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.

Keywords: click-chemistry; cricket paralysis virus; flock house virus; next-generation sequencing; recombination.

Figure 1

ClickSeq uses bio-orthogonal click-chemistry to generate RNAseq libraries from stochastically terminated 3’-azido blocked cDNA fragments. A) Chemical structure of a 3’-azido-2’,3’-dideoxynucleotide (AzNTP). B) ‘Click-ligation’: Copper-catalyzed cycloaddition of a 5’-hexynyl modified adaptor DNA to a 3’-azido blocked cDNA fragment. C) Schematic illustration of ClickSeq. Template RNA is in red, and cDNA fragments are in black. The color of the sequence adaptors corresponds to those in Table S1.

Figure 2

Electrophoretic analysis of click-reaction components, products and PCR amplification. A) Schematic illustration of primer extension by RT-PCR using FHV RNA and the click-ligation of the products to form a triazole-linked ssDNA fragment. B) The components and products were analysed by denaturing urea PAGE and quantified by densitometry. C) cDNA libraries with an increasing distribution of insert lengths were generated from Flock House Virus RNA using a range of AzNTP:dNTPs. The PCR amplified libraries were visualized by electrophoresis on a 1.5% agarose gel stained with Ethidium Bromide.

Figure 3

Read mapping to Flock House Virus (FHV), Cricket Paralysis Virus (CrPV), Drosophila melanogaster mRNAs and recombinations events using ClickSeq and NEBNext are compared. Coverage of sequences reads over A) FHV RNA 1, B) FHV RNA 2 and C) CrPV by NEBNext (red) and ClickSeq (blue). Scatter plots show the correlation between the frequencies of mapping events detected by NEBNext and ClickSeq: D) host mRNA in the FHV RNA datasets; E) Intra-molecular recombination in FHV; F) Inter-molecular recombination in FHV; and G) recombination in CrPV. Multiple overlapping points are merged to form a single larger point, as illustrated in the key.