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
