Phylophlan: error: unrecognized arguments: --strainphlan

Microbial community profiling PhyloPhlAn
1

Hello,

I’m trying to run StrainPhlan from (MetaPhlan version 4.1.0 (23 Aug 2023), installed via conda) using the following command:

strainphlan -s consensus_markers/*.json.bz2 -m db_markers/t__SGB4608.fna -r reference/RT/GCF_000153925.1_ASM15392v1_genomic.fna -o output -n 8 -c t__SGB4608 --mutation_rates -d metaphlan_databases/mpa_vJan21_CHOCOPhlAnSGB_202103.pkl

The output is as follows:

Tue Oct 10 19:25:36 2023: Start StrainPhlAn 4.1.0 execution
Tue Oct 10 19:25:36 2023: Loading MetaPhlAn mpa_vJan21_CHOCOPhlAnSGB_202103 database…
Tue Oct 10 19:25:55 2023: Done.
Tue Oct 10 19:25:55 2023: Creating temporary directory…
Tue Oct 10 19:25:55 2023: Done.
Tue Oct 10 19:25:55 2023: Filtering markers and samples…
Tue Oct 10 19:25:55 2023: Getting markers from samples…
Tue Oct 10 19:26:00 2023: Done.
Tue Oct 10 19:26:00 2023: Getting markers from references…
Tue Oct 10 19:26:01 2023: Done.
Tue Oct 10 19:26:01 2023: Removing bad markers / samples…
Tue Oct 10 19:26:01 2023: Done.
Tue Oct 10 19:26:01 2023: Done.
Tue Oct 10 19:26:01 2023: Writing samples as markers’ FASTA files…
Tue Oct 10 19:26:02 2023: Done.
Tue Oct 10 19:26:02 2023: Calculating polymorphic rates…
Tue Oct 10 19:26:06 2023: Done.
Tue Oct 10 19:26:06 2023: Computing phylogeny…
Tue Oct 10 19:26:06 2023:
Tue Oct 10 19:26:06 2023:
Tue Oct 10 19:26:06 2023:

stdout:

stderr:
usage: phylophlan [-h] [-i INPUT | -c CLEAN] [-o OUTPUT] [-d DATABASE]
[-t {n,a}] [-f CONFIG_FILE] --diversity {low,medium,high}
[–accurate | --fast] [–clean_all] [–database_list]
[-s SUBMAT] [–submat_list] [–submod_list] [–nproc NPROC]
[–min_num_proteins MIN_NUM_PROTEINS]
[–min_len_protein MIN_LEN_PROTEIN]
[–min_num_markers MIN_NUM_MARKERS]
[–trim {gap_trim,gap_perc,not_variant,greedy}]
[–gap_perc_threshold GAP_PERC_THRESHOLD]
[–not_variant_threshold NOT_VARIANT_THRESHOLD]
[–subsample {phylophlan,onethousand,sevenhundred,fivehundred,threehundred,onehundred,fifty,twentyfive,tenpercent,twentyfivepercent,fiftypercent,full}]
[–unknown_fraction UNKNOWN_FRACTION]
[–scoring_function {trident,muscle,random}] [–sort]
[–remove_fragmentary_entries]
[–fragmentary_threshold FRAGMENTARY_THRESHOLD]
[–min_num_entries MIN_NUM_ENTRIES] [–maas MAAS]
[–remove_only_gaps_entries] [–mutation_rates]
[–force_nucleotides] [–input_folder INPUT_FOLDER]
[–data_folder DATA_FOLDER]
[–databases_folder DATABASES_FOLDER]
[–submat_folder SUBMAT_FOLDER]
[–submod_folder SUBMOD_FOLDER]
[–configs_folder CONFIGS_FOLDER]
[–output_folder OUTPUT_FOLDER]
[–genome_extension GENOME_EXTENSION]
[–proteome_extension PROTEOME_EXTENSION] [–update]
[–citation] [–verbose] [-v]
phylophlan: error: unrecognized arguments: --strainphlan

It is halting at the PhyloPhlan step because it doesn’t recognise the “–strainphlan” argument… but I am not sure where this is coming in.

The output directory contains the following files:

phylophlan.cfg
reference_markers/GCF_000153925.1_ASM15392v1_genomic.fna.bz2
t__SGB4608.StrainPhlAn4/sample1.fastq.fna.bz2
t__SGB4608.StrainPhlAn4/sample2.fastq.fna.bz2
t__SGB4608.StrainPhlAn4/sample3.fastq.fna.bz2

Finally, the phylophlan config file looks as below:

[db_dna]
program_name = /home/users/egrant/miniconda3/envs/biobakery3/bin/makeblastdb
params = -parse_seqids -dbtype nucl
input = -in
output = -out
version = -version
command_line = #program_name# #params# #input# #output#

[map_dna]
program_name = /home/users/egrant/miniconda3/envs/biobakery3/bin/blastn
params = -outfmt 6 -evalue 0.1 -max_target_seqs 1000000 -perc_identity 75
input = -query
database = -db
output = -out
version = -version
command_line = #program_name# #params# #input# #database# #output#

[msa]
program_name = /home/users/egrant/miniconda3/envs/biobakery3/bin/mafft
params = --quiet --anysymbol --thread 1 --auto
version = --version
command_line = #program_name# #params# #input# > #output#
environment = TMPDIR=/tmp

[trim]
program_name = /home/users/egrant/miniconda3/envs/biobakery3/bin/trimal
params = -gappyout
input = -in
output = -out
version = --version
command_line = #program_name# #params# #input# #output#

[tree1]
program_name = /home/users/egrant/miniconda3/envs/biobakery3/bin/raxmlHPC-PTHREADS-SSE3
params = -p 1989 -m GTRCAT
input = -s
output_path = -w
output = -n
version = -v
command_line = #program_name# #params# #threads# #output_path# #input# #output#
threads = -T

I am a bit stumped and can’t find any similar errors reported. Any ideas as to why I am getting this odd error or how to troubleshoot would be much appreciated!

2

Hello @erica5, it might be due to a problem with the conda installation. Have you tried re-installing MetaPhlAn/StrainPhlAn to see if the problem persist?

Thanks,
Francesco

3

Hi @erica5 ,
Dear @f.asnicar ,

I got the same error message as you/Erica.
“phylophlan: error: unrecognized arguments: --strainphlan”
Erica, did you solve it ? Any tips would be appreciated as I didn’t see any other discussion with this error message.

I am working on an HPC cluster with MetaPhlAn 4.1.1 and PhyloPhlAn version 3.0.67.
Full error message below.

Best wishes,

Laure

Wed Jul 17 10:59:34 2024: Generating PhyloPhlAn configuration file…
Wed Jul 17 10:59:35 2024: Done.
Wed Jul 17 10:59:35 2024: Executing PhyloPhlAn…Wed Jul 17 10:59:37 2024: [Error] Execution failed for command phylophlan -i /storage/research/ldri/mnut-lb/shared/Laure/Metagenomics/metagenomics_MicroAML/6_tracking_strainphlan/output/tmp3m7skeom/t__SGB5082.StrainPhlAn4 -o . --output_folder /storage/research/ldri/mnut-lb/shared/Laure/Metagenomics/metagenomics_MicroAML/6_tracking_strainphlan/output --nproc 16 --strainphlan --fast --data_folder /storage/research/ldri/mnut-lb/shared/Laure/Metagenomics/metagenomics_MicroAML/6_tracking_strainphlan/output/tmp3m7skeom -f /storage/research/ldri/mnut-lb/shared/Laure/Metagenomics/metagenomics_MicroAML/6_tracking_strainphlan/output/tmp3m7skeom/phylophlan.cfg -t n --diversity low --genome_extension fna --min_num_entries 1 --min_num_markers 1 --fragmentary_threshold 1.0 --not_variant_threshold 1.0 --gap_perc_threshold 0.8 --mutation_ratesWed Jul 17 10:59:37 2024: [Error] stdout:
Wed Jul 17 10:59:37 2024: [Error] stderr:
usage: phylophlan [-h] [-i INPUT | -c CLEAN] [-o OUTPUT] [-d DATABASE]
[-t {n,a}] [-f CONFIG_FILE] --diversity {low,medium,high}
[–accurate | --fast] [–clean_all] [–database_list]
[-s SUBMAT] [–submat_list] [–submod_list] [–nproc NPROC]
[–min_num_proteins MIN_NUM_PROTEINS]
[–min_len_protein MIN_LEN_PROTEIN]
[–min_num_markers MIN_NUM_MARKERS]
[–trim {gap_trim,gap_perc,not_variant,greedy}]
[–gap_perc_threshold GAP_PERC_THRESHOLD]
[–not_variant_threshold NOT_VARIANT_THRESHOLD]
[–subsample {phylophlan,onethousand,sevenhundred,fivehundred,threehundred,onehundred,fifty,twentyfive,tenpercent,twentyfivepercent,fiftypercent,full}]
[–unknown_fraction UNKNOWN_FRACTION]
[–scoring_function {trident,muscle,random}] [–sort]
[–remove_fragmentary_entries]
[–fragmentary_threshold FRAGMENTARY_THRESHOLD]
[–min_num_entries MIN_NUM_ENTRIES] [–maas MAAS]
[–remove_only_gaps_entries] [–mutation_rates]
[–force_nucleotides] [–convert_N2gap]
[–input_folder INPUT_FOLDER] [–data_folder DATA_FOLDER]
[–databases_folder DATABASES_FOLDER]
[–submat_folder SUBMAT_FOLDER]
[–submod_folder SUBMOD_FOLDER]
[–configs_folder CONFIGS_FOLDER]
[–output_folder OUTPUT_FOLDER]
[–genome_extension GENOME_EXTENSION]
[–proteome_extension PROTEOME_EXTENSION] [–update]
[–citation] [–verbose] [-v]
phylophlan: error: unrecognized arguments: --strainphlan
Wed Jul 17 10:59:37 2024: [Error] ExitingWed Jul 17 10:59:37 2024: Stop execution.

4

Hi Laure (and also Francesco–apologies for the late reply), unfortunately I set this aside and haven’t come back to it so can’t confirm whether the issue would be resolved with a reinstall of MetaPhlAn/StrainPhlAn. I suppose trying this would be a good start if you haven’t already, but would be very interested in hearing whether/how you resolve this error, as I will have to confront it eventually :wink: Good luck!

1 Like
5

Hi Erica,

Thanks for the swift reply. I am working with modules on a HPC cluster. Metaphlan4.1.1 module was generated with easybuild (the module was already available on github). So I didn’t ask our HPC cluster admin to reinstall it. I launched the same command line (strainphlan -s …) with Metaphlan4.1.0 and I got the same error message (phylophlan: error: unrecognized arguments: --strainphlan).

I am trying now to launch phylophlan from the temp folder generated by the strainphlan command. I will let you know how this works.

Hi Francesco,

If you have any other suggestions, I would be very greatful.

Best wishes,

Laure

6

Hi all, the next PhyloPhlAn version brings that parameter (ver 3.0.68), so I would say that re-installing will solve the issue. If you cannot/it’s not easy to re-install MetaPhlAn/StrainPhlAn, you can install the latest PhyloPhlAn version and then make sure that StrainPhlAn points to the “right” PhyloPhlAn and not the one (the older version) that you have in your system.

I hope this helps,
Francesco