My research in 60 seconds — Improvements in direct RNA sequencing and its potential to transform clinical research

Background to direct RNA sequencing

As Martin highlights, the capacity to sequence native RNAs is one of the unique features of nanopore sequencing and a revolution for RNA biology. "Sequencing native RNA has opened up a whole new field of research in RNA biology that was difficult to access before.”

As read length is only limited by the size of the RNA fragment presented to the nanopore, it is possible to sequence and quantify extremely long, full-length, native RNA transcripts, with nanopore technology. Sequencing native RNA also means that RNA modifications can be detected within the same dataset and removes the need for amplification or reverse transcription and so their associated biases.

A further benefit of direct RNA sequencing is the ability to accurately measure poly-A tail length. Poly-A tail length is an important factor in post-transcriptional regulation¹⁰, and further studies may provide new insights into gene expression and disease states.

"The benefits of sequencing RNA directly with nanopore [technology] are pretty awesome. You can look at RNA modifications, poly-A tail dynamics, splicing diversity... it’s a really clean way to observe native biological molecules.”

Improvements in RNA sequencing: New RNA004 sequencing kit

Two of the challenges Martin highlighted with direct RNA sequencing have been the low yield and high input requirements — which he believes have hindered its widespread adoption. The new, improved direct RNA sequencing chemistry (SQK-RNA004) looks to have overcome these challenges.

“With RNA004 flow cells… we got 14.75 million reads which knocked my socks off. This was amazing. This is the update we were waiting for.”

The chemistry upgrades include a new nanopore, optimised for RNA base discrimination, and improvements to the basecaller architecture, providing increased raw-read accuracy. Along with the development of a faster motor protein and sequencing software upgrades, output has also increased. These advances combined provide the ability to generate rich transcriptome information.

Advancing clinical research with RNA004

Martin’s long-standing interest is understanding long non-coding RNAs (lncRNAs) — a large class of RNA molecules with lengths over 200 nucleotides. They are involved in various regulatory and catalytic roles within the cell. Although they have no protein-coding capability, evidence suggests that lncRNAs play crucial roles in cancer and other diseases^4,5,6.

In a leukaemia cell line, Martin and his team used Cas9 and Cas13 technology to inhibit the expression of more than 700 lncRNA genes that had previously been identified as dysregulated in leukaemia. Using direct RNA sequencing, they then conducted differential expression analysis.

Their goal was to characterise the biological functions of these non-coding genes to gain a greater insight into the transcriptome of leukaemia. They identified novel isoforms and splicing diversity, including isoforms known to be involved in cell proliferation.

In this talk (below), Martin shared one example of how his team had identified a “second isoform” of MALAT1 — a gene often overexpressed in cancer cells and associated with several cancers^8,9 — which was not represented in the reference transcriptome.

Find out more about Martins research by watching his talk from London Calling 2023 below.