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.

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

“…While this assay is generally intended for higher quality specimens harboring pristine DNA, it has been shown to be highly effective on low quality and quantity samples [44]. As a result, two hair extracts with relatively high qPCR-determined mtDNA quantities were selected for enrichment with this method.…”

Whole mitochondrial genome genetic diversity in an Estonian population sample

“…While more than 25,000 forensic mtDNA sequences and almost 35,000 mtDNA sequences in total are available currently in the EMPOP database [11,12], these data include information only from the mtDNA control region. In addition to STS, a number of other technologies permit practical access to mtDNA coding region data through single nucleotide polymorphism assays, sequence-specific oligonucleotide probes, mass spectroscopy, and MPS technology that emphasize the importance of a whole mtGenome database [13]. Recently, 588 forensic-quality whole mtGenomes from three major US populations have been determined with Sanger sequencing and will be available for query [14].…”

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

“…These DNA extracts were previously processed with STS to generate full mtGenome haplotypes [9,24]. Samples for this study were chosen based on U.S. population group (African American, U.S. Caucasian, and U.S. Hispanic), haplogroup, and unique sequence features (e.g.…”