# Full-length sequencing of individual RNAs from heterogeneous samples

> **NIH NIH R43** · PHAENO INC · 2022 · $398,040

## Abstract

Summary
Despite large investments in nucleic acid technology, the ability to sequence large numbers of full-length
individual RNAs, from complex samples, with highest accuracy, has remained out of reach. Pheno introduces
an advance for large scale Next Generation DNA Sequencing (NGS) sequencing of effectively unlimited numbers
of individual RNA molecules from heterogeneous mixtures. We seek to prove that these methods can be
implemented with the scale and precision to justify translation into commercially viable products and services.
The aims of this proposal address two key steps that determine scale and accuracy at which the technology can
be applied, exploiting recent discoveries in nucleic acid enzymology. Aim #1 focuses on reverse transcription,
prompted by creation, with in vitro directed evolution, of a highly accurate, proof-reading reverse transcriptase,
with high processivity and devoid of integral RNase H activity and other sources of RT artefacts. This advance
over even the best retroviral and enzymes derived from retrotransposons or group-ii introns promises accurate
replication of even the longest RNA viral genomes. Conventional retroviral enzymes, for example, even modified
by site directed mutagenesis, are prone to at least 11 transcriptional artefacts in addition to simple base-reading
errors. Aim #2 exploits a recently discovered class of primer-polymerases used in DNA repair. A key step in our
sequencing chemistry exploits topological advantages of homo-concatamers of tagged cDNAs produced by
Rolling Circle Amplification (RCA) of circularized single-stranded templates. The lowest scale at which the
technology can be applied is limited by conventional RCA protocols that depend on exogenous random DNA
primers. and generate artifactual sequences with samples of small size. New protocols use a PrimPol
polymerase to synthesize RNA primers directly from the template, preventing de novo artefacts and
simultaneously improving amplification by ~ 5 orders of magnitude. This presents the exciting possibility of
applying the technology to samples below the scale of a single-cell transcriptome. Collectively these studies will
seek to set new industry standards for RNA sequencing. This could help accelerate a wide range of precision
medicine, viz. precision cancer diagnostics, immunotherapy; therapeutic gene editing; new drug discovery and
validation. The technology could provide transformational advances in battling infectious diseases, including
HIV/AIDS and SARS-CoV-2-mediated COVID 19.

## Key facts

- **NIH application ID:** 10482321
- **Project number:** 1R43HG012527-01
- **Recipient organization:** PHAENO INC
- **Principal Investigator:** William S Agnew
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $398,040
- **Award type:** 1
- **Project period:** 2022-03-15 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10482321

## Citation

> US National Institutes of Health, RePORTER application 10482321, Full-length sequencing of individual RNAs from heterogeneous samples (1R43HG012527-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10482321. Licensed CC0.

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