Dear Jane.
The error message is because the countcovariates-tool of GATK is no longer supported. You have to use BaseRecalibrator and PrintReads tool as described on the GATK page:
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Hello everybody,
I am using the pipeline of ulz_peter (thanks ) to perform realignment around indels and recalibration.
1) I am stuck at the third step of realignment:
java -Djava.io.tmpdir=/tmp/flx-auswerter -jar picard/FixMateInformation.jar INPUT=input.marked.realigned.bam OUTPUT=input_bam.marked.realigned.fixed.bam SO=coordinate VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=truejava -Djava.io.tmpdir=/tmp -jar /share/apps/picard-tools-1.76/FixMateInformation.jar INPUT=$path/$i/$i.realigned.bam OUTPUT=$path/$i/$i.realigned.fixed.bam SO=coordinate VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=true
Code:##### ERROR ------------------------------------------------------------------------------------------ ##### ERROR A USER ERROR has occurred (version 2.0-39-gd091f72): ##### ERROR The invalid arguments or inputs must be corrected before the GATK can proceed ##### ERROR Please do not post this error to the GATK forum ##### ERROR ##### ERROR See the documentation (rerun with -h) for this tool to view allowable command-line arguments. ##### ERROR Visit our website and forum for extensive documentation and answers to ##### ERROR commonly asked questions http://www.broadinstitute.org/gatk ##### ERROR ##### ERROR MESSAGE: Walker CountCovariates is no longer available in the GATK; it has been deprecated since version 2.0
Code:/share/apps/samtools-0.1.18/samtools rmdup ${bam}.bak $bam java -jar $GA/GenomeAnalysisTK.jar -T RealignerTargetCreator -R $db -o ${bam}.list -I $bam java -Djava.io.tmpdir=/tmp -jar $GA/GenomeAnalysisTK.jar -I $bam -R $db -T IndelRealigner -targetIntervals ${bam}.list -o $path/$i/$i.realigned.bam
2) I haven't run yet the recalibration but I saw, in the picard output:
Code:##### ERROR MESSAGE: Walker CountCovariates is no longer available in the GATK; it has been deprecated since version 2.0 ##### ERROR MESSAGE: Walker TableRecalibration is no longer available in the GATK; it has been deprecated since version 2.0
3) Finally, did I understand well: the recalibration of variant quality score, after variant calling, is rather not working when using a single bam? In this case, I won't perform this recalibration.
Thank you in advance,
JaneLast edited by Jane M; 09-04-2012, 05:22 AM.
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Originally posted by frewise View PostWhen I realigned reads around indels, an error occured:
HTML Code:ERROR MESSAGE: Invalid command line: Cannot process the provided BAM file(s) because they were not indexed.
Code:java -Xmx4g -Djava.io.tmpdir=./ \ -jar picard-tools-1.57/SortSam.jar \ SORT_ORDER=coordinate \ INPUT=MK-5.sam \ OUTPUT=MK-5.bam \ VALIDATION_STRINGENCY=LENIENT \ CREATE_INDEX=true java -Xmx4g -Djava.io.tmpdir=./ \ -jar picard-tools-1.57/MarkDuplicates.jar \ INPUT=MK-5.bam \ OUTPUT=MK-5.marked.bam \ METRICS_FILE=metrics \ VALIDATION_STRINGENCY=LENIENT java -Xmx4g -jar GenomeAnalysisTK-1.6-9-g47df7bb/GenomeAnalysisTK.jar \ -T RealignerTargetCreator \ -R hg19.fa \ -o MK-5.bam.list \ -I MK-5.marked.bam java -Xmx4g -Djava.io.tmpdir=./ \ -jar GenomeAnalysisTK-1.6-9-g47df7bb/GenomeAnalysisTK.jar \ -I MK-5.marked.bam \ -R hg19.fa \ -T IndelRealigner \ -targetIntervals MK-5.bam.list \ -o MK-5.marked.realigned.bam
Try adding a CREATE_INDEX=true to your markduplicates-command as well, and I think it will work.
Ø
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BAM file not indexed
When I realigned reads around indels, an error occured:
HTML Code:ERROR MESSAGE: Invalid command line: Cannot process the provided BAM file(s) because they were not indexed.
Code:java -Xmx4g -Djava.io.tmpdir=./ \ -jar picard-tools-1.57/SortSam.jar \ SORT_ORDER=coordinate \ INPUT=MK-5.sam \ OUTPUT=MK-5.bam \ VALIDATION_STRINGENCY=LENIENT \ CREATE_INDEX=true java -Xmx4g -Djava.io.tmpdir=./ \ -jar picard-tools-1.57/MarkDuplicates.jar \ INPUT=MK-5.bam \ OUTPUT=MK-5.marked.bam \ METRICS_FILE=metrics \ VALIDATION_STRINGENCY=LENIENT java -Xmx4g -jar GenomeAnalysisTK-1.6-9-g47df7bb/GenomeAnalysisTK.jar \ -T RealignerTargetCreator \ -R hg19.fa \ -o MK-5.bam.list \ -I MK-5.marked.bam java -Xmx4g -Djava.io.tmpdir=./ \ -jar GenomeAnalysisTK-1.6-9-g47df7bb/GenomeAnalysisTK.jar \ -I MK-5.marked.bam \ -R hg19.fa \ -T IndelRealigner \ -targetIntervals MK-5.bam.list \ -o MK-5.marked.realigned.bam
Last edited by frewise; 08-17-2012, 12:29 AM.
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why remove genes with multiple variants
Originally posted by raonyguimaraes View PostHello all, I have good news ...
After using annovar, I finally got to the number of 22709 variants on my data.
From there I'm now trying to filter based on this approach:
The numbers are pretty close so I think I'm on the right track
22709 Variants
11.179 Variants
4766 Variants
4222 Variants
removed frequency > 0.01
878 Variants
427 Variants
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Sini, it says you have an out of memory error. Try:
java -Xmx4000m -jar
instead of
java -Xmx4m -jar
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I’m a bit embarrassed to ask help again this soon. I’m now trying to use GATK. This is a new program to me and I run into problems straight away…. I followed the instructions in the pdf. file. and used the command:
java -Xmx4m -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R hg19.fa -o input.bam.list -I input.AS.bam
and got huge error message (below) If anyone could tell me what this error message means I would be very greatful!
##### ERROR ------------------------------------------------------------------------------------------
##### ERROR stack trace
java.lang.ExceptionInInitializerError
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.<init>(GenomeAnalysisEngine.java:146)
at org.broadinstitute.sting.gatk.CommandLineExecutable.<init>(CommandLineExecutable.java:53)
at org.broadinstitute.sting.gatk.CommandLineGATK.<init>(CommandLineGATK.java:55)
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:91)
Caused by: java.lang.RuntimeException: java.util.concurrent.ExecutionException: java.lang.OutOfMemoryError: Java heap space
at org.reflections.Reflections.scan(Reflections.java:166)
at org.reflections.Reflections.<init>(Reflections.java:91)
at org.broadinstitute.sting.utils.classloader.PluginManager.<clinit>(PluginManager.java:79)
... 4 more
Caused by: java.util.concurrent.ExecutionException: java.lang.OutOfMemoryError: Java heap space
at java.util.concurrent.FutureTask$Sync.innerGet(Unknown Source)
at java.util.concurrent.FutureTask.get(Unknown Source)
at org.reflections.Reflections.scan(Reflections.java:162)
... 6 more
Caused by: java.lang.OutOfMemoryError: Java heap space
at javassist.bytecode.ExceptionTable.<init>(ExceptionTable.java:58)
at javassist.bytecode.CodeAttribute.<init>(CodeAttribute.java:108)
at javassist.bytecode.AttributeInfo.read(AttributeInfo.java:78)
at javassist.bytecode.MethodInfo.read(MethodInfo.java:498)
at javassist.bytecode.MethodInfo.<init>(MethodInfo.java:79)
at javassist.bytecode.ClassFile.read(ClassFile.java:716)
at javassist.bytecode.ClassFile.<init>(ClassFile.java:85)
at org.reflections.adapters.JavassistAdapter.createClassObject(JavassistAdapter.java:86)
at org.reflections.adapters.JavassistAdapter.createClassObject(JavassistAdapter.java:22)
at org.reflections.scanners.AbstractScanner.scan(AbstractScanner.java:38)
at org.reflections.Reflections$2.run(Reflections.java:149)
at java.util.concurrent.Executors$RunnableAdapter.call(Unknown Source)
at java.util.concurrent.FutureTask$Sync.innerRun(Unknown Source)
at java.util.concurrent.FutureTask.run(Unknown Source)
at java.util.concurrent.ThreadPoolExecutor$Worker.runTask(Unknown Source)
at java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source)
at java.lang.Thread.run(Unknown Source)
##### ERROR ------------------------------------------------------------------------------------------
Exception in thread "main" java.lang.OutOfMemoryError: Java heap space
at java.util.Arrays.copyOfRange(Unknown Source)
at java.lang.String.<init>(Unknown Source)
at java.util.Properties.loadConvert(Unknown Source)
at java.util.Properties.load0(Unknown Source)
at java.util.Properties.load(Unknown Source)
at java.util.PropertyResourceBundle.<init>(Unknown Source)
at java.util.ResourceBundle$Control.newBundle(Unknown Source)
at java.util.ResourceBundle.loadBundle(Unknown Source)
at java.util.ResourceBundle.findBundle(Unknown Source)
at java.util.ResourceBundle.findBundle(Unknown Source)
at java.util.ResourceBundle.findBundle(Unknown Source)
at java.util.ResourceBundle.getBundleImpl(Unknown Source)
at java.util.ResourceBundle.getBundle(Unknown Source)
at org.broadinstitute.sting.utils.text.TextFormattingUtils.loadResourceBundle(TextFormattingUtils.java:104)
at org.broadinstitute.sting.gatk.CommandLineGATK.getVersionNumber(CommandLineGATK.java:135)
at org.broadinstitute.sting.commandline.CommandLineProgram.exitSystemWithError(CommandLineProgram.java:342)
at org.broadinstitute.sting.commandline.CommandLineProgram.exitSystemWithError(CommandLineProgram.java:398)
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:110)
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Originally posted by sdvie View PostHi!
Make sure you have the whole list of .fa files without any line breaks in between, as each line break will submit what is written as a command. So e.g. if you have prepared your command "offline", like
cat chr1.fa chr2.fa chr3.fa chr4.fa chr5.fa chr6.fa chr7.fa chr8.fa chr9.fa [enter]
chr10.fa chr11.fa chr12.fa chr13.fa chr14.fa chr15.fa chr16.fa chr17.fa chr18.fa [enter]
chr19.fa chr20.fa chr21.fa chr22.fa chrX.fa chrY.fa chrM.fa > hg19.fa
the shell will consider every first string in the line (in bold) as the command to execute and that is why you get these errors.
cheers,
Sophia
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Originally posted by Sini View PostHi!
I have problem continating the single-choromosome files to a single file.
I used the command given in the exome analysis.pdf :
cat chr1.fa chr2.fa chr3.fa chr4.fa chr5.fa chr6.fa chr7.fa chr8.fa chr9.fa
chr10.fa chr11.fa chr12.fa chr13.fa chr14.fa chr15.fa chr16.fa chr17.fa chr18.fa chr19.fa chr20.fa chr21.fa chr22.fa chrX.fa chrY.fa chrM.fa > hg19.fa
I did check (several times) that the files were in this order.
I get this error message:
$ chr10.fa chr11.fa chr12.fa chr13.fa chr14.fa chr15.fa chr16.fa chr17.fafahr18.
-sh: chr10.fa: command not found
and
$ chr19.fa chr20.fa chr21.fa chr22.fa chrX.fa chrY.fa chrM.fa > hg19.fa
-sh: chr19.fa: command not found
I have no idea how to fix this, since i'm not sure what the problem is. can anyone help?
Make sure you have the whole list of .fa files without any line breaks in between, as each line break will submit what is written as a command. So e.g. if you have prepared your command "offline", like
cat chr1.fa chr2.fa chr3.fa chr4.fa chr5.fa chr6.fa chr7.fa chr8.fa chr9.fa [enter]
chr10.fa chr11.fa chr12.fa chr13.fa chr14.fa chr15.fa chr16.fa chr17.fa chr18.fa [enter]
chr19.fa chr20.fa chr21.fa chr22.fa chrX.fa chrY.fa chrM.fa > hg19.fa
the shell will consider every first string in the line (in bold) as the command to execute and that is why you get these errors.
cheers,
Sophia
Leave a comment:
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Hi!
I have problem continating the single-choromosome files to a single file.
I used the command given in the exome analysis.pdf :
cat chr1.fa chr2.fa chr3.fa chr4.fa chr5.fa chr6.fa chr7.fa chr8.fa chr9.fa
chr10.fa chr11.fa chr12.fa chr13.fa chr14.fa chr15.fa chr16.fa chr17.fa chr18.fa chr19.fa chr20.fa chr21.fa chr22.fa chrX.fa chrY.fa chrM.fa > hg19.fa
I did check (several times) that the files were in this order.
I get this error message:
$ chr10.fa chr11.fa chr12.fa chr13.fa chr14.fa chr15.fa chr16.fa chr17.fafahr18.
-sh: chr10.fa: command not found
and
$ chr19.fa chr20.fa chr21.fa chr22.fa chrX.fa chrY.fa chrM.fa > hg19.fa
-sh: chr19.fa: command not found
I have no idea how to fix this, since i'm not sure what the problem is. can anyone help?
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Hi, Guys
I know there are many experts here, hoping someone help me out this question.
I got the .csra file for the accession number on NCBI
From the Metadata it is said as the PAIRED.
First, i fastq-dump this csra file, it should come out 2 .fastq files, one is for forward and the other is for the reverse sequence. Right?! But i only got one .fastq file.
Then, i sam-dump/samtools the csra file into .bam file.
Following ulz_peter's great manual, i did the local realignment around indels using the reference hg19 i download from UCSC, an error occurred, saying that input file reads and reference have incompatible contigs. No overlapping contigs found.
Read contigs=[1,2,...,22, GL000235.1,GL000201.1....]
Reference contigs=[chr1,chr2,...,chrM]
GL000235.1,GL000201.1 ....are the files i download using SRA tool. Cause in the csra file there are linked references.
So what kind of reference i should use? How can i do that?
Thank you !
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Originally posted by sdvie View PostI am experiencing the same error as described below by blackgore, with bam files that have data and work with samtools and several picard commands.
The recalibration .csv file is not generated.
Has there been any explanation or possible solution concerning this issue?
cheers,
Sophia
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CountCovariates error on BAM files
I am experiencing the same error as described below by blackgore, with bam files that have data and work with samtools and several picard commands.
The recalibration .csv file is not generated.
Has there been any explanation or possible solution concerning this issue?
cheers,
Sophia
Originally posted by blackgore View PostIn following the workflow mentioned above, I've come up against an error, and I'm wondering if I'm alone in this. Has anyone experienced difficulty with using CountCovariates tool, specifically with errors regarding accessing information from the input BAM file? I've tried this with several samples, but keep getting the same error, "Bad input: Could not find any usable data in the input BAM file(s)"
(for those interested, the BAM files in question are not empty, and work just fine with samtools view).
Code:java -Xmx16g -jar /$Software/GenomeAnalysisTK-1.3-17-gc62082b/GenomeAnalysisTK.jar -T CountCovariates -R /$Genomes/Broad/Human/b37/human_g1k_v37.fasta -I $Projects/data/SampleA_bowtie.gatk.realign.bam -nt 8 -l INFO -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate -log RECAL.log -recalFile RECAL.csv --knownSites $Genomes/Broad/Human/b37/dbsnp_132.b37.vcf INFO 14:01:25,870 HelpFormatter - --------------------------------------------------------------------------------- INFO 14:01:25,875 HelpFormatter - The Genome Analysis Toolkit (GATK) v1.3-17-gc62082b, Compiled 2011/11/18 15:24:46 INFO 14:01:25,875 HelpFormatter - Copyright (c) 2010 The Broad Institute INFO 14:01:25,876 HelpFormatter - Please view our documentation at [url]http://www.broadinstitute.org/gsa/wiki[/url] INFO 14:01:25,876 HelpFormatter - For support, please view our support site at [url]http://getsatisfaction.com/gsa[/url] INFO 14:01:25,877 HelpFormatter - Program Args: -T CountCovariates -R /$Genomes/Broad/Human/b37/human_g1k_v37.fasta -I $Projects/data/SampleA_bowtie.gatk.realign.bam -nt 8 -l INFO -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate -log RECAL.log -recalFile RECAL.csv --knownSites $Genomes/Broad/Human/b37/dbsnp_132.b37.vcf INFO 14:01:25,878 HelpFormatter - Date/Time: 2011/11/24 14:01:25 INFO 14:01:25,878 HelpFormatter - --------------------------------------------------------------------------------- INFO 14:01:25,878 HelpFormatter - --------------------------------------------------------------------------------- INFO 14:01:26,052 RodBindingArgumentTypeDescriptor - Dynamically determined type of $Genomes/Broad/Human/b37/dbsnp_132.b37.vcf to be VCF INFO 14:01:26,064 GenomeAnalysisEngine - Strictness is SILENT INFO 14:01:26,815 RMDTrackBuilder - Loading Tribble index from disk for file $Genomes/Broad/Human/b37/dbsnp_132.b37.vcf INFO 14:01:30,532 MicroScheduler - Running the GATK in parallel mode with 8 concurrent threads INFO 14:01:32,326 CountCovariatesWalker - The covariates being used here: INFO 14:01:32,327 CountCovariatesWalker - ReadGroupCovariate INFO 14:01:32,327 CountCovariatesWalker - QualityScoreCovariate INFO 14:01:32,327 CountCovariatesWalker - CycleCovariate INFO 14:01:32,328 CountCovariatesWalker - DinucCovariate INFO 14:01:41,189 CountCovariatesWalker - Writing raw recalibration data... INFO 14:01:44,145 HttpMethodDirector - I/O exception (java.net.ConnectException) caught when processing request: Connection refused INFO 14:01:44,146 HttpMethodDirector - Retrying request INFO 14:01:44,149 HttpMethodDirector - I/O exception (java.net.ConnectException) caught when processing request: Connection refused INFO 14:01:44,149 HttpMethodDirector - Retrying request INFO 14:01:44,152 HttpMethodDirector - I/O exception (java.net.ConnectException) caught when processing request: Connection refused INFO 14:01:44,153 HttpMethodDirector - Retrying request INFO 14:01:44,155 HttpMethodDirector - I/O exception (java.net.ConnectException) caught when processing request: Connection refused INFO 14:01:44,155 HttpMethodDirector - Retrying request INFO 14:01:44,158 HttpMethodDirector - I/O exception (java.net.ConnectException) caught when processing request: Connection refused INFO 14:01:44,158 HttpMethodDirector - Retrying request ##### ERROR ------------------------------------------------------------------------------------------ ##### ERROR A USER ERROR has occurred (version 1.3-17-gc62082b): ##### ERROR The invalid arguments or inputs must be corrected before the GATK can proceed ##### ERROR Please do not post this error to the GATK forum ##### ERROR ##### ERROR See the documentation (rerun with -h) for this tool to view allowable command-line arguments. ##### ERROR Visit our wiki for extensive documentation [url]http://www.broadinstitute.org/gsa/wiki[/url] ##### ERROR Visit our forum to view answers to commonly asked questions [url]http://getsatisfaction.com/gsa[/url] ##### ERROR ##### ERROR MESSAGE: Bad input: Could not find any usable data in the input BAM file(s). ##### ERROR ------------------------------------------------------------------------------------------
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