DP for each variant bulk is skewed towards 50 and multiples of 50

Follow

Lauren Fedenia

• April 21, 2021 19:02

When using the GATK pipeline to call variants, I end up obtaining a DP for each variant  skewed towards 50 and multiples of 50 (see photo below) when my average read depth from my sequencing files should be much less than 50.  What could be causing this bias and how might I fix it?

 

If you are seeing an error, please provide(REQUIRED) :
a) GATK version used: gatk-4.1.6.0
b) Exact command used: I am not sure which command in my script is causing the bias.
c) Entire error log: N/A

If not an error, choose a category for your question(REQUIRED):
a)How do I (......)?
b) What does (......) mean?
c) Why do I see (......)?
d) Where do I find (......)?
e) Will (......) be in future releases?

0

• 

  Genevieve Brandt (she/her)

  • April 22, 2021 21:04

  Hi Lauren Fedenia,

  Do you have multiple samples? DP is the unfiltered depth of coverage across all samples.

  You can read more about coverage here: https://gatk.broadinstitute.org/hc/en-us/articles/360035532112-Coverage-Read-depth-metrics

  And the DP calculation: https://gatk.broadinstitute.org/hc/en-us/articles/360057440391-Coverage

  Best,

  Genevieve

  0

  Comment actions Permalink
• 

  Lauren Fedenia

  • April 22, 2021 21:15

  Genevieve,

   

  Yes, we are concatenating 70 fastq files/samples per bulk pool to perform variant calling on.  When I we analyze the depth and coverage outside of the GATK pipeline, we do not obtain a skewed read depth towards 50 or multiples of 50.  Is there a filtering step in the GATK pipeline that might be causing this?

   

  Thanks,
  Lauren

  0

  Comment actions Permalink
• 

  Genevieve Brandt (she/her)

  • April 22, 2021 21:39

  What steps are you running in your GATK pipeline? There are multiple read filters applied with most GATK Tools, for example with HaplotypeCaller, these filters are applied:

  • NotSecondaryAlignmentReadFilter
  • GoodCigarReadFilter
  • NonZeroReferenceLengthAlignmentReadFilter
  • PassesVendorQualityCheckReadFilter
  • MappedReadFilter
  • MappingQualityAvailableReadFilter
  • NotDuplicateReadFilter
  • MappingQualityReadFilter
  • WellformedReadFilter
  0

  Comment actions Permalink
• 

  Lauren Fedenia

  • April 23, 2021 13:57

  java -jar picard.jar FastqToSam F1=Red_sorghum_bulk_FseI_130bp_seqs.txt.trimmed_seqs.fastq O=red-bulk_unaligned_reads.bam READ_GROUP_NAME=@D00572.1 SAMPLE_NAME=RedBulk LIBRARY_NAME=Illumina-111111 PLATFORM_UNIT=D00572:40:CAL35ANXX.1 PLATFORM=Illumina

   

  java -jar picard.jar MarkIlluminaAdapters I=red-bulk_unaligned_reads.bam O=red-bulk_markilluminaadapters.bam M=red-bulk_markilluminaadapters_metrics.txt

   

  java -Xmx48G -jar picard.jar SamToFastq I=red-bulk_markilluminaadapters.bam FASTQ=red-bulk_samtofastq.fq CLIPPING_ATTRIBUTE=XT CLIPPING_ACTION=2 NON_PF=true

   

  ../scripts/Read_Mapping_softwares/bwa/bwa mem -M -t 40 ../scripts/Read_Mapping_softwares/ref_bwa/Sbicolor_454_v3.0.1.fa red-bulk_samtofastq.fq > red-bulk_bwa_mem.sam

   

  java -Xmx16G -jar picard.jar MergeBamAlignment R=../scripts/Read_Mapping_softwares/ref_bwa/Sbicolor_454_v3.0.1.fa UNMAPPED_BAM=red-bulk_unaligned_reads.bam ALIGNED_BAM=red-bulk_bwa_mem.sam O=red_bulk_mergebamalignment.bam CREATE_INDEX=true CLIP_ADAPTERS=false INCLUDE_SECONDARY_ALIGNMENTS=true MAX_INSERTIONS_OR_DELETIONS=-1 PRIMARY_ALIGNMENT_STRATEGY=BestMapq ATTRIBUTES_TO_RETAIN=XS PAIRED_RUN=false CLIP_OVERLAPPING_READS=true

   

  ./gatk --java-options "-Xmx40G" HaplotypeCaller -DF NotDuplicateReadFilter -R ../../scripts/Read_Mapping_softwares/ref_bwa/Sbicolor_454_v3.0.1.fa -I ../red_bulk_mergebamalignment.bam -ERC GVCF -O red_bulk_output.raw.snps.indels.g.vcf

   

  All steps above are repeated for the other bulk file.

  0

  Comment actions Permalink
• 

  Genevieve Brandt (she/her)

  • April 23, 2021 19:08

  Lauren Fedenia, what is the expected depth of coverage for your samples? Here is the GATK Tool DepthofCoverage if you want to check: https://gatk.broadinstitute.org/hc/en-us/articles/360056970332-DepthOfCoverage-BETA-

  0

  Comment actions Permalink
• 

  Lauren Fedenia

  • April 23, 2021 21:09

  The expected depth of coverage is 40 for my samples.

  0

  Comment actions Permalink
• 

  Genevieve Brandt (she/her)

  • May 04, 2021 17:35

  Hi Lauren Fedenia,

  Could you clarify what is being shown on the X and Y axis of the plot?

  Thank you, 

  Genevieve

  0

  Comment actions Permalink

Please sign in to leave a comment.