Reduced amplification and amplicon dropout in PCR-based mitochondrial DNA massively parallel sequencing kits

Area of Science:

• Forensic genetics
• Population genetics
• Molecular biology

Background:

• Mitochondrial DNA (mtDNA) exhibits high polymorphism, complicating PCR primer design.
• Primer-mtDNA mismatches can cause reduced amplification, lower sequencing depth, or amplicon dropout.
• Degenerate primers are used to mitigate but cannot fully prevent issues with rare variants.

Purpose of the Study:

• To evaluate the impact of mtDNA variants on PCR amplification efficiency.
• To assess the performance of two Massively Parallel Sequencing (MPS) kits: Precision ID and ForenSeq.
• To examine the phylogenetic and forensic relevance of amplification dropouts.

Main Methods:

• Analysis of routine casework and population genetics samples.
• Evaluation of Precision ID MtDNA Control Region / Whole Genome Panels.
• Evaluation of ForenSeq mtDNA Whole Genome Kit.
• Examination of variant impact on amplification efficiency and sequencing read depth.

Main Results:

• Observed instances of amplicon dropout and reduced read depth due to mtDNA variants.
• Variants were analyzed for their phylogenetic background, abundance, and forensic relevance.
• The study identified specific challenges posed by polymorphic regions in mtDNA sequencing.

Conclusions:

• mtDNA variants significantly impact PCR amplification efficiency in MPS assays.
• Understanding variant effects is crucial for accurate forensic analysis and population studies.
• Solutions like overlapping amplicons and increased primer degeneracy are proposed to improve mtDNA sequencing robustness.