本文综述了线粒体基因组测序策略和方法，在传统测序方法中介绍了基于物理分离线粒体DNA的克隆文库测序方法和基于PCR扩增产物的直接测序方法，后者重点介绍了基于长PCR扩增产物的引物步移法和基于总DNA的引物步移法；应用新一代高通量测序方法有基于总DNA样品的方法，

包括需要预扩增mtDNA的多物种平行高通量和无需预扩增mtDNA的高通量方法，基于总RNA样品的转录组测序方法等。在实际工作中，选择哪种方法取决于研究规模、样品大小和保存状态、经费情况等。总的来说，基于长PCR扩增产物的引物步移法尤其适合小规模昆虫线粒体基因组研

究，而对于大规模线粒体基因组研究来说，NGS技术无疑是省时省力的最佳选择。

This paper reviews strategy and methods for sequencing mitochondrial genomes. Two traditional sequencing methods are introduced; the clone librarybased sequencing method from physically purified mtDNA, and the PCR ampliconbased sequencing method, which includes primerwalking sequencing from long PCR amplicons and primerwalking sequencing from total DNA. For the application of nextgeneration sequencing strategies, there are total DNAbased methods, which include the parallel tagged sequencing (PTS) method based on preamplifying mtDNA and total DNA, and the transcriptomic sequencing method based on total RNA. From a practical perspective, which method is chosen depends on the scale of the research, size and preservative status of the sample, and financial support. The primer walking sequencing method based on longPCR amplicons is particularly suitable for small scale research on insect mitochondrial genomes. For large scale mitochondrial genome research, NGS technology is the best choice for saving time and effort.