---
id: epigenetics-methylation-seq
name: Galaxy Tour
description: In this tour we will perform the methylation and metric extraction and detect differentially methylated regions (DMRs).
title_default: epigenetics-methylation-seq
steps:
  - title: DNA Methylation data analysis
    content: >-
      In this tour we will perform the methylation and metric extraction and detect differentially methylated regions (DMRs).
    backdrop: true
  - title: Step 1. Load  data and quality control
    element: '#shared .dropdown a[href$="/library/index"]'
    content: >-
      We load now the example dataset which will be used for the tutorial. Click on Shared Data.
    placement: right
  - title: Step 2. Load  data and quality control
    element: 'li a[href$="/library/list"]'
    content: In the dropdown menu click on Data Libraries. In the new window search for MethylSeq_2017. Select the uploaded datasets subset_1.fastq.gz and subset_2.fastq.gz as the fastq files.
    placement: right
  - title: Estimating quality with FastQC
    content: >-
      To estimate sequence quality and treatments to do on the data we will use FastQC open-source tool.
      For each studied indicators, FastQC providing a quick overview to tell in which areas there may be problems.
    backdrop: true
  - title: Step 3. Estimating quality with FastQC
    element: '#tool-search-query'
    content: 'Search for FastQC tool'
    placement: right
    textinsert: 'fastQC'
  - title: Step 4. Estimating quality with FastQC
    element: '#tool-search'
    content: Click on the "FastQC" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fdevteam%2Ffastqc%2Ffastqc%2F0.69"]
  - title: Step 5. Estimating quality with FastQC
    element: '#tool-search'
    content: Execute the tool on the input data with default settings.
    position: left
  - title: Step 6. Estimating quality with FastQC
    element: '.history-right-panel .list-items > *:first'
    content: |-
      Inspect the FastQC report on the webpage <ul>
        <li>have a closer look at ‘Per base sequence content’</li>
      </ul>
    position: left
  - title: Questions. Estimating quality with FastQC
    content: |-
      Inspect the FastQC report on the webpage  <ul>
        <li>Note the GC distribution and percentage of “T” and “C”. Why is this so weird?</li>
        <li>Is everything as expected?</li>
      </ul>
    backdrop: true
  - title: Alignment
    content: We will map now the imported dataset against a reference genome.
    backdrop: true
  - title: Step 7. Mapping with bwameth
    element: '#tool-search-query'
    content: 'Search for bwameth tool'
    placement: right
    textinsert: 'bwameth'
  - title: Step 8. Mapping with bwameth
    element: '#tool-search'
    content: Click on the "bwameth" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fiuc%2Fbwameth%2Fbwameth%2F0.2.0.3"]
  - title: Step 9. Mapping with bwameth
    element: '#tool-search'
    content: |-
      Execute the tool on two imported datasets with  <ul>
        <li>"Select a genome reference from your history or a built-in index?"to "Use a built-in index"</li>
        <li>Set the human genome to "hg38"</li>
        <li>Set the option "Is this library mate-paired?" to "Paired-end"</li>
      </ul>
    position: left
  - title: Mapping with bwameth
    content: Please notice that depending on your system this computation can take some time. If you want to skip this, we provide for you a precomputed alignment. Import aligned_subset.bam to your history.
    backdrop: true
  - title: Questions. Mapping with bwameth
    content: |-
      <ul>
        <li>Why we need other alignment tools for bisulfite sequencing data?</li>
      </ul>
    backdrop: true
  - title: Methylation bias
    content: We will extract the methylation on the resulting BAM file of the alignment step.
    backdrop: true
  - title: Step 10. Methylation bias
    element: '#tool-search-query'
    content: 'Search for MethylDackel tool'
    placement: right
    textinsert: 'MethylDackel'
  - title: Step 11. Methylation bias
    element: '#tool-search'
    content: Click on the "MethylDackel" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Fpileometh%2Fpileometh%2F0.3.0"]
  - title: Step 12. Methylation bias
    element: '#center-panel'
    content: |-
      Execute the tool on two imported datasets with  <ul>
        <li>Set "Load reference genome from"to "Local cache"</li>
        <li>Set for the "Using reference genome" to "hg38"</li>
        <li>Select for the option "sorted_alignments.bam" the computed bam file of step 9 of the bwameth alignment.</li>
        <li>Set "What do you want to do?" to "Determine the position-dependent methylation bias in the dataset, producing diagnostic SVG images"</li>
        <li>Set the parameters "keepSingleton" and "keepDiscordant" to Yes</li>
      </ul>
    position: left
  - title: Remarks. Methylation bias
    content: |-
      Note that biases of less than 5% can be ignored due to the fact that 5% difference is well within the range of biological insignificance.
    backdrop: true
  - title: Questions. Methylation bias
    content: |-
      Inspect the results<ul>
        <li>Consider the original top strand output. Is there a methylation bias?</li>
        <li>If we would trim, what would be the start and the end positions?</li>
      </ul>
    backdrop: true
  - title: Step 13. Methylation extraction with MethylDackel
    element: '#tool-search-query'
    content: 'Search for MethylDackel tool'
    placement: right
    textinsert: 'MethylDackel'
  - title: Step 14. Methylation extraction with MethylDackel
    element: '#tool-search'
    content: Click on the "MethylDackel" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Fpileometh%2Fpileometh%2F0.3.0"]
  - title: Step 15. Methylation extraction with MethylDackel
    element: '#center-panel'
    content: |-
      Execute the tool on the datasets with  <ul>
        <li>Set "Load reference genome from"to "Local cache"</li>
        <li>Set for the "Using reference genome" to "hg38"</li>
        <li>Select for the option "sorted_alignments.bam" the computed bam file of step 9 of the bwameth alignment.</li>
        <li>Set "What do you want to do?" to "Extract methylation metrics from an alignment file in BAM/CRAN format"</li>
        <li>Set "Merge per-Cytosine metrics from CpG and CHG contexts into per-CPG or per-CHG metrics" to Yes</li>
        <li> Set the parameter "fraction" to Yes</li>
      </ul>
    position: left
  - title: Visualization
    content: We will visualize the example with the help of deepTools.
    backdrop: true
  - title: Step 16. Visualization
    element: '#tool-search-query'
    content: 'Search for Wig/BedGraph-to-bigWig tool'
    placement: right
    textinsert: 'Wig/BedGraph-to-bigWig'
  - title: Step 17. Visualization
    element: '#tool-search'
    content: Click on the "Wig/BedGraph-to-bigWig" tool to open it
    placement: right
    postclick:
      - 'a[href$="/tool_runner?tool_id=wig_to_bigWig"]'
  - title: Step 18. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool on two imported datasets with  default settings.
    position: left
  - title: Questions
    content: The execution fails. Do you have an idea why?
    backdrop: true
  - title: Step 19. Visualization
    element: '#tool-search-query'
    content: 'Search for tail tool'
    placement: right
    textinsert: 'tail'
  - title: Step 20. Visualization
    element: '#tool-search'
    content: Click on the "Select last lines from a dataset (tail)" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Ftext_processing%2Ftp_tail_tool%2F1.1.0"]
  - title: Step 21. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool on the datasets with  <ul>
        <li>Use the mode "Operation" the value "Keep everything from this line on" and choose "2" as a value.</li>
      </ul>
    position: left
  - title: Step 22. Visualization
    element: '#tool-search-query'
    content: 'Search for awk tool'
    placement: right
    textinsert: 'awk'
  - title: Step 23. Visualization
    element: '#tool-search'
    content: Click on the "Text reformatting with awk" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Ftext_processing%2Ftp_awk_tool%2F1.1.0"]
  - title: Step 24. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Use as AWK Program: 'BEGIN{OFS="\t"}{print $1, $2, $3, $4}'</li>
      </ul>
    position: left
  - title: Step 25. Visualization
    element: '#tool-search-query'
    content: 'Search for computeMatrix tool'
    placement: right
    textinsert: 'computeMatrix'
  - title: Step 26. Visualization
    element: '#tool-search'
    content: Click on the "computeMatrix" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Fdeeptools_compute_matrix%2Fdeeptools_compute_matrix%2F2.5.1.1.0"]
  - title: Step 27. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Use the file 'CpGIslands.bed' as 'Regions to plot'</li>
        <li>and the in the previous step created bigwig file as the 'score file'</li>
        <li>Use for the option 'computeMatrix has two main output options' the value 'reference-point'.</li>
      </ul>
    position: left
  - title: Step 28. Visualization
    element: '#tool-search-query'
    content: 'Search for plotProfile tool'
    placement: right
    textinsert: 'plotProfile'
  - title: Step 29. Visualization
    element: '#tool-search'
    content: Click on the "plotProfile" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Fdeeptools_plot_profile%2Fdeeptools_plot_profile%2F2.5.1.1.0"]
  - title: Step 30. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Choose for 'Matrix file from the computeMatrix tool' the file computed at step 27</li>
      </ul>
    position: left
  - title: Step 31. Visualization
    content: Import from the data library the files NB1_CpG.meth.bedGraph
    backdrop: true
  - title: Step 32. Visualization
    element: '#tool-search-query'
    content: 'Search for Wig/BedGraph-to-bigWig tool'
    placement: right
    textinsert: 'Wig/BedGraph-to-bigWig'
  - title: Step 33. Visualization
    element: '#tool-search'
    content: Click on the "Wig/BedGraph-to-bigWig" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=wig_to_bigWig"]
  - title: Step 34. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool on the imported file with  default settings.
    position: left
  - title: Questions
    content: The execution fails. Do you have an idea why?
  - title: Visualization
    content: Every chromosome is named different, the list to transfer the ensembl notation to ucsc notation is having more than 500 entries. To convert it you can use the tool Replace column.
    backdrop: true
  - title: Step 35. Uploading the new data
    element: '#tool-panel-upload-button .fa.fa-upload'
    content: We need to upload the conversion file. Open the Galaxy Upload Manager
    placement: right
    postclick:
      - '#tool-panel-upload-button .fa.fa-upload'
      - '#btn-reset'
  - title: Step 36. Uploading the input data
    element: '#btn-new'
    content: Click on Paste/Fetch Data
    placement: right
    postclick:
      - '#btn-new'
  - title: Step 37. Uploading the input data
    element: .upload-text-column .upload-text .upload-text-content.form-control
    content: Insert the link here.<br> The input is available on <a
      href="https://raw.githubusercontent.com/dpryan79/ChromosomeMappings/master/GRCh38_ensembl2UCSC.txt">link</a> in the tutorial. Right click it and then "Copy Link Address"
    placement: right
    textinsert: >-
      https://raw.githubusercontent.com/dpryan79/ChromosomeMappings/master/GRCh38_ensembl2UCSC.txt
  - title: Step 38. Uploading the input data
    element: '#btn-start'
    content: Click on "Start" to start loading the data to history
    placement: right
    postclick:
      - '#btn-start'
  - title: Step 39. Uploading the input data
    element: '#btn-close'
    content: >-
      The upload may take a while.<br> Hit the close button to close this
      window.
    placement: right
    postclick:
      - '#btn-close'
  - title: Step 40. Visualization
    element: '#tool-search-query'
    content: Search for 'replace column' tool
    placement: right
    textinsert: 'replace column'
  - title: Step 41. Visualization
    element: '#tool-search'
    content: Click on the "Replace column" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Freplace_column_by_key_value_file%2Freplace_column_with_key_value_file%2F0.1"]
  - title: Step 42. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Choose for "File in which you want to replace some values" the bedGraph file</li>
        <li>Choose for "Replace information file" the new imported file.</li>
        <li>Choose for "Which column should be replaced?" the value "1"</li>
        <li>Choose for "Skip this many starting lines" the value "1"</li>
        <li>Set "Delimited by" to "Tab"</li>
      </ul>
    position: left
  - title: Visualization
    content: In case this tool is not available in your galaxy instance, we precomputed the files for you. Please import NB1_CpG.meth_ucsc.bedGraph, NB2_CpG.meth_ucsc.bedGraph, BT089_CpG.meth_ucsc.bedGraph, BT126_CpG.meth_ucsc.bedGraph and BT198_CpG.meth_ucsc.bedGraph.
    backdrop: true
  - title: Step 43. Visualization
    element: '#tool-search-query'
    content: 'Search for computeMatrix tool'
    placement: right
    textinsert: 'computeMatrix'
  - title: Step 44. Visualization
    element: '#tool-search'
    content: Click on the "computeMatrix" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Fdeeptools_compute_matrix%2Fdeeptools_compute_matrix%2F2.5.1.1.0"]
  - title: Step 45. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Use the file 'CpGIslands.bed' as 'Regions to plot'</li>
        <li>and the in the previous step created bigwig file as the 'score file'</li>
        <li>Use for the option 'computeMatrix has two main output options' the value 'reference-point'.</li>
      </ul>
    position: left
  - title: Step 46. Visualization
    element: '#tool-search-query'
    content: 'Search for plotProfile tool'
    placement: right
    textinsert: 'plotProfile'
  - title: Step 47. Visualization
    element: '#tool-search'
    content: Click on the "plotProfile" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fbgruening%2Fdeeptools_plot_profile%2Fdeeptools_plot_profile%2F2.5.1.1.0"]
  - title: Step 48. Visualization
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Choose for 'Matrix file from the computeMatrix tool' the file computed at step 27</li>
      </ul>
    position: left
  - title: Metilene
    content: |-
      With metilene it is possible to detect differentially methylated regions (DMRs) which is a necessary prerequisite for characterizing different epigenetic states.
    backdrop: true
  - title: Metilene
    content: |-
      Import from the data library if you have not the files NB1_CpG.meth.bedGraph, NB2_CpG.meth.bedGraph, BT089_CpG.meth.bedGraph, BT126_CpG.meth.bedGraph and BT198_CpG.meth.bedGraph.
    backdrop: true
  - title: Step 49. Metilene
    element: '#tool-search-query'
    content: 'Search for metilene tool'
    placement: right
    textinsert: 'metilene'
  - title: Step 50. Metilene
    element: '#tool-search'
    content: Click on the "metilene" tool to open it
    placement: right
    postclick:
      - >-
        a[href$="/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Frnateam%2Fmetilene%2Fmetilene%2F0.2.6.1"]
  - title: Step 51. Metilene
    element: '#center-panel'
    content: |-
      Execute the tool with  <ul>
        <li>Choose for the first option "Input group 1" the imported files starting with NB</li>
        <li>Choose for the first option "Input group 2" the imported files starting with BT</li>
        <li>Select for the option "BED file containing regions of interest" the imported BAM file CpGIslands.bed</li>
      </ul>
    position: left
  - title: Questions. Metilene
    content: |-
      Have a look at the produced pdf document. What is the data showing?
    backdrop: true
  - title: Key Points
    content: |-
      <ul>
        <li>The output of a methylation NGS is having a different distribution of the four bases. This is caused by the bisulfite treatment of the DNA.</li>
        <li>If there is a different level of methylation in the loci of a gene this can be a hint that something is wrong.</li>
        <li>To get useful results you need -- data, data and data!</li>
      </ul>
    backdrop: true