IGV Tutorial -- GVA2016
Overview
The Integrative Genomics Viewer (IGV) from the Broad Center allows you to view several types of data files involved in any NGS analysis that employs a reference genome, including how reads from a dataset are mapped, gene annotations, and predicted genetic variants.
Learning Objectives
In this tutorial, we're going to learn how to do the following in IGV:
- Create a custom genome database (usually used for microbial genomes) or load a pre-existing genome assembly (usually used for the genomes of model organisms and higher Eukaryotes).
- Load output from mapping reads to a reference genome.
- Load output from calling genetic variants.
- Navigate the view of the genome and interpret the display of this data.
Theory
Because NGS datasets are very large, it is often impossible or inefficient to read them entirely into a computer's memory when searching for a specific piece of data. In order to more quickly retrieve the data we are interested in analyzing or viewing, most programs have a way of treating these data files as databases. Database indexes enable one to rapidly pull specific subsets of the data from them.
The Integrative Genomics Viewer is a program for reading several types of indexed database information, including mapped reads and variant calls, and displaying them on a reference genome. It is invaluable as a tool for viewing and interpreting the "raw data" of many NGS data analysis pipelines.
Workflow 1: Viewing E. coli data in IGV
Data files
You can start this tutorial two ways:
- If you have a
mapping
directory with output from the Mapping tutorial and the SNV calling tutorial, then you should use those files for part 1 of this tutorial. You can proceed with either one alone or with both.
Prepare a GFF feature file for the reference sequence
IGV likes its reference genome files in GFF (Gene Feature Format). Unfortunately, our old friend bp_seqconvert.pl
doesn't do GFF. So, we're going to show you another tool for sequence format conversion called Readseq. We've already installed it into the $BI/bin directory so you don't have to, but here we provide the steps that can be used to install it in a local directory.
Readseq is written in java which makes it a little more complicated to use, but the general command to run the software is one of these (note that you do need to include the entire path, not just the "readseq.jar" name):
java -jar /corral-repl/utexas/BioITeam/bin/readseq.jar java -cp /corral-repl/utexas/BioITeam/bin/readseq.jar run
This should return the help for Readseq.
To do the conversion that we want, use this command:
cds mkdir BDIB_IGV_Tutorial cd BDIB_IGV_Tutorial java -cp /corral-repl/utexas/BioITeam/bin/readseq.jar run $SCRATCH/BDIB_bowtie2_mapping/NC_012967.1.gbk -f GFF -o NC_012967.1.gbk.gff
It's a bit hard to figure out because, unlike most conventions, it takes the unnamed arguments before the optional flag arguments, there is no example command, and you have to switch -jar
to -cp
. Search online for usage examples when you can't figure something out from the help. Take a look at the contents of the original Genbank file and the new GFF file and try to get a handle on what is going on in this conversion using commands like head and tail.
Copy files to your desktop
IGV is an interactive graphical viewer program. You can't run it on TACC, so we need to get the relevant files back to your desktop machine.
They include:
- Indexed reference FASTA files
- GFF reference sequence feature files
- Sorted and indexed mapped read BAM files
- VCF result files
- ... and possibly many other types of files.
The easiest way to to this is probably to copy everything you want to transfer into a new directory called IGV_export
. Since many of the tutorial output files had the same names (but resided in different directories) be careful to give them unique destination names when you copy them into the new directory together. To ensure you don't overwrite things be sure to use the -n or -i option with the cp command. The difference comes from different versions of linux having slightly different cp command options. The -n command will not allow you to overwrite files, while the -i command will prompt you before overwriting anything.
mkdir BDIB_IGV_export cp -i NC_012967.1.gbk.gff BDIB_IGV_export # copy the new file you just converted to the export directory cp -i $SCRATCH/BDIB_bowtie2_mapping/NC_012967.1.fasta BDIB_IGV_export cp -i $SCRATCH/BDIB_samtools_tutorial/NC_012967.1.fasta.fai BDIB_IGV_export cp -i $SCRATCH/BDIB_samtools_tutorial/SRR030257.vcf BDIB_IGV_export cp -i $SCRATCH/BDIB_samtools_tutorial/SRR030257.sorted.bam BDIB_IGV_export/bowtie2.sorted.bam cp -i $SCRATCH/BDIB_samtools_tutorial/SRR030257.sorted.bam.bai BDIB_IGV_export/bowtie2.sorted.bam.bai tar -czvf BDIB_IGV_export.tar.gz BDIB_IGV_export
Now, copy the entire compressed IGV directory back to your local Desktop machine.