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

Bioconjug Chem. 2022 Oct 19;33(10):1789-1795. doi: 10.1021/acs.bioconjchem.2c00353. Epub 2022 Sep 26.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Click Chemistry*
  • DNA Primers
  • DNA, Complementary / genetics
  • High-Throughput Nucleotide Sequencing* / methods
  • RNA / genetics
  • RNA, Messenger / genetics
  • Triazoles


  • DNA, Complementary
  • DNA Primers
  • RNA
  • RNA, Messenger
  • Triazoles