Yasuhiro Murakawa from Kyoto University in Japan presented at London Calling 2025 on “A compendium of human RNA structures and modifications.” Kazuhiro Takeuchi from the Murakawa lab generated a de novo human transcriptome assembly using “full-length” cDNA sequencing. Murakawa noted that there are many RNA forms and regulation events. Long-read sequencing methods, while useful in capturing long transcripts, may not always capture the full-length structure of RNA, explained Murakawa. there may be internal priming sites with a poly dT primer, for example. The research team developed a cap-to-tail sequencing (CT-seq) that ligates overhang adapter for “full-length” RNA sequence analysis. They extracted reads that only contained a polyA tail and called all isoforms. The team combined this approach with short read sequencing methods to identify isoforms from 482 samples. Over 2,400 novel gene isoforms were identified. Most novel gene candidates are expressed in a cell-specific manner. To determine whether the novel gene candidates were translated into proteins, MS/MS analysis was performed. Next, Murakawa wants to use Oxford Nanopore Ultra-long sequencing with CT-seq to resolve the BRCA2 gene structure, for example. Next, Murakawa used the SQK-RNA004 kit and began with 2-5 ug of RNA to attach the sequencing adapter. There was a high correlation between replicates and CT-seq. Additionally, direct RNA sequencing allowed the team to identify modifications and compare these in different cell types. This presentation highlighted the power of different transcriptomic approaches and the massive amount of data generated by this lab group!
