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

Published

July 23, 2026

Author(s)

Kevin Kiesler, Erica Romsos, Peter Vallone, Walther Parson

Abstract

This EDNAP collaborative study evaluated the detection and reporting of mitochondrial DNA 125 (mtDNA) point heteroplasmy (PHP) and length heteroplasmy (LHP) across forensic 126 laboratories using Sanger, Ion Torrent, and Illumina sequencing platforms. Standardized DNA 127 extracts were centrally prepared and distributed to participating laboratories to assess inter128 laboratory concordance and platform-specific (platform = instrument + software) effects. Raw 129 sequence data showed high overall consistency across platforms, and observed mitotype 130 discrepancies were due to transcription errors. PHP detection was generally concordant 131 across laboratories and technologies. MPS data provided relatively stable mixture ratios 132 across platforms. Low-level PHPs were often not reported when they fell below laboratory133 specific minor allele frequency thresholds, although raw data review confirmed their 134 presence. In contrast, LHP interpretation showed substantially greater variation. Sanger135 derived LHP patterns could be harmonized through review under current ISFG guidelines, 136 whereas MPS-based LHP results differed both between and within sequencing technologies. 137 Standardization of the alignment workflow improved concordance to some extent, but 138 important discrepancies remained, particularly in Ion Torrent HVS-I data. These differences 139 were attributable to sequencing technology, analysis software, and human interpretation.

Citation

Forensic Science International: Genetics

Volume

86

Pub Type

Journals

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Keywords

Mitochondrial DNA, Next Generation Sequencing, Homopolymeric poly(C)-tracts, Interpretation, Forensic Genetics

Citation

Kiesler, K. , Romsos, E. , Vallone, P. and Parson, W. (2026), The EDNAP collaborative study on mtDNA heteroplasmy detection using Sanger and Massively Parallel Sequencing technologies, Forensic Science International: Genetics, [online], https://nps-ext.nist.gov/nps/nps_public_api/api/Publication/file/962246 (Accessed October 23, 2026)

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