Sensitivity of mitochondrial DNA heteroplasmy detection using Next Generation Sequencing

Although the use of Next Generation Sequencing (NGS) in mitochondrial DNA (mtDNA) studies related to forensic and human genetics has contributed to the report of heteroplasmy at very low levels (lower than 1% and even 0.5%), their detection is not a straightforward process. Our purpose is to establish mitochondrial heteroplasmy detection limits, generating mixed bases at low frequencies by the PCR co-amplification of mtDNA and a nuclear insertion of mitochondrial origin (NUMT). A primer set that presents a perfect annealing with both mitochondrial and nuclear DNA was used to amplify the mitochondrial DNA region located between positions 6739 and 8910 and the corresponding region located inside a NUMT located in chromosome 1 (GRCh38.p12 Primary Assembly 631910-634079) that presents an identity of 98% with the corresponding region of mtDNA in two samples. Independent Nextera XT® (Illumina) NGS libraries were generated for each sample and sequenced in different MiSeq (Illumina) runs. Non-identical and identical positions between individual mtDNA and NUMT sequences were detected, and heteroplasmy detections limits were established: a) with a minor allele frequency <1.5%, false positive and negative can occur, even with a depth higher than 3000X; b) with a minor allele frequency >3%, no presence of false positive and negative were detected with a depth of ~1000X; and c) with a minor allele frequency between 1.5% and 3%, a minimal depth of 3000X was necessary to avoid false positives or negatives detection. Our results demonstrate an alternative strategy to establish a reliable limit of mitochondrial heteroplasmy detection.