Content Comparison

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You will need to index your reference FASTA and convert your SAM output files into sorted and indexed BAM files. The "why?" behind these steps is described more fully in the Variant calling tutorial. If you are in your intro_to_mapping directory, these commands will perform the necessary steps.

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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wget http://www.broadinstitute.org/igv/projects/downloads/IGV_2.13.225.zip
unzip IGV_2.13.225.zip
cd IGV_2.13.225
java -Xmx2g -jar igv.jar

Locally on your own Mac or Windows computer

Use this link to download IGV:

http://www.broadinstitute.org/igv/projects/downloads/IGV_2.13.225.zip

After unzipping, you should be able to click on igv.bat for Windows or igv.command on MacOSX to lauch IGV. If this is not working, you might need to try the web start.

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Enter the ID and Name of the Genome you are working with (these can be anything) 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.

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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... Some interesting locations to look at...
  Coordinate 2,698,092 Coordinate 2,698,092

Load variant calls into IGV

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• Check out the rbsA gene region? What's going on here?
• What is going on in the pykF gene region? You might see red read pairs. What does that mean?
• Go to coordinate 2,698,092. Compare the bowtie and BWA alignments. Can you explain what's going on here?

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  Answer Answer

• Go to coordinate 475,263. Compare the bowtie and BWA alignments. What's the story here?

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  Answer Answer

Workflow

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2: Viewing Human Genome Data in IGV

Advanced exercise: human data scavenger hunt

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scavenger scavenger

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1. Download and install the Integrative Genome Viewer from the Broad Institute.
2. Select "Human hg18" as the reference genome
3. Get some data: File -> “Load from Server…” -> 1000 genomes -> CEU and YRI trios
4. Find Navigate to the GABBR1 gene and the right rightmost exons
5. Zoom in until you find the SNP
6. Load and look at the SNP track: File -> Load from server -> Annotations -> Variants -> dbSNP

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From here...

You can also use IGV to visualize RNA-seq data in later tutorials.

Check out alternative genome browsers:

• • Tablet - a lightweight NGS data browser