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Old 02-24-2015, 08:37 AM   #1
sfh838t
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Default variant calling in plant

I am trying to get all variants of all types for a sequence I put together through an assembly step followed by consensus building using a reference. Now I am looking for variants. I have used samtools/bcf/vcfutils steps and the first time I did so accidentally using contigs, which gave me a list of only indels as a result, and I can visually (IGV) verify these. then I tried to correct this and used the actual reads, again with samtools. this time I got a list of SNPs only, which again I can locate in IGV.
so now I am wondering what is going on? I was under the impression that samtools would locate both indels and SNPs using reads??? would it be legitimate to use indels found by using contigs in a write up?
the subject is a plant chloroplast sequence and in the end I will need some locations I can use in the lab to find differences between two related species. I am learning this as I go so any information even links to further information would be most appreciated.
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Old 02-24-2015, 09:22 AM   #2
Brian Bushnell
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Whether or not indels are found depends on the aligner (and, perhaps, ploidy). How did you align the reads, and how did you align the contigs?
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Old 02-24-2015, 09:53 AM   #3
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All my alignments are done using BWA, and the same file of reads was used for both GATK and samtools.
I used the samtools mpileup/bcftools/vcfutils steps both times:
samtools +reads = only SNPs
samtools + contigs = only indels
GATK tool: UnifiedGenotyper + reads = only SNPs
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Old 02-24-2015, 09:56 AM   #4
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Do you see indels in the reads when you look at the mapped bam in IGV? And how long are these indels?
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Old 02-24-2015, 10:03 AM   #5
sfh838t
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yes, I can see both indels and SNPs in IGV. most of them are 3-7 bp long. And the indels found are at different locations than the SNPs found.
Is it legitimate to use contigs in calling variants?
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Old 02-24-2015, 10:07 AM   #6
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I look at contigs in IGV and see both SNPs and indels. Have not looked at reads.
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Old 02-24-2015, 10:18 AM   #7
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ok, I just went and looked at reads and see the same insert that I identified in contigs. this is important for the project, because this insert is present in one possible parent but not the other.
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Old 02-24-2015, 11:33 AM   #8
Brian Bushnell
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Calling indels from the contigs is probably a valid approach as long as these are homozygous events; I'm not really sure how chloroplast genomes work. Also, if the assembly is good. Possibly, someone who knows more about GATK or mpileup can comment on why they seem to be missing the indels in reads.
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Old 02-24-2015, 11:43 AM   #9
sfh838t
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I did get the GATK answer and now have a file with both together. working with plants and all bets are off the samtools output might remain a mystery .
Probably a new question: does anyone know how to build a consensus from an alignment which DOES include indels??
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Old 02-24-2015, 11:44 PM   #10
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Did you try the FastaAlternateReferenceMaker in GATK? You need called variants in a VCF file however, not just an alignment - and read the documentation carefully, there are some limitations.
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Old 03-02-2015, 11:54 AM   #11
sfh838t
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Thanks, I will try that.
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