Question: Standard Practice of making VCF file from SAM file
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gravatar for hafiz.talhamalik
11 weeks ago by
Pakistan
hafiz.talhamalik40 wrote:

Can any one guide me to about what standard GATK tools to use when making a vcf file from allignment sam file ? I'm kinda stuck. confused which one to use and when. ???

snp gatk • 203 views
ADD COMMENTlink modified 10 weeks ago by Biostar ♦♦ 20 • written 11 weeks ago by hafiz.talhamalik40

Where are you stuck? What confuses you? Please tell us how far you've gotten in your quest to solve this.

ADD REPLYlink written 11 weeks ago by RamRS20k

I have used PICARD for SortSam, and MarkDuplicates. Now which one to use next ?

ADD REPLYlink modified 11 weeks ago • written 11 weeks ago by hafiz.talhamalik40

GATK Best Practices says BaseRecalibration is next. Have you looked at that resource?

ADD REPLYlink written 11 weeks ago by RamRS20k

I am trying to follow this one. But BaseRecalibration requires input of -knownSites latest_dbsnp.vcf. and i have whole genome of bacteria so what should i do with this parameter ? and what about next step ?

ADD REPLYlink written 11 weeks ago by hafiz.talhamalik40
1

Ah, bacterial genome - I think you should have mentioned that in your original question, I'd have warned you against GATK - I think GATK might not be great for non-human genomes. Maybe try samtools/bcftools?

ADD REPLYlink written 11 weeks ago by RamRS20k

oh okz. I used samtools/bcftools pipeline. I made a vcf file too but i wanted to counter check that with GATK pipeline. one more thing I have a genome of around mb, and samtools pipeline gave me a vcf file with around 2300 variants. Is it normal ? or did I made a mistake ?

ADD REPLYlink written 11 weeks ago by hafiz.talhamalik40

If you were sequencing what was supposed to be the exact same strain of bacteria as your reference, that's kind of high. If it's not, that number might be appropriate. Eyeball some of the putative SNPs in IGV, see if you can verify visually that the reads show the SNPs in the vcf.

ADD REPLYlink written 11 weeks ago by swbarnes24.7k

okz. that helped. Thanks alot

ADD REPLYlink written 11 weeks ago by hafiz.talhamalik40
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