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

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Next-generation sequencing (NGS) is crucial for mitochondrial genome (mtDNA) mutation detection.
• PCR amplification enriches mtDNA but often results in variable sequencing depth, hindering accuracy.

Purpose of the Study:

• To optimize PCR-based strategies for uniform mtDNA sequencing coverage.
• To improve the accuracy of mutation calling in mtDNA sequencing.

Main Methods:

• Assessed DNA quality impact on PCR amplification efficiency.
• Evaluated primer design: number of pairs, amplicon overlap, and primer modification (5'-block).
• Analyzed sticky-end ligation of amplicons.

Main Results:

• DNA quality significantly influences PCR efficiency.
• Short amplicon overlap (<800 bp) caused coverage depth variations (over- and under-representation).
• Increased overlap and 5'-block primer modification/sticky-end ligation improved coverage uniformity and mutation calling accuracy.

Conclusions:

• Optimized PCR strategies enhance mtDNA enrichment and sequencing.
• Established methods provide a foundation for accurate mtDNA mutation detection in disease research.