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Hello,

I'm having some trouble running MarkerScanner.pl I believe I have all the dependencies installed but I'm a bit lost.

AMPHORA2_home is set

Here's my error message:

$ perl /workdir/users/dcd97/home-dcd97/AMPHORA2/Scripts/MarkerScanner.pl -DNA -Bacteria genomes.fasta                                                                                                             
Error: Failed to open sequence file genomes.fasta.orf for reading                                                                                                                                                 

Can't open 5630.hmmsearch at /workdir/users/dcd97/home-dcd97/AMPHORA2/Scripts/MarkerScanner.pl line 100. 

Dear authors of Amphora,

Thank you for making this software available.

The taxonomic output format seems to not handle that some taxonomic levels are incertae sedis. This shifts all the taxonomic labels making it impossible to parse downstream. In this example the class is missing, and so the genus is shifted into the family field, the species into the genus field etc.

cat -T phylotypes.list|head
Query^IMarker^ISuperkingdom^IPhylum^IClass^IOrder^IFamily^IGenus^ISpecies
k87_338378_3^Iadk^IArchaea(1.00)^IThaumarchaeota(1.00)^INitrosopumilales(0.84)^INitrosopumilaceae(0.84)^INitrosopumilus(0.84)^INitrosopumilus maritimus(0.84)
k87_19798_2^Iadk^IArchaea(1.00)^IThaumarchaeota(1.00)^INitrosopumilales(0.82)^INitrosopumilaceae(0.82)^INitrosopumilus(0.82)^INitrosopumilus maritimus(0.82)
k87_19402_1^Iadk^IArchaea(0.97)^IThaumarchaeota(0.97)^INitrosopumilales(0.93)^INitrosopumilaceae(0.93)^INitrosopumilus(0.93)^INitrosopumilus maritimus(0.93)

Is it possible to work around this in any way?

Regards,

Asker Brejnrod

Hello,

I tried to install amphora2 today. Unfortunately during running the Phylotyping.pl script I get the following errors:

Phylotyping.pl -Method ml:

Can't call method "get_dup" on an undefined value at /opt/apps/resif/devel/v1.0-20150402/core/software/bio/BioPerl/1.6.1-ictce-5.3.0-Perl-5.16.3/lib/perl5/site_perl/5.16.3/Bio/DB/Taxonomy/flatfile.pm line 310, line 1.

In Phylotyping.pl this is probably from:
traverse($root, \%ref_taxon);(~L160)

and in sub traverse(... :
for ($node->each_Descendent) (~L205)

Phylotyping.pl -Method mp:

Cannot open RAxML_equallyParsimoniousPlacements.rplS at /home/users/mherold/TOOLS/AMPHORA2-master/Scripts/Phylotyping.pl line 108.

When I try to run the following system call from the shell, substituting the variables:
system ("$raxml -f y -t $AMPHORA_home/Marker/$marker.tre -s $marker.aln -m PROTGAMMAWAG -n $marker -p 132 1>/dev/null 2>/dev/null")

ERROR: Expecting ':' in tree; found: character ')'
08747-NC_011750:0.0)):1.4E-4,REF-YP_0023
RAxML may be expecting to read a tree that contains branch lengths
raxmlHPC: treeIO.c:1425: treeReadLen: Assertion `0' failed.
Aborted

Do you know what I could do to fix this? I thought this might be related to RAxML. I have tried versions 7.4.2 and 8.1.20, obtaining 7.3.0 was not possible anymore.
Any feedback would be appreciated.

Cheers,

Malte

Dear Martin Wu,

I recently installed Phyla_AMPHORA on the HPC cluster of ETH Zurich. Then one of our users reported problems when running the script Phylotyping.pl, because it crashed with the following error:

[samapps@euler01 ~]$ module load gcc/4.8.2 gdc raxml/8.2.4 hmmer/3.1 emboss/6.5.7 perl/5.18.4 phyla_amphora
Now run 'perl-init' to initialize your environment for Perl
[samapps@euler01 ~]$ perl-init
[samapps@euler01 ~]$ Phylotyping.pl 

------------- EXCEPTION: Bio::Root::Exception -------------
MSG: Cannot open file '/cluster/apps/gdc/phyla_amphora/Taxonomy/nodes': Permission denied
STACK: Error::throw
STACK: Bio::Root::Root::throw /cluster/apps/gdc/perl5/perls/perl-5.18.4/lib/site_perl/5.18.4/Bio/Root/Root.pm:449
STACK: Bio::DB::Taxonomy::flatfile::_build_index /cluster/apps/gdc/perl5/perls/perl-5.18.4/lib/site_perl/5.18.4/Bio/DB/Taxonomy/flatfile.pm:370
STACK: Bio::DB::Taxonomy::flatfile::new /cluster/apps/gdc/perl5/perls/perl-5.18.4/lib/site_perl/5.18.4/Bio/DB/Taxonomy/flatfile.pm:138
STACK: Bio::DB::Taxonomy::new /cluster/apps/gdc/perl5/perls/perl-5.18.4/lib/site_perl/5.18.4/Bio/DB/Taxonomy.pm:119
STACK: /cluster/apps/gdc/phyla_amphora/Scripts/Phylotyping.pl:59
-----------------------------------------------------------
[samapps@euler01 ~]$ 

After investigating the problem, I figured out that the program tries to write files into the directory $Phyla_AMPHORA_home/Taxonomy, which is part of the installation directory of the software. On a HPC cluster, this is a problem. We strictly separate installation directories from the data, that is created during a job. The installation directory is owned by a technical user, and the users running jobs on the cluster do not have write permission in any of the software installation directories.

Is there a way to redirect the files "nodes", "parents", "id2names" and "names2id" to a different directory in user space ?

Re: the Bioperl issue, if you can provide a test case or file a bug regarding the errors you have we can try to address them. Thanks!

Hi Martin,

I was using AMPHORA2 to determine the taxonomy of genomes in the Candidate phyla radiation and DPANN archaea but seems like the database is not up to date? How could I update the database?

Cheers

Alan