Targeted nanopore sequencing for forensic SNP genotyping and CpG methylation profiling

Abstract

Forensic DNA phenotyping can provide intelligence leads by inferring externally visible characteristics, biogeographical ancestry, and the chronological age of an unidentified subject of an investigation. Current analyses are based on the detection of informative single nucleotide polymorphisms (SNPs) and DNA methylation markers, using a broad spectrum of laboratory techniques and detection technologies. Currently, these SNP and DNA methylation markers must be detected in separate assays using specific primers for every single marker. Oxford Nanopore Technologies (ONT) sequencing platforms offer the advantage of detecting genetic and epigenetic markers simultaneously using biochemical-free targeted approaches, without the need for conventional laboratory techniques such as polymerase chain reaction (PCR). While base modifications can be revealed by the current signals from nanopore sequencing, the prediction accuracy of current nanopore-based methylation calling tools is still unclear, and no systematic comparison of these tools has been performed to date. The aim of this study is to evaluate nanopore sequencing applied to the field of forensic DNA phenotyping, specifically SNP genotyping and DNA methylation profiling. Firstly, a pipeline called METEORE is presented that provides a standardized workflow for methylation detection using six different nanopore-based methylation calling tools. The per-site and per-read prediction performance of these tools was benchmarked and different strategies to improve prediction accuracy are described. Among six individual tools, Megalodon achieved the best performance overall. Two PCR-free targeted nanopore sequencing approaches - Cas9 enrichment and adaptive sampling were then validated. Cas9-based targeted nanopore sequencing (nCATS) involved sequencing of ten forensically relevant regions and evaluate the six tested tools further using public bisulfite sequencing dataset. Adaptive sampling involves in-silico enrichment of regions of interest (ROI) during sequencing, recovering key methylation information from the targeted loci in a cost-effective, simple, and direct way. Although adaptive sampling resulted in lower coverage than the biochemical methods, it was nevertheless accurate for SNP genotyping and methylation profiling. This study represents a proof of concept for the potential of nanopore sequencing for forensic applications. Nanopore sequencing is expected to become the new gold standard for methylation detection as it not only correlates with sequencing of bisulfite-treated DNA, but it also detects methylation without the need for high coverage. Despite its great potential, the technology requires key improvements for its implementation in routine forensic workflows, especially to work with low-input or mixed DNA samples. The thesis concludes with a discussion of the trends in ongoing improvements to both bioinformatic tools and library preparation methods, which are rapidly making new techniques and new opportunities available for forensic operation.