ClickSeq Library Prep Kit 12 Reactions

Introduction to ClickSeq Technology

ClickSeq is a novel approach to Next-Generation Sequencing (NGS) library preparation that replaces enzymatic ligation with a bioorthogonal chemical reaction, specifically, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This highly efficient and specific reaction is used to attach sequencing adapters to newly synthesized cDNA molecules.

ClickSeq is designed to eliminate common bottlenecks and artifacts in standard RNA-sequencing workflows, such as sequence chimeras, recombination, fragmentation bias, and ligation inefficiencies, all while preserving library complexity and improving reproducibility.

RNA ClickSeq Library Preparation Kit

The RNA ClickSeq Kit is a complete solution for preparing high-quality NGS libraries directly from RNA via a fragmentation-free, ligation-free process.

The protocol begins with a reverse transcription (RT) step using a mixture of standard dNTPs and 3′-azido-2′,3′-dideoxynucleotides (AzNTPs). These modified nucleotides stochastically terminate the cDNA, blocking the 3′ end with an azide group. Instead of relying on enzyme-based adapter ligation, the cDNA fragments are subsequently click-ligated to alkyne-functionalized sequencing adapters.

This click-ligation step forms a stable triazole linkage, which is biocompatible with downstream PCR amplification using standard polymerases.

Problems with traditional RNA-Seq Workflows

Current RNA-seq methods are built on workflows that rely on enzymatic steps for adapter ligation and RNA fragmentation. These steps present multiple limitations:

• Enzymatic ligation introduces recombination artifacts, chimeras, and sequence biases
• Fragmentation steps require optimization and can damage RNA integrity
• Workflow complexity increases time, cost, and risk of failure
• High sample input is often required for sufficient library quality
• Chimera formation leads to data misinterpretation and false discovery

The advantages of Clickseq RNA library preparation assay

The ClickSeq protocol replaces this step by utilizing highly efficient and high yielding Cu(I) catalyzed alkyne azide cycloaddition (CuAAC) for ligation. Therefore, the incorporated 3`-Azido stop nucleotides are click ligated by CuAAC with an alkyne containing adapter Oligo. This results in a ssDNA molecule with a triazole linked DNA backbone, which is biocompatible for PCR amplification. For the defined 3`end by the stop azido nucleotide, no dual primers for both directions are needed.

These facts in combination with the usage of click chemistry instead of enzymatic ligation also significantly reduce the costs for preparing your NGS library. Additionally, ClickSeq with 6-8 h worktime is a lot faster than comparable procedures and due to the randomized stop nucleotides, no fragmentation of DNA is necessary.

Workflow of this Next Generation Sequencing (NGS) Library Prep Kit:

1. Reverse Transcription: Incorporation of 3′-azido nucleotides into the cDNA.
2. Click-Ligation: Connection of cDNA fragments with alkyne-functionalized sequencing adapters.
3. PCR Amplification: Amplification of cDNA-adapter fragments to create the NGS library.

LITERATURE

https://doi.org/10.1016/j.jmb.2015.06.011