X-ClickSeq Library Prep Kit with dual indexing

Product Information

Technical Overview

X-ClickSeq Library Prep with Dual Indexing is a highly flexible kit for constructing next generation sequencing (NGS) libraries from RNA (or DNA), using click chemistry for adapter ligation. Unlike standard kits, X-ClickSeq™ empowers users to design and use their own reverse transcription (RT) primers, enabling targeted, tiled, or custom assays for applications such as viral genome sequencing, recombination analysis, and minority variant detection.

How It Works:

User Defined Priming: RNA is reverse transcribed using user-provided primers (e.g., random, tiled, or gene-specific), each containing a partial Illumina p7 adapter sequence. This enables targeted sequencing of specific regions or entire genomes.

1. Azido-Termination: 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 primed using user-provided primers with a partial p7 adapter, 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).

Library Topology

Final libraries include:

• Illumina p5 and p7 adapters
• Dual indices (i5 and i7)
• 4 nt UMI (unique molecular identifier)
• cDNA fragment

Kit Components

• x-Primer Mix (xPM) – no RT primer included user provides custom primer(s)
• Click Mix p5 (CM)
• Click Accelerant (CA)
• Click Catalyst (CC)
• Elution Buffers (EB1, EB2)
• Note: Enzymes (SuperScript III, RNase H, OneTaq Master Mix), user-provided RT primers, and SPRI beads are user-supplied. Dual Indexing Primers (i5 Index Primers (D501–D512), i7 Index Primers (D701–D712)) can be purchased here.

Key Features & Advantages

• Customizable Priming: Use any primer design—random, tiled, or gene-specific—for targeted sequencing or full-genome coverage.
• 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 and recombination detection.
• 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.

Application Areas

Targeted Viral Genome Sequencing: Proven for complete SARS-CoV-2 genome sequencing with tiled primers.

Recombination & Minority Variant Detection: Simultaneous capture of recombination events and low-frequency variants (Jaworski et al., eLife 2021).

Flexible Targeted NGS: Design custom assays for any region, gene, or organism.

High-Throughput Screening: Dual indexing enables efficient multiplexing for large cohorts.

Technical Specifications

LITERATURE

1. Jaworski et al., Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants. eLife 2021;10:e68479. https://doi.org/10.7554/eLife.68479
2. 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
3. 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