identical .gff file names from different genome, and then issue with mcl groups

[bhclement]

Hello,
I was testing Prokka and Roary for my genomes.
I followed the Prokka command for genome annotations but all .gff files of different samples have the identical .gff file names! Is this normal?
Also there were some error message when I run the Roary commands.
I will appreciate for advice.
Thanks,
KT
p.s. command and result copied below.

[ctsui@grl-salk E_coli] prokka --kingdom Bacteria --outdir Ecoli_V29 --locustag ecoli_V_29 --addgenes --force --cpus 8 V_29.contigs.fasta
[ctsui@grl-salk E_coli]$ roary -e --mafft -p 8 ./Ecoli*/*.gff
2017/08/17 17:52:10 Extracting proteins from GFF files
Extracting proteins from /Ecoli_V5/PROKKA_08172017.gff
Extracting proteins from Ecoli_V29/PROKKA_08172017.gff
Extracting proteins from Ecoli_U16/PROKKA_08172017.gff
Extracting proteins from /Ecoli_V7/PROKKA_08172017.gff
Extracting proteins from /Ecoli_V21/PROKKA_08172017.gff
Combine proteins into a single file
Iteratively run cd-hit
Parallel all against all blast
BLAST Database error: No alias or index file found for protein database [/projects/roary_test/_1503017512/DJ3228qGRO/output_contigs] in search path [/projects/roary_test/_1503017512::]
Cluster with MCL
2017/08/17 17:53:05 Running command: pan_genome_post_analysis -o clustered_proteins -p pan_genome.fa -s gene_presence_absence.csv -c _clustered.clstr --output_multifasta_files -i /projects/E_coli/roary_test/_1503017512/ZV2aUQOBho//_gff_files -f /projects/E_coli/roary_test/_1503017512/ZV2aUQOBho//_fasta_files -t 11 --dont_create_rplots -v --mafft -j Local --processors 1 --group_limit 50000 -cd 99
2017/08/17 17:53:07 Reinflate clusters
Cant open file: _uninflated_mcl_groups

[menright99]

You need to use --prefix 'strain_name' in the command line - this will give you strain_name.gff

I also use --locustag 'strain_name' as this names the genes after your strain and not the default

Hope this works.

[bhclement]

Thanks. I did Prokka again with --prelix in the command.
But the message "Cant open file: _uninflated_mcl_groups" still persist! Is some dependency missing?

[tseemann]

@bhclement sounds like your installation of mcl failed to produce the correct output files.

thank you @menright99 for helping with Prokka.
I tend to use --prefix STRAIN --locustag STRAIN --outdir STRAIN
I should add an --auto mode to do this sort of pattern - maybe in Prokka 2.0

[bhclement]

Thanks Torsten! So I need re-install mcl?
which version should I install?

[bhclement]

Roary was installed in the server, while mcl was installed in a personal dir. Is this the issue?
I already export the path in which mcl was installed.

[tseemann]

Can you provide us with the output of roary -a ?

[bhclement]

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

2017/08/21 21:05:45 Looking for 'Rscript' - found /usr/local/bin/Rscript
2017/08/21 21:05:45 Determined Rscript version is 2.15
2017/08/21 21:05:45 Roary needs Rscript 3 or higher. Please upgrade and try again.
2017/08/21 21:05:45 Looking for 'awk' - found /bin/awk
2017/08/21 21:05:45 Looking for 'bedtools' - found /usr/local/bin/bedtools
2017/08/21 21:05:45 Determined bedtools version is 2.24
2017/08/21 21:05:45 Looking for 'blastp' - found /usr/bin/blastp
2017/08/21 21:05:45 Determined blastp version is 2.2.28
2017/08/21 21:05:45 Looking for 'grep' - found /bin/grep
2017/08/21 21:05:45 Looking for 'kraken' - found /usr/local/bin/kraken
Use of uninitialized value in concatenation (.) or string at /usr/local/share/perl5/Bio/Roary/External/CheckTools.pm li ne 129.
2017/08/21 21:05:46 Determined kraken version is
2017/08/21 21:05:46 Looking for 'kraken-report' - found /usr/local/bin/kraken-report
Use of uninitialized value in concatenation (.) or string at /usr/local/share/perl5/Bio/Roary/External/CheckTools.pm li ne 129.
2017/08/21 21:05:46 Determined kraken-report version is
2017/08/21 21:05:46 Looking for 'mafft' - found /usr/bin/mafft
Use of uninitialized value in concatenation (.) or string at /usr/local/share/perl5/Bio/Roary/External/CheckTools.pm li ne 129.
2017/08/21 21:05:46 Determined mafft version is
2017/08/21 21:05:46 Looking for 'makeblastdb' - found /usr/bin/makeblastdb
2017/08/21 21:05:46 Determined makeblastdb version is 2.2.28
2017/08/21 21:05:46 ERROR: Can't find required 'mcl' in your $PATH
2017/08/21 21:05:46 Looking for 'parallel' - found /usr/bin/parallel
2017/08/21 21:05:46 Determined parallel version is 20130722
2017/08/21 21:05:46 ERROR: Can't find required 'prank' in your $PATH
2017/08/21 21:05:46 Looking for 'sed' - found /bin/sed
2017/08/21 21:05:46 Looking for 'cd-hit' - found /usr/local/bin/cd-hit
2017/08/21 21:05:46 Determined cd-hit version is 4.6
2017/08/21 21:05:46 Looking for 'FastTree' - found /usr/local/bin/FastTree
2017/08/21 21:05:46 Determined FastTree version is 2.1
2017/08/21 21:05:46 Roary version 3.6.0
2017/08/21 21:05:46 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

[bhclement]

Roary did not recognize the local path I export:

export PATH=/projects3/giardia/TB_pilot/4Dec2016/Spade_genomes/Roary_data/mcl-0 5-090/src/alien/oxygen/src/:$PATH

[andrewjpage]

If you type 'mcl' what do you get? Andrew

…

On 22 Aug 2017 5:09 am, "bhclement" ***@***.***> wrote: Roary did not recognize the local path I export: export PATH=/projects3/giardia/TB_pilot/4Dec2016/Spade_genomes/Roary_data/mcl-0 5-090/src/alien/oxygen/src/:$PATH — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#341 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AABeV64X3wcK0NKBxFrBIIhX161JuY4Mks5salSMgaJpZM4O789L> .

[andrewjpage]

Also '*mclblastline'*

…

On 22 Aug 2017 7:46 am, "Andrew Page" ***@***.***> wrote: If you type 'mcl' what do you get? Andrew On 22 Aug 2017 5:09 am, "bhclement" ***@***.***> wrote: > Roary did not recognize the local path I export: > > export PATH=/projects3/giardia/TB_pilot/4Dec2016/Spade_genomes/Roary_data/mcl-0 > 5-090/src/alien/oxygen/src/:$PATH > > — > You are receiving this because you are subscribed to this thread. > Reply to this email directly, view it on GitHub > <#341 (comment)>, > or mute the thread > <https://github.com/notifications/unsubscribe-auth/AABeV64X3wcK0NKBxFrBIIhX161JuY4Mks5salSMgaJpZM4O789L> > . >

[bhclement]

[ctsui@grl-salk 4Dec2016]$ mcl
-bash: mcl: command not found
[ctsui@grl-salk 4Dec2016]$ mclblastline
/usr/bin/env: perl: No such file or directory
[ctsui@grl-salk 4Dec2016]$

[bhclement]

So I did not install mcl properly?

[andrewjpage]

There does appear to be a problem with mcl. Is that space character in the PATH intentional or a typo?
Andrew

[bhclement]

which space?

[tseemann]

@bhclement what does echo $PATH | tr ":" "\n" | nl say?

[bhclement]

 **1	/TB_pilot/4Dec2016/Spade_genomes/Roary_data/mcl-14-137/src/alien/oxygen/src/
 2	/TB_pilot/4Dec2016/Spade_genomes/Roary_data/mcl-05-090/src/alien/oxygen/src/
 3	.
 4	/opt/miniconda2/bin
 5	/usr/local/bin
 6	/bin
 7	/usr/bin
 8	/usr/local/sbin
 9	/usr/sbin
10	/sbin
11	/usr/local/rvm/bin
12	/home/ctsui/bin

any insights? Thanks.

[tseemann]

Seems you are using miniconda. Can you just conda install mcl and get rid of PATH items 1, 2, 3 ?

See https://bioconda.github.io/recipes/mcl/README.html

[bhclement]

Hello, I reinstalled mcl and it appeared to be working now.
Many thanks

[tseemann]

@bhclement that is great news! can you please close this issue now?