Optimization of a duplex amplification and sequencing strategy for the HVI/HVII regions of human mitochondrial DNA for forensic casework

The ABI Prism® BigDye™ version 1.1 chemistry provided high quality sequencing data, with little or no background noise and uniform peak heights, outcomes that favored reliable detection of heteroplasmy, particularly at early sequence reads (<40 bases). Optimal compromise between sensitivity and sequence accuracy in the absence of noise was achieved starting at 150 mitochondrial genome copies. The protocol is effective (no sequence errors) with highly degraded DNA (average detectable template size of 200 bp). Dual artificial template mixtures with the minor component at 15% suggests that heteroplasmy should be detected at this level with confidence.