Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method

Maja Sidstedt; Arvid H Gynnå; Kevin M Kiesler; Linda Jansson; Carolyn R Steffen; Joakim Håkansson; Gustav Johansson; Tobias Österlund; Yalda Bogestål; Andreas Tillmar; Peter Rådström; Anders Ståhlberg; Peter M Vallone; Johannes Hedman

doi:10.1016/j.fsigen.2024.103047

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method

Forensic Sci Int Genet. 2024 Apr 3:71:103047. doi: 10.1016/j.fsigen.2024.103047. Online ahead of print.

Authors

Affiliations

¹ National Forensic Centre, Swedish Police Authority, Linköping SE-581 94, Sweden.
² National Institute of Standards and Technology, 100 Bureau Drive, M/S 8314, Gaithersburg, MD 20899, USA.
³ National Forensic Centre, Swedish Police Authority, Linköping SE-581 94, Sweden; Applied Microbiology, Department of Chemistry, Lund University, Lund SE-221 00, Sweden.
⁴ RISE Unit of Biological Function, Division Materials and Production, Box 857, Borås SE-501 15, Sweden; Department of Laboratory Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg SE-405 30, Sweden; Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg SE-405 30, Sweden.
⁵ SIMSEN Diagnostics, Sahlgrenska Science Park, Gothenburg, Sweden.
⁶ Department of Laboratory Medicine, Sahlgrenska Center for Cancer Research, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Medicinaregatan 1F, Gothenburg 41390, Sweden; Wallenberg Center for Molecular and Translational Medicine, University of Gothenburg, Gothenburg 41390, Sweden; Department of Clinical Genetics and Genomics, Sahlgrenska University Hospital, Gothenburg, Region Västra Götaland 41390, Sweden.
⁷ RISE Unit of Biological Function, Division Materials and Production, Box 857, Borås SE-501 15, Sweden.
⁸ Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping SE-587 58, Sweden.
⁹ Applied Microbiology, Department of Chemistry, Lund University, Lund SE-221 00, Sweden.
¹⁰ National Forensic Centre, Swedish Police Authority, Linköping SE-581 94, Sweden; Applied Microbiology, Department of Chemistry, Lund University, Lund SE-221 00, Sweden. Electronic address: johannes.hedman@tmb.lth.se.

PMID: 38598919
DOI: 10.1016/j.fsigen.2024.103047

Abstract

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1-15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

Keywords: Forensic DNA; Machine learning; Massively parallel sequencing; Short tandem repeats; Targeted PCR; UMI.