X-ClickSeq: Custom-Primed Protocol with Single Indexing using ClickSeq Kit v1

ClickSeq is a simple, fragmentation-free method for the synthesis of Next-Generation Sequencing (NGS) libraries. ClickSeq derives its name by using ‘Click-Chemistry‘ in the place of common ligation enzymes to ‘click-ligate’ nucleic acids to sequencing adaptors – an essential and often problematic step in the synthesis of Next-Generation Sequencing cDNA libraries. The process takes advantage of the chain-terminating properties of 3′-azido-nucleotides, which are included the initial in vitro reverse-transcription reactions uniformly required for RNAseq. The modified nucleotides are stochastically incorporated into the nascent cDNA, yielding cDNA fragments blocked at their 3′ ends with azido groups. The 3′-azido-blocked cDNA fragments are ‘click-ligated’ onto alkyne-functionalized sequencing adaptors, which can subsequently be PCR-amplified to yield a sequencing-ready NGS library. ClickSeq is a highly flexible and modular platform for NGS library synthesis where both RNA and DNA templates can be used as input material. ClickSeq offers many advantages over standard RNA NGS protocols as click-chemistry is utilized to attach on the required sequencing adapter, rather than commonly used enzymatic reactions. Overall, this results in increased efficiency of the protocol, fewer processing steps, and reduced time from RNA to sequencing ready libraries. Additionally, since there are no fragmentation steps typically required in common RNA-seq approaches and because in vitro template switching during RT is limited by the chain terminating azido-nucleotides, ClickSeq offers the benefit of ultra-low artifactual chimera rate, with only 3 chimeric events per million reads. X-ClickSeq kits provide the same reagents as the ClickSeq kits, except that no primers are provided for the Reverse Transcription step. Rather, the user may use their own RT primer as per the requirements of their own assay. For example, a 9N random primer might be used instead of a 6N random primer; or a set of SARS-CoV-2 targeting tiled-primers might be used (see Jaworski et al. eLife, 2021). All other aspects of the library prep are otherwise identical to ClickSeq. The data analysis pipeline will also be altered as per the assay design.