Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA

Abstract

Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3′-azide-modified dideoxyribonucleotide onto the 3′-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3′-end to a 5′-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.

High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

“…The successful use of ‘click’ chemistry as a means of adapter addition was previously reported [41, 42]. Here, we executed ‘click’ reactions to generate oligo-modified nucleotides with oligonucleotide attached via the 5′ terminus prior to their incorporation into the growing DNA strand ( and S4, see Methods).…”

“…The resulting extension products are amplified via PCR and subjected to standard Illumina paired-end sequencing. The forward sequencing read (R1) contains 16S rRNA V1-V2 regions starting from the specific priming site while the reverse read The successful use of 'click' chemistry as a means of adapter addition was previously reported [41,42]. Here, we executed 'click' reactions to generate oligo-modified nucleotides with oligonucleotide attached via the 5′ terminus prior to their incorporation into the growing DNA strand (Fig.…”

Artificial nucleic acid backbones and their applications in therapeutics, synthetic biology and biotechnology

“…A combinatorial approach for the discovery of splint-templated chemical ligations has been reported to identify DNA-compatible reactions to ligate terminally functionalised ONs [57]. Moreover, the generation of artificial backbone mimics has been shown for bridging 5 0 -S-phosphorothioester linkages (Ps) [58], PA [59][60][61][62], AM [61], urea [63], SQAM [63], TL1 [64] and TL3 [61]. Indeed, copper-catalysed azide-alkyne cycloaddition (CuAAC) to form TL3 was reported for the assembly of whole genes [12,65] and long RNA [9,10] from azide and alkyne modified shorter ONs.…”

A New 1,5-Disubstituted Triazole DNA Backbone Mimic with Enhanced Polymerase Compatibility

“…For example, the CuAAC click ligation of 3′-azido ONs with 5′-alkyne adaptor ONs to form TL 1 was recently described for the preparation of next-generation sequencing libraries. 17 However, the authors reported low replication efficiencies by several polymerases through TL 1 . Furthermore, click ligation has been used to assemble long DNA templates with isolated TL 6 modifications which can be replicated 9 , 29 or transcribed 8 , 9 by polymerases in bacterial 9 or mammalian 8 cells while retaining high fidelity read-through.…”