Mitochondrial DNA (mtDNA) is an indispensable genetic marker in forensic genetics. The emergence and development of massively parallel sequencing (MPS) makes it possible to obtain complete mitochondrial genome sequences more quickly and accurately. The study evaluated the advantages and limitations of the ForenSeq mtDNA Whole Genome Kit in the practical application of forensic genetics by detecting human genomic DNA standards and thirty-three case samples. We used control DNA with different amount to determine sensitivity of the assay. Even when the input DNA is as low as 2.5 pg, most of the mitochondrial genome sequences could still be covered. For the detection of buccal swabs and aged case samples (bloodstains, bones, teeth), most samples could achieve complete coverage of mitochondrial genome. However, when ancient samples and hair samples without hair follicles were sequenced by the kit, it failed to obtain sequence information. In general, the ForenSeq mtDNA Whole Genome Kit has certain applicability to forensic low template and degradation samples, and these results provide the data basis for subsequent forensic applications of the assay. The overall detection process and subsequent analysis are easy to standardize, and it has certain application potential in forensic cases.
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