Optimized PCR-Based Enrichment Improves Coverage Uniformity and Mutation Detection in Mitochondrial DNA Next-Generation Sequencing.

Next-generation sequencing-based methods have been commonly used for detecting mutations of mitochondrial genome (mtDNA). PCR amplification is a highly effective method of mtDNA enrichment before sequencing. However, it has been observed that highly variable sequencing depth within PCR amplicons severely reduces the coverage uniformity and accuracy of mutation calling. Therefore, it is necessary to develop an optimized PCR-based strategy for mtDNA sequencing. Herein, the effect of DNA quality on the efficiency of PCR amplification was analyzed and the effects of different primer-design methods, including the number of primer pairs, overlap length of amplicons, and modification of primers, on coverage uniformity and mutation calling in mtDNA sequencing were assessed. Results showed that DNA quality significantly affected the efficiency of PCR amplification. Importantly, over- and under-representation of coverage depth at overlap regions of amplicons were observed when amplicons were not modified and overlap was shorter than two sequencing fragment sizes (800 bp). Then, under-representation was overcome by increasing the overlap length of the amplicons, and over-representation was effectively reduced by 5'-block modification of primers and sticky-end ligation of amplicons. Moreover, findings indicated that these two optimized PCR-based sequencing strategies effectively improved mutation calling in primer-binding regions. Optimized PCR-based mtDNA enrichment and sequencing approaches have been established, which laid a foundation for accurate mutation detection of mtDNA in diseases.