Forum: How To Provide Reference Sequence Dictionary To Reordersam?
3
gravatar for Charles Warden
5.1 years ago by
Charles Warden5.8k
Duarte, CA
Charles Warden5.8k wrote:

Problem: I am trying to use MuTect to analyze some exome capture data for paired tumor and normal samples. I am working with Ion Torrent Proton data.

Basically, MuTect requires .bam files to be ordered in karyotypic order (just like GATK).

If I try to run MuTect on the .bam files produced by TMAP, I get the following error message:

##### ERROR ------------------------------------------------------------------------------------------
##### ERROR A USER ERROR has occurred (version 2.2-25-g2a68eab):
##### 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: Lexicographically sorted human genome sequence detected in reference.
##### ERROR For safety's sake the GATK requires human contigs in karyotypic order: 1, 2, ..., 10, 11, ..., 20, 21, 22, X, Y with M either leading or trailing these contigs.
 ##### ERROR This is because all distributed GATK resources are sorted in karyotypic order, and your processing will fail when you need to use these files.
 ##### ERROR You can use the ReorderSam utility to fix this problem: http://www.broadinstitute.org/gsa/wiki/index.php/ReorderSam
 ##### ERROR   reference contigs = [chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr1, chr20, chr21, chr22, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chrM, chrX, chrY]
 ##### ERROR ------------------------------------------------------------------------------------------

When I try to run ReorderSam in Picard (using the reference I downloaded from ftp://ftp.broadinstitute.org/bundle/2.5/hg19/), I get the following error message:

ERROR 2013-11-22 11:34:20 ReorderSam No reference sequence dictionary found. Aborting.  You can create a sequence dictionary for the reference fasta using CreateSequenceDictionary.jar.

I have no problem creating the sequence dictionary with CreateSequenceDictionary.jar, but I can't get ReorderSam to recognize the sequence dictionary. I have tried "samtools reheader" on the .bam file (using the @SQ values from CreateSequenceDictionary.jar), but I still get the same error message.

Solution: The Sequence Dictionary needs to have a .dict extension. In fact, this file is available for download from ftp://ftp.broadinstitute.org/bundle/2.5/hg19/

This solves the problem for this specific error message, but the final solution will require extra work. For example, once the sequence dictionary is recognized, I see a new error message:

Exception in thread "main" net.sf.picard.PicardException: Discordant contig lengths: read chrM LN=16569, ref chrM LN=16571

So, I might have to find the reference used by TMAP (the .bam file is automatically produced) and sort it myself. Nevertheless, I don't typically have to use .dict files, so I thought it might be helpful to share this with others.

gatk forum picard • 6.6k views
ADD COMMENTlink modified 4.2 years ago by Chirag Nepal2.1k • written 5.1 years ago by Charles Warden5.8k
0
gravatar for Chirag Nepal
4.2 years ago by
Chirag Nepal2.1k
Copenhagen
Chirag Nepal2.1k wrote:

Thanks Charles for sharing ! Now, i am running into the similar problem.

 

It says, failed to load reference dictionary, seems like it is not taking .fa file. I tried to download the file from the link u provided, but the link does not work anymore. this is how the erros looks.  Any help will be appreciated.

My command:

java -Xmx2g -jar ~/unixTools/muTect-1.1.4-bin/muTect-1.1.4.jar --analysis_type MuTect --reference_sequence /steno-internal/chirag/data/ucsc/goldenPath/hg19/assembly.fa --cosmic ~/unixTools/muTect-1.1.4-bin/inputFiles/b37_cosmic_v54_120711.vcf --dbsnp ~/unixTools/muTect-1.1.4-bin/inputFiles/00-All.vcf --intervals 17:1577100-7577200 --input_file:normal /NextGenSeqData/project-data/chirag/rawReadsMapped/chan_et_al_2013_liverFluke/bam/P10N_3001N_merged_sort.bam --input_file:tumor /NextGenSeqData/project-data/chirag/rawReadsMapped/chan_et_al_2013_liverFluke/bam/P10T_3001T_merged_sort.bam --out /NextGenSeqData/project-data/chirag/projects/exomeLiver/testOut --vcf /NextGenSeqData/project-data/chirag/projects/exomeLiver/testname.SNP.vcf

INFO  15:54:15,264 ArgumentTypeDescriptor - Dynamically determined type of /home/chirag/unixTools/muTect-1.1.4-bin/inputFiles/00-All.vcf to be VCF
INFO  15:54:15,285 ArgumentTypeDescriptor - Dynamically determined type of /home/chirag/unixTools/muTect-1.1.4-bin/inputFiles/b37_cosmic_v54_120711.vcf to be VCF
INFO  15:54:15,301 GenomeAnalysisEngine - Strictness is SILENT
WARN  15:54:18,323 RestStorageService - Error Response: PUT '/GATK_Run_Reports/YBzRxqdRvTVRacYj2VLuLRCQyIxjGsf5.report.xml.gz' -- ResponseCode: 403, ResponseStatus: Forbidden, Request Headers: [Content-Length: 733, Content-MD5: 7NBpw4F1jkCwVeC5+8FwDg==, Content-Type: application/octet-stream, x-amz-meta-md5-hash: ecd069c381758e40b055e0b9fbc1700e, Date: Mon, 29 Sep 2014 13:54:16 GMT, Authorization: AWS AKIAJXU7VIHBPDW4TDSQ:dXqD6yDLe5iY1wNeAaL+3FkR3KY=, User-Agent: JetS3t/0.8.1 (Linux/3.2.0-4-amd64; amd64; en; JVM 1.7.0_67), Host: s3.amazonaws.com, Expect: 100-continue], Response Headers: [x-amz-request-id: 46D9A3E8A7B498D7, x-amz-id-2: 0g3fl8lY8s/xh2M443KR7UzkuDshanWb27GfNts6/hpDYnYAwQWYt0bMP6Vq7Nca, Content-Type: application/xml, Transfer-Encoding: chunked, Date: Mon, 29 Sep 2014 13:54:18 GMT, Connection: close, Server: AmazonS3]
##### ERROR ------------------------------------------------------------------------------------------
##### ERROR A USER ERROR has occurred (version 2.2-25-g2a68eab):
##### 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: Invalid command line: Failed to load reference dictionary
##### ERROR ------------------------------------------------------------------------------------------

 

Thanks !

ADD COMMENTlink written 4.2 years ago by Chirag Nepal2.1k

Unfortunately, I figured the out the solution that I posted through trail and error.  I don't actually work on the Picard development team.  I would recommend seeing if one of them can help you.

Perhaps this can help get you started in the right direction:

http://broadinstitute.github.io/picard/ (says to use samtools mailing list for questions)

Also, here are some possibly relevant discussion groups:

http://gatkforums.broadinstitute.org/discussion/58/companion-utilities-reordersam

http://seqanswers.com/forums/showthread.php?t=13909

ADD REPLYlink written 4.2 years ago by Charles Warden5.8k
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