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SplitNCigarReads generates badCigar reads
For our RNA variant calling pipeline, we follow the GATK best practices workflow (STAR 2-pass -> mark duplicates & sort -> SplitNTrim -> indel realignement -> base recalibration -> variantcalling).
After SplitNCigarReads is succesfully run, the InderRealigner filters out reads because of failing BadCigarFilter. That seems strange to me, because I don't think that SplitNCigarReads should cause the reads with a BadCigarFilter.
Can someone explain to me what is happening in SplitNCigarReads to the reads that are filtered out?
For example, here are the number of reads after each steps for a Sample:
So for this sample, 621 reads are filtered out, which is also reported in the logfile of indelRealigner:
INFO 11:10:40,726 MicroScheduler - 621 reads were filtered out during the traversal out of approximately 22177873 total reads (0.00%)
INFO 11:10:40,726 MicroScheduler - -> 621 reads (0.00% of total) failing BadCigarFilter
INFO 11:10:40,727 MicroScheduler - -> 0 reads (0.00% of total) failing MalformedReadFilter
These are the commands (GATK v3.4) I used (untill realignment):
STAR --genomeDir Homo_sapiens.GRCh37 --runThreadN 4 --outFileNamePrefix sample_ --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --readFilesCommand zcat --outSJfilterIntronMaxVsReadN 10000000 --chimJunctionOverhangMin 15 --chimSegmentMin 15 --twopassMode Basic --readFilesIn Sample_L001_R1_001.fastq.gz,Sample_L002_R1_001.fastq.gz,Sample_L003_R1_001.fastq.gz,Sample_L004_R1_001.fastq.gz
java -jar AddOrReplaceReadGroups.jar INPUT=Sample_Aligned.sortedByCoord.out.bam OUTPUT=Sample_sorted.bam RGID=Sample_L004_R1 RGLB=R1 RGPL=ILLUMINA RGPU=L004 RGSM=Sample
java -jar MarkDuplicates.jar INPUT=Sample_sorted.bam OUTPUT=Sample_sorted_dedupped.bam CREATE_INDEX=true VALIDATION_STRINGENCY=SILENT M=Sample_markDup_metrics.txt
java -jar GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T SplitNCigarReads -R Homo_sapiens.GRCh37.GATK.illumina.fa -I Sample_sorted_dedupped.bam -o Sample_sorted_dedupped_splitN.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS
java -jar GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T RealignerTargetCreator -R Homo_sapiens.GRCh37.GATK.illumina.fa -I Sample_sorted_dedupped_splitN.bam -o Sample_target.intervals
java -jar GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T IndelRealigner -RHomo_sapiens.GRCh37.GATK.illumina.fa -I Sample_sorted_dedupped_splitN.bam -targetIntervals Sample_target.intervals -o Sample_sorted_dedupped_splitN_realigned.bam