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ClickSeq: Replacing Fragmentation and Enzymatic Ligation with Click-Chemistry to Prevent Sequence Chimeras

  • Elizabeth Jaworski
  • Andrew RouthEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1712)

Abstract

We recently reported a fragmentation-free method for the synthesis of Next-Generation Sequencing libraries called “ClickSeq” that uses biorthogonal click-chemistry in place of enzymes for the ligation of sequencing adaptors. We found that this approach dramatically reduces artifactual chimera formation, allowing the study of rare recombination events that include viral replication intermediates and defective-interfering viral RNAs. ClickSeq illustrates how robust, bio-orthogonal chemistry can be harnessed in vitro to capture and dissect complex biological processes. Here, we describe an updated protocol for the synthesis of “ClickSeq” libraries.

Key words

ClickSeq RNAseq Click-chemistry Next-generation sequencing Flock house virus 

Notes

Acknowledgments

This work was supported by UTMB start-up funds and a University of Texas System Rising STARs Award to A.R.

References

  1. 1.
    Birts CN et al (2014) Transcription of click-linked DNA in human cells. Angew Chem Int Ed Engl 53(9):2362–2365CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Chen X, El-Sagheer AH, Brown T (2014) Reverse transcription through a bulky triazole linkage in RNA: implications for RNA sequencing. Chem Commun (Camb) 50(57):7597–7600CrossRefGoogle Scholar
  3. 3.
    Dallmann A et al (2011) Structure and dynamics of triazole-linked DNA: biocompatibility explained. Chemistry 17(52):14714–14717CrossRefPubMedGoogle Scholar
  4. 4.
    El-Sagheer AH, Sanzone AP, Gao R, Tavassoli A, Brown T (2011) Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia Coli. Proc Natl Acad Sci U S A 108(28):11338–11343CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    el-Sagheer AH, Brown T (2011) Efficient RNA synthesis by in vitro transcription of a triazole-modified DNA template. Chem Commun (Camb) 47(44):12057–12058CrossRefGoogle Scholar
  6. 6.
    Qiu J, El-Sagheer AH, Brown T (2013) Solid phase click ligation for the synthesis of very long oligonucleotides. Chem Commun (Camb) 49(62):6959–6961CrossRefGoogle Scholar
  7. 7.
    Sanzone AP, El-Sagheer AH, Brown T, Tavassoli A (2012) Assessing the biocompatibility of click-linked DNA in Escherichia Coli. Nucleic Acids Res 40(20):10567–10575CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Isobe H, Fujino T, Yamazaki N, Guillot-Nieckowski M, Nakamura E (2008) Triazole-linked analogue of deoxyribonucleic acid ((TL)DNA): design, synthesis, and double-strand formation with natural DNA. Org Lett 10(17):3729–3732CrossRefPubMedGoogle Scholar
  9. 9.
    Isobe H, Fujino T (2014) Triazole-linked analogues of DNA and RNA ((TL)DNA and (TL)RNA): synthesis and functions. Chem Rec 14(1):41–51CrossRefPubMedGoogle Scholar
  10. 10.
    Fujino T et al (2011) Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA. Chem Asian J 6(11):2956–2960CrossRefPubMedGoogle Scholar
  11. 11.
    Shivalingam A, Tyburn AE, El-Sagheer AH, Brown T (2017) Molecular requirements of high-fidelity replication-competent DNA backbones for orthogonal chemical ligation. J Am Chem Soc 139(4):1575–1583CrossRefPubMedGoogle Scholar
  12. 12.
    Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl 40(11):2004–2021CrossRefPubMedGoogle Scholar
  13. 13.
    Baskin JM et al (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A 104(43):16793–16797CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    El-Sagheer AH, Brown T (2009) Synthesis and polymerase chain reaction amplification of DNA strands containing an unnatural triazole linkage. J Am Chem Soc 131(11):3958–3964CrossRefPubMedGoogle Scholar
  15. 15.
    Routh A, Head SR, Ordoukhanian P, Johnson JE (2015) ClickSeq: fragmentation-free next-generation sequencing via click ligation of adaptors to stochastically terminated 3′-Azido cDNAs. J Mol Biol 427(16):2610–2616CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Gorzer I, Guelly C, Trajanoski S, Puchhammer-Stockl E (2010) The impact of PCR-generated recombination on diversity estimation of mixed viral populations by deep sequencing. J Virol Methods 169(1):248–252CrossRefPubMedGoogle Scholar
  17. 17.
    Meyerhans A, Vartanian JP, Wain-Hobson S (1990) DNA recombination during PCR. Nucleic Acids Res 18(7):1687–1691CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Routh A, Ordoukhanian P, Johnson JE (2012) Nucleotide-resolution profiling of RNA recombination in the encapsidated genome of a eukaryotic RNA virus by next-generation sequencing. J Mol Biol 424(5):257–269CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Routh A, Johnson JE (2014) Discovery of functional genomic motifs in viruses with ViReMa-a virus recombination mapper-for analysis of next-generation sequencing data. Nucleic Acids Res 42(2):e11CrossRefPubMedGoogle Scholar
  20. 20.
    Jaworski E, Routh A (2017) Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in flock house virus. PLoS Pathog 13(5):e1006365CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Routh A et al (2017) Poly(a)-ClickSeq: click-chemistry for next-generation 3-end sequencing without RNA enrichment or fragmentation. Nucleic Acids Res 45(12):e112CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Hong V, Presolski SI, Ma C, Finn MG (2009) Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew Chem Int Ed Engl 48(52):9879–9883CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Abel GR, Calabrese ZA, Ayco J, Hein JE, Ye T (2016) Measuring and suppressing the oxidative damage to DNA during Cu(I)-catalyzed azide-alkyne cycloaddition. Bioconjug Chem 27(3):698–704CrossRefPubMedGoogle Scholar
  24. 24.
    Litovchick A et al (2015) Encoded library synthesis using chemical ligation and the discovery of sEH inhibitors from a 334-million member library. Sci Rep 5:10916CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyThe University of Texas Medical BranchGalvestonUSA
  2. 2.Sealy Center for Structural Biology and Molecular BiophysicsUniversity of Texas Medical BranchGalvestonUSA

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