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Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling