PubReading [222] - Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing - E. Schönegger, T. Frischmuth

<p>Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length <strong>cDNA libraries</strong> 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 <strong>click chemistry</strong>-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 <strong>PAC-seq</strong>, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts. </p><p><em>DOI: </em><a href="https://doi.org/10.1021/acs.bioconjchem.2c00353"><em>10.1021/acs.bioconjchem.2c00353 - </em></a><em>2022</em></p>