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.

doi:10.1016/j.jmb.2015.06.011

Cited by 64 publications

(101 citation statements)

References 43 publications

(50 reference statements)

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“…The encapsidated genomic RNA was then extracted and prepared for RNAseq using ClickSeq (a cDNA library generation method we recently developed that considerably reduces artifactual recombination[21]). Final cDNA sequencing libraries were prepared for paired-end sequencing with an average fragment length of 150–200bps and sequenced on an Illumina NextSeq giving 150bp for each read.…”

Section: Resultsmentioning

confidence: 99%

CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

Routh

Chang

Okulicz

et al. 2015

Methods

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Next-generation sequencing (NGS) has transformed our understanding of the dynamics and diversity of virus populations for human pathogens and model systems alike. Due to the sensitivity and depth of coverage in NGS, it is possible to measure the frequency of mutations that may be present even at vanishingly low frequencies within the viral population. Here, we describe a simple bioinformatic pipeline called CoVaMa (Co-Variation Mapper) scripted in Python that detects correlated patterns of mutations in a viral sample. Our algorithm takes NGS alignment data and populates large matrices of contingency tables that correspond to every possible pairwise interaction of nucleotides in the viral genome or amino acids in the chosen open reading frame. These tables are then analysed using classical linkage disequilibrium to detect and report evidence of epistasis. We test our analysis with simulated data and then apply the approach to find epistatically linked loci in Flock House Virus genomic RNA grown under controlled cell culture conditions. We also reanalyze NGS data from a large cohort of HIV infected patients and find correlated amino acid substitution events in the protease gene that have arisen in response to anti-viral therapy. This both confirms previous findings and suggests new pairs of interactions within HIV protease. The script is publically available at http://sourceforge.net/projects/covama

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Section: Resultsmentioning

confidence: 99%

CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

Routh

Chang

Okulicz

et al. 2015

Methods

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“…This design allows for cDNA chain termination to occur only in the residues just upstream of poly(A) tail, essentially 'homing-in' on the junction of the 3´UTR and the poly(A) tail. We have found that a ratio of 1:5 AzVTPs:dNTPs reliably yields cDNA fragments ranging from 50-400 nts in length (42).…”

Section: Poly(a)-clickseq Library Generationmentioning

confidence: 98%

“…We recently reported a technique called 'ClickSeq' that uses azido-nucleotide terminators in randomlyprimed RT reactions to produce cDNA fragments from non-fragmented template RNA (42). Azidonucleotides are stochastically incorporated during cDNA synthesis inducing chain-termination yielding a distribution of cDNA fragment lengths, which is determined by the ratio of AzNTPs to dNTPs.…”

Section: Poly(a)-clickseq Library Generationmentioning

confidence: 99%

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Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

Routh

Jaworski

et al. 2017

Preprint

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The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture, and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3´ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error, and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called "ClickSeq". Here, we adapt this method to direct the cDNA synthesis specifically toward the 3´UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PACseq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.

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“…Instead, adapter sequences can be ligated to the ends of RNA before reverse transcription, included in the reverse transcription primer or ligated to the cDNA 3′ end. Alternatively, the terminal transferase activity of M-MLV reverse transcriptase can be used in combination with its ability to switch template to add a 3′ cDNA adapter sequence during reverse transcription [6], or reverse transcription can be terminated stochastically with 3′ azido labeled dideoxynucleotides followed by click chemistry-mediated adapter addition [7]. …”

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Excess Primer Degradation by Exo I Improves the Preparation of 3′ cDNA Ligation-Based Sequencing Libraries

et al. 2019

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RNA sequencing library construction using single-stranded ligation of a DNA adapter to 3′ ends of cDNAs often produces primer–adapter byproducts, which compete with cDNA–adapter ligation products during library amplification and, therefore, reduces the number of informative sequencing reads. We find that Escherichia coli Exo I digestion efficiently and selectively removes surplus reverse transcription primer and thereby reduces the primer–adapter product contamination in 3′ cDNA ligation-based sequencing libraries, including small RNA libraries, which are typically similar in size to the primer–adapter products. We further demonstrate that Exo I treatment does not lead to trimming of the cDNA 3′ end when duplexed with the RNA template. Exo I digestion is easy to perform and implement in other protocols and could facilitate a more widespread use of 3′ cDNA ligation for sequencing-based applications.

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

Cited by 64 publications

References 43 publications

CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

Excess Primer Degradation by Exo I Improves the Preparation of 3′ cDNA Ligation-Based Sequencing Libraries

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