Abstract
Background5′-end sequencing assays, and Cap Analysis of Gene Expression (CAGE) in particular, have been instrumental in studying transcriptional regulation. 5′-end methods provide genome-wide maps of transcription start sites (TSSs) with base pair resolution. Because active enhancers often feature bidirectional TSSs, such data can also be used to predict enhancer candidates. The current availability of mature and comprehensive computational tools for the analysis of 5′-end data is limited, preventing efficient analysis of new and existing 5′-end data.ResultsWe present CAGEfightR, a framework for analysis of CAGE and other 5′-end data implemented as an R/Bioconductor-package. CAGEfightR can import data from BigWig files and allows for fast and memory efficient prediction and analysis of TSSs and enhancers. Downstream analyses include quantification, normalization, annotation with transcript and gene models, TSS shape statistics, linking TSSs to enhancers via co-expression, identification of enhancer clusters, and genome-browser style visualization. While built to analyze CAGE data, we demonstrate the utility of CAGEfightR in analyzing nascent RNA 5′-data (PRO-Cap). CAGEfightR is implemented using standard Bioconductor classes, making it easy to learn, use and combine with other Bioconductor packages, for example popular differential expression tools such as limma, DESeq2 and edgeR.ConclusionsCAGEfightR provides a single, scalable and easy-to-use framework for comprehensive downstream analysis of 5′-end data. CAGEfightR is designed to be interoperable with other Bioconductor packages, thereby unlocking hundreds of mature transcriptomic analysis tools for 5′-end data. CAGEfightR is freely available via Bioconductor: bioconductor.org/packages/CAGEfightR.
Highlights
Transcription start sites (TSSs) are central entities of transcriptional regulation, where a wide range of cues from surrounding factors such as core promoter elements, transcription factor binding sites, chromatin modifications, and distal elements such as enhancers and silencers are integrated to decide whether transcription initiation takes place, and with what rate [1,2,3]
Names of the main CAGEfightR functions for each analysis are indicated in Fig. 1, 2, 3, 4, 5
As an initial validation step, we investigated whether enhancer candidates had the expected chromatin patterns compared to Tag Cluster or Unidirectional Cluster (TC), by overlapping with DNase I hypersensitive sites sequencing (DNase-Seq), H3K27ac, H3K4me3 and H3K4me1 ChIP-Seq signals from the same cell type
Summary
Transcription start sites (TSSs) are central entities of transcriptional regulation, where a wide range of cues from surrounding factors such as core promoter elements, transcription factor binding sites, chromatin modifications, and distal elements such as enhancers and silencers are integrated to decide whether transcription initiation takes place, and with what rate [1,2,3]. Accurate identification of TSSs and their activity is a prerequisite for understanding gene regulation. Several genome-wide, high-throughput sequencing assays have been developed for identifying TSS activity, all based on the idea of capturing and sequencing only the. In terms of TSS identification, such methods have distinct advantages over other assays, e.g. RNA-sequencing (RNA-Seq) and Chromatin Immunoprecipitation Sequencing (ChIP-Seq). 5′-end methods effectively pile up reads at TSSs, providing high local coverage for accurate prediction of TSSs. ChIP-Seq targeting RNA polymerase II or pre-initiation complex proteins has low positional resolution due to the length of ChIP-Seq fragments, and does not explicitly measure TSS usage
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
