Picard: problem with Java?

[aleidenroth]

Hi all,

I have the following problem when I run the Picard tool CollectInsertSizeMetrics.jar on a SAM file:

>java -jar
INPUT=alignedsorted.bam
OUTPUT=collectgcbiasmetricsoutfile
HISTOGRAMFILE=chartoutputoutfile.pdf VALIDATION_STRINGENCY=LENIENT

This command successfully creates an outfile with the insert size metric raw numbers, but fails to create the histogram file and gives the following error:


---------------------------------------------------------------------------------
Exception in thread "main" net.sf.picard.PicardException: Unexpected exception executing [Rscript /var/folders/LQ/LQtAvdk7FwmOLyjOs72FKU+++TI/-Tmp-/Andy/script7235887036363822816.R /Users/Andy/ngs/pipetest/refgen/collectgcbiasmetricsoutfile /Users/Andy/ngs/pipetest/refgen/chartoutputoutfile.pdf alignedsorted.bam]
at net.sf.picard.util.ProcessExecutor.execute(ProcessExecutor.java:102)
at net.sf.picard.util.ProcessExecutor.execute(ProcessExecutor.java:78)
at net.sf.picard.util.RExecutor.executeFromFile(RExecutor.java:73)
at net.sf.picard.util.RExecutor.executeFromClasspath(RExecutor.java:55)
at net.sf.picard.analysis.CollectInsertSizeMetrics.finish(CollectInsertSizeMetrics.java:207)
at net.sf.picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:145)
at net.sf.picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:54)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:156)
at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:117)
at net.sf.picard.analysis.CollectInsertSizeMetrics.main(CollectInsertSizeMetrics.java:81)
Caused by: java.io.IOException: Cannot run program "Rscript": error=2, No such file or directory
at java.lang.ProcessBuilder.start(ProcessBuilder.java:460)
at java.lang.Runtime.exec(Runtime.java:593)
at java.lang.Runtime.exec(Runtime.java:466)
at net.sf.picard.util.ProcessExecutor.execute(ProcessExecutor.java:93)
... 9 more
Caused by: java.io.IOException: error=2, No such file or directory
at java.lang.UNIXProcess.forkAndExec(Native Method)
at java.lang.UNIXProcess.<init>(UNIXProcess.java:53)
at java.lang.ProcessImpl.start(ProcessImpl.java:91)
at java.lang.ProcessBuilder.start(ProcessBuilder.java:453)
... 12 more
system cd ~/ngs/pipetest/refgen/; java -jar /users/andy/ngs/applications/picard-tools-1.38/CollectInsertSizeMetrics.jar INPUT=alignedsorted.bam OUTPUT=collectgcbiasmetricsoutfile HISTOGRAM_FILE=chartoutputoutfile.pdf VALIDATION_STRINGENCY=LENIENT
---------------------------------------------------------------------------------


If I run the command as above but with

>java jvm-args -jar

The program crashes right away with error message:


---------------------------------------------------------------------------------
Exception in thread "main" java.lang.NoClassDefFoundError: jvm-args
Caused by: java.lang.ClassNotFoundException: jvm-args
at java.net.URLClassLoader$1.run(URLClassLoader.java:202)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:190)
at java.lang.ClassLoader.loadClass(ClassLoader.java:307)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:301)
at java.lang.ClassLoader.loadClass(ClassLoader.java:248)
system cd ~/ngs/pipetest/refgen/; java jvm-args -jar /users/andy/ngs/applications/picard-tools-1.38/CollectInsertSizeMetrics.jar INPUT=alignedsorted.bam OUTPUT=collectgcbiasmetricsoutfile HISTOGRAM_FILE=chartoutputoutfile.pdf VALIDATION_STRINGENCY=LENIENT
---------------------------------------------------------------------------------


I'm a newbie currently learning unix/perl and use of NGS tools. Any ideas what my problem could be?

Any help would be greatly appreciated.

Andy

[drio]

1. Tell your sysadmins to install R in your cluster/machine.

2. Change jvm-args for whatever parameters you want to use. Typically: -Xmx2g for
picard.

[aleidenroth]

Hi,

Thanks a lot, I installed R and it works like a charm now!

[pd]

Hi

I am trying to use picard tool - CollectMultipleMetrics.jar to find out the insert size distribution. Unfortunately when i run the following command
"java -jar CollectMultipleMetrics.jar I=myfile.sam R=mouse_genome.fa STOP_AFTER=1000000 O=output_file"

I end with following error:

WARNING 2011-11-14 13:13:16 SinglePassSamProgram File reports sort order 'unsorted', assuming it's coordinate sorted anyway.
[Mon Nov 14 13:13:46 CET 2011] net.sf.picard.analysis.CollectMultipleMetrics done. Elapsed time: 0.50 minutes.
Runtime.totalMemory()=1953366016
Exception in thread "main" net.sf.picard.PicardException: Requesting earlier reference sequence: 11 < 14
at net.sf.picard.reference.ReferenceSequenceFileWalker.get(ReferenceSequenceFileWalker.java:78)
at net.sf.picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:115)
at net.sf.picard.analysis.CollectMultipleMetrics.doWork(CollectMultipleMetrics.java:107)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:175)
at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:118)
at net.sf.picard.analysis.CollectMultipleMetrics.main(CollectMultipleMetrics.java:85)




Could anyone guide me, why reference file is showing error, inspite of using the same reference file to generate .bam file?

With Regards
Parveen Kumar

[nilshomer]

Is your SAM file sorted?

[pd]

yes the file was unsorted. Once i sorted it, worked excellently. But facing one more problem now. :-).. WHICH IS
SAM validation error: ERROR: Record 7181005, Read name HWI-ST571:95:C04NEACXX:5:1106:13509:190206, MAPQ should be 0 for unmapped read

What should i do? After googling i found to add VALIDATION_STRINGENCY=SILENT as an option but hw much good this option is?

[swbarnes2]

The problem is just what it says; the sam file is formatted a little differently than Picard is expecting, specifically, unmapped reads have been given mapping qualities, which they really shouldn't have. Perhaps the .bam can be mended by Picard, but it's not a serious problem.

The other option is to use VALIDATION_STRINGENCY=LENIENT; then you'll see all the reads that it is complaining about, and why.

[pd]

Hello

I have some CIGAR length problem in one of my sequence in .sam file. Anyone had any idea about the picard CleanSam.jar option from picard tools.. What actually it does?? Or it would be good to remove that particular sequence from .sam file?

[jay2008]

I have another problem using picard: can anyone tell me how to get org.xerial.snappy?


Exception in thread "main" java.lang.NoClassDefFoundError: org/xerial/snappy/SnappyError
at net.sf.samtools.util.TempStreamFactory.getSnappyLoader(TempStreamFactory.java:43)
at net.sf.samtools.util.TempStreamFactory.wrapTempOutputStream(TempStreamFactory.java:74)
at net.sf.samtools.util.SortingCollection.spillToDisk(SortingCollection.java:207)
at net.sf.samtools.util.SortingCollection.add(SortingCollection.java:150)
at net.sf.samtools.SAMFileWriterImpl.addAlignment(SAMFileWriterImpl.java:157)
at

[nilshomer]

Originally Posted by jay2008

I have another problem using picard: can anyone tell me how to get org.xerial.snappy?


Exception in thread "main" java.lang.NoClassDefFoundError: org/xerial/snappy/SnappyError
at net.sf.samtools.util.TempStreamFactory.getSnappyLoader(TempStreamFactory.java:43)
at net.sf.samtools.util.TempStreamFactory.wrapTempOutputStream(TempStreamFactory.java:74)
at net.sf.samtools.util.SortingCollection.spillToDisk(SortingCollection.java:207)
at net.sf.samtools.util.SortingCollection.add(SortingCollection.java:150)
at net.sf.samtools.SAMFileWriterImpl.addAlignment(SAMFileWriterImpl.java:157)
at

For those who may come to this thread through a web search:
http://sourceforge.net/apps/mediawik...appy_in_Picard