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Visualizing data using IGV

References

One of our goals is to visualize our RNA-seq data using a genome browser.

To prepare alignment data for upload to IGV, the RNAseq_analyzer script simply performed some file format conversions.

file.sam –> file.bam This compresses the .sam file into a binary file
file.bam –> file_sort.bam This sorts the .bam file by chromosome location
file_sort.bam.bai This makes a simple indexing reference file for the sorted .bam file
file.bam –> file.bx This creates a bigwig file

These files can be downloaded from summit using Cyberduck. They can then be opened with IGV.

First, we'll go through how to do this using IGV.


IGV Exercise

  • Open IGV
  • From the drop down menu, select S.cerevisiae (SacCer3)
  • From File, select Load from File
  • Select your sample01_sort.bam file, making sure that the file sample01_sort.bam.bai is located in the same directory.
  • Load sample03_sort.bam also.

A cautionary note about .bam files and scaling

You can not compare the heights of any genome browser plots until you have normalized their heights. This is because the height of each plot is proportional to the number of fragments that were sequenced over all. Until you normalize, .bam files from samples that were sequenced to 50 million reads will look twice as tall as those sequenced to 25 million reads.

To normalize the samples:

  • Go to View
  • Go to Preferences
  • Go to Tracks
  • Go to Normalize Coverage Data
  • Click on Normalize Coverage Data
  • Navigate to the OK box that is typically not visible at the bottom of the window.
  • Click OK

To remove autoscaling

  • Right click on the header for the track called sample01_sort.bam Coverage
  • Unclick autoscale
  • Click on Set Data Range..
  • Select the desired height

Exercise

  • Play around with the browser.
  • Check out cool genes: AGP1, HXT1, CAN1
  • Read about interesting genes you find on SGD

Visualizing data using UCSC

wiki/igv_visualization.1543506577.txt.gz · Last modified: 2018/11/29 08:49 by erin