ChIPseq Practical 2: Evaluating ChIP seq data

Shoko Hirosue, Joanna Krupka, Dora Bihary, Shamith Samarajiiwa

Contents

1 Introduction

In this practical session, we will familiarize ourselves to IGV (Integrative Genomics Viewer) to assess the ChIP seq quality. IGV is used to visualize next-generation sequencing data and annotations. Open the virtual desktop and type igv in the terminal. A window of IGV should open.

This material was adapted from the practical material written by Dora Bihary in 2019.

1.1 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)”). If it’s not listed, select Genomes > Load Genome from Server, select hg38 and load the genome.

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.

1.2 Loading your data into IGV

1. Now click File > Load from File… and open the bam files and peak files. Let’s open “tp53_r1.fastq_trimmed.fastq_sorted.bam” and its input file “input.fastq_trimmed.fastq_sorted.bam”, as well as the narrowPeak file “tp53_r1.fastq_trimmed.fastq_sorted_peaks.narrowPeak” and summit bed file “tp53_r1.fastq_trimmed.fastq_sorted_summite.bed” in the data directory (/home/ubuntu/Course_Materials/ChIPSeq/practicals/data).

2. Zoom into one of the peak regions.

3. Select bam files you have loaded, right click them and select “Group Autoscale”.

4. 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.

5. Remove all the files (just so it’s easier to see.) Load BigWig files (“tp53_r1.fastq_trimmed.fastq_sorted_standard_treat_pileup.bw” and “tp53_r1.fastq_trimmed.fastq_sorted_standard_control_lambda.bw”)

6. Group autoscale the two tracks so they are comparable.

7. Set different colours for each of the tracks (Right click at the file name, choose Change Track Color (Positive values)…).

8. Export an image from File > Save Image and have a look at the saved file.