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Some metadata columns have special meanings: the score column accesible via the score function and thick column, as these columns can be exported to a standard BED-format file via the export function from the rtracklayer package. A RangedSummarizedExperiment can store several matrices of the same shape, e. Storing all information as a single RangedSummarizedExperiment helps keeping data organized, for example when extracting subsets of the data, where subset can be used to simultaneously extract requested data from all three tables at once.

The most important groups are:. In all cases, data is a stored as a RangedSummarizedExperiment :. The majority of functions in CAGEfightR are endomorphisms , meaning they returned modified versions of the input objects. This often works by adding calculated values as new metadata columns, accesible via rowData or mcols.


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While not used in this vignette, this means that CAGEfightR is highly compatible with the pipes from the magrittr package. The following section contains a detailed walkthrough of most of the functions in CAGEfightR , using the built-in dataset. To keep this vignette compact, the example dataset is extremely small - for a more realistic analysis of a full CAGE dataset using both CAGEfightR and additional packages, additional workflows will be added in the future.

Rather than repeating this, we use the built-in exampleCTSSs object:. This is a somewhat special RangedSummarizedExperiment : The assay is not a normal matrix , but rather a sparse matrix dgCMatrix from the Matrix package. Since CAGE data is inherently sparse vast majority sites in the genome have no counts , storing CTSSs as a sparse matrix allows for much faster and memory efficient processing of data:.

The ranges are stored as GPos , where all ranges a one basepair wide. Again, this is much more memory-efficient way of storing CTSSs:. However, since the different samples have different library sizes, CTSS counts must first be normalized. As this is just a subset of the original dataset, we instead use the total number tags from the the complete dataset, by specifying the name of the column in colData Note that a warning is passed that we are overwritting the previous TPM assay :.

To alleviate this issue, CTSSs appearing in only a single or few samples can be discarded. We refer to this as calculating the support : the number of samples expressing a feature above some threshold:.


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The majority of CTSSs are only expressed in a single sample. We can discard these sites using subset , and then recalculate TPM values:. The subsetBySupport function wraps the common task of calling calcSupport and subset.

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The simplest way of doing this is to group nearby CTSSs on the same strand into Tag Clusters TCs , which are likely candidates for real TSSs although some post-filtering is almost always a good idea, see next section. CAGEfightR does this by a slice-reduce approach: It finds sites above some value slice and then merges nearby sites reduce.

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In the simplest form this can be run as:. This is often the result of a just few spread-out tags that link several major TCs, which does not represent the TSS structure very well.

One way of dealing with this is to increase the the pooled cutoff for calling clusters. Another way is to first filter the CTSSs based on support:. Both approaches reduces the overall width of the TCs identified, in particular the most wide TCs. In case you want to save TCs to a bed file, you can use the export function from the rtracklayer package. Active enhancers produce weak but consistent bidirectional transcription of capped eRNAs, resulting in a characteristic CTSS pattern of two diverging peaks approximally basepairs apart.

Again, a slice-reduce approach can be use to identify invidividual enhancers based on the balance scores. We refer to this approach as bidirectional clustering as opposed to the conventional unidirectional clustering used to identify TSSs: and hence the name of the cluster functions: clusterBidirectionally and clusterUnidirectionally :. The GRanges includes the chromosome, start and end positions of each Bidirectional Cluster BC , as well as two other key values: the score column holds the sum of pooled CTSSs of the BC on both strands and the BC midpoint in the thick column: This is the maximally balanced site in the BC Again, the export function from the rtracklayer package can be used to export enhancers to a BED-file.

As balance here is solely defined on the pooled CTSSs, a useful filter is to make sure the BC is observed to be bidirectional in at least a single sample.

A step-by-step guide to analyzing CAGE data using R/Bioconductor

Similarly to how we calculated support, we can calculate the sample-wise bidirectionality of BCs:. Many BCs are not observed to be bidirectional in one or more samples. We can remove these using subset The subsetBySupport function wraps the common task of calling calcBidirectionality and subset :.

This bidirectional clustering approach identifies any bidirectional site in the genome. This means that for example bidirectional promoters will also detected given that they are sufficiently balanced. To remove these cases, annotation with transcript models can be used to remove bidirectional clusters overlapping known promoters and exons.

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This is described in the next section. The Trust is open year-round by appointment to visitors to assist ongoing work involving, in diverse ways, the legacy of John Cage. Peters , in promoting new performances and recordings and providing licensing and rights. And, for nearly two decades, it worked intimately with the Cunningham Dance Foundation to assist its continued use of live music for dance, a trademark tradition initiated by Cage and Cunningham in with the formation of the Merce Cunningham Dance Company.

CAGEfightR: analysis of 5′-end data using R/Bioconductor | BMC Bioinformatics | Full Text

A particularly interesting example, from , involved the sampling of prepared piano sounds that were subsequently released by Big Fish Audio in a format suitable for MIDI systems. This work, which Kuhn directed, was seen in venues around the world, including Edinburgh, Berlin, Dublin, Perth, and California. In , the John Cage Trust became a permanent member of the Bard community, and in the organization moved into a new building on campus, known familiarly as The Wilson House, significantly expanding its on-site archives and working space.

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CAGEfightR: analysis of 5′-end data using R/Bioconductor

Launching Xcode If nothing happens, download Xcode and try again. Fixes Latest commit 51e6c76 Jan 6, Cage: a Wayland kiosk This is Cage, a Wayland kiosk. A kiosk runs a single, maximized application. Building and running Cage You can build Cage with the meson build system. Cage is based on the annotated source of tinywl and rootston. Bugs For any bug, please create an issue on GitHub.

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