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

Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3'-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts.

Efficient DNA Click Reaction Replaces Enzymatic Ligation.

We report a chemical DNA-DNA ligation method based on copper-catalyzed azide-alkyne cycloaddition (CuAAC). We demonstrate that ion addition dramatically influences the efficiency of the so-called click reaction. Even without any further additions, such as typically splint oligonucleotides for preorganization, the "click ligation" yields up to ∼83% product without any byproducts. Additionally, purification of the desired product is straightforward. In comparison to enzymatic ligation methods used to introduce adapters into, e.g., mRNA library preparation, this targeted chemical ligation method exhibits several advantages: increased ligated product and no adapter or cDNA oligomers byproducts. The advantages of the click ligation method were demonstrated by incorporation of azide modified nucleotides by several enzymes as well as broad polymerase acceptance of the obtained triazole linkage in PCR.