The EDNAP collaborative study on mtDNA heteroplasmy detection using Sanger and Massively Parallel Sequencing technologies.

This EDNAP (European DNA Profiling Group) collaborative study evaluated the detection and reporting of mitochondrial DNA (mtDNA) point heteroplasmy (PHP) and length heteroplasmy (LHP) across forensic laboratories using Sanger, Ion Torrent, and Illumina sequencing platforms. Standardized DNA extracts were centrally prepared and distributed to participating laboratories to assess inter-laboratory concordance and platform-specific (platform = instrument + software) effects. Raw sequence data showed high overall consistency across platforms, and observed mitotype discrepancies were due to transcription errors. PHP detection was generally concordant across laboratories and technologies. MPS data provided relatively stable mixture ratios across platforms. Low-level PHPs were often not reported in this study when they fell below laboratory-specific minor allele frequency thresholds, although raw data review confirmed their presence. In contrast, LHP interpretation showed substantially greater variation. Sanger-derived LHP patterns could be harmonized through review under current ISFG guidelines, whereas MPS-based LHP results differed both between and within sequencing technologies. Standardization of the alignment workflow improved concordance to some extent, but important discrepancies remained, particularly in Ion Torrent HVS-I (Hypervariable Segment I) poly(C)-tract data. These differences were attributable to sequencing technology, analysis software, and human interpretation.