GATK IndelRealigner error

[Heisman]

Hey all,
I am attempting to use this for the first time. Here are my commands:

java -Xmx2000M -jar /Tools/GenomeAnalysisTK-1.0.2885/GenomeAnalysisTK.jar -T RealignerTargetCreator -R Path/hg18_reference_seq.fa -o Test_Output_File_21_Step1 -I Test_ID.21.bam


java -Xmx4000M -jar /Tools/GenomeAnalysisTK-1.0.2885/GenomeAnalysisTK.jar -T IndelRealigner -I Test_ID.21.bam -R Path/hg18_reference_seq.fa -targetIntervals Test_Output_File_21_Step1 -o Test_21_newAligned.bam

The first command runs fine. The second command gives this error:

The following error has occurred:

org.broadinstitute.sting.utils.StingException: First element of the alt consensus cigar must be M or I. Actual: 3H7M1D91M:

I'm pretty new to all of this. What is an "alt consensus cigar", and how should I go about trying to fix this? My first data set were a sample of 1 million paired end reads that I aligned with Novoalign, output in SAM format, sorted, removed duplicates using Picard, sorted, and then tried to do this.

[swbarnes2]

Hey all,
I am attempting to use this for the first time. Here are my commands:

java -Xmx2000M -jar /Tools/GenomeAnalysisTK-1.0.2885/GenomeAnalysisTK.jar -T RealignerTargetCreator -R Path/hg18_reference_seq.fa -o Test_Output_File_21_Step1 -I Test_ID.21.bam


java -Xmx4000M -jar /Tools/GenomeAnalysisTK-1.0.2885/GenomeAnalysisTK.jar -T IndelRealigner -I Test_ID.21.bam -R Path/hg18_reference_seq.fa -targetIntervals Test_Output_File_21_Step1 -o Test_21_newAligned.bam

The first command runs fine. The second command gives this error:

The following error has occurred:

org.broadinstitute.sting.utils.StingException: First element of the alt consensus cigar must be M or I. Actual: 3H7M1D91M:

I'm pretty new to all of this. What is an "alt consensus cigar", and how should I go about trying to fix this? My first data set were a sample of 1 million paired end reads that I aligned with Novoalign, output in SAM format, sorted, removed duplicates using Picard, sorted, and then tried to do this.

Well, try the obvious thing first. GATK is claiming not to like the fact that your read was hard clipped. That's what the H in the first bit of the CIGAR means.

So redo the .sam file without hard clipping.

[Heisman]

Well, try the obvious thing first. GATK is claiming not to like the fact that your read was hard clipped. That's what the H in the first bit of the CIGAR means.

So redo the .sam file without hard clipping.

Thanks for the reply. Assuming hard clipping is only done by adding -H when running Novoalign, I'm not doing any hard clipping. Here are the commands I used:

novoalign -o SAM -r none -e 1 -k -t 200 -a AGATCGGAAGAGCG -d ref_seq.novoindex -f Read1 Read2 1> Aligned.sam 2> Aligned.txt

samtools import ref_seq.samtoolsIndex Aligned.sam Aligned.bam

samtools sort Aligned.bam Aligned_sort

Java -Xmx16000m -jar picard-tools-1.26/MarkDuplicates.jar INPUT=Aligned_sort.bam OUTPUT=Aligned_sort_noDup.bam METRICS_FILE=Aligned_sort_noDup.txt REMOVE_DUPLICATES=True ASSUME_Sorted=True &

samtools sort Aligned_sort_noDup.bam Aligned_sort_noDup_sort

samtools index Aligned_sort_noDup_sort.bam

Are there any good links to explain what CIGAR means? I tried googling it in a sequencing context and found nothing.