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. This technology can be used to learn what is happening in an organism. Traditionally, people have first converted RNA to cDNA to sequence. One limitation is that the length of the poly(A) tail is lost with cDNA conversion. This information has recently been found to be important to learn about dynamic expression patterns. Reverse transcription has its limitations too: the reverse transcriptase can have limited processivity. With direct RNA sequencing, one obtains RNA modification, poly(A) tail information, complete isoform mapping, and transcript quantitation. Snell noted that the ONT cDNA sequencing library prep provides poly(A) tail length information. Also, Snell suggested using PromethION flow cells for transcriptomics because of the amount of data that can be obtained. For RNA sequencing, the motor protein works at 100-124 base pairs per second instead of the 400 bps rate for DNA sequencing. The direct RNA sequencing kit was first released in 2017. The RNA 004 kit was improved using RP4 with an RNA specific pore and improved motor protein. Combined, this provides increased accuracy. Snell spoke about improving the flow cells that are specific for RNA. They also emphasized careful RNA extraction, handling, and storage. Some suggestions Snell shared were to perform poly(A) enrichment or include a poly(A) step for 30-90 seconds. When it comes to RNA Integrity Numbers, Snell recommends at least 7. This session shared several tools and updates!
