Re-Reading RNA to Obtain High Accuracy - Project Summary
The ultimate goal in the field of RNA sequencing (transcriptomics and epitranscriptomics) is single-molecule,
high-accuracy, de novo RNA sequencing, including the ability to sequence modified bases without the need for
additional techniques/methods that add time, cost, and sample requirements. Unfortunately, current
transcriptomic sequencing technologies are costly, have low accuracy (specifically relative to DNA sequencing
approaches), they can require reverse transcription that removes the identity of modified bases
(limiting/prohibiting epitranscriptomic investigations), and/or they can use or require processive enzymes that fail
to traverse certain types of sequences. During this Phase I SBIR project, we will develop a sequencing
technology/methodology that overcomes these short comings, and is capable of state-of-the-art, RNA
sequencing and characterization. More specifically, we will develop and demonstrate an approach for
controllably and iteratively translocating single RNA molecules back-and-forth through a nanopore reader such
that single molecules can be directly and repeatedly sequenced, and corresponding ultra-high accuracy
consensus sequence determinations can be made. Furthermore, during this project, we will demonstrate the
ability to multiplex the associated measurement, significantly reducing the risk associated with developing a high-
throughput, single-molecule sequencing system following this effort. Nanopore-based technology has the
potential to enable complete and comprehensive RNA sequencing, and to be a field-leading technology in the
future. During this project, we will take steps toward making that future a reality by developing and optimizing all
components of our technology and then demonstrating RNA sequencing, including the ability to sequence N6-
methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ). We will then show the functionality and
advantages of the developed technology by benchmarking our approach against current nanopore-based
sequencing approaches.
