Forensic DNA phenotyping using Oxford Nanopore Sequencing system

Abstract

AbstractIn forensic science, the demand for precision, consistency, and cost‐effectiveness has driven the exploration of next‐generation sequencing technologies. This study investigates the potential of Oxford Nanopore Sequencing (ONT) Technology for analyzing the HIrisPlex‐S panel, a set of 41 single nucleotide polymorphism (SNP) markers used to predict eye, hair, and skin color. Using ONT sequencing, we assessed the accuracy and reliability of ONT‐generated data by comparing it with conventional capillary electrophoresis (CE) in 18 samples. The Guppy v6.1 was used as a basecaller, and sample profiles were obtained using Burrows–Wheeler Aligner, Samtools, BCFtools, and Python. Comparing accuracy with CE, we found that 62% of SNPs in ONT‐unligated samples were correctly genotyped, with 36% showing allele dropout, and 2% being incorrectly genotyped. In the ONT‐ligated samples, 85% of SNPs were correctly genotyped, with 10% showing allele dropout, and 5% being incorrectly genotyped. Our findings indicate that ONT, particularly when combined with ligation, enhances genotyping accuracy and coverage, thereby reducing allele dropouts. However, challenges associated with the technology's error rates and the impact on genotyping accuracy are recognized. Phenotype predictions based on ONT data demonstrate varying degrees of success, with the technology showing high accuracy in several cases. Although ONT technology holds promise in forensic genetics, further optimization and quality control measures are essential to harness its full potential. This study contributes to the ongoing efforts to refine sequence read tuning and improve correction tools in the context of ONT technology's application in forensic genetics.