Platinum-Quality Mitogenome Haplotypes from United States Populations

Abstract

A total of 1327 platinum-quality mitochondrial DNA haplotypes from United States (U.S.) populations were generated using a robust, semi-automated next-generation sequencing (NGS) workflow with rigorous quality control (QC). The laboratory workflow involved long-range PCR to minimize the co-amplification of nuclear mitochondrial DNA segments (NUMTs), PCR-free library preparation to reduce amplification bias, and high-coverage Illumina MiSeq sequencing to produce an average per-sample read depth of 1000 × for low-frequency (5%) variant detection. Point heteroplasmies below 10% frequency were confirmed through replicate amplification, and length heteroplasmy was quantitatively assessed using a custom read count analysis tool. Data analysis involved a redundant, dual-analyst review to minimize errors in haplotype reporting with additional QC checks performed by EMPOP. Applying these methods, eight sample sets were processed from five U.S. metapopulations (African American, Caucasian, Hispanic, Asian American, and Native American) corresponding to self-reported identity at the time of sample collection. Population analyses (e.g., haplotype frequencies, random match probabilities, and genetic distance estimates) were performed to evaluate the eight datasets, with over 95% of haplotypes unique per dataset. The platinum-quality mitogenome haplotypes presented in this study will enable forensic statistical calculations and thereby support the usage of mitogenome sequencing in forensic laboratories.

A multi-dimensional evaluation of the ‘NIST 1032’ sample set across four forensic Y-STR multiplexes

“…This study used the well-established 'NIST 1036' sample set from four main U.S. populations which was previously characterized for other forensic markers [4][5][6]11,12,40] as well as for the Y-STRs in CE-based studies in previous publications [2,3,13,14,41]. To adhere to the high standards of data quality associated with this sample set, here a complete set of Y-STR haplotypes for 30 Y-STR loci is given, including revisions of Y-STR calls published earlier, which are now further confirmed by sequencing, as well as the currently available commercial Y-STR CE kits.…”

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

“…NUMTs are likely to be observed at known NUMT locations [29,30,42,[47][48][49]56], whereas point heteroplasmyparticularly in the codR - [57][58][59][60][61] and stochastic error occur at random locations across the mitogenome [62][63][64]. Furthermore, point heteroplasmy is not observed in a majority of mitogenome haplotypes when applying variant detection thresholds realistic to forensic samples, i.e., a minimum of 5-10% [57][58][59][60][61]. NUMTs (including mega-NUMTs [16,31,32,34]) and point heteroplasmy are reproducible across PCR events from the same DNA sample (or library events, if performing WGS or capture), whereas stochastic error is not reproducible.…”

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

“…To avoid NUMT confounding biases, this study established an assay to sequence the entire mtDNA from a single amplicon produced by long-range PCR, a method that was used before for this purpose. Most long-range PCR approaches covered the entire mitogenome with one to three overlapping amplicons ( Zhang et al, 2012 ; Burgstaller et al, 2014 ; Kloss-Brandstätter et al, 2015 ; Seneca et al, 2015 ; Paijmans et al, 2016 ; Deiner et al, 2017 ; Taylor et al, 2020 ). Nowadays, NGS-based determination of the entire mitogenome from a single long-range PCR product has become routine practice in human medicine, e.g., for diagnosing patients with clinically suspected mtDNA disease ( Zhang et al, 2012 ; Luo et al, 2018 ).…”