London Calling 2023: Direct RNA sequencing update (SQK-RNA004): initial impressions of RNA004 and its application for functional profiling of cancer-associated long non-coding RNAs

Oxford Nanopore Technologies’ direct RNA sequencing chemistry is unique as it determines the sequence of long-read RNA transcripts, capturing full-length isoforms, UTRs, and poly(A) tail lengths, without the requirement of first converting to DNA, nor PCR amplification. Here, we present our new, improved direct RNA sequencing chemistry (SQK-RNA004). Recent chemistry upgrades include a new nanopore optimized for RNA base discrimination and improvements to the basecaller architecture, providing increased raw-read accuracy. Furthermore, through the development of a faster motor protein and sequencing software upgrades, we have boosted sequencing output. Combined, these augmentations provide the ability to generate rich transcriptome information.

The capacity to sequence native RNAs is one of the unique features of nanopore sequencing and a revolution for RNA biology. However, the low yield and high input requirement have hindered the widespread adoption of this sequencing chemistry. I will first present our initial impressions of the updated RNA004 kit, comparing yields from poly(A) enriched and total RNA input, quality scores, and various observations on synthetic spike-in standards. I will then present preliminary results from over 18 RNA004 PromethION Flow Cells applied to functional cancer genomics. Specifically, we generated native transcriptomes from CRISPR-Cas13 knockdowns of four cancer-associated long non-coding RNAs identified through a CRISPR-Cas9 screen, seeking to characterise the biological functions of these non-coding genes.