Advances in genome technologies ushered in vast cost reductions in DNA sequencing and increased read
lengths, the latter afforded by development of new single-molecule sequencing technologies. As a result, much
of the genome’s “dark matter” has been elucidated, and higher-quality reference genomes were made available.
In addition to genome sequencing, these single-molecule methods have enabled new applications for probing
chemical modifications in DNA, by either probing the kinetics of sequencing-by-synthesis using optical
waveguides, or by electrically distinguishing modified bases using nanopores.
Despite progress, a critical barrier in genomics is understanding the roles of RNA in biology, which demands
methods for quantitative analysis of RNA molecules in a cell. The myriad of types of RNAs in a cell, their dynamic
chemical modifications, and their elaborate structural and functional diversity, all hint at a tremendous level of
regulation and biological significance. Traditional RNA sequencing methods have primarily relied on conversion
to complementary DNA (cDNA) followed by cDNA sequencing using either high-throughput second-generation
methods or third generation single-molecule methods, the latter of which offers long reads. Using these methods,
some RNA modifications can be read through prior chemical functionalization of the RNA prior to conversion to
cDNA (for example, m6A, pseudouridine, A-to-I editing, 1-methyluridine, and dihydrouridine). However, the
chemical reactions involved in these methods are not 100% quantitative or specific, and further, detection is
often done through incomplete reads due to reverse transcription blocks, which precludes detection of multiple
modifications. The only available method for direct RNA sequencing, the Oxford Nanopore Technologies
platform, suffers from several drawbacks that include high input requirements, limited ability to probe RNA
modifications, and incomplete reads, particularly near the RNA 5’ end.
We address these limitations by developing a new single-molecule method that can be scaled to allow long read
direct RNA sequencing at high throughputs, all with very low input requirements of several picograms. Building
on zero-mode waveguides (ZMWs) originally developed by Pacific Biosciences, we have recently developed
electro-optical ZMWs (eZMWs) that allow low-input capture of DNA and RNA molecules. We demonstrated using
these devices identification of DNA fragments from low inputs by rapid capture of single molecules and their
flash sequencing. Together with the Pyle group, we are developing the integration of MarathonRT, an ultra-
processive reverse transcriptase that converts RNA molecules to cDNA molecules with high processivity and
accuracy, into our electro-optical eZMWs for direct RNA sequencing. We have already fused MarathonRT to a
streptavidin protein and demonstrated its functionality in eZMWs. Here we will build on these developments to
develop a direct RNA sequencing method that can detect single base edits and chemical modifications, all with
high coverage from single-cell inputs of a few pg per run.
