title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Hanna
authors.initials: H
authors.lastname: Kim
authors.forename: Henry A
authors.initials: HA
authors.lastname: Erlich
authors.affiliation: 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.
authors.forename: Cassandra D
authors.initials: CD
authors.lastname: Calloway
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D053803:Forensic Genetics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial