A strategy for a high enrichment of insect mitochondrial DNA for mitogenomic analysis

Next-generation sequencing has dramatically fostered insect mitogenomic research in recent years. However, studies on the insect mitochondrial genome (mitogenome) assembly mainly rely on the sequencing data from total DNA, which is not cost-effective as a huge data from nuclear DNA are wasted. Besides, many mitogenomic studies require genomic information from individual organisms, whereas the DNA yield from small individual insects is too low to meet the sequencing requirements. Here, we describe a strategy for a high enrichment of insect mitochondrial DNA (mtDNA) using rolling circle amplification (RCA) technique. This strategy consists of standard DNA extraction, RCA enrichment, next-generation sequencing and mitogenome assembly. We have evaluated the performance of this strategy on nine insect species representing eight families of insecta, three other invertebrates, and even two vertebrate specimens. Results show that our strategy is especially suitable for insects, which allows almost all tested insect mtDNA contents to reach 80% and above. A further examination of enrichment efficiency of our strategy among different taxa shows that it is also applicable to other invertebrates and even some vertebrates such as Rhacophorus and ptyas species, although its enrichment efficiency in these groups is lower than that of insects. After treatment with our strategy, small flux sequencing data can realize the assembly of mitogenome with deep coverage, providing a solid base for subsequent mitogenome-based studies.