Induro™, a Novel Reverse Transcriptase for Nanopore Direct RNA Sequencing with Significantly Improved RNA 5’ Coverage
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Transcriptome analysis based on cDNA sequencing of direct or PCR amplified cDNA has limitations, including generation of cDNA chimeras during library preparation, artifacts associated with template switching, DNA contamination and size bias during PCR amplification. Recent advances from Oxford Nanopore Technologies (ONT) have enabled direct RNA sequencing without PCR. This method not only interrogates full-length RNA molecules and generates higher resolution data on complex isoforms, but also allows for the detection of RNA modifications. This opens up new opportunities for studying RNA stability and regulation. To perform direct RNA sequencing, one approach is to first convert RNA into an RNA/cDNA hybrid molecule using a reverse transcriptase (RT). The RNA/cDNA hybrid molecule reduces RNA secondary structure formation, leading to higher sequencing throughput and longer sequencing reads without sacrificing RNA modification information. To accomplish this, an ideal reverse transcriptase would be highly processive and generate high cDNA yields. Here we introduce Induro, a novel reverse transcriptase that delivers superior performance in the cDNA synthesis reaction. Using this novel RT, we generated a RNA/cDNA hybrid library starting with mouse brain polyA-enriched RNA. After ligation with ONT sequencing adapter following the SQK-RNA002 protocol, this library was sequenced on the GridION. When compared with other commonly used RTs, Induro produced longer average read lengths and better 5’ end coverage of RNA transcripts. Similar results were also obtained by performing direct sequencing on a cDNA library generated using this RT enzyme. In summary, Induro is a significant improvement compared to other available RT enzymes. It can produce longer cDNA molecules, and exhibits higher cDNA yields, better full-length coverage, leading to more accurate transcriptome analysis.