Full mtGenome reference data: Development and characterization of 588 forensic-quality haplotypes representing three U.S. populations

Abstract

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

Whole mitochondrial genome genetic diversity in an Estonian population sample

“…RMP for mtGenome data was 1.20 versus 4.99 % for HVI/HVII. Compared to RMP values of other populations [14,6,36], the RMP results presented herein for HVI/HVII data are higher. This finding might be explained with 24 HVI/HVII haplotypes having ≥4 identical matches in the population sample.…”

“…Of these mtDNA profiles, 100 (87.7 %) were unique within the data set, and 12.3 % of sequenced mtGenomes were observed twice. Compared to the previous whole mtGenome population studies [6,14], the number of shared haplotypes in the current study is relatively high. This might be a reflection of lower mtGenome diversity in Estonia or just may arise from sampling variance.…”

“…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]. However, with MPS, population data can be generated far more expeditiously and at a lower cost per nucleotide.…”

Importância do DNA em Investigações Forenses: Análise de DNA Mitocondrial

“…Podemos perceber uma tendência nos últimos cinco anos no intuito de realizar a tipagem total do genoma mitocondrial 7,9,12,13 . Esta tendência é importante pois a tipagem do genoma mitocondrial total nos traz, além das informações já amplamente pesquisadas das regiões não codificantes, informações sobre as regiões codificantes que podem ser de interesse forense.…”

“…Em contrapartida, a abordagem de sequenciamento total do genoma mitocondrial é uma solução universal para a obtenção de dados de mtDNA de alta resolução, que podem discriminar entre linhagens maternas relacionadas. No entanto, embora a metodologia forneça um mecanismo para gerar sequências do genoma inteiro de mtDNA de amostras difíceis e degradadas, há uma clara necessidade para o desenvolvimento paralelo de bases de dados de alta qualidade do genoma mitocondrial 7 .Podemos perceber uma tendência nos últimos cinco anos no intuito de realizar a tipagem total do genoma mitocondrial 7,9,12,13 . Esta tendência é importante pois a tipagem do genoma mitocondrial total nos traz, além das informações já amplamente pesquisadas das regiões não codificantes, informações sobre as regiões codificantes que podem ser de interesse forense.…”