A survey of RNA modifications across the human transcriptome by direct RNA nanopore sequencing


Oxford Nanopore Technologies Direct RNA Sequencing Kit has the ability to sequence RNA molecules directly without first making cDNA. This removes bias from this intermediate synthesis step and preserves RNA modifications during sequencing, which is proven to be useful for many applications. Recently, Oxford Nanopore Technologies has released an updated version of their Direct RNA Sequencing Kit, RNA004, which addresses the lower throughput and per-read accuracy when compared to nanopore cDNA. We were part of the RNA004 beta-testing period and sequenced human transcriptome from HEK293T cells at the baseline and upon the treatment with the METTL3 inhibitor, STM2457. We assessed the performance of the RNA004 kit in multiple replicates (N=3) and in comparison with the RNA002 kit, comparing the data from the two sequencing kits on the very same RNA. We found that from RNA004, the throughput increased by ~2.5 fold using half the recommended input RNA, and the per-read median accuracy increased from ~93% to ~97% when compared to RNA002. Furthermore, we evaluated changes in m6A detection in both RNA002 and RNA004 data using existing software tools: m6Anet, ELIGOS, and Nanocompore; and the new model released with Remora only on RNA004 data. Overall, the new RNA004 Direct RNA Sequencing Kit represents a significant improvement for sequencing RNA molecules.


Logan Mulroney is an EMBL-IIT post-doctoral (ETPOD) fellow at EMBL-EBI and the Center for Genomic Science at the Italian Institute of Technology where he is working with Ewan Birney and Francesco Nicassio. His research interests include developing computational methods to detect nucleotide modifications in direct RNA nanopore sequencing data. Logan completed his PhD at the University of California, Santa Cruz (UCSC) in the nanopore group with Mark Akeson. At UCSC, he developed nanopore sequencing methods for protein substrates and full-length RNA strands.

Authors: Logan Mulroney