ClickSeq

ClickSeq is a click-chemistry based method for generating next generation sequencing libraries for deep-sequencing platforms including Illumina, HiSeq, MiSeq and NextSeq. ^{[1] [2] [3]} Its function is similar to most other techniques for generating RNAseq or DNAseq libraries in that it aims to generate random fragments of biological samples of RNA or DNA and append specific sequencing adaptors to either end of every fragment, as per the requirements of the particular sequencing platform to be used (e.g. HiSeq).

In ClickSeq, reverse transcription (RT) reactions are supplemented with small amounts of 3'-azido-nucleotides (AzNTPs) at defined ratios to deoxyribonucleotides (dNTPs). AzNTPs are chain-terminators and therefore induce the stochastic termination of cDNA synthesis at an average length determined by the ratio of AzNTPs to dNTPs. This results in the production of single-stranded cDNA fragments that contain an azido-group at their 3' ends. These 3'-azido-blocked cDNA molecules are purified away from the components of the RT reaction, and subsequently 'click-ligated' ^{[4] [5]} to 5' alkyne-modified DNA adaptors via copper-catalysed azide-alkyne cycloaddition (CuAAC). ^[6] This generates ssDNA molecules with unnatural triazole-linked DNA backbones. Nevertheless, these templates are used in PCR reactions and amplified to generate a cDNA sequencing library with the appropriate 5' and 3' sequencing adapters and indices required for Next-Generation Sequencing. ClickSeq has predominantly been used to sequence viral RNA genomes such as Flock House virus, ^{[7] [8]} cricket paralysis virus, ^[9] and Zika virus, ^[10] due to its resilience to artifactual chimera formation.

Poly(A)-ClickSeq

Poly(A)-ClickSeq is a variant of ClickSeq designed to target the junction of the three prime untranslated region (UTRs) and poly(A)-tails of the messenger RNAs (mRNAs) of higher-order organisms and of RNA viruses infecting these cells types. ^[11] The core principle is similar to ClickSeq, however, the reverse-transcription step uses an oligo-dT primer (unanchored) to initiate cDNA synthesis from within the poly(A) tail and only three 3'azido-nucleotides (AzATP, AzGTP and AzCTP, collectively referred to as AzVTPs) are supplemented. Due to the omission of AzTTP, stochastic termination of cDNA synthesis cannot occur during reverse transcription of the poly(A)-tail. Rather, termination can only occur in the 3'UTR at a distance upstream of the poly(A) tail defined by the ratio of AzVTPs to dNTPs.

Applications

ClickSeq and Poly(A)-ClickSeq provide specific applications over other common RNA-seq techniques. These include:

• Removal of RNA fragmentation steps: When the reverse-transcription step is random-primed and cDNA synthesis is terminated by the 3'-azido-nucleotides, cDNA fragments can be generated without chemical, mechanical or enzymatic fragmentation of the sample RNA
• Removal of RNA/DNA ligase enzymes: In ClickSeq, there are no RNA or DNA ligation steps, as are commonly required in most next generation sequencing library synthesis strategies ^[12]
• Reduction of artifactual recombination: In the original ClickSeq publication, ^[13] Routh et al. demonstrated that the artifactual generation of cDNA chimeras was substantially reduced when using ClickSeq. This allowed the authors to detect rare RNA recombination events that arise during the replication of Flock House virus.
• Poly(A)-ClickSeq does not require enrichment or purification of mRNA or viral RNAs from biological specimens. Rather, Poly(A)-ClickSeq can be performed in a simple manner directly from crude RNA or total cellular RNA extracted from biological specimens.
• The copper-catalyst required for CuAAC may induce oxidative damage of the template DNA ^[14]

References

1. "ClickSeq". Illumina. Retrieved 16 February 2018.
2. Jaworski, E; Routh, A (2018). "ClickSeq: Replacing Fragmentation and Enzymatic Ligation with Click-Chemistry to Prevent Sequence Chimeras". Next Generation Sequencing. Methods in Molecular Biology. Vol. 1712. pp. 71–85. doi:10.1007/978-1-4939-7514-3_6. ISBN   978-1-4939-7512-9. PMID   29224069.
3. Routh, A; Head, SR; Ordoukhanian, P; Johnson, JE (14 August 2015). "ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs". Journal of Molecular Biology. 427 (16): 2610–6. doi:10.1016/j.jmb.2015.06.011. PMC   4523409 . PMID   26116762.
4. Qiu, J; El-Sagheer, AH; Brown, T (11 August 2013). "Solid phase click ligation for the synthesis of very long oligonucleotides". Chemical Communications. 49 (62): 6959–61. doi: 10.1039/c3cc42451k . PMID   23814786.
5. El-Sagheer, AH; Brown, T (21 August 2012). "Click nucleic acid ligation: applications in biology and nanotechnology". Accounts of Chemical Research. 45 (8): 1258–67. doi:10.1021/ar200321n. PMC   3423825 . PMID   22439702.
6. Kolb, HC; Finn, MG; Sharpless, KB (2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie. 40 (11): 2004–2021. doi:10.1002/1521-3773(20010601)40:11<2004::aid-anie2004>3.0.co;2-5. PMID   11433435.
7. Routh, A; Chang, MW; Okulicz, JF; Johnson, JE; Torbett, BE (December 2015). "CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data". Methods. 91: 40–47. doi:10.1016/j.ymeth.2015.09.021. PMC   4684750 . PMID   26408523.
8. Jaworski, E; Routh, A (May 2017). "Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus". PLOS Pathogens. 13 (5) e1006365. doi: 10.1371/journal.ppat.1006365 . PMC   5435362 . PMID   28475646.
9. Routh, A; Head, SR; Ordoukhanian, P; Johnson, JE (14 August 2015). "ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs". Journal of Molecular Biology. 427 (16): 2610–6. doi:10.1016/j.jmb.2015.06.011. PMC   4523409 . PMID   26116762.
10. Barrows, NJ; Campos, RK; Powell, ST; Prasanth, KR; Schott-Lerner, G; Soto-Acosta, R; Galarza-Muñoz, G; McGrath, EL; Urrabaz-Garza, R; Gao, J; Wu, P; Menon, R; Saade, G; Fernandez-Salas, I; Rossi, SL; Vasilakis, N; Routh, A; Bradrick, SS; Garcia-Blanco, MA (10 August 2016). "A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection". Cell Host & Microbe. 20 (2): 259–70. doi:10.1016/j.chom.2016.07.004. PMC   4993926 . PMID   27476412.
11. Routh, A; Ji, P; Jaworski, E; Xia, Z; Li, W; Wagner, EJ (7 July 2017). "Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation". Nucleic Acids Research. 45 (12): e112. doi:10.1093/nar/gkx286. PMC   5499544 . PMID   28449108.
12. Head, SR; Komori, HK; LaMere, SA; Whisenant, T; Van Nieuwerburgh, F; Salomon, DR; Ordoukhanian, P (2014). "Library construction for next-generation sequencing: overviews and challenges". BioTechniques. 56 (2): 61–4, 66, 68, passim. doi:10.2144/000114133. PMC   4351865 . PMID   24502796.
13. Routh, A; Head, SR; Ordoukhanian, P; Johnson, JE (14 August 2015). "ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs". Journal of Molecular Biology. 427 (16): 2610–6. doi:10.1016/j.jmb.2015.06.011. PMC   4523409 . PMID   26116762.
14. Abel GR, Jr; Calabrese, ZA; Ayco, J; Hein, JE; Ye, T (16 March 2016). "Measuring and Suppressing the Oxidative Damage to DNA During Cu(I)-Catalyzed Azide-Alkyne Cycloaddition" (PDF). Bioconjugate Chemistry. 27 (3): 698–704. doi:10.1021/acs.bioconjchem.5b00665. PMID   26829457. S2CID   255636.