Daniel P. Depledge from New York University presented at London Calling 2019 on “Redefining the transcriptional complexity of viral pathogens using direct RNA sequencing.” Depledge started with a slide and the question: Got herpes? They are fascinated with herpes viruses and their multiple features and applications. Herpes simplex virus type 1 (HSV-1) is a 152 kb dsDNA virus with 90+ proteins and 100+ polyadenylated RNAs! Depledge spoke about how HSV-1 infects a cell and begins three waves of replication. The genome of HSV-1 is compact: the virus can have, for example, three distinct transcripts with a conserved RNA cleavage site. Depledge has used the Oxford Nanopore Technologies MinION to learn about splicing, transcription start sites (TSS), and polyadenylation sites (CPAS). They have used read pileups to learn about cleavage and polyadenylation sites. CAGE-Seq was performed to learn about transcription start sites with Illumina sequences and verified with ONT long-read sequencing. Depledge uses Illumina reads for error correction. Error correction increases the alignment proportion of the read but also may result in some 5′ and 3′ clipping. This information has helped Depledge learn about transcript lengths and timing. Accumulation of late transcripts during infection can then be studied using compounds to inhibit steps. Depledge explained that direct RNA sequencing helps uncover new biology. I agree and find it fascinating. I hope to learn more about direct RNA sequencing in bacteria.
