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Analysis of Mixtures Using Next Generation Sequencing of Mitochondrial DNA Hypervariable Regions

NCJ Number
Date Published
June 2015
10 pages
This article reports on a study that applied massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.
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 (&#8201;~&#8201;100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants (&#8201;~&#8201;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)
Date Published: June 1, 2015