Thermostable group II intron reverse transcriptases and their applications in next generation RNA sequencing, diagnostics, and precision medicine

Thermostable group II intron reverse transcriptases (TGIRTs) from thermophilic bacteria are advantageous for biotechnological applications that require cDNA synthesis, such as RT-qPCR and RNA-seq. TGIRTs have higher thermostability, processivity and fidelity than conventional retroviral RTs, along with a novel end-to-end template-switching activity that attaches RNA-seq adapters to target RNAs without RNA ligation. First, I optimized the TGIRT template-switching method for RNA-seq analysis of small non-coding RNAs (ncRNAs). I showed that TGIRT-seq gives full-length reads of tRNAs, which are refractory to retroviral RTs, and enables identification of a variety of base modifications in tRNAs by distinctive patterns of misincorporated nucleotides. With collaborators, I developed an efficient and quantitative high-throughput tRNA sequencing method, identified RNAs bound by the human interferon-induced protein IFIT5, yielding new insights into its functions in tRNA quality control and innate immunity, and uncovered a novel mRNA-independent mechanism for elongation of nascent peptides. Second, I developed a new, streamlined TGIRT-seq method for comprehensive analysis of all RNA size classes in a single RNA-seq. This method enables RNA-seq library construction from <1 ng of fragmented RNAs in