Researchers Develop Long-read-based Strategy for circRNA Analysis

Researchers in the Center for Computational and Genomic Medicine at Children’s Hospital of Philadelphia are eager to understand circRNAs’ unique set of properties and their functional roles.

The path to discovery is a circular one for researchers looking to describe fully a unique type of ribonucleic acid (RNA) molecule.

Scientists usually study linear RNA molecules composed of ribonucleotide bases that convert genetic information to build proteins. In the last few years, advances in sequencing technology revealed that our genes also generate an abundance of circular RNAs (circRNAs) that have the potential to influence cellular processes.

Researchers are eager to understand circRNAs’ unique set of properties and their functional roles. CircRNAs can range in size from very small to more than 4,000 bases, and they mostly contain their parental genes’ exonic or intronic sequences. They appear to be primarily non-coding, frequently tissue specific, and can be detected in body fluids. CircRNAs’ continuous loop also seems to make them stable, but many unknowns about their function remain.

A new sequencing technology and its companion data analysis tool, called isoCirc, developed by Yi Xing, PhD, the Francis West Lewis chair and director of the Center for Computational and Genomic Medicine at Children’s Hospital of Philadelphia, and his team is paving the way for the scientific community to characterize circRNAs and study their function by more precisely identifying their full-length sequences.

This week in Nature Communications, they demonstrated how the sequencing technology and data analysis tool provide researchers a new method to determine the sequences and expression profiles of circRNAs related to various biological states and diseases. Postdoctoral fellow Ruijiao Xin, PhD, and graduate student Yan Gao, are first authors of the paper, “isoCirc Catalogs Full-length Circular RNA Isoforms in Human Transcriptomes.”

CircRNAs form through a process called back-splicing that covalently joins exons head-to-tail. Scientists can detect these back-splicing junctions using conventional short-read RNA sequencing, but it is difficult to determine the entire sequence of a circRNA when it is longer than 500 bases.

“Let’s say you have a very large circular RNA,” said Dr. Xing, who is also professor of Pathology and Laboratory Medicine at the University of Pennsylvania. “If you use short-read RNA-seq to find the circular RNAs, you can see the back-splice junctions, but you don’t know the distinct internal sequences. Our motivation was to find a new approach that allows us to look at the entire circular RNA transcript.”

Dr. Xing’s team developed isoCirc as a long-read-based strategy for circRNA analysis. Their strategy merges rolling circle amplification of circRNA templates and nanopore long-read sequencing to reveal circRNAs’ exact full-length sequences.

Nanopore sequencing works by registering differences to an electrical current as nucleic acids pass through a small protein channel called a nanopore. Unlike traditional “second generation” sequencing, which generates short sequences of typically 100 to 200 bases, nanopore sequencing can generate sequences of longer than 10,000 bases. This long read length makes nanopore sequencing a powerful strategy for characterizing circRNAs.

The CHOP researchers tested the technology by performing isoCirc of 12 human tissues and a human cell line, and they used the dataset to create a catalog of full-length circRNA isoforms.

“This will be a helpful resource for scientists to more accurately characterize circRNAs’ roles and facilitate future functional studies,” Dr. Xing said. “In pediatrics, determining full-length sequences of circular RNAs will be an important first step to explore how they may act as biomarkers of disease state and progression, and if they could be novel therapeutic targets.”

Learn more about isoCirc, and view the catalog of full-length circRNA isoforms of human genes.