Josie Gleeson from the University of Melbourne, Australia presented at London Calling 2022 the ten-minute session entitled “Accurate expression quantification from nanopore direct RNA sequencing with NanoCount.” I am really interested in learning about transcriptomics with Nanopore devices and didn’t know about NanoCount. Gleeson spoke about how “long-read sequencing with Oxford Nanopore Technologies (ONT) can sequence RNA molecules directly… to capture novel isoforms.” Gene quantification is a challenge at the isoform level. Thus, Gleeson emphasized improving isoform quantification with direct RNA sequencing. They developed the tool NanoCount that uses read alignment to the transcriptome (BAM file) and performs filtering on alignments prior to quantifying them. NanoCount is available for download from GitHub. Gleeson’s experiment had the goal of quantifying a couple of isoforms that have been studied previously. Filtering includes 3′ end filtering and alignment score filtering. The tema compared NanoCount to other isoform quantification tools. They tested quantification of differential expression in a synthetic sample and between human cell populations. Next, FLAIR can be used to identify novel isoforms. After optimizing, they identify forty novel isoforms. Gleeson concluded that direct RNA sequencing and NanoCount can help accurately count and identify transcripts. i was aware of direct RNA sequencing, yet had not considered why it is difficult to quantify isoforms. This session shared software and how it was developed.
