Massively parallel sequencing (MPS) is increasingly being adopted in forensic laboratories for the analysis of short tandem repeats (STRs) and mitochondrial DNA (mtDNA). With the rapid development of novel sequencing technologies, each offering distinct chemistries and read characteristics, a systematic comparison of available platforms for forensic applications is warranted.

This study presents a comparative evaluation of five MPS platforms — Illumina MiSeq, Illumina MiSeq i100, Oxford Nanopore Technologies PromethION 24, Element Biosciences AVITI, and Pacific Biosciences Revio — using sequencing data from single-source human DNA samples. Data were analyzed using FDSTools, applying a uniform workflow across all platforms. For STR analysis, performance was assessed using concordance with capillary electrophoresis (CE) results, allele coverage ratio, coefficient of variation of coverage, stutter ratio, and signal-to-noise ratio. For mtDNA analysis, concordance with proficiency test results, quantification of cytosine-tract length, and detection of heteroplasmy were evaluated.

We will present an overview of platform-specific strengths and limitations for both STR and mtDNA analysis. For platforms not yet validated for forensic use, the results additionally serve as proof of concept for their suitability in casework applications. The findings aim to support informed decisions regarding the implementation and validation of MPS technologies in forensic laboratories and to contribute to the development of a standardized interpretation framework for MPS-based forensic DNA analysis.

Authors

• Susan Kloet (Leiden University Medical Center, Netherlands)
• Fenne Schuurmans (Leiden University Medical Center, Netherlands)
• Rick de Leeuw (Leiden University Medical Center, Netherlands)
• Marije te Raa (Leiden University Medical Center, Netherlands)
• Thirsa Kraaijenbrink (Leiden University Medical Center, Netherlands)