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marcdelpino edited this page May 2, 2019 · 4 revisions

Table of contents

READ MNase-SEQ DATA

Once you have all your data at the Galaxy server (read the previous section to learn how to do so) you are ready to run Nucleosome Dynamics tools. Before running any analysis, first thing to do is convert your BAM files into RData using the tool readBAM.

  1. Select Nucleosome Dynamics and click on readBAM from the toolbar at the left side.

READBAM SELECTED

  1. Select files and the parameters that you want to apply:
  • MNase-seq / ATAC-seq BAM file : Choose the BAM file you just imported in your history
  • Type : Select the type of library, "Paired" or "Single".

Execute the tool and wait while the process runs. The job will be shown at your history. Force "refresh" to update the history items to make sure the output is displayed.

refresh history

Your output file is on RData format. Now, you can run any other analyses, like NucleR, for calling nucleosome positions from your reads coverage . Here there is one example, about how to run NucleR.

RUN nucleR FOR NUCLEOSOME POSITIONING

  1. Select nucleR tool from the tool's left menu in the 'Nucleosome Dynamics' section.
  2. Select the files you want to analyse and parameters to execute the tool. You can either keep the default parameters, or enter new ones

nucleR

ℹ️ Learn more about each of the arguments for NucleR at the usage section

  1. After the execution, a GFF named "NR__.gff" will be shown at history. The GFF will be displayed clicking on the eye icon.

nucleR_eye

VISUALIZE RESULTS

Nucleosome Dynamics mainly generate sequence annotation files in BIGWIG or GFF3 formats. They both can be downloaded and processed locally. Yet, Galaxy allows to export via URL these files to be shown at some sequence browsers, which are either locally installed in your PC (Integrative Genome Viewer (IGV), Integrated Genome Browser(IGB)), or remotely accessed (UCSC Genome Browser). Galaxy mostly facilitates this installation in one click. The number of available browsers depends on the reference genome of your data. Here an example of the nucleosome calls generated by nucleR displayed using the IGV sequence browser:

igv vsiualizer

For installing IGV, just follow the Galaxy instructions. Note that IGV has to be running in your PC for being able to automatically import Galaxy data.

For plain or tabular files like GFFs, Galaxy directly display the file by clicking the "eye" icon. Each entry in the nucleR GFF represents a predicted nucleosome. The last column contains extra information fully documented here.

stats

ℹ️ Learn more about each the GFF annotations at the results section

RUN STATISTICAL ANALYSES

Once you have your output files generated, you can run statistical tools to visualize the results. Notice that there is one statistical tool for each analysis tool. For example, we have run 'nucleR' tool, so now we can visualize the statistics for our calling (averages, gene mapping, etc) running 'nucleR Statistics'.

  1. Select Nucleosome Dynamics and click on 'nucleR Statistics' from the toolbar at the left side.

nucleR-selected

  1. Configure tool's parameters:
  • Nucleosome Calls (GFF): select the files that nucleR GFF you want to analyse, named "NR__.gff".
  • Built-in reference genome: For extracting statistics based on the genes positions, select your reference genome. The Galaxy server have a list of available reference genomes, but if your genome is not listed, you can upload our own assembly importing to your history the following files:
    • Gene positions for the reference genome (GFF)
    • Size and chromosomes for the reference genome (CHROM_SIZE)

Check the format for these files (here)[]

For the example dataset, select the 'Saccharomyces Cerevisiae R64-1-1'. Once selected, execute the tool.

nucleR_stats

ℹ️ Learn more about each of the arguments for nucleR Statistics at the usage section

  1. After the execution, the statistical reports will appear at your history. They correspond to histograms or other plots (PNGs) or tabular data (CSV) with the frequencies or averages. For 'nucleR Statistics', two CSVs files are generated:
  • "NR__stats__.genes.csv" CSV format: Nucleosome properties per gene
  • "NR__stats__.gw.csv" CSV file: Predicted nucleosome feature frequencies

These files will be displayed by clicking on the eye icon.

stats

RUN OTHER ANALYSES TOOLS

The rest of tools offered by the Nucleosome Dynamics toolkit are executed in a similar way. They all are eligible from the tool's left-hand menu inside Nucleosome Dynamics section, and they all run in two phases, one running the analysis per se, and a second one, where some statistics are extracted. The other analyses are:

  • NucDyn (and NuclDyn Statistics)
  • NFR (and NFR Statistics)
  • Periodicity (and Periodicity Statistics)
  • Stiffness (and Stiffness Statistics)
  • TSS (and TSS Statistics)

ℹ️ Learn more about the particular arguments for each analyses in the usage section

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