Abstract
Abstract 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 labelling 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. 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 labelling protocol for cost‐effectively ligating barcode sequences to DNA samples and used PCR‐generated baits for mtDNA enrichment. The widely adopted ‘direct pooling’ sequencing strategy is the fastest but performs the worst among the four strategies. Of the 96 samples analysed, 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. We hope that our comparisons will assist researchers in selecting suitable strategies for large‐scale sequencing of mitogenomes from museum specimens, thereby advancing museomics.
Published Version
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