The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.

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