Highlights
- •ForenSeq™ mtDNA Control Region Kit concordant with other mtDNA sequencing assays.
- •The robust chemistry can reliably recover profiles from challenging forensic samples.
- •Sequencing of mitochondrial whole genome assays can benefit forensic casework.
Abstract
The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software
(UAS) were assessed to better define the performance and limitations of the system
with forensically relevant samples to provide data for its transition into practice.
A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three
runs of additional orthogonal sequencing chemistries, and Sanger sequencing results
for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture
detection studies, as well as studies to measure the performance of amplification
and sequencing controls were performed. The use and reliability of the UAS for data
analysis was also examined. With a variety of sample types and controls representing
many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with
the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible,
and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which
allows the system to offer low limits of detection for better interrogation of potential
heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS)
for mtDNA in laboratories were addressed in this research, including initial template
quantification and confirmation of haplotypes generated by UAS software regarding
length-based polymorphisms. To improve performance with forensic samples, laboratories
could implement mitochondrial-specific qPCR assays for quantification and perform
the optional manual normalization protocol. Additional optimization on sample multiplexing
can provide methods that either increase sensitivity or cost efficiency of the assay.
Keywords
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Article info
Publication history
Published online: May 11, 2022
Accepted:
May 8,
2022
Received in revised form:
April 13,
2022
Received:
January 16,
2022
Identification
Copyright
© 2022 Elsevier B.V. All rights reserved.
