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Schönegger, Eva S.; Crisp, Antony; Müller, Markus ORCID logoORCID: https://orcid.org/0000-0002-3579-3317; Fertl, Jessica; Serdjukow, Sascha; Croce, Stefano; Kollaschinski, Michael; Carell, Thomas ORCID logoORCID: https://orcid.org/0000-0001-7898-2831 and Frischmuth, Thomas (26. September 2022): Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing. In: Bioconjugate Chemistry, Vol. 33, No. 10: pp. 1789-1795 [PDF, 660kB]

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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.

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