RUNTIME ERROR (version 2.5-2-gf57256b): Duplicate allele added to VariantContext:T (UnifiedGenotype)

Answers

• Geraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

  January 2014

  Can you please describe what this SNPIR pipeline entails and what command line produced the error?

  0 · Share on Twitter Share on Google+
• navonil13 Member ✭

  January 2014 edited January 2014

  a

  Post edited by navonil13 on January 2014

  0 · Share on Twitter Share on Google+
• navonil13 Member ✭

  January 2014 edited January 2014

  after that the rest part is like this (thought its from different sample)

  java -Xmx10g -jar packages/GenomeAnalysisTK-2.5-2/GenomeAnalysisTK.jar -I 19N_c_sorted_removedup.bam -R packages/hg19genome.fa -T RealignerTargetCreator -o 19N_c_sorted_removedup.bam_forIndelRealigner.intervals -known packages/1000G_indels_for_realignment.hg19.vcf -known packages/dbsnp_132.hg19.vcf

      java -Xmx4g -Djava.io.tmpdir=/dev/shm -jar packages/GenomeAnalysisTK-2.5-2/GenomeAnalysisTK.jar -I 19N_c_sorted_removedup.bam -R packages/hg19genome.fa -T IndelRealigner -targetIntervals 19N_c_sorted_removedup.bam_forIndelRealigner.intervals -o 19N_c_sorted_removedup.bam_realignedBam.bam -known packages/1000G_indels_for_realignment.hg19.vcf -known packages/dbsnp_132.hg19.vcf
  
  
      java -Xmx4g -jar packages/GenomeAnalysisTK-2.5-2/GenomeAnalysisTK.jar -R packages/hg19genome.fa -knownSites packages/dbsnp_132.hg19.vcf -I 19N_c_sorted_removedup.bam_realignedBam.bam -T BaseRecalibrator -o 19N_c_sorted_removedup.bam.pre_recal_data.csv
  
  
      java -Xmx4g -Djava.io.tmpdir=/dev/shm -jar packages/GenomeAnalysisTK-2.5-2/GenomeAnalysisTK.jar -R packages/hg19genome.fa -I 19N_c_sorted_removedup.bam_realignedBam.bam -T PrintReads -BQSR 19N_c_sorted_removedup.bam.pre_recal_data.csv -o 19N_c_sorted_removedup.bam_recal.bam
  
  
      ./packages/samtools-0.1.19/samtools index 19N_c_sorted_removedup.bam_recal.bam 19N_c_sorted_removedup.bam_recal.bam.bai
  
  
      java -jar packages/picard-tools-1.92/AddOrReplaceReadGroups.jar I=19N_c_sorted_removedup.bam_recal.bam O=19N_c_sorted_removedup.bam_recal_RG.bam SORT_ORDER=coordinate TMP_DIR=/dev/shm RGID=1 RGLB=1 RGPL=illumina RGSM=ICGC RGPU=NAVONIL CREATE_INDEX=True
  
  
      java -jar  packages/GenomeAnalysisTK-2.5-2/GenomeAnalysisTK.jar -R /packages/hg19genome.fa -T UnifiedGenotyper -I 19N_c_sorted_removedup.bam_recal_RG.bam --dbsnp packages/dbsnp_135.hg19.vcf -o 19N_c_sorted_removedup.bam_WG_snp_indel.raw.vcf -stand_call_conf 0.0 -stand_emit_conf 0.0 -glm BOTH
  

  this last command has created the error

  Post edited by navonil13 on January 2014

  0 · Share on Twitter Share on Google+
• navonil13 Member ✭

  January 2014 edited January 2014

  SNPIR:

  SNPiR: Reliable Identification of Genomic Variants Using RNA-seq Data

  Getting Ready:
  Step 1. download necessary files:
  from UCSC browser: hg19 genome (make sure to get ALL the chromosomes),
  hg19 repeat masker annotation (in bed format),
  Refseq, UCSC, Gencode, Ensembl gene annotation files
  - use "all fields from selected table" output format
  - concatenate all gene annotation files
  (careful: the UCSC gene annotation file is missing the first field, so make sure to add it - awk '{OFS="\t";print "1",$0}')
  - sort concatenated gene annotation file based on chromosome and transcript start (sort -k3,3 -5,5n)
  from rnaedit.com : list of known RNA editing sites (in bed format),
  Step 2. download required software: samtools, bedtools, command line blat, perl, bwa, GATK, picardtools
  Step 3. change paths to your samtools, bedtools and blat executables in file config.pm in the SNPiR package

  Mapping reads:

  Step 1. Concatenate splice junction sequence file with hg19 reference genome (junction files are provided at http://lilab.stanford.edu/SNPiR/junctions/)

  Step 2. Create bwa index for this concatenated file

  Step 3. Map reads with bwa as single end sequences to this reference file

  Step 4. Convert the position of reads that map across splicing junctions onto the genome: java -Xmx2g convertCoordinates < in.sam > out.sam

  Step 5. Use Picard MarkDuplicates to remove duplicate reads

  Step 6. Filter out unmapped reads and reads with mapping quality < 20 using samtools

  Step 7. Index this mapping file with samtools

  Step 8. Perform indel realignment and base quality score recalibration using IndelRealigner, CountCovariates, and TableRecalibration in GATK (see GATK webpage for best practices)

  Call/filter Variants (see EXAMPLES.sh for more information):

  Step 1. Initial variant calling with GATK UnifiedGenotyper: options of -stand_call_conf 0 -stand_emit_conf 0

  Step 2. Convert VCF format to our custom variant format and filter variants with low quality: ConvertVCF.sh in.vcf out.txt MINQUAL

  Step 3. Remove mismatches in first 6 bp of reads: perl filter_mismatch_first6bp.pl

  Step 4. Use bedtools to remove sites in repetitive regions based on RepeatMasker annotation

  Further filtering:
  Step 1. Filter intronic candidates that are within 4 bp of splicing junctions: perl filter_intron_near_splicejuncts.pl

  Step 2. Filter candidates in homopolymer runs: perl filter_homopolymer_nucleotides.pl

  Step 3. Use BLAT to ensure unique mapping: perl BLAT_candidates.pl

  Step 4. Use bedtools to separate out candidates that are known RNA editing sites

  0 · Share on Twitter Share on Google+
• Geraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

  January 2014 Accepted Answer

  OK, I see. Well, this is a little different from our best practices but nothing stands out as potentially causing the error. Since you're using an older version that is no longer supported, you'll need to repeat the UnifiedGenotyper run with the latest version of GATK (2.8). If the error persists, try validating your input files using Picard tools (ValidateSAMFile).

  5 · Share on Twitter Share on Google+
• navonil13 Member ✭

  January 2014

  Ok.. Thanks. I will try and revert

  0 · Share on Twitter Share on Google+