GATK complains that my bam file isn't indexed

[efoss]

I am running GATK's RealignerTargetCreator with this command:

java -Xmx36g -jar GenomeAnalysisTK.jar -S LENIENT -T RealignerTargetCreator -R human_g1k_v37.fasta -o SRR098359.interval_list -I SRR098359.bam -B:snps,VCF 00-All.vcf

The process quits with an error that includes this:

Cannot process the provided BAM file(s) because they were not indexed.

However, the bam file WAS indexed. I see the .bai file there. I recreated the index with the following command (in case something had gone wrong creating it):

samtools index SRR098359_sorted.bam

It created an identical .bai file and I ran RealignerTargetCreator again, and the same thing happened. Does anyone know what I'm doing wrong?

Thank you.

Eric

[cedance]

With a quick glance of what they require, it seems you may require your bam file to be coordinate sorted (before .bai file creation). You should have a look at picard tools.

[efoss]

With a quick glance of what they require, it seems you may require your bam file to be coordinate sorted (before .bai file creation). You should have a look at picard tools.

Hi cedance,

Thanks for the suggestion, but I don't think this is my problem. My previous command coordinate-sorted them:

java -jar /home/efoss/sequencing/picard-tools-1.52/SortSam.jar VALIDATION_STRINGENCY=LENIENT INPUT=SRR098359.bam OUTPUT=SRR098359_sorted.bam SORT_ORDER=coordinate

Eric

[cedance]

One last thing I could think of (the documentation says 1 or more aligned bam files as input). After you mapped with the software of your choice (the reads to your reference), did you obtain aligned reads alone? Maybe you should try using picard tools "ViewSam" with ALIGNMENT_STATUS=aligned to obtain the aligned reads from the bam file and then sort and index it. I would use picard tools for every operation instead of samtools. Sorry, I couldn't be of more help, but I guess this is worth a try.

[maubp]

Maybe a typo, but why are you not using the SRR098359_sorted.bam file when you call GATK? Your command says you are using the unsorted BAM file.

[efoss]

Hi maubp,

THANK YOU, THANK YOU, THANK YOU!!!!!!!!! I stared at that so long without seeing my mistake. I feel very stupid, but also very grateful that you caught it.

Best wishes,

Eric

[maubp]

Happy to help.

[carolW]

I get 2 different error messages when I run gatk

If I use the output of picard markedduplicate, I get error message on unindexed bam file whereas the bam file is already indexed as it is already generated by picard samsort before invoking picard markedduplicate. bai file exist too.

And if I use the output of picard sortsam directly, I get
ERROR MESSAGE: Bad input: We encountered a non-standard non-IUPAC base in the provided reference: '10'

What would you advise?

Thanks,

Carol
-----------------------------------
java -jar SortSam.jar SO=coordinate INPUT=~/NGS/data/SRR062641.filt.sam OUTPUT=~/NGS/data/SRR062641.filt.bam VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=true

- no error is generated

~/NGS/pgm/GenomeAnalysisTK-2.4-9-g532efad$ java -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R /home/carolw/NGS/hg19/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa -o ~/NGS/data/SRR062641.filt.bam.list -I ~/NGS/data/SRR062641.filt.bam

ERROR MESSAGE: Bad input: We encountered a non-standard non-IUPAC base in the provided reference: '10'

-----------------------------------------------------------
java -jar MarkDuplicates.jar INPUT=~/NGS/data/SRR062641.filt.bam OUTPUT=~/NGS/data/SRR062641.filt.marked.bam METRICS_FILE=metrics VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=true

- no error is generated

java -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R /home/carolw/NGS/hg19/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa -o ~/NGS/data/SRR062641.filt.bam.list -I ~/NGS/data/SRR062641.filt.marked.bam

ERROR MESSAGE: Invalid command line: Cannot process the provided BAM file(s) because they were not indexed. The GATK does offer limited processing of unindexed BAMs in --unsafe mode, but this GATK feature is currently unsupported.

[efoss]

Hi CarolW,

Sorry - I don't know what to suggest other than to look very carefully at the name of the index file compared to the name of the bam file.

Good luck.

Eric

[1520191]

I used "samtools index bamfile" created a bam.bai file, then i ran again.it was successful. thanks a lot.