Optimized PCR-based enrichment improves coverage uniformity and mutation detection in mitochondrial DNA next generation sequencing

Publication date: Available online 7 February 2020Source: The Journal of Molecular DiagnosticsAuthor(s): Yang Liu, Shanshan Guo, Chun Yin, Xu Guo, Manling Liu, Zhidong Yuan, Zheng Zhao, Yongfeng Jia, Jinliang XingABSTRACTNext generation sequencing -based methods have been commonly used to detect mutations of mitochondrial genome. PCR amplification is a highly effective way for 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. We analyzed the effect of DNA quality on efficiency of PCR amplification and assessed the effects of different primer design methods including the number of primer pairs, overlap length of amplicon and modification of primers on coverage uniformity and mutation calling in mtDNA sequencing. Our results showed that the DNA quality significantly affected the PCR amplification efficiency. Importantly, the over- and under-representation of coverage depth at overlap region of amplicons was observed when amplicons were not modified and overlap was shorter than two sequencing fragment sizes (800bp). Then, under-representation was overcome by increasing overlap length of amplicons, and over-representation was effectively reduced by 5’-block modification of primers and sticky-end ligation of amplicons. Moreover, we found tha...

Source: The Journal of Molecular Diagnostics - February 8, 2020 Category: Pathology Source Type: research