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Updated: Apr 9, 2026

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Hanna Kim, Henry A Erlich, Cassandra D Calloway1
1Cassandra D. Calloway, Children's Hospital Oakland Research Institute, Center for Genetics, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA, scalloway@chori.org.
Aim:
To apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.
Methods:
A duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel.
Results:
This assay was shown to be highly sensitive for sequencing limited DNA amounts (~100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants (~1%) as well as "complex" mixtures (≥3 contributors). PCR artifact "hybrid" sequences generated by jumping PCR or template switching were observed at a low level (<2%) in the analysis of mixed samples but could be eliminated by reducing the PCR cycle number.
Conclusion:
This study demonstrates the power of NGS technologies targeting the mtDNA HVI/HVII regions for analysis of challenging forensic samples, such as mixtures and specimens with limited DNA.
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