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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
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Workflow 1: Viewing E. coli data in IGV
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- 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. 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
Expand 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:
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wget http://iubio.bio.indiana.edu/soft/molbio/readseq/java/readseq.jar
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The general command to run the software is one of these:
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java -jar readseq.jar
java -cp readseq.jar run
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To do the conversion that we want, use this command:
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java -cp readseq.jar run NC_012967.1.gbk -f GFF -o NC_012967.1.gbk.gff
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Another useful trick with either IGV or UCSC: displaying your own BLAST results: BioPerl allows for super-easy conversion from blast output to a gff file; IGV and the UCSC browser both understand GFF files. The short script Let's use the blast result we had from the earlier test for the JAG1 gene to show you how. You'll need to provide the input file - it's the ".oNNNNNN" output file from your blast job.
The resulting jag1_blast.out.gff can be moved to your local machine and opened in IGV. Load the human reference first though! |
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Repeat the last three commands for each SAM output file that you want to visualize in IGV. |
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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:
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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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In the terminal connected to Lonestar, figure out the complete path to the IGV directory.
Open a new terminal window on your Desktop. Fill in the parts in brackets <> in this command:
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This downloads the IGV executable and tells the command line to launch it (via the java command).
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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.
Expand 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.
Expand 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?
Expand 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
, andbowtie2
alignments. Can you explain what's going on here?Expand 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?
Expand 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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- Download and install the Integrative Genome Viewer from the Broad Institute.
- Select "Human hg18" or "Human hg19" as the reference genome
- Get some data: File ? -> Load from Server… ? -> 1000 genomes ? CEU ? -> Alignments -> CEU Trio WGS -> select those 3 samples
- Navigate to the rightmost exons of the GABBR1 gene and the rightmost exons
- 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
- 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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rs29220, rs29222, rs28359988, rs76688565, there might be more in the locus; I got tired of looking. |
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