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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Reduced amplification and amplicon dropout in PCR-based mitochondrial DNA massively parallel sequencing kits
Lourdes Prieto1, Masinda Nguidi2, Christina Amory3
1Comisaría General de Policía Científica, DNA Laboratory, Madrid, Spain.
Abstract:
The highly polymorphic nature of mitochondrial DNA (mtDNA) poses a significant challenge for primer design in PCR-based assays, because mismatches under primer binding sites can lead to reduced amplification and thus lower sequencing read depth or even complete amplicon dropout. While manufacturers aim to prevent this by adding variant-specific combinations to the primer pools (also known as degenerate primers), it cannot be entirely avoided that some samples are still affected, particularly when rare variants appear in the primer target region. In this study, we evaluated the impact of such variants on the amplification efficiency using two widely implemented Massively Parallel Sequencing kits, the Precision ID MtDNA Control Region / Whole Genome Panels (Thermo Fisher Scientific) and the ForenSeq mtDNA Whole Genome Kit (Verogen). The samples used in this study came from routine casework applications (analysed in the Control Region) and from a study of population genetics and phylogeography (encompassing the entire mitogenome). The observed instances of drop-out or reduced read depth were examined for their phylogenetic background and for their expected abundance and relevance in forensic casework. We discuss possible solutions to mitigate these issues, including the use of overlapping amplicons and an increased number of degenerate primers.
Insights
Mitochondrial DNA (mtDNA) primer design is challenging due to high polymorphism. This study evaluated how variants affect amplification efficiency in Massively Parallel Sequencing kits, impacting forensic casework and population genetics.
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.
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