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Old 07-17-2017, 11:46 AM   #1
gglike
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Question Finding mutations in MiSeq data?

Hello,

First time posting here. I'm a beginner in computational biology. I am working with a bacterium that already has a reference genome.

Basically, I have a mutant of that bacterium that lacks a certain protein. In my experiments, I saw a peculiar phenotype with certain colonies of the mutant. I think these colonies might have some interesting mutations in them.

I sent the parent mutant, as well as the colonies of that mutant that exhibited the interesting phenotype for MiSeq sequencing.

I have 2x301bp paired end data for the mutant parent as well as the colonies. How would I go about finding the mutations in the colonies compared to the mutant parent?

Here is what I have done so far using Geneious:

1.) I selected the two read files for the mutant parent and colonies and set paired reads with the insert size I got from the sequencing company.
2.) I mapped the mutant parent and the colonies to the reference genome of my organism using Geneious.
3.) I then did "Find Variations/SNPs" on Geneious on the parent mutant as well as the colonies.

Doing this, I got csv files with a list of mutations. The first file I got is the mutations in the mutant parent compared to the reference genome. The other files I got are mutations in the colonies compared to the reference genome.

Am I on the right track? Is Geneious the best tool for me to find mutations? Is there anything else I should be doing?
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Old 07-17-2017, 04:04 PM   #2
GenoMax
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You are on a track that can get you an answer. You could now compare the two VCF files to see mutations that are unique to the parent and the mutant to get an idea of differences. I am not familiar with geneious but is there vcf-compare like functionality (available via vcftools)?
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Old 07-18-2017, 12:51 PM   #3
gglike
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I don't think Geneious has a vcf-compare like functionality. I can export the files I have as vcf though.

Would vcftools help me get common mutations in all of my colonies? I have lists of SNPs for the parent mutant and the colonies.

Thanks again!

EDIT: Also, would vcftools work with bacterial vcf files? Or does it only work with eukaryotic organisms.

Last edited by gglike; 07-18-2017 at 01:31 PM.
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Old 07-19-2017, 03:17 AM   #4
GenoMax
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vcftools should work with any valid VCF file.
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