RNA-Seq Libraries from Stochastically Terminated 3′-azido-blocked cDNA Fragments

ClickSeq is an RNA sequencing technique that uses bioconjugation as an alternative to fragmentation in the library preparation step, to produce lower error rates than standard sequencing methods (Routh et al., 2015). First, RNA is reverse-transcribed into cDNA, in a process similar to Sanger sequencing, with 3′-azido-2′,3’dideoxynucleotides (AzNTPs). This process induces chain termination and semirandom primers (6 random nucleotides followed by an Illumina 3′ P7 adapter) to sequence random positions on the RNA template. Single-stranded cDNA is purified, and the 3′-ends are click-ligated with alkyne-modified (5’hexynyl) P5 adapters. After PCR amplification, the cDNA library is ready to be sequenced.


  • Significantly reduced artifactual recombination rate due to elimination of the fragmentation step
  • Highly suitable for detecting rare recombination events
  • No fragmentation step


  • 3′-azido blocked cDNA fragments are converted into double-stranded DNA with low efficiency
  • Read-through of AzNTP is still highly inefficient
  • Further optimization required for single-cell or single-molecule studies


Illumina Library prep and Array Kit Selector


None available yet


Routh A., Head S. R., Ordoukhanian P. and Johnson J. E. ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs. J Mol Biol. 2015;427:2610-2616