Mitochondrial genome sequencing with ForenSeq™ mtDNA Whole Genome Kit.

Mitochondrial DNA (mtDNA) possesses unique genetic characteristics and plays a crucial role in forensic DNA analysis. Based on the massively parallel sequencing (MPS) technology alongside the short overlapping amplicon method, the ForenSeq™ mtDNA Whole Genome Kit is specifically designed for mtDNA analysis. In this study, we employ the ForenSeq™ mtDNA Whole Genome Kit on the MiSeq FGx® Sequencing System for mitochondrial genome (mtGenome) sequencing across nine consecutive runs and assess its MPS performance, such as read depth (RD), forward/reverse strand bias (SB), and mtGenome coverage. Furthermore, we conduct internal validations to evaluate its routine application in forensic sciences, including sensitivity, repeatability, concordance, degraded samples, inhibitor samples, case-type samples, and contamination. As a result, the Real-Time Analysis (RTA) and Universal Analysis Software (UAS) demonstrate proficient run metrics and MPS performance when 12-14 libraries are sequenced within a standard flow cell, achieving > 80 % of reads passing filter, > 80 % bases with ≥Q30, > 5000 × of the average RD, ∼1.0 of the average SB, > 70 % of the inter-amplicon balance, and > 99 % of the mtGenome coverage. The five most vulnerable amplicons, exhibiting low RD and high SB, are identified as nucleotide positions (nps) 1094-1177, 5858-5975, 6109-6149, 6718-6810, and 7021-7090. For tertiary data analysis, the substitutions are accurately reported by UAS, while insertions and deletions (indels), point heteroplasmies (PHPs), and/or length heteroplasmies (LHPs) still necessitate manual inspection. On average, 40 variants were found in 60 samples, ranging from 27 to 54. A total of 2426 variants are observed at 491 nps. Moreover, the workflow can yield repeatable and reproducible results, generate complete mtGenome profiles from ≥ 2 pg input gDNA for high quality samples/control DNA or ≥ 0.5 cm hair shafts, and recover more/complete mtGenome information from severely degraded samples (degradation index >10) and various types of case samples. If two rounds of purification are conducted, it can more effectively remove additional reaction components and enhance data recovery from the mtGenome, especially for low-input samples. The negative controls in three runs cover some reads, but these contaminations cannot compromise the mitochondrial analyses. In conclusion, the ForenSeq™ mtDNA Whole Genome Kit, including 234 short overlapping amplicons with an average size of 131 bp, can meet forensic needs on the whole mtGenome sequencing in real scenarios. In addition, the ten insights gained from this study may serve as a valuable reference for forensic scientists who are utilizing this kit.