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# Base recalibration & realignment without known sites

Member Posts: 19
edited October 2012

Dear GATK team,

Thanks a lot for the new GATK version and GATK forum!

I am trying to use GATK for yeast strains. I do not have files of known sites of SNPs/indels.
I understand that the BaseRecalibrator must get such a file.
Do you suggest to skip calibration and realignment, or is there another way to go here?

Post edited by Geraldine_VdAuwera on
Tagged:

Hi Gilgi,

Glad you like the new digs!

Skipping Base recalibration and realignment are never a good option. Theere is an article on Base Quality Score Recalibration in the Methods & Workflows section of the Guide that you need to read. Towards the end, in the "Troubleshooting" section, there is a discussion of what to do if you don't have a set of known variants for your organism.

Good luck!

Post edited by Geraldine_VdAuwera on

Geraldine Van der Auwera, PhD

Hi Gilgi,

Glad you like the new digs!

Skipping Base recalibration and realignment are never a good option. Theere is an article on Base Quality Score Recalibration in the Methods & Workflows section of the Guide that you need to read. Towards the end, in the "Troubleshooting" section, there is a discussion of what to do if you don't have a set of known variants for your organism.

Good luck!

Post edited by Geraldine_VdAuwera on

Geraldine Van der Auwera, PhD

• Member Posts: 19

Thanks a lot!!!

• Member Posts: 2

The link is broken right now.

Geraldine Van der Auwera, PhD

• Member Posts: 8

Hi, do you have any solution for the IndelRealigner as well? I have a non-model organism as reference and there are no known indel sites/document which I can use.

If you don't have any known indels you can run without.

Geraldine Van der Auwera, PhD

• Member Posts: 8

Hi, I ran it without any known indels, but some regions didn't seem to realign as expected. As a result, GATK is calling both a SNP and single base insertion at the same location (the true variant is the insert). Any idea why this is happening?

Eric Banks, PhD -- Director, Data Sciences and Data Engineering, Broad Institute of Harvard and MIT

• Member Posts: 8

Hi Eric,

to identify intervals:
java -Xmx1g -jar GenomeAnalysisTKLite.jar -T RealignerTargetCreator -R reference.fa -I sampleA_clean_paired.sorted.dedup.bam -o sampleA_clean_paired.sorted.dedup.intervals

to realign:
java -Xmx2g -jar GenomeAnalysisTKLite.jar -T IndelRealigner -R reference.fa -I sampleA_clean_paired.sorted.dedup.bam -targetIntervals sampleA_clean_paired.sorted.dedup.intervals -o sampleA_clean_paired.sorted.dedup.realigned.bam

Okay, good. Those look reasonable.
Now why do you think some regions didn't realign properly? Could you please post an example IGV screenshot with an explanation? Thanks.

Eric Banks, PhD -- Director, Data Sciences and Data Engineering, Broad Institute of Harvard and MIT

• Member Posts: 8

Hi Eric,

I've attached a screenshot of a region as an example. (see: http://imageshack.us/photo/my-images/803/realigned.png)
Also, I noticed some messages during running GATK that goes like this:

INFO 15:56:32,707 IndelRealigner - Not attempting realignment in interval contig_31331:6142-6275 because there are too many reads.

I'm wondering if this is the reason why... and if so, any suggestions on how to deal with this?

Thanks so much for your help.

That is certainly a possible reason. Please take a look at the documentation for command-line arguments that allow you to increase the maximum number of reads allowed in a given realignment region.

Eric Banks, PhD -- Director, Data Sciences and Data Engineering, Broad Institute of Harvard and MIT

• Member Posts: 8

Hi Eric,

I found the --maxReadsForRealignment option and I set it at a higher number (~ 100000) and am getting less of those messages. There are, however, still a few of these cases. There is a note in the documentation which says "For expert users only!". Is there other harms of increasing this number, other than the toll it takes on memory? How do I decide what to set this number at?

Much thanks.

Just memory. The value to use really depends on your average coverage and whether it's important to realign every single region that might need it.

Eric Banks, PhD -- Director, Data Sciences and Data Engineering, Broad Institute of Harvard and MIT

• Member Posts: 9

Hi, I would like to use the BaseRecalibrator but I do not have a database of known SNPs for the organism I am studying. I have a VCF files with potential real SNPs and I want to use the best ones as known sites. How many SNPS at least do we need? Are 500 enough?

There is no hard number, it depends how divergent you expect your organism samples to be. Do you have any way to estimate this?

Geraldine Van der Auwera, PhD

• Member Posts: 9
edited November 2012

We will study 40 genotypes. So far, the genetic differentiation between 2 genotypes has been assessed from microsatellite markers (Fsc), and varies from 50% to 70%. We currently have GATK results on 7 genotypes from a previous study reporting at least 14500 polymorphic sites present on each genotype with a quality between 1329 and 4867. Maybe we can use these ones or a part of them?

Yes, you can use a high-confidence subset of those.

Geraldine Van der Auwera, PhD

• Member Posts: 2

Hi
I also work on nonmodel organisms and don't have a set of known polymorphisms. I have 1-4 individuals each from several quite divergent species. Would you use high-confidence SNPs found with Unified Genotyper run over all individuals of all species together or would you define the set of high-confidence SNPs for each species separately, even though I only have few individuals each?
Thanks a lot.