Recalibration step from SNP calling

Question

[mostafarafiepour]

Hello everyone,

I am trying to run Recalibration stage from SNP calling for whole genome sequencing data. But, my reference genome do not have a known sites VCF file. So the -knownSites option is removed from my command line and i encounter the following error (Picture in Attachment):

My question is here, is it necessary for the reference genomes that do not have a known sites VCF file to perform the Recalibration step?

Cod i run:

java -jar /home/m.rafiepour222/GenomeAnalysisTK-3.8-1-0-gf15c1c3ef/GenomeAnalysisTK.jar -R /home/m.rafiepour222/GCF_000471725.1_UMD_CASPUR_WB_2.0_genomic.fa -T BaseRecalibrator -I /home/m.rafiepour222/1_BBKHU01_F/1_BBKHU01_F.sort.rmdup.bam -o /home/m.rafiepour222/1_BBKHU01_F/1_BBKHU01_F.grp enter code here

My Error:

As seen in the image, the error is associated with the same known sites VCF file...

[cpad0112]

It can be skipped as discussed here (http://evodify.com/gatk-the-best-practice-for-genotype-calling-in-a-non-model-organism/). OP raised the similar issue (base recalibration using variant information for non-model organism) in gatk forum: https://gatkforums.broadinstitute.org/gatk/discussion/4164/base-re-calibration-when-i-dont-have-a-publicly-available-dbsnp. OP proposed a method. However it didn't work. Suggestion in OP's post (mentioned above) was to skip base recalibration. I guess same would work for Variant recalibration as both of them use variant information.

[mostafarafiepour]

many thanks for your reply,

yes, OP suggested a similar issue, but I think the proposal is both difficult and not working. I have tried a lot, but I could not get any result And this has caused me concern. I do not know what to do?

[cpad0112]

OP suggested to skip base recalibration.

[mostafarafiepour]

Is this subject approved by the GATK team ?? If confirmed, can you submit a document ?

Answer 1

[Pierre Lindenbaum]

https://software.broadinstitute.org/gatk/documentation/tooldocs/3.8-0/org_broadinstitute_gatk_tools_walkers_bqsr_BaseRecalibrator.php

Inputs

A BAM file containing data that needs to be recalibrated.

A database of known polymorphic sites to mask out.

you didn't provide "A database of known polymorphic sites to mask out. "

[mostafarafiepour]

many thanks for your reply,

yes i didn't provide "A database of known polymorphic sites to mask out " , Because my reference genome do not have a known sites VCF file.

[Pierre Lindenbaum]

then try to provide an empty VCF file as the database....

[mostafarafiepour]

an empty VCF file ? Sorry, I do not understand what you mean ?

[Pierre Lindenbaum]

just the header of a VCF, no variant.

[mostafarafiepour]

I have tried to create a VCF file, but when I put it as an input file, i am again encountering error.

If possible, I request you to send me a VCF file (The same VCF, which is your opinion)??

[mostafarafiepour]

Excuse me, is the Recalibration stage really necessary or can it be ignored?