Interlaboratory exercise to establish proficiency testing for sequencing of forensic STR and SNP markers

Massively parallel sequencing (MPS) is increasingly used in forensic DNA analysis for SNP and STR genotyping, though accredited proficiency tests remain limited. To address this, five forensic DNA laboratories from four countries participated in a study to assess MPS methods across different kits and platforms. In this study ForenSeq DNA Signature Prep Kit, ForenSeq MainstAY kit, Precision ID GlobalFiler NGS STR Panel v2, Precision ID Identity Panel and Precision ID Ancestry Panel were used to analyze four reference samples and three mock stain samples with different number and proportion of contributors (3:1, 3:1:1, 6:3:1). Performance for autosomal, Y-chromosomal, and X-chromosomal STRs, as well as for identity-, ancestry-, and phenotype-informative SNPs was evaluated. In addition, appearance and ancestry prediction for unknown sample donors was compared between the laboratories. Overall, the results from the participating laboratories showed a high level of agreement, regardless of the platform employed. The issues leading to unsuccessful genotyping were mainly related to different characteristics of the library preparation kits and sequencing technologies, software algorithms used for genotyping (e.g. noise and artefact filtering), or in-house interpretation rules (such as thresholds for allele calling or imbalance). The findings also emphasized the importance of using multiple software tools for accurate ancestry and phenotype prediction. The outcome of the study will help to standardize MPS practices, ensuring reliable and consistent results across laboratories. These findings contribute with valuable knowledge for developing future proficiency tests in forensic MPS analysis, offering insights into analytical variability and result reliability. This study identified key issues affecting genotyping accuracy, critical for developing effective proficiency tests. The insights gained apply broadly to current and future MPS kits.