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

Sadam, Maarja ; Sidstedt, Maja LU ; Järving, Reet ; Kampmann, Marie Louise ; Mogensen, Helle Smidt ; Hanssen, Eirik Natås ; Janssen, Kirstin ; Salvo, Nina Mjølsnes ; Hedman, Johannes LU and Väli, Marika (2025) In Forensic Science International: Genetics 78.
Abstract

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... (More)

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.

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; ; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Forensic genetics, Massively parallel sequencing (MPS), Next generation sequencing (NGS), Proficiency test, Short tandem repeat (STR), Single nucleotide polymorphism (SNP)
in
Forensic Science International: Genetics
volume
78
article number
103285
publisher
Elsevier
external identifiers
  • scopus:105004222561
  • pmid:40319668
ISSN
1872-4973
DOI
10.1016/j.fsigen.2025.103285
language
English
LU publication?
yes
id
8d0f3590-7762-44e4-a7e0-51004c59b6ee
date added to LUP
2025-07-18 10:42:45
date last changed
2025-07-18 10:43:38
@article{8d0f3590-7762-44e4-a7e0-51004c59b6ee,
  abstract     = {{<p>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.</p>}},
  author       = {{Sadam, Maarja and Sidstedt, Maja and Järving, Reet and Kampmann, Marie Louise and Mogensen, Helle Smidt and Hanssen, Eirik Natås and Janssen, Kirstin and Salvo, Nina Mjølsnes and Hedman, Johannes and Väli, Marika}},
  issn         = {{1872-4973}},
  keywords     = {{Forensic genetics; Massively parallel sequencing (MPS); Next generation sequencing (NGS); Proficiency test; Short tandem repeat (STR); Single nucleotide polymorphism (SNP)}},
  language     = {{eng}},
  publisher    = {{Elsevier}},
  series       = {{Forensic Science International: Genetics}},
  title        = {{Interlaboratory exercise to establish proficiency testing for sequencing of forensic STR and SNP markers}},
  url          = {{http://dx.doi.org/10.1016/j.fsigen.2025.103285}},
  doi          = {{10.1016/j.fsigen.2025.103285}},
  volume       = {{78}},
  year         = {{2025}},
}