RNA-ClickSeq Library Prep Kit with dual indexing

Product Information

Technical Overview

RNA-ClickSeq™ Library Prep with Dual Indexing is an innovative kit for constructing next-generation sequencing (NGS) libraries from RNA, using click chemistry for adapter ligation. This method eliminates the need for enzymatic ligation and fragmentation, streamlining the workflow and minimizing bias and artifacts.

How It Works:

1. Azido-Termination: RNA is reverse transcribed using a random primer containing a partial Illumina p7 adapter. The presence of 3′-azido-nucleotides stochastically terminates cDNA synthesis, generating fragments terminated with 3′-azido groups.
2. Click-Ligation: Azido-terminated cDNA fragments are covalently linked to alkyne-functionalized sequencing adapters via copper-catalyzed azide–alkyne cycloaddition (CuAAC), also known as click chemistry. This chemical ligation is highly efficient and enzyme-free.
3. Dual Indexing PCR Amplification: Click-ligated fragments are PCR-amplified using unique pairs of i5 and i7 index primers, enabling robust sample multiplexing and error correction. The final libraries are fully compatible with Illumina sequencing workflows.

Workflow Summary

• Reverse Transcription: RNA is randomly primed and copied in the presence of azido-nucleotides, generating 3′-azido-terminated cDNA fragments.
• Optional RNA Removal: RNase H can be used to remove template RNA.
• First Bead Purification: SPRI beads purify cDNA fragments.
• Click-Ligation: Alkyne-functionalized adapters are covalently attached to cDNA via click chemistry.
• Second Bead Purification: Removes reaction components, leaving adapter-flanked cDNA.
• PCR Amplification with Dual Indexing: Unique i5 and i7 index primers amplify and barcode each sample.
• Final Bead Purification & Size Selection: Yields sequencing-ready libraries (optimal size: 300–600 bp).

Key Features & Advantages

• Fragmentation-Free: No need for mechanical or enzymatic fragmentation, preserving sample integrity and reducing workflow complexity.
• Ultra-Low Chimera Rate: Peer-reviewed studies report only ~3 chimeric events per million reads, supporting high-confidence variant detection and structural analysis ⁽¹⁾
• Ligation-Free: Click chemistry replaces enzymatic ligation, minimizing bias and unwanted byproducts.
• Dual Indexing: Supports both i5 and i7 indices for robust sample multiplexing and accurate sample tracking.
• Flexible Input Range: Works with 10 ng to 4 µg of RNA (optimal: >100 ng).
• Fast Protocol: Complete workflow in approximately 6 hours.
• Ready for Illumina Platforms: Generates libraries compatible with all major Illumina sequencers (NextSeq, NovaSeq, MiSeq, HiSeq).
• 12 Unique Dual-Index Primer Pairs: Enables multiplexing of up to 12 samples per kit.

Kit Components

• Click Primer Mix (CPM)
• Click Mix p5 (CM)
• Click Accelerant (CA)
• Click Catalyst (CC)
• Elution Buffers (EB1, EB2)
• Note: Enzymes (SuperScript III, RNase H, OneTaq Master Mix), SPRI beads are user-supplied. Dual Indexing Primers (i5 Index Primers (D501–D512), i7 Index Primers (D701–D712)) can be purchased here.

Library Topology

Final libraries include:

Illumina p5 and p7 adapters

Dual indices (i5 and i7)

4 nt UMI (unique molecular identifier)

cDNA fragment

Application Areas

• Transcriptome Profiling: High-quality RNA-seq with minimal bias and artifact.
• Variant Detection & Structural Analysis: Ultra-low chimera rates support confident detection of rare events.
• High-Throughput Screening: Dual indexing enables efficient multiplexing for large cohorts.

Technical Specifications

Technical Data from Publications

Chimera Suppression: Only ~3 chimeric events per million reads ⁽¹⁾

Coverage & Error: Comparable error rates and coverage to standard RNA-seq methods.

Chemical Ligation: CuAAC click chemistry forms biocompatible triazole linkages, efficiently read-through by polymerases ⁽²⁾.

Flexible Input: Works with both RNA and DNA templates.

LITERATURE

1. Routh, A., Head, S. R., Ordoukhanian, P., & Johnson, J. E. (2015). ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs. Journal of Molecular Biology, 427(16), 2610-2616. https://doi.org/10.1016/j.jmb.2015.06.011
2. Jaworski, E., & Routh, A. (2017). ClickSeq: Replacing Fragmentation and Enzymatic Ligation with Click-Chemistry to Prevent Sequence Chimeras. In Next Generation Sequencing: Methods and Protocols (pp. 71-85). Springer New York. https://doi.org/10.1007/978-1-4939-7514-3_6

For a detailed list of publications utilizing ClickSeq technology, please visit: ClickSeq Publications