ClickSeq

ClickSeq

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 induces chain termination and semi-random primers (6 random nucleotides followed by an Illumina 3' adapter (P7) to sequence random positions on the RNA template. Single-stranded cDNA are then purified and their 3'-ends click-ligated with alkyne-modified (5'hexynyl) P5 adapters. After PCR amplification, the cDNA library is ready to be sequenced.

Pros:

• Significantly reduced artefactual recombination rate (2.5 events per million reads) due to removal of fragmentation step
• Highly suitable for detecting rare recombination events

Cons:

• 3'-azido blocked cDNA fragments have low efficiency when converted into double stranded DNA
• Read-through of AzNTP is still highly inefficient
• Needs further optimizations for single-cell or single-molecule studies

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

1. Routh A., Chang M. W., Okulicz J. F., Johnson J. E. and Torbett B. E. (2015) CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data. Methods 91: 40-47