Nanopore sequencing of a forensic combined STR and SNP multiplex.

Nanopore sequencing for forensic purposes has gained attention, as it yields added discriminatory power compared to capillary electrophoresis (CE), without the need for a high up-front capital investment. Besides enabling the detection of iso-alleles, Massively Parallel Sequencing (MPS) facilitates the analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in parallel. In this research, six single-contributor samples were amplified by such a combined multiplex of 58 STR and 94 SNP loci, followed by nanopore sequencing using an R10.3 flowcell. Basecalling was performed using two state-of-the-art basecallers, Guppy and Bonito. An advanced alignment-based analysis method was developed, which lowered the noise after alignment of the STR reads to a reference library. Although STR genotyping by nanopore sequencing is more challenging, correct genotyping was obtained for all autosomal and all but two non-autosomal STR loci. Moreover, genotyping of iso-alleles proved to be very accurate. SNP genotyping yielded an accuracy of 99% for both basecallers. The use of novel basecallers, in combination with the newly developed alignment-based analysis method, yields results with a pronouncedly higher STR genotyping accuracy compared to previous studies.

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