R2C2: Improving nanopore read accuracy enables the sequencing of highly-multiplexed full-length single-cell cDNA

Abstract

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms.Because of this shortfall, long-read sequencing is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms.However, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption.Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here we introduce and validate a new long-read ONT based sequencing method. At the same cost, our R olling Circle Amplification to C oncatemeric C onsensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method.These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single cell samples. Significance StatementSubtle changes in RNA transcript isoform expression can have dramatic effects on cellular behaviors in both health and disease. As such, comprehensive and quantitative analysis of isoform-level transcriptomes would open an entirely new window into cellular diversity in fields ranging from developmental to cancer biology. The R2C2 method we are presenting here is the first method with sufficient throughput and accuracy to make the comprehensive and quantitative analysis of RNA transcript isoforms in bulk and single cell samples economically feasible.

MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

“…A number of methods have been developed that increase the accuracy of these platforms, all sharing the general tactic of generating multiple repeats of each template and using these repetitive units for error correction during the computational processing step. Two such approaches were initially published, R2C2 [37] and INC-Seq [38], both resulting in 2-3% error rates in Nanopore platforms, but they are optimized for spliced transcript detection, or use chemistries that are no longer supported. Moreover, both these approaches do not have a way to curb or detect template switching events between similar templates, as would be present in the viral swarm of circulating HIV RNAs.…”

isoCirc catalogs full-length circular RNA isoforms in human transcriptomes

“…In fact, circular consensus sequencing is a well-established approach to improve the accuracy of PacBio long reads 36,37 . Recently, a similar approach was developed for long-read nanopore RNA-seq of linear transcripts with the R2C2 method, in which linear transcripts are circularized followed by RCA and nanopore sequencing 38 .…”

The how and why of lncRNA function: An innate immune perspective

“…As a long cDNA or RNA strand translocates through the nanopore at single nucleotide precision from enzymatic regulation, the ionic current across the membrane is recorded. This technology can sequence full-length transcripts and can yield up to 10 million reads on the MinION or up to 60 million reads on the PromethION for cDNA [257,258]. An initial limitation of ONT was the 5-10% per read error rate, which has been overcome with a new technology called Rolling Circle to Concatemeric Consensus (R2C2) bringing the error rate down to 2.5% by increasing the read coverage.…”