Abstract
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.
Highlights
Forensic DNA genotyping for identification purposes is routinely performed by analysis of short tandem repeats (STRs) [1]
The results presented in this paper were obtained by analyzing three samples, consisting of two single-contributor samples and a mixture sample
Illumina sequencing resulted in 316,450 reads, of which 36.6% could be categorized by locus based on the primer sequence
Summary
Forensic DNA genotyping for identification purposes is routinely performed by analysis of short tandem repeats (STRs) [1]. Those regions in the genome, characterized by short repeating sequences, are polymorphous among individuals regarding the number of repeats. MPS technologies yield a considerably more informative output, as besides the number of repeats, other types of variation (e.g., single nucleotide polymorphisms (SNPs) and isoalleles) can be assessed. This increased discriminative power is, in particular, beneficial for low-input samples, where dropouts are more common.
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