Hi, I'm sorry if this is a repeat question. I have been searching through old questions and have yet to find an answer which has fixed the issue. Thank you for your time!
I am trying to create realigned intervals to realign radseq DNA data around indels to ultimately create variants. I have indexed the reference file and created the sequence dictionary as well as indexed the input file and they are all in the same folder. I have run this sequence many times before on similar versions of my data and it has worked and all the sudden it just stopped working and giving me this error message. I realize I am working with an older version of GATK, it is what I downloaded when I started the project and up till now has faithfully worked every time. I thought perhaps it would just be easiest to download the newest version of GATK and did so, but quickly found out that the RealignerTargetCreator and IndelRealigner tools were not included in this new version. If there are similar tools that I could use from the new version I would appreciate suggestions.
Code I ran:
java -jar /Users/taylermassey/Programs/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -R reference.fasta \
-T RealignerTargetCreator \
-I cat_sortPM.bam -o cat_realigner.intervals -nt 2
Output from running above code:
INFO 11:03:39,146 HelpFormatter - ---------------------------------------------------------------------------------
INFO 11:03:39,151 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.4-46-gbc02625, Compiled 2015/07/09 17:38:12
INFO 11:03:39,151 HelpFormatter - Copyright (c) 2010 The Broad Institute
INFO 11:03:39,152 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk
INFO 11:03:39,158 HelpFormatter - Program Args: -R reference.fasta -T RealignerTargetCreator -I cat_sortPM.bam -o cat_realigner.intervals -nt 2
INFO 11:03:39,165 HelpFormatter - Executing as taylermassey@Tayler-Masseys-MacBook-Pro.local on Mac OS X 10.10.5 x86_64; Java HotSpot(TM) 64-Bit Server VM 1.8.0_25-b17.
INFO 11:03:39,165 HelpFormatter - Date/Time: 2018/02/23 11:03:39
INFO 11:03:39,165 HelpFormatter - ---------------------------------------------------------------------------------
INFO 11:03:39,166 HelpFormatter - ---------------------------------------------------------------------------------
INFO 11:03:39,857 GenomeAnalysisEngine - Strictness is SILENT
INFO 11:03:44,924 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 1000
INFO 11:03:44,935 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
INFO 11:03:48,270 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 3.33
INFO 11:03:51,564 GATKRunReport - Uploaded run statistics report to AWS S3
ERROR ------------------------------------------------------------------------------------------
ERROR stack trace
java.lang.NullPointerException
at java.util.TreeMap.put(TreeMap.java:563)
at java.util.TreeSet.add(TreeSet.java:255)
at org.broadinstitute.gatk.utils.sam.ReadUtils.getSAMFileSamples(ReadUtils.java:70)
at org.broadinstitute.gatk.engine.samples.SampleDBBuilder.addSamplesFromSAMHeader(SampleDBBuilder.java:66)
at org.broadinstitute.gatk.engine.GenomeAnalysisEngine.initializeSampleDB(GenomeAnalysisEngine.java:846)
at org.broadinstitute.gatk.engine.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:296)
at org.broadinstitute.gatk.engine.CommandLineExecutable.execute(CommandLineExecutable.java:121)
at org.broadinstitute.gatk.utils.commandline.CommandLineProgram.start(CommandLineProgram.java:248)
at org.broadinstitute.gatk.utils.commandline.CommandLineProgram.start(CommandLineProgram.java:155)
at org.broadinstitute.gatk.engine.CommandLineGATK.main(CommandLineGATK.java:106)
ERROR ------------------------------------------------------------------------------------------
ERROR A GATK RUNTIME ERROR has occurred (version 3.4-46-gbc02625):
ERROR
ERROR This might be a bug. Please check the documentation guide to see if this is a known problem.
ERROR If not, please post the error message, with stack trace, to the GATK forum.
ERROR Visit our website and forum for extensive documentation and answers to
ERROR commonly asked questions http://www.broadinstitute.org/gatk
ERROR
ERROR MESSAGE: Code exception (see stack trace for error itself)
ERROR ------------------------------------------------------------------------------------------
Additionally, this is the output when I run ValidateSameFile:
java -jar /Users/taylermassey/Programs/picard-tools-1.140/picard.jar ValidateSamFile \
I=cat_sortPM.bam \
MODE=SUMMARY
[Fri Feb 23 11:49:48 HST 2018] picard.sam.ValidateSamFile INPUT=cat_sortPM.bam MODE=SUMMARY MAX_OUTPUT=100 IGNORE_WARNINGS=false VALIDATE_INDEX=true IS_BISULFITE_SEQUENCED=false MAX_OPEN_TEMP_FILES=8000 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json
[Fri Feb 23 11:49:48 HST 2018] Executing as taylermassey@Tayler-Masseys-MacBook-Pro.local on Mac OS X 10.10.5 x86_64; Java HotSpot(TM) 64-Bit Server VM 1.8.0_25-b17; Picard version: 1.140(a81bc82e781dae05c922d1dbcee737334612399f_1444244284) JdkDeflater
[Fri Feb 23 12:03:11 HST 2018] picard.sam.ValidateSamFile done. Elapsed time: 13.38 minutes.
Runtime.totalMemory()=1908932608
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" java.lang.OutOfMemoryError: GC overhead limit exceeded
at java.io.BufferedOutputStream.(BufferedOutputStream.java:76)
at htsjdk.samtools.util.IOUtil.maybeBufferOutputStream(IOUtil.java:151)
at htsjdk.samtools.util.IOUtil.maybeBufferOutputStream(IOUtil.java:144)
at htsjdk.samtools.util.FileAppendStreamLRUCache$Functor.makeValue(FileAppendStreamLRUCache.java:52)
at htsjdk.samtools.util.FileAppendStreamLRUCache$Functor.makeValue(FileAppendStreamLRUCache.java:49)
at htsjdk.samtools.util.ResourceLimitedMap.get(ResourceLimitedMap.java:76)
at htsjdk.samtools.CoordinateSortedPairInfoMap.getOutputStreamForSequence(CoordinateSortedPairInfoMap.java:180)
at htsjdk.samtools.CoordinateSortedPairInfoMap.ensureSequenceLoaded(CoordinateSortedPairInfoMap.java:102)
at htsjdk.samtools.CoordinateSortedPairInfoMap.access$300(CoordinateSortedPairInfoMap.java:53)
at htsjdk.samtools.CoordinateSortedPairInfoMap$MapIterator.advanceToNextNonEmptyReferenceIndex(CoordinateSortedPairInfoMap.java:227)
at htsjdk.samtools.CoordinateSortedPairInfoMap$MapIterator.next(CoordinateSortedPairInfoMap.java:257)
at htsjdk.samtools.CoordinateSortedPairInfoMap$MapIterator.next(CoordinateSortedPairInfoMap.java:211)
at htsjdk.samtools.SamFileValidator.validateUnmatchedPairs(SamFileValidator.java:229)
at htsjdk.samtools.SamFileValidator.validateSamFile(SamFileValidator.java:200)
at htsjdk.samtools.SamFileValidator.validateSamFileSummary(SamFileValidator.java:127)
at picard.sam.ValidateSamFile.doWork(ValidateSamFile.java:163)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:206)
at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:95)
at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:105)
Any help would be greatly appreciated!!!!