Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase


Posted: 2021-10-15 19:00:00
Proc Natl Acad Sci U S A . 2021 Oct 19;118(42):e2107900118. doi: 10.1073/pnas.2107900118. Affiliations Expand Affiliations 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. 2 Bakar Innovation Fellow, University of California, Berkeley, CA 94720. 3 Center for Computational Biology, University of California, Berkeley, CA 94720. 4 Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720. 5 HHMI, University of California, Berkeley, CA 94720. 6 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; kcollins@berkeley.edu. 7 Bakar Fellow, University of California, Berkeley, CA 94720. Item in Clipboard Heather E Upton et al. Proc Natl Acad Sci U S A. 2021. Show details Display options Display options Format Proc Natl Acad Sci U S A . 2021 Oct 19;118(42):e2107900118. doi: 10.1073/pnas.2107900118. Affiliations 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. 2 Bakar Innovation Fellow, University of California, Berkeley, CA 94720. 3 Center for Computational Biology, University of California, Berkeley, CA 94720. 4 Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720. 5 HHMI, University of California, Berkeley, CA 94720. 6 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; kcollins@berkeley.edu. 7 Bakar Fellow, University of California, Berkeley, CA 94720. Item in Clipboard CiteDisplay options Display options Format Abstract Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3' ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples. Keywords: RNA sequencing; miRNA; non-LTR retroelement reverse transcriptase; noncoding RNA; tRNA. Copyright © 2021 the Author(s). Published by PNAS. Conflict of interest statement Competing interest statement: H.E.U., L.F., S.C.P., and K.C. are named inventors on University of California, Berkeley patent applications describing OTTR technology. H.E.U. and K.C. are founders of Karnateq, Inc., which licensed this technology. References Lander E. S., et alInternational Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921. Eickbush T. H., Jamburuthugoda V. K.. The diversity of retrotransposons and the properties of their reverse transcriptases. Virus Res.. 2008;134:221–234. Sultana T., Zamborlini A., Cristofari G., Lesage P.. Integration site selection by retroviruses and transposable elements in eukaryotes. Nat. Rev. Genet.. 2017;18:292–308. Eickbush T. H., Eickbush D. G.. Integration, regulation, and long-term stability of R2 retrotransposons. Microbiol. Spect.. 2015;3. Fujiwara H.. Site-specific non-LTR retrotransposons. Microbiol. Spect.. 2015;3. Show all 56 references [x] Cite Copy Format: Send To [x]

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