Detection of RNA methylation patterns in forensically relevant transcripts in dried bloodstains

Abstract

RNA degradation kinetics can be used to estimate the age of a biological sample found at a crime scene. RNA sequencing of transcripts from various tissue types shows that degradation occurs faster at the 5’ end than the 3’ end. This discovery led to the development of the 5’-3’ assay, which quantifies and compares each end of a transcript to estimate sample age. This assay has been validated on dried bloodstains, however why the 5’ end of the transcript degrades faster than the 3’ end remains unknown. As this phenomenon is being observed in dried bloodstains, we hypothesize that chemical modifications of RNA molecules may be playing a role. Increasing research shows that methylation of RNA molecules can alter the kinetics of RNA degradation, leading to either increased or decreased transcript stability, dependent on the RNA binding proteins (RBPs) present. This study aims to investigate the methylation patterns in forensically relevant transcripts in dried bloodstains via next generation sequencing (NGS) using Oxford Nanopore Technologies’ (ONT) native RNA sequencing protocol followed by bioinformatic analysis. We also developed a novel RNA enrichment technique that utilizes 120 nucleotide DNA probes designed to hybridize to the 5’ or 3’ end of a transcript for selected target enrichment. We detected possible modifications on twelve transcripts sequenced from dried bloodstains, five of which were identified on more than one sample. Although the results of this study are preliminary due to the lack of controls for statistical comparison, the duplicate modifications identified strengthens the overall findings. The results provided here can serve as the basis for future studies aiming to interrogate the epitranscriptome of forensically relevant samples to further optimize RNA-based assays for implementation into the field of forensic science.