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.

Narrative In the past 20 years the technology for extremely high throughput DNA sequencing has not taken into account that for human genes, the dictum that applies is one-gene:multiple-proteins. Phaeno NGS based RNA sequencing can reveal the full diversity of individual RNA molecules in a complex sample, at almost unlimited scale. This technology provides the foundation to entirely new approaches to gene-based diseases such as Cancer or Alzheimer’s diseases – and can provide tremendous leverage in the battle against RNA viruses such as the novel SARS-CoV-2 coronavirus.