Visualizing the peaks in a genome browser

From BITS wiki
Jump to: navigation, search

Choosing a genome browser

There are several options for genome browsers, divided between the local browsers (need to install the program, eg. IGV) and the online web browsers (eg. UCSC genome browser, Ensembl). We often use both types, depending on the aim and the localisation of the data.

Note that if you're working on a non-model organism, the local viewer will be the only choice. If the aim is to share the results with your collaborators, view many tracks in the context of many existing annotations, then the online genome browsers are more suitable.

Viewing the aligned reads in IGV

Open IGV. Be patient, it might take a few minutes for the program to start.

Change the genome in IGV from Human hg19 to the one you used in the mapping.

You can also visualize the annotation (genes) in IGV. You can obtain a file with annotations from the Refseq record.

You can also download the GFF3 file from our website.

If you want to load the .gff3 file and visualize the annotation properly in IGV, it’s necessary to comment (or remove) the third line:

##sequence-region NC_000913.3 1 4641652
##species https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=511145
## NC_000913.3	RefSeq	region	1	4641652	.	+	.	ID=NC_000913.3:1..4641652;Dbxref=taxon:511145;Is_circular=...
NC_000913.3	RefSeq	gene	190	255	.	+	.	ID=gene-b0001;Dbxref=ASAP:ABE-0000006,ECOCYC:EG11277...

You can visualize reads in IGV as long as they are sorted according to genomic location. Download the two sorted and indexed bam files (for SRR576933 and SRR576938) from GenePattern to your computer and load them in IGV.

Browse around in the genome. Do you see peaks?

Do the same for gene ycfP.

Looking at .bam files does not allow to directly compare the two samples as data are not normalized. To generate normalized data for visualization you can use bamCoverage from deepTools (it's available in GenePattern). It generates BigWig files out of .bam files.

Repeat for the control (again you see the benefit of creating a pipeline for repeating the same steps on multiple samples).

Download the BigWig files, start a new session in IGV and load the BigWig files in IGV.

Go back to the genes we looked at earlier: pepT, ycfP. Look at the shape of the signal.

Viewing the peaks in IGV

Download the bdg files generated by MACS from GenePattern to your computer and rename them with the extension .bedgraph.

Open a new session in IGV. Reload the .ggf3 file with the annotation.

Download and view the BED file containing the peak locations.

The end result should look like this: 3 tracks with data (the bedgraph files of the 2 samples and the peaks file) and 1 track with annotation:

IGVLoadFile5.png


Go back again to the genes we looked at earlier: pepT, ycfP. Do you see peaks?