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Hello,

sorry if i missed the same problem in other threads in the forum... but we are having trouble running BaseRecalibrator in a sample and i couldn't find the solution.

I tried many steps and here is what i've found until now:

1 - Other samples work fine

2 - Running picard ValidateSamFile in realigned.bam (after IndelRealigner) gives many erros :
2a - Mate negative strand flag does not match read negative strand flag of mate
2b - Mate alignment does not match alignment start of mate
3c - Value was put into PairInfoMap more than once. (fatal)

3 - Running BaseRecalibrator with option -L 1:428-249250621 works fine!

After the fact that -L works fine i discarded the problem in vcf files and reference file. I don't know how to go further in this investigation since GATK 1 realined.bam also gives me the errors in (2) and those error are peanuts comparing the total number of reads.

The big difference here is that we're are using bwa7.

Any ideas? Thanks!

(i'm filtering out "secondary hits" given by bwa7 and will update this thread, if it works it may be helpful in the future)

GATK output:

INFO 14:11:47,441 HelpFormatter - -------------------------------------------------------------------------------- INFO 14:11:47,443 HelpFormatter - The Genome Analysis Toolkit (GATK) v2.4-9-g532efad, Compiled 2013/03/19 07:35:36 INFO 14:11:47,443 HelpFormatter - Copyright (c) 2010 The Broad Institute INFO 14:11:47,443 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk INFO 14:11:47,447 HelpFormatter - Program Args: -nct 8 -T BaseRecalibrator -I /mnt/work/rlb/pac661825//OUT_661825.realigned.bam -R ../data/databases//1KGP/GRCh37_female_exome_mt1kg.fasta --knownSites ../data/databases//dbSNP/dbSNP_137/00-All.vcf -o /mnt/work/rlb/pac661825//OUT_661825.grp INFO 14:11:47,447 HelpFormatter - Date/Time: 2013/03/26 14:11:47 INFO 14:11:47,447 HelpFormatter - -------------------------------------------------------------------------------- INFO 14:11:47,447 HelpFormatter - -------------------------------------------------------------------------------- INFO 14:11:47,458 ArgumentTypeDescriptor - Dynamically determined type of ../data/databases/dbSNP/dbSNP_137/00-All.vcf to be VCF INFO 14:11:47,500 GenomeAnalysisEngine - Strictness is SILENT INFO 14:11:47,558 GenomeAnalysisEngine - Downsampling Settings: No downsampling INFO 14:11:47,565 SAMDataSource$SAMReaders - Initializing SAMRecords in serial INFO 14:11:47,577 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.01 INFO 14:11:47,587 RMDTrackBuilder - Loading Tribble index from disk for file ../data/databases/dbSNP/dbSNP_137/00-All.vcf INFO 14:11:47,704 MicroScheduler - Running the GATK in parallel mode with 8 total threads, 8 CPU thread(s) for each of 1 data thread(s), of 8 processors available on this machine INFO 14:11:47,745 GenomeAnalysisEngine - Creating shard strategy for 1 BAM files INFO 14:11:47,750 GenomeAnalysisEngine - Done creating shard strategy INFO 14:11:47,750 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING] INFO 14:11:47,750 ProgressMeter - Location processed.reads runtime per.1M.reads completed total.runtime remaining INFO 14:11:47,773 BaseRecalibrator - The covariates being used here:
INFO 14:11:47,773 BaseRecalibrator - ReadGroupCovariate INFO 14:11:47,773 BaseRecalibrator - QualityScoreCovariate INFO 14:11:47,773 BaseRecalibrator - ContextCovariate INFO 14:11:47,774 ContextCovariate - Context sizes: base substitution model 2, indel substitution model 3 INFO 14:11:47,774 BaseRecalibrator - CycleCovariate INFO 14:11:47,776 ReadShardBalancer$1 - Loading BAM index data for next contig INFO 14:11:47,777 ReadShardBalancer$1 - Done loading BAM index data for next contig INFO 14:12:18,626 ProgressMeter - 1:15956928 1.10e+06 30.0 s 28.0 s 0.5% 95.1 m 94.6 m INFO 14:12:48,655 ProgressMeter - 1:34102053 2.70e+06 60.0 s 22.0 s 1.1% 89.0 m 88.0 m INFO 14:13:18,685 ProgressMeter - 1:59096606 4.50e+06 90.0 s 20.0 s 1.9% 77.1 m 75.6 m INFO 14:13:48,714 ProgressMeter - 1:103467532 5.90e+06 120.0 s 20.0 s 3.4% 58.7 m 56.7 m INFO 14:14:18,745 ProgressMeter - 1:153234111 7.50e+06 2.5 m 20.0 s 5.0% 49.5 m 47.0 m INFO 14:14:48,774 ProgressMeter - 1:172414433 9.30e+06 3.0 m 19.0 s 5.7% 53.1 m 50.1 m INFO 14:15:19,054 ProgressMeter - 1:208266349 1.10e+07 3.5 m 19.0 s 6.9% 51.3 m 47.8 m INFO 14:15:49,095 ProgressMeter - 1:247611815 1.27e+07 4.0 m 19.0 s 8.2% 49.3 m 45.2 m INFO 14:15:56,507 GATKRunReport - Uploaded run statistics report to AWS S3

ERROR ------------------------------------------------------------------------------------------
ERROR stack trace

org.broadinstitute.sting.utils.exceptions.ReviewedStingException: START (0) > (-1) STOP -- this should never happen -- call Mauricio! at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:537) at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesLeftTail(ReadClipper.java:176) at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:389) at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:392) at org.broadinstitute.sting.gatk.walkers.bqsr.BaseRecalibrator.map(BaseRecalibrator.java:244) at org.broadinstitute.sting.gatk.walkers.bqsr.BaseRecalibrator.map(BaseRecalibrator.java:131) at org.broadinstitute.sting.gatk.traversals.TraverseReadsNano$TraverseReadsMap.apply(TraverseReadsNano.java:230) at org.broadinstitute.sting.gatk.traversals.TraverseReadsNano$TraverseReadsMap.apply(TraverseReadsNano.java:218) at org.broadinstitute.sting.utils.nanoScheduler.NanoScheduler$ReadMapReduceJob.run(NanoScheduler.java:471) at java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:471) at java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:334) at java.util.concurrent.FutureTask.run(FutureTask.java:166) at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1146) at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:615) at java.lang.Thread.run(Thread.java:679)

ERROR ------------------------------------------------------------------------------------------
ERROR A GATK RUNTIME ERROR has occurred (version 2.4-9-g532efad):
ERROR
ERROR Please visit the wiki to see if this is a known problem
ERROR If not, please post the error, 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: START (0) > (-1) STOP -- this should never happen -- call Mauricio!
ERROR ------------------------------------------------------------------------------------------