time, and the length and the base error rate of the obtained mitochondrial sequences. The four sequencing strategies mainly differ in: (1) whether to label the DNA of each specimen and (2) whether to perform mtDNA enrichment. To reduce experiment difficulty and expenses, we presented an "in-situ" DNA labeling protocol for cost-effectively ligating barcode sequences to DNA samples and used PCR-generated baits for mtDNA enrichment. 3. The widely adopted "direct pooling" sequencing strategy is the fastest but performs the worst among the four strategies. Of the 96 samples analyzed, this approach yields averagely only 670 bp of mitochondrial sequences per sample. The most complicated sequencing strategy, which labels the DNA of each specimen and performs mtDNA enrichment, performs the best, producing mitogenome sequences of ~9.3 kb per sample. The sequencing accuracy is highly related to the sequencing depths of mtDNA but not the storage time of samples. 4. We hope that our comparisons will assist researchers in selecting suitable strategies for large-scale sequencing of mitogenomes from museum specimens, thereby advancing museomics.

1. 近年来，从馆藏标本中获取线粒体基因组（mitochondrial genome）序列的研究受到越来越多的关注。目前已有多种针对该场景的测序策略被提出，包括直接DNA混合法、DNA标记法以及线粒体DNA（mtDNA）富集法。在开展大规模测序工作前，明确这些方法的优势与局限至关重要，但目前尚无针对这些测序策略性能的全面对比与评估。2. 本研究通过对比试剂成本、实验耗时、生物信息学分析耗时，以及所得线粒体序列的长度与碱基错误率，评估了四种不同的线粒体基因组（mitogenome）测序策略的性能。这四种测序策略的核心差异在于：(1) 是否对每份标本的DNA进行标记；(2) 是否开展线粒体DNA富集实验。为降低实验难度与成本，我们开发了一种"原位（in-situ）"DNA标记方案，可高效将条形码（barcode）序列连接至DNA样本中，并采用PCR合成的诱饵探针完成线粒体DNA富集。3. 当前应用广泛的"直接混合法"测序策略是四种策略中耗时最短的，但性能最差。在本次分析的96份样本中，该方法每份样本仅能平均获得670 bp的线粒体序列。而最为复杂的测序策略（即对每份标本DNA进行标记并开展线粒体DNA富集）性能最优，每份样本可获得约9.3 kb的线粒体基因组序列。测序准确率与线粒体DNA的测序深度高度相关，但与样本的保存时长无关。4. 我们期望本研究的对比结果能够帮助研究人员为馆藏标本线粒体基因组的大规模测序选择合适的策略，进而推动博物馆基因组学（museomics）的发展。