GATK Error SAM/BAM file SAMFileReader is malformed BAM header

dashti

Hi there,

I get an error when I try to run GATK with the following command:

java -jar GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar -T RealignerTargetCreator -R reference.fa -I  merged_bam_files_indexed_markduplicate.bam -o reads.intervals

However I get this error:

SAM/BAM file SAMFileReader{/merged_bam_files_indexed_markduplicate.bam} is malformed: Read HWI-ST303_0093:5:5:13416:34802#0 is either missing the read group or its read group is not defined in the BAM header, both of which are required by the GATK.  Please use http://gatkforums.broadinstitute.org/discussion/59/companion-utilities-replacereadgroups to fix this problem

It suggest that it a header issue however my bam file has a header:

samtools view -h merged_bam_files_indexed_markduplicate.bam | grep ^@RG
@RG     ID:test1      PL:Illumina     PU:HWI-ST303    LB:test     PI:75   SM:test   CN:japan
@RG     ID:test2      PL:Illumina     PU:HWI-ST303    LB:test     PI:75   SM:test    CN:japan

when I grep the read within the error:

HWI-ST303_0093:5:5:13416:34802#0        99      1       1090    29      23S60M17S       =       1150    160     TGTTTGGGTTGAAGATTGATACTGGAAGAAGATTAGAATTGTAGAAAGGGGAAAACGATGTTAGAAAGTTAATACGGCTTACTCCAGATCCTTGGATCTC        GGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGFGGGGGGGGGDGFGFGGGGGFEDFGEGGGDGEG?FGGDDGFFDGGEDDFFFFEDG?E        MD:Z:60 PG:Z:MarkDuplicates     RG:Z:test1      XG:i:0  AM:i:29 NM:i:0  SM:i:29 XM:i:0  XO:i:0  XT:A:M

Following Picard solution:

java -XX:MaxDirectMemorySize=4G -jar picard-tools-1.85/AddOrReplaceReadGroups.jar I= test.bam O= test.header.bam SORT_ORDER=coordinate RGID=test RGLB=test  RGPL=Illumina RGSM=test/ RGPU=HWI-ST303  RGCN=japan CREATE_INDEX=True 

I get this error after 2 min.:

Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Record 12247781, Read name HWI-ST303_0093:5:26:10129:50409#0, MAPQ should be 0 for unmapped read.`

Any recommendation on how to solve this issue ?

My plan is to do the following to resolve the issue:

picard/MarkDuplicates.jar I=test.bam O=test_markduplicate.bam M=test.matrix AS=true VALIDATION_STRINGENCY=LENIANT
samtools  index test_markduplicate.bam

I see a lot of messages like below but the command still running:

Ignoring SAM validation error: ERROR: Record (number), Read name HWI-ST303_0093:5:5:13416:34802#0, RG ID on SAMRecord not found in header: test1

while running the command

then try the GATK RealignerTargetCreator

I already tried to do the following

java -jar GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar -T RealignerTargetCreator -R reference.fa -I  merged_bam_files_indexed_markduplicate.bam -o reads.intervals --validation_strictness LENIENT

But I still got the same error

N.B: the same command run with no issue with GATK version (1.2)

My pipeline in short: mapping the paired end reads with

bwa aln -q 20 ref.fa read > files.sai
bwa sampe ref.fa file1.sai file2.sai read1 read2 > test1.sam
samtools view -bS test1.sam | samtools sort - test
samtools  index test1.bam
samtools merge -rh RG.txt test test1.bam test2.bam

RG.txt

@RG     ID:test1      PL:Illumina     PU:HWI-ST303    LB:test     PI:75   SM:test   CN:japan
@RG     ID:test2      PL:Illumina     PU:HWI-ST303    LB:test     PI:75   SM:test    CN:japan

samtools  index test.bam
picard/MarkDuplicates.jar I=test.bam O=test_markduplicate.bam M=test.matrix AS=true VALIDATION_STRINGENCY=SILENT
samtools  index test_markduplicate.bam

Geraldine_VdAuwera

You need to fix your SAM file before you can proceed with any GATK analysis. I would recommend not using lenient validation with the Picard tools -- you should use strict validation and fix any problems that come up. Otherwise you're just going to get more problems later on.

If you can't figure out how to fix your sam file, I would recommend going back to the original data and reprocessing it. Validate your files at every step.

Jack

see http://sourceforge.net/apps/mediawiki/picard/index.php?title=Main_Page#Q:Why_am_I_getting_errors_from_Picard_like.22MAPQ_should_be_0_for_unmapped_read.22_or_.22CIGAR_should_have_zero_elements_for_unmapped_read.3F.22