Hi Starr,

Are you sure that all the sequences in ucsc.hg19.fasta appear in the header of My_SOCO.mrkdup_ARG.bam?  This program will only re-order the existing sequences.  If your new sequence dictionary has a sequence that doesn't appear in your current BAM, it won't work.

-Alec

On 7/26/14, 1:25 PM, Hazard, E. Starr wrote:

I am using and PicardTools 1.117 to prep files for GATK 3.2.

ReorderSam throws an exception so it may be failing to complete the output bam/bai files. Is there some way to get some traction on this exception? When I use PicardTools 1.109 on another LINUX system I do not see this problem.

I ask this because  when I go ahead and  run GATK on the final  reordered bam/bai files, GATK runs through the file but writes no output.I am trying to decipher what is happening. Obviously the ReorderSam exception is suspect

java -Djava.io.tmpdir=/shared/app/tmp -jar ${PICARDPATH}/ReorderSam.jar INPUT=/PATH/My_SOCO.mrkdup_ARG.bam OUTPUT=/PATH/My_SOCO.mrkdup_ARG_karyotypesort.bam REFERENCE=$BOWTIE_INDEXES/GATK/ucsc.hg19.fasta CREATE_INDEX=true

...
INFO 2014-07-23 12:45:53 ReorderSam Wrote 1826605 reads
INFO 2014-07-23 12:45:53 ReorderSam  Processing chrY
INFO 2014-07-23 12:45:58 ReorderSam Wrote 450501 reads
[Wed Jul 23 12:45:58 EDT 2014] picard.sam.ReorderSam done. Elapsed time: 15.07 minutes.
Runtime.totalMemory()=337117184
To get help, see http://picard.sourceforge.net/index.shtml#GettingHelp
Exception in thread "main" java.lang.IllegalArgumentException: Invalid reference index -1
at htsjdk.samtools.QueryInterval.<init>(QueryInterval.java:24)
at htsjdk.samtools.SAMFileReader.query(SAMFileReader.java:397)
at picard.sam.ReorderSam.doWork(ReorderSam.java:118)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:183)
at picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:124)
at picard.sam.ReorderSam.main(ReorderSam.java:85)

In spite of posting the error, both a BAM file and a BAI file are created and accepted by GATK

java -Djava.io.tmpdir=/shared/app/tmp -jar  /shared/app/GATK_Queue/GenomeAnalysisTK.jar -T UnifiedGenotyper -R $BOWTIE_INDEXES/GATK/ucsc.hg19.fasta -I /PATH/My_SOCO.mrkdup_ARG_karyotypesort.bam --dbsnp $BOWTIE_INDEXES/GATK/dbsnp_138.hg19.vcf -o /PATH/My_Sample_936_snps.raw.vcf -stand_call_conf 50 -stand_emit_conf 10.0 -dcov 200  --filter_reads_with_N_cigar

INFO  12:31:47,649 HelpFormatter - -------------------------------------------------------------------------------- 
INFO  12:31:47,651 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.2-2-gec30cee, Compiled 2014/07/17 15:22:03 
INFO  12:31:47,652 HelpFormatter - Copyright (c) 2010 The Broad Institute 
INFO  12:31:47,652 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk 
INFO  12:31:47,656 HelpFormatter - Program Args: -T UnifiedGenotyper -R /shared/app/BOWTIE/indexes//GATK/ucsc.hg19.fasta -I /PATH/My_SOCO.mrkdup_ARG_karyotypesort.bam --dbsnp /shared/app/BOWTIE/indexes//GATK/dbsnp_138.hg19.vcf -o /PATH/MySample_936_snps.raw.vcf -stand_call_conf 50 -stand_emit_conf 10.0 -dcov 200 --filter_reads_with_N_cigar 
INFO  12:31:47,661 HelpFormatter - Executing as cgc@hpcc3 on Linux 2.6.32-358.el6.x86_64 amd64; OpenJDK 64-Bit Server VM 1.7.0_65-mockbuild_2014_07_14_06_19-b00. 
INFO  12:31:47,661 HelpFormatter - Date/Time: 2014/07/26 12:31:47 
INFO  12:31:47,661 HelpFormatter - -------------------------------------------------------------------------------- 
INFO  12:31:47,662 HelpFormatter - -------------------------------------------------------------------------------- 
INFO  12:31:47,783 GenomeAnalysisEngine - Strictness is SILENT 
INFO  12:31:47,879 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 200 
INFO  12:31:47,887 SAMDataSource$SAMReaders - Initializing SAMRecords in serial 
INFO  12:31:47,936 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.04 

The program conducts a full search, creates an output vcf file but the file is absolutely empty.
 If there was something wrong with the input bam/bai files why would this not get flagged immediately?

I have submitted this to GATK as well
What can you  suggest from the PicardTools perspective?


Starr


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