Hello everyone,

I want to use Realigner Target Creator provided in the toolshed with the GATK version2.8. Unfortunately, I have a strange error :

"Failed runtime validation of Using reference genome (A built-in reference genome is not available for the build associated with the selected input file)"

The strange thing is that I am using hg19 as reference and when I fill the form I can actually choose hg19. So I guess it means that it exists.

When I look into the details I have this first line in the table :

• Input parameter : Choose the source for the reference list
• Value : Choose the source for the reference list 
• Note for rerun : None

And lastly, if I try to rerun the job (on the failed job) I have a different error :

Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/DATAS/tmp/galaxy
##### ERROR ------------------------------------------------------------------------------------------
##### ERROR A USER ERROR has occurred (version 2.8-1-g932cd3a):
##### ERROR
##### ERROR This means that one or more arguments or inputs in your command are incorrect.
##### ERROR The error message below tells you what is the problem.
##### ERROR
##### ERROR If the problem is an invalid argument, please check the online documentation guide
##### ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
##### ERROR
##### ERROR Visit our website and forum for extensive documentation and answers to
##### ERROR commonly asked questions http://www.broadinstitute.org/gatk
##### ERROR
##### ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
##### ERROR
##### ERROR MESSAGE: Input files reads and reference have incompatible contigs: Relative ordering of overlapping contigs differs, which is unsafe.
##### ERROR   reads contigs = [chr1, chr2, etc...]
##### ERROR ------------------------------------------------------------------------------------------

Does that mean that the picked up option is wrong?

Thank you for your help.

Christ

Hello,

If you are working at http://usegalaxy.org, then the only reference genome that will show up in the list "Using reference genome" will be hg_g1k_v37. But this also means that GATK version 1.4 is in use .. so this work is being done elsewhere?

If another public Galaxy instance, contacting the administrators is the way to resolve the problem. The version of hg19 loaded does not appear to be "GATK" sorted, given the error message.

If this is your own instance, then installing the genome, with correct sorting, will most likely resolve the problem.

Thanks, Jen, Galaxy team

Hi Jen,

Thank you for your answer.

What do you mean by correct sorting? I used the ""Generate GATK-sorted Picard indexes" data manager to prepare the reference genome, and I specified the location in the gatk2_picard_index.loc file.

Did I miss something ?

Christ

Hi Jen,

I still can't use this tool in a workflow or alone. I forgot to say that I am working on my own instance.

I have these lines written by me or by the data manager :

hg19    hg19    hg19    /home/galaxy/galaxypath/tool-data/hg19/gatk_picard_index/hg19/hg19.fa
hg38    hg38    hg38    /home/galaxy/galaxypath/tool-data/hg19/gatk_picard_index/hg38/hg38.fa

In these files :

./tool-data/toolshed.g2.bx.psu.edu/repos/iuc/gatk2/84584664264c/gatk2_picard_index.loc
./tool-data/toolshed.g2.bx.psu.edu/repos/devteam/data_manager_gatk_picard_index_builder/700f2df51eb0/gatk_sorted_picard_index.loc

And I tried to add them in this file as well :

./tool-data/gatk2_picard_index.loc

Can you tell me the files that are supposed to be filled to have my reference genome considered as installed?

Thank you for your help

Christ

Hi Jen,

Thank you for your suggestion. I followed what you suggested and created a new GATKhg19 genomes using "Create DBKey and Reference Genome" with the GATK sort option. Then I used BWA index builder and Generate GATK-sorted Picard indexes builder.

My workflow still stop on Realigner Target Creator with the very same errors than in my first message. I have to add that we have this in the log file :

INFO  11:34:10,574 HelpFormatter - -------------------------------------------------------------------------------- 
INFO  11:34:10,576 HelpFormatter - The Genome Analysis Toolkit (GATK) v2.8-1-g932cd3a, Compiled 2013/12/06 16:47:15 
INFO  11:34:10,576 HelpFormatter - Copyright (c) 2010 The Broad Institute 
INFO  11:34:10,576 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk 
INFO  11:34:10,580 HelpFormatter - Program Args: -T RealignerTargetCreator -o /home/galaxy/galaxy_21juil2015/galaxy/database/files/002/dataset_2608.dat --num_cpu_threads_per_data_thread 1 --num_threads 1 -R /home/galaxy/galaxy_21juil2015/galaxy/tool-data/GATKhg19/gatk_picard_index/GATKhg19/GATKhg19.fa -I /DATAS/tmp/galaxy/tmp-gatk-SqZohx/gatk_input.bam 
INFO  11:34:10,580 HelpFormatter - Date/Time: 2015/09/01 11:34:10 
INFO  11:34:10,580 HelpFormatter - -------------------------------------------------------------------------------- 
INFO  11:34:10,580 HelpFormatter - -------------------------------------------------------------------------------- 
INFO  11:34:11,294 GenomeAnalysisEngine - Strictness is SILENT 
INFO  11:34:11,447 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 1000 
INFO  11:34:11,461 SAMDataSource$SAMReaders - Initializing SAMRecords in serial 
INFO  11:34:11,495 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.03 
INFO  11:34:11,979 HttpMethodDirector - I/O exception (java.net.SocketException) caught when processing request: java.security.NoSuchAlgorithmException: Error constructing implementation (algorithm: Default, provider: SunJSSE, class: sun.security.ssl.SSLContextImpl$DefaultSSLContext) 
INFO  11:34:11,979 HttpMethodDirector - Retrying request 
INFO  11:34:11,980 HttpMethodDirector - I/O exception (java.net.SocketException) caught when processing request: java.security.NoSuchAlgorithmException: Error constructing implementation (algorithm: Default, provider: SunJSSE, class: sun.security.ssl.SSLContextImpl$DefaultSSLContext) 
INFO  11:34:11,980 HttpMethodDirector - Retrying request 
INFO  11:34:11,982 HttpMethodDirector - I/O exception (java.net.SocketException) caught when processing request: java.security.NoSuchAlgorithmException: Error constructing implementation (algorithm: Default, provider: SunJSSE, class: sun.security.ssl.SSLContextImpl$DefaultSSLContext) 
INFO  11:34:11,982 HttpMethodDirector - Retrying request 
INFO  11:34:11,984 HttpMethodDirector - I/O exception (java.net.SocketException) caught when processing request: java.security.NoSuchAlgorithmException: Error constructing implementation (algorithm: Default, provider: SunJSSE, class: sun.security.ssl.SSLContextImpl$DefaultSSLContext) 
INFO  11:34:11,984 HttpMethodDirector - Retrying request 
INFO  11:34:11,985 HttpMethodDirector - I/O exception (java.net.SocketException) caught when processing request: java.security.NoSuchAlgorithmException: Error constructing implementation (algorithm: Default, provider: SunJSSE, class: sun.security.ssl.SSLContextImpl$DefaultSSLContext) 
INFO  11:34:11,985 HttpMethodDirector - Retrying request 

I don't know how to fix this, it is really annoying for me. Do you have any suggestion?

Thank you,

Christ

Is it possible that the builder for GATK is not properly preparing the genome for GATK2?

I finally found what happened with my jobs. When I looked to the fasta files for these tools, it appeared that the ChrM was not sorted in the same way.

The issue was about the data manager for GATK. Apparently he didn't use the genome.fa with the GATK sorting options. I took the right file, GATK sorted, and I generated the .dict and .fai files. I placed them in the path written in the loc file. Now it works fine.

I don't really get why BWA data managers did used the good reference genome file while the GATK data manager did used the wrong one...

Well I think I should not delete anything right now since I'm not sure that I found the real issue.

I tried to launch my workflow with the GATKhg19 genome, once again the realigner target creator failed. But when I try to "run this job again" it works. I didn't change anything.

I'm lost... Could this problem be related to the workflow and not to the tools? Do I have to clean the cache and delete the content of the tmp folder to avoid conflicts

Hi Jen,

I come back for this very same problem. I created a new instance, with the very last version of Galaxy, and I used all the data manager properly. I mean, i used the "GATK sorting" option on the first step, and the next step refered the the previous one.

When i use GATK (here 3.4, but it does not matter), I still have the error :

##### ERROR MESSAGE: Input files reads and reference have incompatible contigs: The contig order in reads and referenceis not the same;

##### ERROR   reads contigs = [chrM, chr1, chr2

##### ERROR   reference contigs = [chr1, chr2, chr3,

I think I found the explanation by looking the files located in tool-data/hg19, which are the files generated by the data_managers.

galaxy@rosetta:~/galaxy/tool-data/hg19$ ls -lrt */*/*
-rw-r--r-- 1 galaxy galaxy       3534 sept. 30 20:22 sam_indexes/hg19/hg19.fa.fai
lrwxrwxrwx 1 galaxy galaxy         17 sept. 30 20:22 sam_indexes/hg19/hg19.fa -> ../../seq/hg19.fa
-rw-r--r-- 1 galaxy galaxy       4035 sept. 30 21:22 bwa_mem_index/hg19/hg19.fa.ann
-rw-r--r-- 1 galaxy galaxy  784290318 sept. 30 21:22 bwa_mem_index/hg19/hg19.fa.pac
-rw-r--r-- 1 galaxy galaxy 1568580688 sept. 30 21:22 bwa_mem_index/hg19/hg19.fa.sa
-rw-r--r-- 1 galaxy galaxy       8595 sept. 30 21:22 bwa_mem_index/hg19/hg19.fa.amb
-rw-r--r-- 1 galaxy galaxy 3137161344 sept. 30 21:22 bwa_mem_index/hg19/hg19.fa.bwt
lrwxrwxrwx 1 galaxy galaxy         17 sept. 30 21:22 bwa_mem_index/hg19/hg19.fa -> ../../seq/hg19.fa
-rw-r--r-- 1 galaxy galaxy       3539 oct.   2 11:52 gatk_picard_index/hg19/hg19.fa.fai
-rw-r--r-- 1 galaxy galaxy 3199905909 oct.   2 11:52 gatk_picard_index/hg19/hg19.fa
-rw-r--r-- 1 galaxy galaxy      14735 oct.   2 11:52 gatk_picard_index/hg19/hg19.dict

As you can see, both sam indexes and BWA_mem fasta files are symbolic links to seq/hg19.fa. This fasta file is NOT gatk sorted, meanwhile gatk_picard_index/hg19.fa is gatk sorted. So I use BWA for the mapping, and then I try to use GATK, but it can't work since their reference file are differents.

I didn't try yet, but it would mean that I need to generate .fai, .dict, etc. from gatk_picard_index/hg19.fa to replace them in all these files so the reference is the same everywhere. Am i right?

Is there a simple way to correct this data manager's behaviour?

Thank you,

Christ

Hi, Thank you for your answer.

Now I have 2 solutions : 

generate all .dict, .fai, .len, etc. files, and replace all of them in my galaxy folders. I think it's going to be quite long and complicated.

delete all my reference genomes files, modify the loc files to erase them, and generate them again with the data managers, from a hg19 fasta file GATK sorted instead of the hg19 coming from UCSC. Do you think it could lead to conflict with the database?

Hi Jen,

I followed your guidance, here is the result.

I used the option "history" for the source in the "Create DBKey and Reference Genome - fetching" data manager with a GATK sorted fasta file, with the option "GATK". But when I check in the tool-data/hg19/seq/hg19.fa, the file sorting is wrong (with chrM as the first sequence), and the tool-data/hg19/gatk_picard_index/hg19/hg19.fa is sorted properly.

Then I tried the same thing, with a good GATK sorted hg38.fa file, but i DID NOT pick the "GATK" option. I picked "as is" option. It works fine, and the sorting is right.

So my guess was right, the data manager is doing a wrong sorting when we pick the GATK option that lead to conflict between references.

I hope this will help to solve this behaviour.

Regards,

Christ

ERROR MESSAGE: Invalid command line: Malformed walker argument: Could not find walker with name: IndelRealigner

having same error again and again. i have tried updating java , also i have latest GATK 3.7. can any one help me ?