A groundbreaking advancement in RNA sequencing, developed at the John Curtin School of Medical Research (JCSMR), promises to transform how scientists study the genetic code and its regulation in health and disease. Dr Alex Sneddon, a Postdoctoral Fellow in the Eyras Group of Computational RNA Biology, has spearheaded the development of RISER (Real-time Identification and Selective Rejection), a neural network-powered software that enables selective RNA sequencing without the use of expensive, harsh biochemicals.

RNA, a thread-like molecule essential for the expression of genetic code, plays a vital role in cell function. However, inside cells, the abundance of certain RNA types, such as messenger RNA (mRNA), often obscures the detection of rarer RNA molecules, including long non-coding RNAs (lncRNAs). These rare RNA types are crucial for understanding cell development and disease mechanisms but are notoriously difficult to study due to the limitations of existing sequencing methods.

Traditional approaches to selective RNA sequencing often involve complex filtering steps in the laboratory that are both time-consuming and expensive, with the potential to damage RNA molecules. RISER offers an innovative solution to these challenges, harnessing the power of Oxford Nanopore’s sequencing technology.

How RISER Works

Nanopore sequencing works by threading RNA strands through a small pore while recording electrical current signals, or “squiggles,” that correspond to the RNA sequence. The technology features a unique capability called “read-until,” which allows researchers to reject unwanted molecules during sequencing. However, standard “read-until” processes are computationally demanding and slow, requiring a sequence readout before decisions can be made.

Enter RISER. This software uses a neural network trained to recognise patterns in the very first part of the squiggle, predicting the RNA type in real-time. RISER can quickly command the sequencing hardware to eject off-target molecules without waiting for a sequence readout, significantly improving efficiency.

“RISER is faster and requires fewer computational resources than traditional read-until approaches,” explains Dr Sneddon. “It empowers RNA researchers with a flexible and efficient strategy for biochemical-free targeted sequencing.”

Applications and Impact

In a recent clinical application, RISER was trained to detect globin mRNA, which dominates blood samples and hampers the detection of other clinically relevant RNAs. By rejecting globin mRNAs in real time, RISER enabled the sequencing of more non-globin mRNAs, demonstrating its potential for advancing disease research and diagnosis.

The software has also been successfully trained to detect mitochondrial RNA, which is abundant in muscle cells, as well as messenger RNA. Its modular design allows researchers to easily adapt RISER for other RNA classes, making it a versatile tool for the scientific community.

Dr Alex Sneddon (right) and Agin Ravindran with the the Oxford Nanopore sequencing device that RISER controls at the JCSMR laboratory. (Photo: Kassapa Senarath / ANU)

A Collaborative Effort

Dr Sneddon emphasises that RISER’s development was a team effort. “This project would not have been possible without Agin Ravindran from the Eyras and Shirokikh groups, who performed all the nanopore sequencing, including testing RISER in real-time. I’m also incredibly grateful to Dr Carolina Correa Ospina from ANU’s Biomolecular Resource Facility for her invaluable guidance and support. Finally, I would like to thank my supervisor Professor Eduardo Eyras for being a wonderful mentor throughout the project.”

Open-Source and Ready for Researchers

RISER is freely available to researchers worldwide. Its code, hosted on GitHub, ensures accessibility and encourages collaboration within the scientific community. The findings were recently published in Nature Communications, a testament to the importance of this breakthrough.

• Access RISER on GitHub: https://github.com/comprna/riser
• Read the full research article: Nature Communications

Dr Sneddon and her team’s innovation is poised to have a lasting impact on RNA research, paving the way for new discoveries in cell biology real-time monitoring of gene expression and a new generation of RNA sequencing control.