Towards direct RNA sequencing with electro-optical waveguides.

Abstract

Long-read sequencing technologies are revolutionizing transcriptome characterization such as decoding full-length transcripts, isoform quantification, splice variants, and RNA containing different modification or mutation sites. Currently, the most widely adopted long-read RNA sequencing technologies are PacBio cDNA isoform sequencing (Iso-Seq) and nanopore-based direct RNA sequencing, both methods having common limitations: First, neither one is capable of sequencing sub-ng levels of DNA/RNA with any practical throughput. Input sample requirements for sequencing (>3 µg/1 Gb genome) are still orders of magnitude higher than the amounts of DNA (∼6 pg) and RNA (10-30 pg) in a human cell. Therefore, typical library preparation from low-input samples involves amplification/conversion steps, which remove any base modifications present in the native strand and biases the sample toward high copy numbers. Second, both technologies suffer from read length bias since they rely on the capture of biomolecules into a nanoscale aperture. Third, PacBio iso-seq detects cDNA products synthesized by a reverse transcriptase rather than the native RNA strand limiting its ability to explore epigenetic modifications. To overcome these issues, we developed electro-optical ZMWs (eZMWs), a novel design of electrically actuatable ZMWs to load biomolecules electrophoretically in order to minimize the length-bias and the input requirements of long-read technologies. Our new devices facilitate high-efficiency length-independent DNA loading into ZMWs and enable rapid identification of DNA molecules. The final goal is to develop a smRNASeq, a single-molecule RNA sequencing system based on electro-optical waveguides that read individual transcripts during reverse transcription in real time using multi-color single-fluorophore detection of labeled nucleotides. Currently, we are in the process of integrating an engineered MarathonRT-streptavidin fusion into eZMWs and developing fluorescently labeled dNTPs compatible with the enzyme to demonstrate MarathonRT binding and smRNA sequencing in eZMWs.