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).

Getting situated

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

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. 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!)

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.

• Indexed reference FASTA files
• GFF reference sequence feature files
• Sorted and indexed mapped read BAM files
• VCF result files

samtools_bowtie
comparison
NC_012967.1.gbk.gff

Import to IGV.

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.

Preparing alignment file.

If you do not have an alignment file in the SAM format you may want to start with Introduction to mapping (bowtie, BWA).

Convert to BAM format.

samtools sort -bS input.sam output.bam

Sort.

samtools sort input.bam sorted_output

Note: The second parameter passed here is a prefix and will be suffixed with .bam by samtools.

Index

samtools index input.bam

Import to IGV.

From the main window of IGV, click on File -> Load from File and select your input.bam file.

IGV

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.