ClickSeq

ClickSeq is a click-chemistry based method for generating next generation sequencing libraries for deep-sequencing platforms including Illumina, HiSeq, MiSeq and NextSeq. Its function is similar to most other techniques for generating RNAseq or DNAseq libraries in that it aims to generate random fragments of biological samples of RNA or DNA and append specific sequencing adaptors to either end of every fragment, as per the requirements of the particular sequencing platform to be used (e.g. HiSeq). In ClickSeq, reverse transcription (RT) reactions are supplemented with small amounts of 3'-azido-nucleotides (AzNTPs) at defined ratios to deoxyribonucleotides (dNTPs). AzNTPs are chain-terminators and therefore induce the stochastic termination of cDNA synthesis at an average length determined by the ratio of AzNTPs to dNTPs. This results in the production of single-stranded cDNA fragments that contain an azido-group at their 3' ends. These 3'-azido-blocked cDNA molecules are purified away from the components of the RT reaction, and subsequently 'click-ligated' to 5' alkyne-modified DNA adaptors via copper-catalysed azide-alkyne cycloaddition (CuAAC). This generates ssDNA molecules with unnatural triazole-linked DNA backbones.…

ClickSeq is a click-chemistry based method for generating next generation sequencing libraries for deep-sequencing platforms including Illumina, HiSeq, MiSeq and NextSeq. Its function is similar to most other techniques for generating RNAseq or DNAseq libraries in that it aims to generate random fragments of biological samples of RNA or DNA and append specific sequencing adaptors to either end of every fragment, as per the requirements of the particular sequencing platform to be used (e.g. HiSeq). In ClickSeq, reverse transcription (RT) reactions are supplemented with small amounts of 3'-azido-nucleotides (AzNTPs) at defined ratios to deoxyribonucleotides (dNTPs). AzNTPs are chain-terminators and therefore induce the stochastic termination of cDNA synthesis at an average length determined by the ratio of AzNTPs to dNTPs. This results in the production of single-stranded cDNA fragments that contain an azido-group at their 3' ends. These 3'-azido-blocked cDNA molecules are purified away from the components of the RT reaction, and subsequently 'click-ligated' to 5' alkyne-modified DNA adaptors via copper-catalysed azide-alkyne cycloaddition (CuAAC). This generates ssDNA molecules with unnatural triazole-linked DNA backbones. Nevertheless, these templates are used in PCR reactions and amplified to generate a cDNA sequencing library with the appropriate 5' and 3' sequencing adapters and indices required for Next-Generation Sequencing. ClickSeq has predominantly been used to sequence viral RNA genomes such as Flock House virus, cricket paralysis virus, and Zika virus, due to its resilience to artifactual chimera formation.

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