Trinotate - problem with rnammer

[M. Olalla Lorenzo-Carballa]

Hello all,

I am trying to run the RnammerTranscriptome.pl script in Trinotate to identify rRNAs in my assembly. I have installed rnammer (v1.2), and have also tested it with the example data, without any apparent error, so all seems to be ok, however, everytime I try to run the Trinotate script on my data, I get the following output in the screen:

perl /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/RnammerTranscriptome.pl --transcriptome /home/olalla/Dropbox/CGR-Server-Results/Hastata_Assembly/Hastata_All_Assembly.fasta.adj --path_to_rnammer /home/olalla/Downloads/rnammer-1.2/rnammer
CMD: /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/superScaffoldGenerator.pl /home/olalla/Dropbox/CGR-Server-Results/Hastata_Assembly/Hastata_All_Assembly.fasta.adj transcriptSuperScaffold 100
acc: TRINITY_DN29184_c0_g2_i1      

Done.

CMD: perl /home/olalla/Downloads/rnammer-1.2/rnammer -S euk -m tsu,lsu,ssu -gff tmp.superscaff.rnammer.gff < transcriptSuperScaffold.fasta
CMD: /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/rnammer_supperscaffold_gff_to_indiv_transcripts.pl -R tmp.superscaff.rnammer.gff -T transcriptSuperScaffold.bed > Hastata_All_Assembly.fasta.adj.rnammer.gff
WARNING: No RNAMMER features are described in file: tmp.superscaff.rnammer.gff at /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/rnammer_supperscaffold_gff_to_indiv_transcripts.pl line 46.
Error, cmd: /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/rnammer_supperscaffold_gff_to_indiv_transcripts.pl -R tmp.superscaff.rnammer.gff -T transcriptSuperScaffold.bed > Hastata_All_Assembly.fasta.adj.rnammer.gff died with ret 65280 at /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/RnammerTranscriptome.pl line 80.


I have found a patch for rnammer (https://sourceforge.net/p/trinotate/mailman/message/31946113/).

I would like to know whether this patch would help to override the error I am getting when running the script.

Also, if this is the case I am not quite sure about the following information regarding hmmsearch on the readme in the patch:

Be sure to have hmmsearch v2 installed, having renamed that v2 hmmsearch binary to 'hmmsearch2', and ensured that hmmsearch2 can be located within your PATH setting. Typing 'which hmmsearch2' in any directory should return the full path to the software as installed on your system, once available via your PATH setting.

My version of hmmer seems to be 3.1b1, so I am not sure which version of hmmsearch I do have. Is it also 3? If so, can I rename the binary file to hmmsearch2 and the patch would work?

Any input would be very appreciated, as I am a bit stuck here :)

Thanks in advance

Olalla


 

[M. Olalla Lorenzo-Carballa]

Sorry for bothering you again with this, but it would be nice if I could get some input (if possible) on this issue with Trinotate (see message below)

Best regards and huge thanks in advance

Olalla

[Brian Haas]

Hi,

When you tested rnammer with the sample data that came with it, did it generate an output file that contained identified rna gene features?     The system used to require hmmer v2 for this and wasn't compatible with hmmer v3.   We have instructions here on how to set it up:

http://trinotate.github.io

• Installation notes:
  RNAMMER requires the older version of hmmsearch (v2).
  You can obtain the hmmsearch_v2 at ftp://selab.janelia.org/pub/software/hmmer/2.3.2/hmmer-2.3.2.tar.gz[ftp://selab.janelia.org/pub/software/hmmer/2.3.2/hmmer-2.3.2.tar.gz].
  After building the software, we suggest you rename this version of hmmsearch as 'hmmsearch2'.

  In the 'rnammer' software configuration, edit the rnammer script to point
  $HMMSEARCH_BINARY = "/path/to/hmmsearch2";

  Be sure that rnammer functions correctly by executing it on their provided sample data.  RNAMMER is quite useful, but the current implementation is not robust to error, so check carefully.

• 


[M. Olalla Lorenzo-Carballa]

Thanks  a lot Brian,

I will install hmmer v2 and let you know if all works fine.

Cheers

Olalla

--

M. Olalla Lorenzo-Carballa, PhD.

Institute of Integrative Biology 
Evolution, Ecology and Behaviour
 
Biosciences Building, 
Crown Street, 
University of Liverpool,
Liverpool, L69 7ZB. 

United Kingdom.

Linkedin: https://www.linkedin.com/pub/m-olalla-lorenzo-carballa/50/92/969

ResearchGate: https://www.researchgate.net/profile/M_Olalla_Lorenzo-Carballa

[Brian Haas]

ok - good luck!

~b

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

[M. Olalla Lorenzo-Carballa]

So, I have installed hmmer v2 and renamed hmmsearch to hmmsearch2, provided the path to the rnammer script, and this is the error I am getting now:

perl Trinotate-3.0.0/util/rnammer_support/RnammerTranscriptome.pl --transcriptome /home/olalla/Dropbox/CGR-Server-Results/Hastata_Assembly/Hastata_All_Assembly.fasta.adj --path_to_rnammer /home/olalla/Downloads/rnammer-1.2/rnammer

CMD: /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/superScaffoldGenerator.pl /home/olalla/Dropbox/CGR-Server-Results/Hastata_Assembly/Hastata_All_Assembly.fasta.adj transcriptSuperScaffold 100
acc: TRINITY_DN29184_c0_g2_i1      

Done.

CMD: perl /home/olalla/Downloads/rnammer-1.2/rnammer -S euk -m tsu,lsu,ssu -gff tmp.superscaff.rnammer.gff < transcriptSuperScaffold.fasta

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

CMD: /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/rnammer_supperscaffold_gff_to_indiv_transcripts.pl -R tmp.superscaff.rnammer.gff -T transcriptSuperScaffold.bed > Hastata_All_Assembly.fasta.adj.rnammer.gff
WARNING: No RNAMMER features are described in file: tmp.superscaff.rnammer.gff at /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/rnammer_supperscaffold_gff_to_indiv_transcripts.pl line 46.

Error, cmd: /home/olalla/Downloads/Trinotate-3.0.0/util/rnammer_support/util/rnammer_supperscaffold_gff_to_indiv_transcripts.pl -R tmp.superscaff.rnammer.gff -T transcriptSuperScaffold.bed > Hastata_All_Assembly.fasta.adj.rnammer.gff died with ret 65280 at Trinotate-3.0.0/util/rnammer_support/RnammerTranscriptome.pl line 80.


When I chech the tmp.superscaff.rnammer.gff file this is what is in there:


##gff-version2
##source-version RNAmmer-1.2
##date 2016-04-04
##Type DNA
# seqname           source                      feature     start      end   score   +/-  frame  attribute
# ---------------------------------------------------------------------------------------------------------
# ---------------------------------------------------------------------------------------------------------

So the file looks actually quite empty...

When I test the rnammer script with the provided example data this is the screen log I do get:

 perl /home/olalla/Downloads/rnammer-1.2/rnammer -S bac -m lsu,ssu,tsu -gff - < /home/olalla/Downloads/rnammer-1.2/example/ecoli.fsa                                  [ 3:20PM]

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect

FATAL: POSIX threads support is not compiled into HMMER; --cpu doesn't have any effect
##gff-version2
##source-version RNAmmer-1.2
##date 2016-04-04
##Type DNA
# seqname           source                      feature     start      end   score   +/-  frame  attribute
# ---------------------------------------------------------------------------------------------------------
# ---------------------------------------------------------------------------------------------------------

Any ideas? I am quite stuck on this at the moment.

Massive thanks in advance

Olalla

[Brian Haas]

Try just removing the --cpu  parameter from the hmmer call in the rnammer script and see if that helps.

best,

~b

[M. Olalla Lorenzo-Carballa]

I did that and it looks like the program is actually doing something and not reporting any error, or at least not by now :)  Fingers crossed

Thanks a lot, Brian!

Cheers

Olalla

[Brian Haas]

great!  Hopefully it works now.

~b

[M. Olalla Lorenzo-Carballa]

yes it worked! I have now an output with some rRNAs identified :D

Thanks again

Hope this is useful for someone else here

Olalla

[Brian Haas]

Great!  I'm sure it will be.

thx,

~b

[Karen Rossmassler]

Hi,

I think I have a related error (if not, I'm sorry, I'll start a new thread). I have followed the directions on the Trinotate installation website for installing rnammer. I've changed the $HMMSEARCH_BINARY and $INSTALL_PATH to the correct paths, I've installed hmmer2.3, and I've removed --cpu 1 from the core-rnammer script in both places, but although rnammer doesn't give any errors, it doesn't quite seem to be running correctly. When running the example command, it doesn't create new ecoli.fsa, ecoli.gff, or ecoli.hmmreport files, instead, it creates these:

21166.lsu.cf

temp.21166.fsa

Any ideas about what is happening?

Thanks for your help,

Karen

[Xio Lee]

If I remember correctly what I did was to install hmmer2.3 in another directory and change hmm2.3 to hmmsearch2, by default make install, this will install to the same directory of the first version of hmmer and the system will be confused.

Xiao

[Karen Rossmassler]

Yes, you're right, rnammer requires hmmer2, and the instructions say to install hmmer2 in a separate directory (hmmsearch2). I have both installations of hmmer (hmmer-2.3, now called hmmsearch2) and hmmer-3.1b2 (required by Trinotate) as separate directories, installed separately, and named different things. I think I'm confused, which is the 'first version' that you are referring to?

Karen

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[Xio Lee]

first version is hmm3.1

xp

[Karen Rossmassler]

Hm. So if I'm understanding correctly, we both have installed hmmer2 and hmmer3 in separate directories, and we are both following the instructions correctly. How were able to solve the error we got?

Karen