• Hanna Kim, Henry A Erlich, and Cassandra D Calloway.
• Cassandra D. Calloway, Children's Hospital Oakland Research Institute, Center for Genetics, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA, scalloway@chori.org.
• Croat. Med. J. 2015 Jun 1; 56 (3): 208-17.

AimTo apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.MethodsA 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.ResultsThis 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.ConclusionThis 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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