BCFtools Mpileup Qual=0

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Hi Galaxy Community members,
I mapped fastq.gz files with ref genome using BWA MEM and then used BCFtools mpileup to generate vcf file for downstream variant detection using varscan. When I look at this vcf file, the qual score is zero for all the location. Am i doing any mistake here? This also happened when i did samtools mpileup.
I will be grateful for all your suggestions.

Thanks
Sanjay

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Hi Sanjay

There is not really enough information in your query to predict what is going on. I suggest that you might want to try out some of the workflows from the Galaxy training materials (e.g. the ones here: https://training.galaxyproject.org/training-material/topics/variant-analysis/) to test your approach against a well-known approach and see if you still get the same results.

Peter

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Hi Sanjay,
i have project, may you have good experince and more knowledge to advice me.
i have 3 accession number for raw reads for 3 whole genome,

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Hi Dina_Yamin,
Can you please write in details abouth your project?
Thank you
Sanjay

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Hi Dina,
This looks doable.
Have you tried assembling your strains with a reference genome? You can do it by using BWA-MEM (usually if your read output is >100 base pairs). After assembling you can use samtools pileup and then BCFtools call to find the SNPs.

Thank you
Sanjay

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hi,
last update
I have fastQ files Downloaded from NCBI by SRA accession number then align (with BWA) to a reference genome. So, I have obtained a .bam file.
i use the samtools pilup to remove the duplication is that right .
how i can use BCFtools to call SNPs.?
can you send any tutorial or screen shot to Know what i should do ?

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Hi Dina,
To operate “BCFtools call”, you need a .vcf file. The current version of samtools pileup in galaxy has no options for .vcf output. Therefore you need to follow this:
search samtools mpileup. Then click version tab on the top right and select version 2.1.3. And execute Mpileup. This was you will get .vcf file. Again selecting the threshold depends on your question and genome quality.
After you get a .vcf file, you can use this for “BCFtools call”. You may find more about variant calling here: https://training.galaxyproject.org/training-material/topics/variant-analysis/
Thank you
Sanjay

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