Highlights
- •The full mtGenome of 90 high quality samples were sequenced on the Illumina MiSeq.
- •Nextera XT library preparation and sequencing was performed at two laboratories.
- •NGS data was 99.9996% concordant with previously generated Sanger data.
- •Variant calls were reproducible and variant frequency (VF) differed by only 0.23%.
- •Replicate analysis resulted in the same variant calls and only 0.01% VF difference.
Abstract
Sanger-type sequencing (STS) of mitochondrial DNA (mtDNA), specifically the control
region (CR), is routinely employed in forensics in human identification and missing
persons scenarios. Yet next-generation sequencing (NGS) has the potential to overcome
some of the major limitations of STS processing, permitting reasonable paths forward
for full mitochondrial genome (mtGenome) sequencing, while also offering higher-throughput
and higher sensitivity capabilities. To establish the accuracy and reproducibility
of NGS for the development of mtDNA data, 90 DNA extracts that were previously used
to generate forensic quality full mtGenomes using STS were sequenced using Nextera
XT library preparation and the Illumina MiSeq. Using the same amplicon product, replicate
library sets were generated and sequenced at different laboratories, and analysis
was performed in replicate using the CLC Genomics Workbench. Both sequencing sets
resulted in 99.998% of positions with greater than 10X coverage when 96 samples (including
controls) were multiplexed. Overall, 99.9996% concordance was observed between the
NGS data and the STS data for the full mtGenome. The only “discordant” calls involved
low level point heteroplasmies, with the differences resulting from stochastic variation
and/or the increased sensitivity of NGS. Higher sensitivity also allowed for the detection
of a mixed sample previously not detected with STS. Additionally, variant calls were
reproducible between sequencing sets and between software analysis versions with the
variant frequency only differing by 0.23% and 0.01%, respectively. Further validation
studies and specialized software functionality tailored to forensic practice should
facilitate the incorporation of NGS processing into standard casework applications.
The data herein comprise the largest, and likely most thoroughly examined, complete
mtGenome STS-NGS concordance dataset available.
Keywords
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Article info
Publication history
Published online: June 06, 2016
Accepted:
June 3,
2016
Received in revised form:
May 27,
2016
Received:
February 29,
2016
Identification
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