Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing

Schönegger, Eva S.; Crisp, Antony; Müller, Markus ; Fertl, Jessica; Serdjukow, Sascha; Croce, Stefano; Kollaschinski, Michael; Carell, Thomas und Frischmuth, Thomas (26. September 2022): Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing. In: Bioconjugate Chemistry, Bd. 33, Nr. 10: S. 1789-1795

Abstract

Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3′-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts.

• Dokumententyp: Zeitschriftenartikel
• EU Funded Grant Agreement Number: 642023
• EU-Projekte: Horizon 2020 > Marie Skłodowska Curie Actions > Marie Skłodowska Curie: Fostering new skills by means of excellent initial training of researchers > 861381: NATURE-ETN - Nucleic Acids for Future Gene Editing, Immunotherapy and Epigenetic Sequence Modification ; Horizon 2020 > Marie Skłodowska Curie Actions > Marie Skłodowska-Curie Innovative Training Networks > 642023: ClickGene - Click Chemistry for Future Gene Therapies to Benefit Citizens, Researchers and Industry
• Publikationsform: Submitted Version
• Fakultät: Chemie und Pharmazie > Department Chemie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• Sprache: Englisch