title: ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs.
abstract: We present a simple method called "ClickSeq" for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2',3'-dideoxynucleotides that randomly terminate DNA synthesis and release 3'-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are "click ligated" via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5'-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address: arouth@scripps.edu.
authors.forename: Andrew
authors.initials: A
authors.lastname: Routh
authors.affiliation: The Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Head
authors.affiliation: The Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Phillip
authors.initials: P
authors.lastname: Ordoukhanian
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Johnson
mesh_terms: D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D057930:Click Chemistry / Q000379:methods*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D054306:Dideoxynucleotides / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D001386:Azides; D018076:DNA, Complementary; D054306:Dideoxynucleotides; D012333:RNA, Messenger; D012367:RNA, Viral
keywords: click-chemistry; cricket paralysis virus; flock house virus; next-generation sequencing; recombination