[Baseclick Newsletter] NGS library preparations_BaseClick - 코아사이언스

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Advancing RNA sequencing by introducing ClickSeq Library Prep

In recent decades, nucleic acid sequencing has become an important tool in molecular biology, diagnostics and biotechnology. While next-generation sequencing (NGS) methods are known for their high performance, they face challenges in certain applications, particularly in transcriptome analysis.

 

The ClickSeq Kit: An innovative solution

The ClickSeq kit offers a simple, reliable and accurate method for transcriptome analysis, using click chemistry. This newsletter explores the features, applications and benefits of ClickSeq compared to traditional mRNA library preparation methods for transcriptome analysis.

 

Key Applications areas:

RNA/DNA Sequencing: Supports both random-primed RNA/DNA sequencing and poly(A)-focused mRNA sequencing for broad transcriptome coverage and gene expression analysis.

• Poly(A)-ClickSeq was used in transcriptomic studies to characterize mechanisms of oncogenesis and to identify the biomarkers of glioblastoma progression and prognosis.

Tiled/Targeted Sequencing: Enables high-resolution analysis of specific genomic regions.

• Tiled-ClickSeq was used for SARS-CoV-2 surveillance during the pandemic from nasopharyngeal swabs collected from infected individual, revealing previously missed 5’UTR variants, minority variants and recombination events.

Viral Genome Analysis: High-precision sequencing of viral RNA for diagnostics, epidemiology, and recombination studies.

• Tiled-ClickSeq was used to characterize the evolution and stability of deletion events found near the furin cleavage site of spike protein in SARS-CoV-2 in experimental and mouse models of viral infection.

Strand-Specific Sequencing: Distinguishes between sense and antisense RNA strands, even in degraded samples.

• ClickSeq was used to emerge RNA virus replication in mosquito-borne viruses such as Chikungunya virus.

Targeted Gene Libraries: Focuses on oncogenes, tumor suppressors, and fusion events for cancer research and molecular diagnostics.

• Targeted DNA-ClickSeq was deployed to capture the integration sites of transposons into Salmonella Typhi genomes, revealing key genes regulating biofilm formation.

Technology behind ClickSeq

Labeling

Nucleic acids are tagged with azido-groups that specifically react with alkyne-modified adapter oligos.

Fragmentation

No sample fragmentation is required. cDNA fragments are generated during the reverse transcription reaction, minimizing artifacts and degradation.

Adapter Ligation

Adapter ligation is achieved using click chemistry, allowing precise and selective reactions. This also provides flexibility in the sequence of the adapter used. Both long and short sequencing adapters can be ligated, with or without barcoding and Unique Molecular Identifiers (UMIs).

Simple Process

Streamlined protocols with fewer steps compared to traditional methods. All clean-up steps can be performed using SPRI bead technology, reducing hands-on time and making the process fully automatable.

Compatibility

Offers flexibility for all Illumina NGS sequencing platforms. Libraries can also be sequenced on Element BioScience flowcells and are compatible with Oxford Nanopore sequencing protocols.

 

Workflow of ClickSeq Prep Kit

The ClickSeq library prep involves three main steps, with bead clean-ups in between:

 

Step 1.

Reverse Transcription: RNA is transcribed using azido-modified nucleotides, stochastically terminating cDNA synthesis.
Step 2.

Click Chemistry Ligation: Azido-terminated cDNAs are chemically ligated to sequencing adapters.
Step 3.

PCR Amplification: Indexed primers generate sequencing-ready NGS libraries. Additional size selection can be performed during PCR-cleanup with SPRI beads. Library prep takes less than 5-6 hours (which includes <2 hours of hands-on time).

 

Success stories
Learn more about the applications of ClickSeq in publictions of the inventors Dr. Andrew Routh and Dr. Elizabeth Jaworski. or visit the official ClickSeq website

 

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