1 Introduction

This tutorial will give you a short introduction into using IGV (Integrative Genomics Viewer) to visualize next-generation sequencing data and annotations. We will use the same ChIP-seq dataset we have aligned and peak called in the previous practicals.

2 Loading IGV and basic navigation on the genome browser

  1. Open IGV and select the genome build we were using for the alignment in the top left corner (“Human (hg38)”).

  2. Select a single chromosome (eg. chr3).

  3. Navigate to chr3:39,000,000-43,000,000.

  4. Double click on one of the genes to recenter the window on that gene.

  5. Navigate to your favorite gene (or ZMAT3).

  6. Zoom out to see the surrounding genes.

  7. Right click the left side panel next to your genome track and select “Expanded” to see all the transcripts.

3 Loading your data into IGV

  1. Now click File > Load from File… and load the THOR differentail binding results that you generated from ~/Course_Materials/ChIPseq/Materials/differential_binding/ folder (Alternatively, if THOR is still running on your computer you can find the same files preprocessed for you under ~/Course_Materials/ChIPseq/Preprocessed/differential_binding/ folder):
    • all the normalised bigWig files (extension: .bw),
    • the differential binding result file ending with “-diffpeaks.bed”.
  2. Visualize all chromosomes - you will see that in our data we only have reads and differentially bound regions on chr3.

  3. Navigate to ZMAT3.

  4. Select the all the .bw files you have loaded, right click them and select “Group Autoscale”.

  5. Set different colours for the tp53 and TAp73beta sample tracks (Change Track Color (Positive values)…).

  6. Zoom out to see the signal of the surrounding genes.

  7. Compare the signal in the two files you have loaded.

  8. Bookmark this region:
    • Go to Regions > Region Navigator. Click Add, and give your region a name (eg. MyFirstRegion) in the “Description” field. Click “View”.
    • This way if you navigate somewhere else on the genome you can always easily access this region from Regions > Region Navigator.
  9. Similarly, load (some of) the .bam files:
    • either from ~/Course_Materials/ChIPseq/Materials/Alignment_BWA/ folder, where you will only find .bam files for tp53,
    • or from from ~/Course_Materials/ChIPseq/Preprocessed/Alignment_BWA/ folder where you can find .bam files for both conditions.
  10. Zoom in until you can see the underlying sequence.

  11. Load the peaks you called with MACS2. These are the .narrowPeak files in:
    • ~/Course_Materials/ChIPseq/Materials/Peaks/ or
    • ~/Course_Materials/ChIPseq/Preprocessed/Peaks/ folder.
  12. Compare the THOR differentially bound regions with the MACS2 peaks.

  13. You can colour the loaded .bam and .narrowPeak files and scale the .bam files the same way you did with the bigWig files.

4 Loading data from server

  1. Go to File > Load From Server… and select “GC %” from Annotations.

5 Running IGVtools (generating .tdf file)

  1. Click Tools > Run igvtools…

  2. Leave the command on “Count” and select the tp53 bam file as input file.

  3. Change the output file name so that you don’t overwrite the already existing .tdf file that you generated using the command line version of igvtools on Monday.

  4. Click Run. This will generate a .tdf file, that you can load later.