This study demonstrated 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. 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. 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 percent) 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 percent) in the analysis of mixed samples but could be eliminated by reducing the PCR cycle number. (Publisher abstract modified)
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