In this tutorial, we're going to view the aligned reads and variants that we called in the past two lessons in the Integrated Genomics Viewer from the Broad Center. You'll need the output from Introduction to mapping (bowtie, BWA) and Introduction to variant calling (Samtools).
Create a fresh directory for this tutorial. We're going to assume that it's inside of the introduction_to_mapping directory, and that you have results in those subdirectories. If not, see us and we can tell you where to copy "canned" results from.
cd $SCRATCH/intro_to_mapping |
IGV likes its reference genome files in GFF (Gene Feature Format). Unfortunately, our old friend bp_seqconvert.pl doesn't do GFF. Fortunately, it's cousin bp_genbank2gff3.pl does.
module load bioperl cp /corral-repl/utexas/BioITeam/ngs_course/scripts/bp_genbank2gff3.pl . ./bp_genbank2gff3.pl NC_012967.1.gbk |
Where's the output? Take a look at the *.gbk and *.gff files and try to get a handle on what is going on in this conversion.
(NB: I had to "fix" bp_genbank2gff3.pl to work on our GenBank file, by removing a line that caused an error. If someone known of an easier way to get a GFF file from a file downloaded from Genbank, please share!)
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.
We're going to copy all of these into a new directory called IGV to make it easier to just transfer the ones that we want.
mkdir IGV cp NC_012967.1.gbk.gff IGV cp samtools_bowtie/NC_012967.1.fasta IGV cp samtools_bowtie/NC_012967.1.fasta.fai IGV cp samtools_bowtie/SRR030257.sorted.bam IGV/bowtie.sorted.bam cp samtools_bowtie/SRR030257.sorted.bam.bai IGV/bowtie.sorted.bam.bai cp samtools_bwa/SRR030257.sorted.bam IGV/bwa.sorted.bam cp samtools_bwa/SRR030257.sorted.bam.bai IGV/bwa.sorted.bam.bai cp comparison/* IGV |
Navigate a web browser to this page: http://www.broadinstitute.org/software/igv/download
Go ahead and click on the "Launch with 2 GB" option. This will download a "Java Web Start" file that you can launch by locating on your Desktop and double-clicking.
From the main window of IGV, click on File -> Import Genome and you should be presented with the following window.
Enter the ID and Name of the Genome you are working with and select the path to your *.fasta file (the index, *.fai file needs to be in the same directory), then select the path to your *.gff file for the Gene File.
If you do not have an alignment file in the SAM format you may want to start with Introduction to mapping (bowtie, BWA).
samtools sort -bS input.sam output.bam |
samtools sort input.bam sorted_output |
Note: The second parameter passed here is a prefix and will be suffixed with .bam by samtools.
samtools index input.bam |
From the main window of IGV, click on File -> Load from File and select your input.bam file.
After importing your reference genome and loading an alignment file, your screen should look similar to the following:
And you are now free to investigate different areas and their alignments in the genome.