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Introduction

PL-Grid instance of Galaxy provides all the tools necessary for creating and executing analysis of Bisulfite-Seq (BS-Seq) data. Tools which are required for every step are marked by info frames. All the form fields that has to be filled are described below the info frames.

Input Dataset

In this tutorial we  will use the file ‘test_dataset.fastq’ which is available for download from the Bismark homepage (it contains 10,000 reads in FastQ format, Phred33 qualities, 50 bp long reads, from a human directional BS-Seq library).

Link to input dataset:

http://www.bioinformatics.babraham.ac.uk/projects/bismark/test_data.fastq

Upload data

To perform the analysis we need to load input dataset. All data import tools are available in Get Data category in Tools side panel.

Click on the Upload File tool in the Get Data section of the leftmost menu. Next, click on the button “Paste/Fetch”, copy and paste input dataset file URLs into text box. You should also set “Type” to “fastqsanger” dataype. Finally, press the Start button.

 

Quality Control

Before using data we must evaluate quality of reads. QC allows us to detect poor quality reads or other characteristics of our data and act accordingly. More information about QC you can find QC tutorial.

For test_data.fastq file we have to execute FastQC:Read Quality Reports from category NGS: QC and manipulation.

To use FastQC you must pick input file and press Execute.

 

Analysis of Bisulfite-Seq (BS-Seq) data

Next, for test_data.fastq file we have to execute Bismark bisulfite mapper (bowtie2) from category Personalized medicine. Bismark requires to select a reference genome. In this tutorial we have to select “Human Feb. 2009 (GRCh37/hg19)” and “Single-end” library.

 

The result of the Bismark bisulfate mapper processing in a single BAM file, which will be necessary in next step of this workflow.

In the last step we have to perform the analysis using Bismark methylation extractor (Bismark Meth. Extractor Reports on methylation status of reads mapped by Bismark from category Personalized medicine).

A typical values for Bismark methylation extractor’s parameters to extract context-dependent (CpG/CHG/CHH) methylation should look like in the form below:

  

To use Bismark methylation extractor you must press Execute.

The final result of the analysis is available by pressing the "eye icon" in the history panel.

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