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# Downsampling

Member Posts: 50
edited July 2012

Dear all,

I would like to ask you about some inconsistencies (maybe!) that I am observing regarding the downsampling of reads in GATK.
This is the first time I am dealing with real high coverage (several 1000/sample) and it starts to matter for me to know how to adjust these parameters.
All of the below is from GenomeAnalysisTK-1.6.596/GenomeAnalysisTKLite.jar.

1. I was anticipating the need for some tuning, so I read the CommandLine GATK documentation, where three options related to this issue are listed:
--downsample_to_coverage
--downsample_to_fraction
--downsampling_type
All of them have NA as default value, so I assumed, that all my reads would count for the variant calling.

2. In the documentation for the UnifiedGenotyper, there are no options related to downsampling.
My command:
java -jar GenomeAnalysisTKLite.jar -T UnifiedGenotyper -R ref.fa -I 1.bam -I 2.bam -o GATK.vcf -glm BOTH -stand_call_conf 1 -stand_emit_conf 1

3. Neither are in the VariantAnnotator which I used subsequently on the UG output.
My command:
java -jar GenomeAnalysisTKLite.jar -T VariantAnnotator -R ref.fa -I 1.bam -I 2.bam -o GATK_anno.vcf --variant GATK.vcf -A DepthPerAlleleBySample -A BaseCounts -A AlleleBalanceBySample -A AlleleBalance -A DepthOfCoverage -A SampleList

Nevertheless, my variant-called and annotated vcf file features the following:

1. In the header, I see the full set of options used for the UG and the annotations:

UnifiedGenotyper="analysis_type=UnifiedGenotyper input_file=[F323/lib.218C/C0TV2ACXX_7_12_1.fastq_process_MQ35.bam, F325/lib.219C/C0TV2ACXX_7_13_1.fastq_process_MQ35.bam] read_buffer_size=null phone_home=STANDA
RD gatk_key=null read_filter=[] intervals=null excludeIntervals=null interval_set_rule=UNION interval_merging=ALL interval_padding=0 reference_sequence=/project/production/Indexes/samtools/hsapiens_v37_chrM.fa no
nDeterministicRandomSeed=false downsampling_type=BY_SAMPLE downsample_to_fraction=null downsample_to_coverage=250 baq=OFF baqGapOpenPenalty=40.0 performanceLog=null useOriginalQualities=false BQSR=null
[etc]

VariantAnnotator="analysis_type=VariantAnnotator input_file=[F323/lib.218C/C0TV2ACXX_7_12_1.fastq_process_MQ35.bam, F325/lib.219C/C0TV2ACXX_7_13_1.fastq_process_MQ35.bam] read_buffer_size=null phone_home=STANDA
RD gatk_key=null read_filter=[] intervals=null excludeIntervals=null interval_set_rule=UNION interval_merging=ALL interval_padding=0 reference_sequence=/project/production/Indexes/samtools/hsapiens_v37_chrM.fa no
nDeterministicRandomSeed=false downsampling_type=BY_SAMPLE downsample_to_fraction=null downsample_to_coverage=1000 baq=OFF baqGapOpenPenalty=40.0 performanceLog=null useOriginalQualities=false BQSR=null
[etc]

1. My INFO and FORMAT fields for one position look like this:

ABHom=0.997;AC=4;AF=1.00;AN=4;BaseCounts=1990,4,5,0;BaseQRankSum=-0.059;DP=2000;DS;Dels=0.00;FS=0.000;HaplotypeScore=6.1837;MLEAC=4;MLEAF=1.00;MQ=35

1/1:1,998:250:99:8592,700,0

1/1:4,992:250:99:8405,719,0

Due to the different thresholds applied by UG and VA, I have now these inconsistenf values for per-sample AD and DP.

Is this supposed to work like this, or am I using a "non-cannonical" sequence of operations by following up the UG with the VA?
Is there a way to switch OFF downsampling?

Many thanks as always for your comments!

cheers,

Sophia

Tagged:

Hi Sophia,

Downsampling is performed by the GATK engine, so the command-line argument that controls it lives in the
CommandLineGATK

The reason you're seeing different behaviors is that some walkers like the UG override the engine's default downsampling setting; for example the UG sets downsampling to 250 by default (sorry that wasn't specified, we'll make sure to add a note about that in the UG docs in the future). But you can manually override this by using the -dcov CommandLineGATK argument (as documented in the link above).

Geraldine Van der Auwera, PhD

Hi Sophia,

Downsampling is performed by the GATK engine, so the command-line argument that controls it lives in the
CommandLineGATK

The reason you're seeing different behaviors is that some walkers like the UG override the engine's default downsampling setting; for example the UG sets downsampling to 250 by default (sorry that wasn't specified, we'll make sure to add a note about that in the UG docs in the future). But you can manually override this by using the -dcov CommandLineGATK argument (as documented in the link above).

Geraldine Van der Auwera, PhD

Turning off downsampling exposes you to major performance problems in regions of excess coverage. We don't recommend you play with this parameter

--
Mark A. DePristo, Ph.D.
Co-Director, Medical and Population Genetics
Broad Institute of MIT and Harvard

• Member Posts: 50
edited August 2012

I tried the following two settings now for both the UG and the VA:

--downsampling_type BY_SAMPLE --downsample_to_coverage 1000

This produced the expected outcome, namely a global DP of 1000xn(samples) in the INFO field, 1000 in the per-sample DP field and AD per sample numbers that added up to 1000.

--downsampling_type ALL_READS --downsample_to_coverage 10000

Here, I was expecting GATK to downsampling all reads from all samples to a total of 10.000 per position. Instead, only positions where any single sample reached a depth of 10.000 were printed, e.g.:
AC=2;AF=1.00;AN=2;BaseQRankSum=6.858;DP=170360;DS;Dels=0.00;FS=8.789;HaplotypeScore=6809.3095;MLEAC=2;MLEAF=1.00;MQ=35.00;MQ0=0;MQRankSum=1.569;QD=0
.19;ReadPosRankSum=1.513;SB=-3.277e+04 GT:AD:DP:GQ:PL ** ./. ** 1/1:63,170165:170355:99:32767,32767,0 ./.

@Mark_DePristo: I am aware of the problems excess coverage can cause; in this case, though, I am dealing with a very short reference sequence, and am interested in mosaik-like variant configurations.