So I've done a bit of homework but I'm still a little confused on the best way to go, hoping someone can point me in the right direction.
I recently sequenced 11 mutant strains and 1 reference strain of Bacillus (~4MB) by NextSeq Mid 2 x 75. I'm ultimately looking to compare the mutants to the reference to detect SNV/MNV as well as InDels.
So far, my understanding is to start with a de novo assembly using something like SPADes. Once I have the contigs, I can use OSLay to map these back to a reference strain, either the one I sequenced, or a previously available (master) one, of which there are several. I still have a few questions though:
1) What application should I be looking at for detecting the mutations? I imagine I'd essentially need a large alignment tool, though one that can take in to account coverage and probability of mutation would be a great feature to have (assuming not all reads give the same SNV, etc).
2) When detecting mutations, is it better to do so against the reference strain that I sequenced or against a downloaded one and just compare my reference to it as well, ignoring any commonalities?
3) Are there better programs to use than the two I listed above? Are there any that are specifically built for my purpose, and that aren't CLC Genomics that would cost me $5k (grad student budget here)?
Much appreciated everyone!
I recently sequenced 11 mutant strains and 1 reference strain of Bacillus (~4MB) by NextSeq Mid 2 x 75. I'm ultimately looking to compare the mutants to the reference to detect SNV/MNV as well as InDels.
So far, my understanding is to start with a de novo assembly using something like SPADes. Once I have the contigs, I can use OSLay to map these back to a reference strain, either the one I sequenced, or a previously available (master) one, of which there are several. I still have a few questions though:
1) What application should I be looking at for detecting the mutations? I imagine I'd essentially need a large alignment tool, though one that can take in to account coverage and probability of mutation would be a great feature to have (assuming not all reads give the same SNV, etc).
2) When detecting mutations, is it better to do so against the reference strain that I sequenced or against a downloaded one and just compare my reference to it as well, ignoring any commonalities?
3) Are there better programs to use than the two I listed above? Are there any that are specifically built for my purpose, and that aren't CLC Genomics that would cost me $5k (grad student budget here)?
Much appreciated everyone!
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