ABRF 2024

Post-transcriptionally deposited chemical modifications of RNA molecules, or epitranscriptomic marks, impart structural and functional consequences to RNA, and can allow hundreds or even thousands of unique RNA molecules to be expressed from a single gene. More than 170 distinct modifications are known to decorate RNA, and while only a small number of these have been characterized by nanopore sequencing, in principle, this technology enables the identification of any modification that generates a differentiable signal distortion. However, distinguishing the signals produced by different RNA modifications is a fast-growing area of research with major implications for the study of RNA biology, as well as mRNA vaccine development. We leveraged the diverse chemical repertoire of tRNAs, the most abundantly modified class of RNA, to evaluate the signals produced at known modification sites across a broad range of viral, prokaryotic, and eukaryotic species. This talk will discuss profiling of more than 45 distinct modifications on RNA molecules using second generation nanopore direct RNA sequencing technology on the P2 solo and P2 integrated platforms.

Long-read sequencing platforms, including Oxford Nanopore (ONT), have the potential to revolutionize personalized medicine through the ability to accurately assess all clinically relevant structural variants (SVs), repeats and rearrangements, often undetectable with short read technologies. True production-scale processing for clinical reporting requires consistent high-quality data, low DNA sample input requirements (1-3ug), platform and software stability, data standards as well as competitive cost models. The Baylor College of Medicine - Human Genome Sequencing Center (BCM-HGSC) has assessed long-read data from the ONT PromethION long-read platform by the generation of >25x whole genome sequence data. Samples were selected from the NIH All of Us Research Program (AoURP) CDRv5 (98K) short read data set and to date we have completed 959 ONT genomes averaging ~34x coverage/PromethION flow cell. Specifically, library and sequencing kits (ONT LSK114/ R10.4.1 kits) were optimized to consistently deliver >80Gb/PromethION flow cell with an insert size >15Kb and more recent optimizations targeting ~25Kb inserts. Optimized protocols for library, sequencing, and analysis pipelines were established, along with performance metrics to provide assessment of the production readiness of the platform. Laboratory optimizations included precision size cuts using Pippin HT, controlled shearing parameters, optimization of all library steps for maximum yield and titration of instrument loading amounts to maximize yield. Key production metrics were established including mean coverage, % genome coverage at 10x, HIFI yields, mean read length, contamination, and Q30 or Q10 mapped bases. Initial accuracy for SNVs was assessed using the standard NIST control (HG002) and 97.1% and SVs we observe similar high accuracies of 92.6%. This work was part of the operational development of platforms and data for the All of Us Research program.