Hi,
Could you send me the output of running 'roary -a' ?

Andrew

On 29 August 2016 at 16:11, mmrec notifications@github.com wrote:

Iteratively run cd-hit
sh: line 1: 58934 Trace/BPT trap: 5 /usr/local/bin/cd-hit -i
_combined_files -o _clustered -T 1 -M 1800 -g 1 -s 1 -d 256 -c 1 >
/dev/null 2>&1
Cant open file: _clustered.clstrParallel all against all blast

------------- EXCEPTION -------------
MSG: Could not read file 'clustered': No such file or directory
STACK Bio::Root::IO::initialize_io /usr/local/Cellar/perl/5.24.0
1/lib/perl5/site_perl/5.24.0/Bio/Root/IO.pm:270
STACK Bio::SeqIO::initialize /usr/local/Cellar/perl/5.24.0
1/lib/perl5/site_perl/5.24.0/Bio/SeqIO.pm:499
STACK Bio::SeqIO::fasta::initialize /usr/local/Cellar/perl/5.24.0
1/lib/perl5/site_perl/5.24.0/Bio/SeqIO/fasta.pm:87
STACK Bio::SeqIO::new /usr/local/Cellar/perl/5.24.0
1/lib/perl5/site_perl/5.24.0/Bio/SeqIO.pm:375
STACK Bio::SeqIO::new /usr/local/Cellar/perl/5.24.0_
1/lib/perl5/site_perl/5.24.0/Bio/SeqIO.pm:421
STACK Bio::Roary::ChunkFastaFile::_build__input_seqio
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/
Bio/Roary/ChunkFastaFile.pm:27
STACK Bio::Roary::ChunkFastaFile::_input_seqio reader
Bio::Roary::ChunkFastaFile::_input_seqio (defined at
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/Bio/Roary/ChunkFastaFile.pm
line 18):8
STACK Bio::Roary::ChunkFastaFile::_build_sequence_file_names
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/
Bio/Roary/ChunkFastaFile.pm:48
STACK Bio::Roary::ChunkFastaFile::sequence_file_names reader
Bio::Roary::ChunkFastaFile::sequence_file_names (defined at
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/Bio/Roary/ChunkFastaFile.pm
line 14):8
STACK Bio::Roary::ParallelAllAgainstAllBlast::_build__sequence_file_names
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/Bio/Roary/
ParallelAllAgainstAllBlast.pm:61
STACK Bio::Roary::ParallelAllAgainstAllBlast::sequence_file_names reader
Bio::Roary::ParallelAllAgainstAllBlast::sequence_file_names (defined at
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/Bio/Roary/ParallelAllAgainstAllBlast.pm
line 23):8
STACK Bio::Roary::ParallelAllAgainstAllBlast::run
/usr/local/Cellar/perl/5.24.0_1/lib/perl5/site_perl/5.24.0/Bio/Roary/
ParallelAllAgainstAllBlast.pm:112
STACK Bio::Roary::run /usr/local/Cellar/perl/5.24.0
1/lib/perl5/site_perl/5.24.0/Bio/Roary.pm:92
STACK Bio::Roary::CommandLine::Roary::run /usr/local/Cellar/perl/5.24.0
1/lib/perl5/site_perl/5.24.0/Bio/Roary/CommandLine/Roary.pm:297
STACK toplevel /usr/local/bin/roary:14

Can You help?

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Unfortunately I am not able to run Roary any more, it just won't start anymore after upgrading with:
sudo cpanm -f Bio::Roary

Could you send me the error message your getting?
Andrew

On 30 August 2016 at 10:47, mmrec notifications@github.com wrote:

Unfortunately I am not able to run Roary any more, it just won't start
anymore after upgrading with:
sudo cpanm -f Bio::Roary

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I am not able to reach computer I used to preform analysis.
After the upgrade roary command was not in the bash. I tried to add path but it didn't help.
Then tried to use brew to install earlier version, but it didn't help either.

Fortunately I had finished my analysis - just tried to preform checks.

Grand, sounds like somethings up with your underlying system. Hope it
doesnt take you too much work to resolve.
Andrew

On 30 August 2016 at 10:59, mmrec notifications@github.com wrote:

I am not able to reach computer I used to preform analysis.
After the upgrade roary command was not in the bash. I tried to add path
but it didn't help.
Then tried to use brew to install earlier version, but it didn't help
either.

Fortunately I had finished my analysis - just tried to preform checks.

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Got roary working but same error.
roary -a output, it became a problem after cd-hit 4.6.6 update:

roary -a

Please cite Roary if you use any of the results it produces:
Andrew J. Page, Carla A. Cummins, Martin Hunt, Vanessa K. Wong, Sandra Reuter, Matthew T. G. Holden, Maria Fookes, Daniel Falush, Jacqueline A. Keane, Julian Parkhill,
"Roary: Rapid large-scale prokaryote pan genome analysis", Bioinformatics, 2015 Nov 15;31(22):3691-3693
doi: http://doi.org/10.1093/bioinformatics/btv421
Pubmed: 26198102

2016/09/08 19:37:51 Looking for 'Rscript' - found /usr/local/bin/Rscript
2016/09/08 19:37:51 Determined Rscript version is 3.2
2016/09/08 19:37:51 Looking for 'awk' - found /usr/bin/awk
2016/09/08 19:37:51 Looking for 'bedtools' - found /usr/local/bin/bedtools
2016/09/08 19:37:51 Determined bedtools version is 2.26
2016/09/08 19:37:51 Looking for 'blastp' - found /usr/local/bin/blastp
2016/09/08 19:37:51 Determined blastp version is 2.4.0
2016/09/08 19:37:51 Looking for 'grep' - found /usr/bin/grep
2016/09/08 19:37:51 Looking for 'kraken' - found /usr/local/bin/kraken
Use of uninitialized value in concatenation (.) or string at /Library/Perl/5.18/Bio/Roary/External/CheckTools.pm line 129.
2016/09/08 19:37:52 Determined kraken version is
2016/09/08 19:37:52 Looking for 'kraken-report' - found /usr/local/bin/kraken-report
Use of uninitialized value in concatenation (.) or string at /Library/Perl/5.18/Bio/Roary/External/CheckTools.pm line 129.
2016/09/08 19:37:52 Determined kraken-report version is
2016/09/08 19:37:52 Looking for 'mafft' - found /usr/local/bin/mafft
2016/09/08 19:37:52 Determined mafft version is 7.221
2016/09/08 19:37:52 Looking for 'makeblastdb' - found /usr/local/bin/makeblastdb
2016/09/08 19:37:52 Determined makeblastdb version is 2.4.0
2016/09/08 19:37:52 Looking for 'mcl' - found /usr/local/bin/mcl
2016/09/08 19:37:52 Determined mcl version is 14-137
2016/09/08 19:37:52 Looking for 'parallel' - found /usr/local/bin/parallel
2016/09/08 19:37:52 Determined parallel version is 20160822
2016/09/08 19:37:52 Looking for 'prank' - found /usr/local/bin/prank
2016/09/08 19:37:52 Looking for 'sed' - found /usr/bin/sed
2016/09/08 19:37:52 Looking for 'cd-hit' - found /usr/local/bin/cd-hit
dyld: Library not loaded: /usr/local/opt/gcc/lib/gcc/6/libgomp.1.dylib
Referenced from: /usr/local/bin/cd-hit
Reason: image not found
2016/09/08 19:37:53 Could not determine version of cd-hit - please install version 4.6 or higher
2016/09/08 19:37:53 Looking for 'FastTree' - found /usr/local/bin/FastTree
2016/09/08 19:37:53 Determined FastTree version is 2.1
2016/09/08 19:37:53 Roary version 3.6.8
2016/09/08 19:37:53 Error: You need to provide at least 2 files to build a pan genome
Usage: roary [options] *.gff

Options: -p INT number of threads [1]
-o STR clusters output filename [clustered_proteins]
-f STR output directory [.]
-e create a multiFASTA alignment of core genes using PRANK
-n fast core gene alignment with MAFFT, use with -e
-i minimum percentage identity for blastp [95]
-cd FLOAT percentage of isolates a gene must be in to be core [99]
-qc generate QC report with Kraken
-k STR path to Kraken database for QC, use with -qc
-a check dependancies and print versions
-b STR blastp executable [blastp]
-c STR mcl executable [mcl]
-d STR mcxdeblast executable [mcxdeblast]
-g INT maximum number of clusters [50000]
-m STR makeblastdb executable [makeblastdb]
-r create R plots, requires R and ggplot2
-s dont split paralogs
-t INT translation table [11]
-z dont delete intermediate files
-v verbose output to STDOUT
-w print version and exit
-y add gene inference information to spreadsheet, doesnt work with -e
-h this help message

Example: Quickly generate a core gene alignment using 8 threads
roary -e --mafft -p 8 *.gff

For further info see: http://sanger-pathogens.github.io/Roary/

Error occured after upgrading cd-hit to version 4.6.6.
After upgrade had to upgrade gcc version 5 to version 6.
Then had to upgrade FastTree.

Now got Roary working again!

One Question, after running roary -a got following for kraken and kraken-report:

2016/09/08 20:06:52 Looking for 'kraken' - found /usr/local/bin/kraken
Use of uninitialized value in concatenation (.) or string at /Library/Perl/5.18/Bio/Roary/External/CheckTools.pm line 129.
2016/09/08 20:06:52 Determined kraken version is 
2016/09/08 20:06:52 Looking for 'kraken-report' - found /usr/local/bin/kraken-report
Use of uninitialized value in concatenation (.) or string at /Library/Perl/5.18/Bio/Roary/External/CheckTools.pm line 129.
2016/09/08 20:06:52 Determined kraken-report version is 

Full Roary -a output:

2016/09/08 20:06:51 Looking for 'Rscript' - found /usr/local/bin/Rscript
2016/09/08 20:06:51 Determined Rscript version is 3.2
2016/09/08 20:06:51 Looking for 'awk' - found /usr/bin/awk
2016/09/08 20:06:51 Looking for 'bedtools' - found /usr/local/bin/bedtools
2016/09/08 20:06:51 Determined bedtools version is 2.26
2016/09/08 20:06:51 Looking for 'blastp' - found /usr/local/bin/blastp
2016/09/08 20:06:52 Determined blastp version is 2.4.0
2016/09/08 20:06:52 Looking for 'grep' - found /usr/bin/grep
2016/09/08 20:06:52 Looking for 'kraken' - found /usr/local/bin/kraken
Use of uninitialized value in concatenation (.) or string at /Library/Perl/5.18/Bio/Roary/External/CheckTools.pm line 129.
2016/09/08 20:06:52 Determined kraken version is 
2016/09/08 20:06:52 Looking for 'kraken-report' - found /usr/local/bin/kraken-report
Use of uninitialized value in concatenation (.) or string at /Library/Perl/5.18/Bio/Roary/External/CheckTools.pm line 129.
2016/09/08 20:06:52 Determined kraken-report version is 
2016/09/08 20:06:52 Looking for 'mafft' - found /usr/local/bin/mafft
2016/09/08 20:06:52 Determined mafft version is 7.221
2016/09/08 20:06:52 Looking for 'makeblastdb' - found /usr/local/bin/makeblastdb
2016/09/08 20:06:52 Determined makeblastdb version is 2.4.0
2016/09/08 20:06:52 Looking for 'mcl' - found /usr/local/bin/mcl
2016/09/08 20:06:52 Determined mcl version is 14-137
2016/09/08 20:06:52 Looking for 'parallel' - found /usr/local/bin/parallel
2016/09/08 20:06:52 Determined parallel version is 20160822
2016/09/08 20:06:52 Looking for 'prank' - found /usr/local/bin/prank
2016/09/08 20:06:52 Looking for 'sed' - found /usr/bin/sed
2016/09/08 20:06:52 Looking for 'cd-hit' - found /usr/local/bin/cd-hit
2016/09/08 20:06:52 Determined cd-hit version is 4.6
2016/09/08 20:06:52 Looking for 'FastTree' - found /usr/local/bin/FastTree
2016/09/08 20:06:52 Determined FastTree version is 2.1
2016/09/08 20:06:52 Roary version 3.6.8
2016/09/08 20:06:52 Error: You need to provide at least 2 files to build a pan genome
Usage:   roary [options] *.gff

Hi andrew,

I am experiencing the same issue and have not been able to find the solution. Here is the error message from roary:

2016/10/06 17:26:26 Output directory created: /nfs/main/Ali/Project_RNA_seq/Ortholog_detection/temp_out_4
2016/10/06 17:26:26 Fixing input GFF files
2016/10/06 17:26:28 Extracting proteins from GFF files
Extracting proteins from /nfs/main/Ali/Project_RNA_seq/Ortholog_detection/temp/new_gff/HM26_10062016.gff
Extracting proteins from /nfs/main/Ali/Project_RNA_seq/Ortholog_detection/temp/new_gff/HM27_10062016.gff
Combine proteins into a single file
Iteratively run cd-hit
Cant open file: _clustered.clstrParallel all against all blast

------------- EXCEPTION -------------
MSG: Could not read file '_clustered': No such file or directory
STACK Bio::Root::IO::_initialize_io /home/username/perl5/perlbrew/perls/perl-5.20.1/lib/site_perl/5.20.1/Bio/Root/IO.pm:270
STACK Bio::SeqIO::_initialize /home/username/perl5/perlbrew/perls/perl-5.20.1/lib/site_perl/5.20.1/Bio/SeqIO.pm:499
STACK Bio::SeqIO::fasta::_initialize /home/username/perl5/perlbrew/perls/perl-5.20.1/lib/site_perl/5.20.1/Bio/SeqIO/fasta.pm:87
STACK Bio::SeqIO::new /home/username/perl5/perlbrew/perls/perl-5.20.1/lib/site_perl/5.20.1/Bio/SeqIO.pm:375
STACK Bio::SeqIO::new /home/username/perl5/perlbrew/perls/perl-5.20.1/lib/site_perl/5.20.1/Bio/SeqIO.pm:421
STACK Bio::Roary::ChunkFastaFile::_build__input_seqio /nfs/main/bin_group/Roary/lib/Bio/Roary/ChunkFastaFile.pm:38
STACK Bio::Roary::ChunkFastaFile::_input_seqio reader Bio::Roary::ChunkFastaFile::_input_seqio (defined at /nfs/main/bin_group/Roary/lib/Bio/Roary/ChunkFastaFile.pm line 29):8
STACK Bio::Roary::ChunkFastaFile::_build_sequence_file_names /nfs/main/bin_group/Roary/lib/Bio/Roary/ChunkFastaFile.pm:59
STACK Bio::Roary::ChunkFastaFile::sequence_file_names reader Bio::Roary::ChunkFastaFile::sequence_file_names (defined at /nfs/main/bin_group/Roary/lib/Bio/Roary/ChunkFastaFile.pm line 25):8
STACK Bio::Roary::ParallelAllAgainstAllBlast::_build__sequence_file_names /nfs/main/bin_group/Roary/lib/Bio/Roary/ParallelAllAgainstAllBlast.pm:72
STACK Bio::Roary::ParallelAllAgainstAllBlast::_sequence_file_names reader Bio::Roary::ParallelAllAgainstAllBlast::_sequence_file_names (defined at /nfs/main/bin_group/Roary/lib/Bio/Roary/ParallelAllAgainstAllBlast.pm line 34):8
STACK Bio::Roary::ParallelAllAgainstAllBlast::run /nfs/main/bin_group/Roary/lib/Bio/Roary/ParallelAllAgainstAllBlast.pm:123
STACK Bio::Roary::run /nfs/main/bin_group/Roary/lib/Bio/Roary.pm:97
STACK Bio::Roary::CommandLine::Roary::run /nfs/main/bin_group/Roary/lib/Bio/Roary/CommandLine/Roary.pm:302
STACK toplevel /nfs/main/bin_group/Roary/bin/roary:19
-------------------------------------

This is the output from roary -a

2016/10/06 17:32:49 Looking for 'Rscript' - found /home/username/anaconda/bin/Rscript
2016/10/06 17:32:49 Determined Rscript version is 3.1
2016/10/06 17:32:49 Looking for 'awk' - found /usr/bin/awk
2016/10/06 17:32:49 Looking for 'bedtools' - found /home/username/bin/bedtools2-master/bin/bedtools
2016/10/06 17:32:49 Determined bedtools version is 2.23
2016/10/06 17:32:49 Looking for 'blastp' - found /home/username/bin/blast/bin/blastp
2016/10/06 17:32:49 Determined blastp version is 2.2.31
2016/10/06 17:32:49 Looking for 'grep' - found /usr/bin/grep
2016/10/06 17:32:49 Looking for 'kraken' - found /nfs/main/bin_group/kraken-master/kraken
Use of uninitialized value in concatenation (.) or string at /nfs/main/bin_group/Roary/lib/Bio/Roary/External/CheckTools.pm line 139.
2016/10/06 17:32:49 Determined kraken version is 
2016/10/06 17:32:49 Looking for 'kraken-report' - found /nfs/main/bin_group/kraken-master/kraken-report
Use of uninitialized value in concatenation (.) or string at /nfs/main/bin_group/Roary/lib/Bio/Roary/External/CheckTools.pm line 139.
2016/10/06 17:32:49 Determined kraken-report version is 
2016/10/06 17:32:49 Looking for 'mafft' - found /nfs/main/bin_group/Roary/build/mafft-7.271-without-extensions/build/bin/mafft
2016/10/06 17:32:49 Determined mafft version is 7.271
2016/10/06 17:32:49 Looking for 'makeblastdb' - found /home/username/bin/blast/bin/makeblastdb
2016/10/06 17:32:49 Determined makeblastdb version is 2.2.31
2016/10/06 17:32:49 Looking for 'mcl' - found /nfs/main/bin_group/Roary/build/mcl-14-137/src/shmcl/mcl
2016/10/06 17:32:49 Determined mcl version is 14-137
2016/10/06 17:32:49 Looking for 'parallel' - found /nfs/main/bin_group/Roary/build/parallel-20160722/src/parallel
2016/10/06 17:32:49 Determined parallel version is 20160722
2016/10/06 17:32:49 Looking for 'prank' - found /nfs/main/bin_group/prank
2016/10/06 17:32:49 Looking for 'sed' - found /usr/bin/sed
2016/10/06 17:32:49 Looking for 'cd-hit' - found /nfs/main/bin_group/cd-hit
2016/10/06 17:32:49 Determined cd-hit version is 4.6
2016/10/06 17:32:49 Looking for 'FastTree' - found /nfs/main/bin_group/Roary/build/fasttree/FastTree
2016/10/06 17:32:49 Determined FastTree version is 2.1
2016/10/06 17:32:49 Roary version 1.006924
2016/10/06 17:32:49 Error: You need to provide at least 2 files to build a pan genome

I would appreciate any help with this regard.
Thanks

Hello
I am working on a project based on all the genomes of enterococcus faecalis.
It's been a week since roary tells me this mistake.

Could not open /home/mamadou/Pangenome/gOwpb0VOYj/FPDW01.gff.proteome.faa: No such file or directory at /usr/share/perl5/Bio/Roary/SplitGroups.pm line 84.

Hi, Its most likely a problem with your input data.
I would double check the QC of the data to remove any obvious contamination, poor quality assemblies, different species, etc...
Then I would double check each assembly was annotated with a unqiue locus tag (so that each gene has a globally unique identifier).
Regards,
Andrew

@andrewjpage thanks for the really helpful package. I'm using gff files downloaded using ncbi-genome-download for all sequences matching a particular taxid. These are not 'my' sequences. Would you still recommend QC check with Kraken? I ask because I start with roary -f *** on my downloaded gff files followed by the same with the 'fixed' gff files. My output files are all empty, and I see some of the errors mentioned above. Not sure as to how to proceed with the pangenome analysis and exploration from here.

I am getting this error
Please help me to fix this

Subhashinies-MacBook-Air:roary indika$ /Users/indika/miniconda3/bin/roary roary /Users/indika/pangenome *.gff
Can't locate Bio/Roary/CommandLine/Roary.pm in @inc (you may need to install the Bio::Roary::CommandLine::Roary module) (@inc contains: /Users/indika/Roary/lib/Bio/Roary/lib /Library/Perl/5.18/darwin-thread-multi-2level /Library/Perl/5.18 /Network/Library/Perl/5.18/darwin-thread-multi-2level /Network/Library/Perl/5.18 /Library/Perl/Updates/5.18.2 /System/Library/Perl/5.18/darwin-thread-multi-2level /System/Library/Perl/5.18 /System/Library/Perl/Extras/5.18/darwin-thread-multi-2level /System/Library/Perl/Extras/5.18 .) at /Users/indika/miniconda3/bin/roary line 12.
BEGIN failed--compilation aborted at /Users/indika/miniconda3/bin/roary line 12.

@senula2017 your error indicates the software is not installed correctly. I would reinstalling (conda install roary) or contact your local IT support/system administrator