Forensic Nanopore Sequencing of STRs and SNPs Using Verogen's ForenSeq DNA Signature Prep Kit and MinION

The MinION nanopore sequencing device (Oxford Nanopore Technologies, Oxford, UK) is the smallest commercially available sequencer and can be used outside of conventional laboratories. The use of the MinION for forensic applications, however, is hindered by the high error rate of nanopore sequencing. One approach to solving this problem is to identify forensic genetic markers that can consistently be typed correctly based on nanopore sequencing. In this pilot study, we explored the use of nanopore sequencing for single nucleotide polymorphism (SNP) and short tandem repeat (STR) profiling using Verogen's (San Diego, CA, USA) ForenSeq DNA Signature Prep Kit. Thirty single-contributor samples and DNA standard material 2800 M were genotyped using the Illumina (San Diego, CA, USA) MiSeq FGx and MinION (with R9.4.1 flow cells) devices. With an optimized cutoff for allelic imbalance, all 94 identity-informative SNP loci could be genotyped reliably using the MinION device, with an overall accuracy of 99.958% (1 error among 2926 genotypes). STR typing was notably error prone, and its accuracy was locus dependent. We developed a custom-made bioinformatics workflow, and finally selected 13 autosomal STRs, 14 Y-STRs, and 4 X-STRs showing high consistency between nanopore and Illumina sequencing among the tested samples. These SNP and STR loci could be candidates for panel design for forensic analysis based on nanopore sequencing.