Tonight, I continued watching a Knowledge Exchange about direct RNA sequencing. Daniel Garalde, Associate Director of Technology Markets with Oxford Nanopore Technologies, introduced the Knowledge Exchange webinar I watched tonight. The title of the Knowledge Exchange is “How direct RNA nanopore sequencing can enhance your research.” Libby Snell, Director of RNA and cDNA Sample Technology, presented an update on the technology. The date of the recording was July 11, 2024. Snell provided history about the RNA sequencing method. Snell explained that nanopore sequencing is “read length agnostic” and sequencing RNA directly is very powerful as it can obtain full length transcripts. The new kit has an updated motor protein and pore. When starting RNA work, Snell emphasized minimizing RNA degradation. Also, Snell noted that “every isoform is important,” and therefore, minimizing degradation during extraction and storage is critical. Nanopore has tested protocols, recommending starting with a RIN between 7-10. Enriching for poly(A) mRNAs is an approach commonly used. This can be done with beads to “yank out” mRNAs or remove non-mRNA components. Snell shared examples of different RIN values. There is also a protocol for non-poly(A) RNA handling that is useful for bacterial transcriptomics, for example. The library prep removes secondary structure by using an RNA/DNA hybrid. The DNA strand is peeled off and never read. The new improvements to the sample prep have allowed for longer transcripts and higher output. Snell shared data from in vitro transcribed RNAs of different lengths and said customers have sequenced the coronavirus! RNA run conditions have also been improved. Now, pore occupancy continues past 14 hr. The short-read sequencing option has increased output. Snell explained that replicates are needed for transcriptomics, and at least three are recommended. Using a Universal Human Reference RNA (total RNA), the ONT team obtained ~20M reads on a PromethION flow cell and ~5M on a MinION. Accuracy improvements have been observed, though the super accuracy base-calling is still computationally intensive. Snell recommends re-base calling with SUP if needed. Snell also shared data from known spike in experiments in which correct gene detection was obtained. Long-read direct RNA nanopore sequencing, along with high coverage, is important for mapping complete isoforms. RNA modifications can be detected with improved models. For example, m6A modifications can be detected. Snell ended by sharing biopharma manufacturing applications for direct RNA sequencing. For example, direct mRNA sequencing can improve mRNA vaccine QC to identify poly(A) tail length variation. An mRNA QC test pack with the GridION Q v1.1 has been developed. Snell ended by sharing several resources on the Resource Center page. Snell did mention that multiplexing and targeted RNA sequencing sample prep systems are in the research and development stages, along with automation and tRNA sequencing options.
