Content Comparison

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Enter the ID and Name of the Genome you are working with (these can be anything that makes sense to you) 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 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 23,698,092894,997. What type of mutation is this? Coordinate 21,698,092733,647. What's going on here? See if you can find more interesting locations. There are ~40 mutations total in this sample.

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 2475,698,092288. Compare the bowtie, BWA, and BWA 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.

• Go to coordinate 4751,733,263647. Compare the bowtie, BWA, and BWA bowtie2 alignments. What's the story here?

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  Answer Answer
  There was an insertion of a new copy of a mobile genetic element (an IS150 element). Note the reads with discordant pair mapping.

Workflow 2: Viewing Human Genome Data in IGV

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