title: Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens.
abstract: Forensic mitochondrial DNA (mtDNA) testing requires appropriate, high quality reference population data for estimating the rarity of questioned haplotypes and, in turn, the strength of the mtDNA evidence. Available reference databases (SWGDAM, EMPOP) currently include information from the mtDNA control region; however, novel methods that quickly and easily recover mtDNA coding region data are becoming increasingly available. Though these assays promise to both facilitate the acquisition of mitochondrial genome (mtGenome) data and maximize the general utility of mtDNA testing in forensics, the appropriate reference data and database tools required for their routine application in forensic casework are lacking. To address this deficiency, we have undertaken an effort to: (1) increase the large-scale availability of high-quality entire mtGenome reference population data, and (2) improve the information technology infrastructure required to access/search mtGenome data and employ them in forensic casework. Here, we describe the application of a data generation and analysis workflow to the development of more than 400 complete, forensic-quality mtGenomes from low DNA quantity blood serum specimens as part of a U.S. National Institute of Justice funded reference population databasing initiative. We discuss the minor modifications made to a published mtGenome Sanger sequencing protocol to maintain a high rate of throughput while minimizing manual reprocessing with these low template samples. The successful use of this semi-automated strategy on forensic-like samples provides practical insight into the feasibility of producing complete mtGenome data in a routine casework environment, and demonstrates that large (>2kb) mtDNA fragments can regularly be recovered from high quality but very low DNA quantity specimens. Further, the detailed empirical data we provide on the amplification success rates across a range of DNA input quantities will be useful moving forward as PCR-based strategies for mtDNA enrichment are considered for targeted next-generation sequencing workflows.
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States; University of Maryland, College Park, 8082 Baltimore Ave., College Park, MD 20740, United States. Electronic address: rebecca.s.just.ctr@mail.mil.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Just
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Scheible
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Spence A
authors.initials: SA
authors.lastname: Fast
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Sturk-Andreaggi
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Higginbotham
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Lyons
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Jocelyn M
authors.initials: JM
authors.lastname: Bush
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Peck
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Ring
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Toni M
authors.initials: TM
authors.lastname: Diegoli
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Innsbruck, Austria.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Röck
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Innsbruck, Austria.
authors.forename: Gabriela E
authors.initials: GE
authors.lastname: Huber
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Innsbruck, Austria.
authors.forename: Simone
authors.initials: S
authors.lastname: Nagl
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Innsbruck, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Strobl
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Innsbruck, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Zimmermann
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Innsbruck, Austria; Penn State Eberly College of Science, 517 Thomas Building, University Park, PA 16802, United States.
authors.forename: Walther
authors.initials: W
authors.lastname: Parson
authors.affiliation: Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, United States; American Registry of Pathology, 120A Old Camden Rd., Camden, DE 19934, United States.
authors.forename: Jodi A
authors.initials: JA
authors.lastname: Irwin
mesh_terms: D004272:DNA, Mitochondrial / Q000235:genetics*; D053803:Forensic Genetics; D015894:Genome, Human; D006239:Haplotypes; D006801:Humans
chemical_list: D004272:DNA, Mitochondrial
keywords: Automation; Low template specimens; Mitochondrial genome; Sequencing; mtDNA