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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.

Table of Contents

Workflow 1: Viewing E. coli data in IGV

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1. If you have an intro_to_mapping directory with output from the Mapping tutorial or the Variant calling tutorial, then you should use those files for part 1 of this tutorial. You can proceed with either one alone or with both.

2. If you do not have any results, you can use some "canned" ones that we provide. Copy the entire contents of this directory back to your local machine:

   Code Block
   /corral-repl/utexas/BioITeam/ngs_course/intro_to_mapping/IGV

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   Help Help
   Code Block scp -r username@lonestar.tacc.utexas.edu:/corral-repl/utexas/BioITeam/ngs_course/intro_to_mapping/IGV .

   Then skip down to #Launching IGV.

Prepare a GFF feature file for the reference sequence

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To get this onto TACC easily, use:

wget http://iubio.bio.indiana.edu/soft/molbio/readseq/java/readseq.jar

The general command to run the software is one of these:

java -jar readseq.jar
java -cp readseq.jar run

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To do the conversion that we want, use this command:

java -cp readseq.jar run NC_012967.1.gbk -f GFF -o NC_012967.1.gbk.gff

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grep '^gi' blast_jag1.o586038 > jag1_blast.out
module load perl
module load bioperl
bl2gff.pl jag1_blast.out > jag1_blast.out.gff

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samtools faidx NC_012967.1.fasta
samtools view -b -S -o bowtie/SRR030257.bam bowtie/SRR030257.sam
samtools sort bowtie/SRR030257.bam bowtie/SRR030257.sorted
samtools index bowtie/SRR030257.sorted.bam

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For starters, you could change into your intro_to_mapping directory and run commands like these if you just came from the Mapping tutorial:

mkdir IGV
cp NC_012967.1.fasta IGV
cp NC_012967.1.fasta.fai IGV
cp NC_012967.1.gbk.gff IGV
cp bowtie/SRR030257.sorted.bam IGV/bowtie.sorted.bam
cp bowtie/SRR030257.sorted.bam.bai IGV/bowtie.sorted.bam.bai
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pwd

scp -r <username>@lonestar.tacc.utexas.edu:</full/path/to/IGV/> .

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This downloads the IGV executable and tells the command line to launch it (via the java command).

wget http://www.broadinstitute.org/igv/projects/downloads/IGV_2.3.5.zip
unzip IGV_2.3.5.zip
cd IGV_2.3.5
java -Xmx2g -jar igv.jar

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• Why are some reads different colors? Hint: Try changing the display options to show read pairs and editing some of the distance constraints.

• What is a typical mapping quality (MQ) for a read? Convert this to the probability that it is mismapped.

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  title Remember the formula for a Phred quality score?
  The estimated probability that a read is mapped incorrectly is 10^(-MQ/10).

• Can you find a variant where the sequenced sample differs from the reference? This is going to be like looking for a needle in a haystack. Fortunately, we are going to learn how to use variant callers tomorrow and then we'll be able to zoom right to areas where there are discrepancies between reads and the reference genome that might indicate there were mutations in the sequenced E. coli.

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  Some interesting locations to look at for the time being... Some interesting locations to look at for the time being...
  Coordinate 161,041. What gene is this in and what is the effect on the protein sequence? Coordinate 3,248,957. What gene is this in and what is the effect on the protein sequence? Coordinate 4,015,892. What is different about the reads mapped to this location? Coordinate 3,894,997. What type of mutation is this? Coordinate 1,733,647. What's going on here? See if you can find more interesting locations. There are ~40 mutations total in this sample.

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Tip: You can also index BAM and FASTA files the same way inside of IGV if you haven't already created indexes for them. But, it's usually easier and quicker to do this on the command line.

Exercises

• Check out the rbsA gene region? What's going on here?

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  Answer Answer
  There was a large deletion. Can you figure out the exact coordinates of the endpoints?

• Navigate to coordinate 475,288. Compare the bowtie, BWA, and bowtie2 alignments. Can you explain what's going on here?

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  Answer Answer
  There is a 16 base deletion in the gltA gene reading frame.

• What is going on in the pykF gene region? You might see red read pairs. What does that mean? Can you guess what type of mutation occurred here?

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  Answer Answer
  The read pairs are discordantly mapped. There was an insertion of a new copy of a mobile genetic element (an IS150 element) that exists at other locations in the reference sequence.

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1. Download and install the Integrative Genome Viewer from the Broad Institute.
2. Select "Human hg18" or "Human hg19" as the reference genome
3. Get some data: File ? -> Load from Server… ? -> 1000 genomes ? CEU ? -> Alignments -> CEU Trio WGS -> select those 3 samples
4. Navigate to the rightmost exons of the GABBR1 gene and the rightmost exons
5. Zoom in until you find some SNPs - they might be in exons or introns; there is also at least one example of a short insertion variant between exons 2 and 3
6. Load and look at the SNP track: File ? -> Load from server ? -> Annotations ? Variants ? -> Variants and Repeats -> dbSNP

This is whole genome coverage data; later we'll look at exome data.

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