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

  title={Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation},
  author={Andrew L. Routh and Ping Ji and Elizabeth Jaworski and Zheng Xia and Wei Li and Eric J. Wagner},
  journal={Nucleic Acids Research},
  pages={e112 - e112}
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… 

DPAC: a tool for Differential Poly(A) Site usage from poly(A)–targeted RNAseq data

Poly(A)-tail targeted RNAseq approaches, such as 3’READS, PAS-seq and Poly(A)-ClickSeq, are becoming popular alternatives to random-primed RNAseq for simplified gene expression analyses as well as to

DPAC: A Tool for Differential Poly(A)–Cluster Usage from Poly(A)–Targeted RNAseq Data

  • A. Routh
  • Biology
    G3: Genes, Genomes, Genetics
  • 2019
Poly(A)-tail targeted RNAseq approaches, such as 3′READS, PAS-Seq and Poly(A)-ClickSeq, are becoming popular alternatives to random-primed RNAseq to focus sequencing reads just to the 3′ ends of

Alternative polyadenylation analysis in animals and plants: newly developed strategies for profiling, processing and validation

New next generation sequencing methods for genome-wide profiling of alternative polyadenylation (APA) sites, bioinformatics pipelines for data processing and both wet and dry laboratory approaches for APA validation are reviewed.

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

An updated protocol for the synthesis of "ClickSeq" libraries is described, finding 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.

Alternative Polyadenylation: a new frontier in post transcriptional regulation

The current knowledge on APA is reviewed and how its regulatory complex factors work together to determine RNA splicing location, cell cycle velocity, microRNA processing, and oncogenesis regulation.

An integrative model for alternative polyadenylation, IntMAP, delineates mTOR-modulated endoplasmic reticulum stress response

A novel bioinformatics algorithm, IntMAP, is developed, which integrates RNA-Seq and PolyA Site (PAS)-Seq data for a comprehensive characterization of APA events, and expands the understanding of the physiological role of mTOR-coordinatedAPA events to ER stress response.

Alternative cleavage and polyadenylation in health and disease

The experimental and computational methods that have enabled the global mapping of mRNA and of long non-coding RNA 3ʹ ends, quantification of the resulting isoforms and the discovery of regulators of alternative cleavage and polyadenylation (APA) are reviewed.

Sensitive and accurate analysis of gene expression signatures enabled by oligonucleotide-labelled cDNA

MTAS-seq (mRNA sequencing via terminator-assisted synthesis) is reported – a novel RNA-seq library preparation method directed towards mRNA 3′ termini and the specific enrichment for 3′-terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance.



3'READS+, a sensitive and accurate method for 3' end sequencing of polyadenylated RNA.

3'READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3' end counting, especially when the amount of input total RNA is limited.

Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq.

PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation and detected significant changes in the global APA profile that lead to lengthening of 3' untranslated regions (UTR) in many mRNAs during stem Cell differentiation.

Ending the message: poly(A) signals then and now.

A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.

A quantitative atlas of polyadenylation in five mammals.

PolyA-seq is shown to be as accurate as existing RNA sequencing approaches for digital gene expression (DGE), enabling simultaneous mapping of polyA sites and quantitative measurement of their usage, and usage is more similar within the same tissues across different species than within a species.

Analysis of alterative cleavage and polyadenylation by 3′ region extraction and deep sequencing

Quantitative analysis of APA isoforms indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pA, suggesting global modulation of the 3′ end–processing activity in development and differentiation.

Genome-wide mapping of polyadenylation sites in fission yeast reveals widespread alternative polyadenylation

It is shown that for both coding and non-coding genes, the most common regulatory motif associated with CSs in fission yeast is the canonical human AAUAAA sequence.

Cleavage factor Im is a key regulator of 3′ UTR length

It is demonstrated that the loss-of-function of CF Im68 and CF Im25 but not ofCF Im59 leads to a transcriptome-wide increase in the use of proximal polyadenylation sites in HEK293 cells.