کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2184287 | 1095822 | 2015 | 7 صفحه PDF | دانلود رایگان |
• ClickSeq produces RNAseq libraries without sample fragmentation or adaptor ligation.
• cDNA fragments are made via stochastic reverse transcription PCR termination with azido-nucleotides.
• Alkyne-modified adaptors are ligated using click chemistry in place of enzymes.
• High-quality unbiased RNAseq libraries are made with low error rates.
• Artifactual recombination is also reduced, resolving rare recombinant viral species.
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.
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Journal: Journal of Molecular Biology - Volume 427, Issue 16, 14 August 2015, Pages 2610–2616