Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation...

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Traditional cDNA library generation methods often rely on template-switching (TS) reactions, which can introduce biases and limit the amplification of certain RNA transcripts.
• Existing click chemistry-based RNA sequencing techniques, while advantageous, face challenges with stochastic cDNA termination and limited read-through compatibility.
• The amplification of full-length cDNA libraries is crucial for comprehensive transcriptomic analysis, including the study of alternative splicing and fusion events.

Purpose of the Study:

• To develop a novel click chemistry-based method for generating and amplifying full-length cDNA libraries from total RNA.
• To overcome limitations of previous methods, specifically avoiding template-switching reactions and stochastic cDNA termination.
• To enhance the amplification of previously inaccessible transcripts and increase the possible insert size for RNA sequencing.

Main Methods:

• Development of a click chemistry-based protocol for cDNA synthesis and amplification.
• Incorporation of modified PCR primers with two 3'-overhanging nucleotides to improve cDNA read-through compatibility.
• Comparative analysis of the novel method against the state-of-the-art PAC-seq technique and a commercial kit using Oxford Nanopore sequencing.

Main Results:

• The novel method successfully generated full-length cDNA libraries without template-switching reactions.
• A significant improvement in read-through compatibility was achieved, more than doubling the possible insert size compared to PAC-seq.
• The method demonstrated comparable performance to a commercial kit in terms of sequencing output and quality.

Conclusions:

• The developed click chemistry-based method offers a robust alternative for full-length cDNA library generation and amplification.
• This technique enhances the accessibility of a broader range of RNA transcripts, including those with complex structures.
• The method holds potential for improved analyses of mRNA splicing variants and fusion transcripts, advancing transcriptomic research.