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Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq

  • Michelle A. Peck
    Correspondence
    Correspondence to: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr Dover AFB, DE 19902, United States.
    Affiliations
    Armed Forces DNA Identification Laboratory, 115 Purple Heart Drive, Dover AFB, DE 19902, United States

    American Registry of Pathology Sciences, 9210 Corporate Boulevard, Suite 120, Rockville, MD 20850, United States
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  • Michael D. Brandhagen
    Affiliations
    Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, United States
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  • Charla Marshall
    Affiliations
    Armed Forces DNA Identification Laboratory, 115 Purple Heart Drive, Dover AFB, DE 19902, United States

    American Registry of Pathology Sciences, 9210 Corporate Boulevard, Suite 120, Rockville, MD 20850, United States
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  • Author Footnotes
    1 Present address: Defense Forensic Science Center, 4930 N. 31st Street, Forest Park, GA 30297, United States.
    ,
    Author Footnotes
    2 Present address: ANSER, 5275 Leesburg Pike, Suite N-5000, Falls Church, VA 22041, United States.
    Toni M. Diegoli
    Footnotes
    1 Present address: Defense Forensic Science Center, 4930 N. 31st Street, Forest Park, GA 30297, United States.
    2 Present address: ANSER, 5275 Leesburg Pike, Suite N-5000, Falls Church, VA 22041, United States.
    Affiliations
    Armed Forces DNA Identification Laboratory, 115 Purple Heart Drive, Dover AFB, DE 19902, United States

    American Registry of Pathology Sciences, 9210 Corporate Boulevard, Suite 120, Rockville, MD 20850, United States
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  • Jodi A. Irwin
    Affiliations
    Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, United States
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  • Kimberly Sturk-Andreaggi
    Affiliations
    Armed Forces DNA Identification Laboratory, 115 Purple Heart Drive, Dover AFB, DE 19902, United States

    American Registry of Pathology Sciences, 9210 Corporate Boulevard, Suite 120, Rockville, MD 20850, United States
    Search for articles by this author
  • Author Footnotes
    1 Present address: Defense Forensic Science Center, 4930 N. 31st Street, Forest Park, GA 30297, United States.
    2 Present address: ANSER, 5275 Leesburg Pike, Suite N-5000, Falls Church, VA 22041, United States.

      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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