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Created 2016-03-02 02:32:26 | Updated 2016-03-02 02:49:03 | Tags: depthofcoverage validation coverage depth-of-coverage qualimap molecular-gender gender

Comments (3)

In our pipeline we try to do relatively quick molecular gender validation using Depth of Coverage on chromosome 1, X and Y and compare the coverage of each, report any anomalies or move on.

For exomes this works fine once we've worked out the bias for a given capture platform.

For genomes though, when running Depth Of Coverage (3.5-0-g36282e4) we get some wildly exaggerated numbers for Y coverage. For example in a male Chr1 is ~40, X is ~20 and Y is ~40!

We think this is due to repetitive sequences on the Y chromosome being mis-handled as additional coverage. The exome .bed files limit the regions looked at so these repeats aren't seen. The genomic .bed file is just the whole Y.

It's just so consistently close to a 1:1 ratio of Chr1 to Y coverage for all our male samples.

QualiMap (v2.1 build 2015-03-19 12:05) on the other-hand does not have this issue. We get the expected coverages of ~40, ~20 and ~18 respectively.

If it is simply a repetition issue, would there be a way to correct this output?

Could it be something different since the Chr1:Y ratio is roughly 1 for every male sample we run.

Is there better way to validate molecular gender with equal or less processing time?

Thanks.

Script snippet:

trait GATK_Arguments extends CommandLineGATK {
    this.reference_sequence = referenceFile
    this.nCoresRequest      = numThreads
    this.isIntermediate     = true
}

trait Finger_Arguments extends CommandLineGATK {
    this.isr = org.broadinstitute.gatk.utils.interval.IntervalSetRule.INTERSECTION
}

trait DoC_Arguments extends DepthOfCoverage {
    this.omitDepthOutputAtEachBase  = true
    this.omitLocusTable             = true
    this.omitIntervals              = true
}

... 

val depthOfACoverage = new DepthOfCoverage with GATK_Arguments with Finger_Arguments with DoC_Arguments
val depthOfXCoverage = new DepthOfCoverage with GATK_Arguments with Finger_Arguments with DoC_Arguments
val depthOfYCoverage = new DepthOfCoverage with GATK_Arguments with Finger_Arguments with DoC_Arguments

depthOfACoverage.input_file = Seq(printReads.out)
depthOfXCoverage.input_file = depthOfACoverage.input_file
depthOfYCoverage.input_file = depthOfACoverage.input_file

depthOfACoverage.intervals = Seq(capPlatforms(platform)) // .bed file for exome chip
depthOfXCoverage.intervals = depthOfACoverage.intervals
depthOfYCoverage.intervals = depthOfACoverage.intervals

depthOfACoverage.intervalsString = Seq("1")
depthOfXCoverage.intervalsString = Seq(trueXRegion) // "X:2699521-154931043"
depthOfYCoverage.intervalsString = Seq(trueYRegion) // "Y:2649521-59034050"

val DoCHead = indiPath + "/" + gvDepth

depthOfACoverage.out = DoCHead + "-A"
depthOfXCoverage.out = DoCHead + "-X"
depthOfYCoverage.out = DoCHead + "-Y"

depthOfACoverage.analysisName = individual
depthOfXCoverage.analysisName = individual
depthOfYCoverage.analysisName = individual

depthOfACoverage.jobName = individual + "_IGV_DoAC"
depthOfXCoverage.jobName = individual + "_IGV_DoXC"
depthOfYCoverage.jobName = individual + "_IGV_DoYC"

add(
  depthOfXCoverage,
  depthOfYCoverage,
  depthOfACoverage
)

/home/ClinGen/Resources/Capture_Platforms/GRCh37/Genomic.bed:

1   1   249250621   chromosome 1    0   .
...
X   1   155270560   chromosome X    0   .
Y   1   59373566    chromosome Y    0   .

Depth of Coverage for Autosomal chromosome 1 command line:

'java'  '-Xmx8192m'  '-XX:+UseParallelOldGC'  '-XX:ParallelGCThreads=4'  '-XX:GCTimeLimit=50'  '-XX:GCHeapFreeLimit=10'  '-Djava.io.tmpdir=/home/shawarden/ClinGen/Dev/ID/.queue/tmp'  '-cp' '/home/ClinGen/bin/queue-3.5-0-g36282e4/Queue.jar'  'org.broadinstitute.gatk.engine.CommandLineGATK'  '-T' 'DepthOfCoverage'  '-I' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/ID.PrintReads.bam'  '-L' '/home/ClinGen/Resources/Capture_Platforms/GRCh37/Genomic.bed'  '-L' '1'  '-isr' 'INTERSECTION'  '-R' '/home/ClinGen/Resources/broad_bundle_b37_v2.5/human_g1k_v37.fasta'  '-o' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/gvDepthOfCoverage-A'  '-omitLocusTable'  '-omitIntervals'  '-omitBaseOutput'  

Depth of Coverage for Autosomal chromosome 1 sample_summary output:

sample_id   total       mean    granular_third_quartile granular_median granular_first_quartile %_bases_above_15
ID          9212399088  40.89   45                      40              36                      99.5
Total       9212399088  40.89   N/A                     N/A             N/A

Depth of Coverage for X chromosome command line:

'java'  '-Xmx8192m'  '-XX:+UseParallelOldGC'  '-XX:ParallelGCThreads=4'  '-XX:GCTimeLimit=50'  '-XX:GCHeapFreeLimit=10'  '-Djava.io.tmpdir=/home/shawarden/ClinGen/Dev/ID/.queue/tmp'  '-cp' '/home/ClinGen/bin/queue-3.5-0-g36282e4/Queue.jar'  'org.broadinstitute.gatk.engine.CommandLineGATK'  '-T' 'DepthOfCoverage'  '-I' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/ID.PrintReads.bam'  '-L' '/home/ClinGen/Resources/Capture_Platforms/GRCh37/Genomic.bed'  '-L' 'X:2699521-154931043'  '-isr' 'INTERSECTION'  '-R' '/home/ClinGen/Resources/broad_bundle_b37_v2.5/human_g1k_v37.fasta'  '-o' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/gvDepthOfCoverage-X'  '-omitLocusTable'  '-omitIntervals'  '-omitBaseOutput'  

Depth of Coverage for X chromosome sample_summary output:

sample_id   total       mean    granular_third_quartile granular_median granular_first_quartile %_bases_above_15
ID          3050588508  20.55   24                      21              18                      87.2
Total       3050588508  20.55   N/A                     N/A             N/A

Depth of Coverage for Y chromosome command line:

'java'  '-Xmx8192m'  '-XX:+UseParallelOldGC'  '-XX:ParallelGCThreads=4'  '-XX:GCTimeLimit=50'  '-XX:GCHeapFreeLimit=10'  '-Djava.io.tmpdir=/home/shawarden/ClinGen/Dev/ID/.queue/tmp'  '-cp' '/home/ClinGen/bin/queue-3.5-0-g36282e4/Queue.jar'  'org.broadinstitute.gatk.engine.CommandLineGATK'  '-T' 'DepthOfCoverage'  '-I' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/ID.PrintReads.bam'  '-L' '/home/ClinGen/Resources/Capture_Platforms/GRCh37/Genomic.bed'  '-L' 'Y:2649521-59034050'  '-isr' 'INTERSECTION'  '-R' '/home/ClinGen/Resources/broad_bundle_b37_v2.5/human_g1k_v37.fasta'  '-o' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/gvDepthOfCoverage-Y'  '-omitLocusTable'  '-omitIntervals'  '-omitBaseOutput' 

Depth of Coverage for Y chromosome sample_summary output:

sample_id   total       mean    granular_third_quartile granular_median granular_first_quartile %_bases_above_15
ID          950208612   41.34   25                      22              18                      90.1
Total       950208612   41.34   N/A                     N/A             N/A

QualiMap Command Line:

'/home/ClinGen/bin/qualimap_v2.1/qualimap'  'bamqc'  '-bam' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/ID.PrintReads.bam'  '-outfile' 'mlQualiMap_genomic.pdf'  '-c'  '--java-mem-size=24G'  '-outformat' 'pdf'  '-outdir' '/mnt/ClinGen/TSD/GRCh37/Active/Genomic/ID/mlQualiMap_genomic'  '-gd' 'HUMAN'  '-nr 500'  '-nt' '16'

QualiMap output:

2.7. Chromosome stats
Name    Length      Mapped bases    Mean coverage   Standard deviation
1       249250621   10494149541     42.1            1,328,790.04
...
X       155270560   3488511556      22.47           3,979
Y       59373566    1085839604      18.29           184,271.52

Created 2013-02-02 15:23:08 | Updated 2013-02-02 22:49:30 | Tags: unifiedgenotyper vqsr haplotypecaller validation statistics sensitivity specificity

Comments (1)

Hi all, I've somewhere in this site that before VQSR the FP rate is expected to be around 10% (I guess for UnifiedGenotyper). Are there some updated statistics for VQRS? For HaplotypeCaller? For Exome/WG data? Another thing: we apply VQRS on all our analysis, we are trying to collect some validation statistics. We suspect that most of the FP have some particular "culprits" in VQRS (especially QD and MQ). Do you have some data about this? Best

d