Original scientific paper
https://doi.org/10.3325/cmj.2015.56.208
Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Hanna Kim
; 1Center for Genetics, Children’s Hospital Oakland Research Institute, Oakland, CA, USA
Henry A. Erlich
; 1Center for Genetics, Children’s Hospital Oakland Research Institute, Oakland, CA, USA
Cassandra D. Calloway
; 1Center for Genetics, Children’s Hospital Oakland Research Institute, Oakland, CA, USA
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 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.
Keywords
Hrčak ID:
144066
URI
Publication date:
15.6.2015.
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