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.
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.