ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs

Andrew Routh, Steven R. Head, Phillip Ordoukhanian, John E. Johnson

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2610-2616
Number of pages7
JournalJournal of Molecular Biology
Volume427
Issue number16
DOIs
StatePublished - Aug 14 2015
Externally publishedYes

Fingerprint

Ligation
Alkynes
Complementary DNA
DNA
Dideoxynucleotides
Click Chemistry
Triazoles
Azides
Cycloaddition Reaction
Viral RNA
Gene Library
Artifacts
Genetic Recombination
Libraries
Reverse Transcription
Copper
Polymerase Chain Reaction
Messenger RNA

Keywords

  • click-chemistry
  • cricket paralysis virus
  • flock house virus
  • next-generation sequencing
  • recombination

ASJC Scopus subject areas

  • Molecular Biology
  • Medicine(all)

Cite this

ClickSeq : Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs. / Routh, Andrew; Head, Steven R.; Ordoukhanian, Phillip; Johnson, John E.

In: Journal of Molecular Biology, Vol. 427, No. 16, 14.08.2015, p. 2610-2616.

Research output: Contribution to journalArticle

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