Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions.

ABSTRACT:

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 genera

SUBMITTER: Kim H 

PROVIDER: S-EPMC4500979 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature