The bioBakery help forum

CRITICAL ERROR: Can not find file metaphlan_bugs_list.tsv

Hi,
I have installed HUMAnN3 via Conda in a cluster, however, when I try to run it I get the following error message:

CRITICAL ERROR: Can not find file name_of_fastq_file_metaphlan_bugs_list.tsv

I can not find the cause of this error, do you know what’s happening and how to solve it?. Thanks in advance for your help.

Hello, Are you possibly providing a taxonomic profile as input? If so, is it possible the path or file name is not an exact match? If not, if you would post the command you ran along with the lead up to the error message then I can help debug.

Thank you,
Lauren

Hello, sorry for the late response. No, I am not providing a taxonomic profile as input, all input files are FASTQ files compressed in gzip. As these are paired-end files, first I join the two files using cat:

cat example_1.fq.gz example_2.fq.gz > example.fq.gz

Then I use the following command:

humann --input example.fq.gz --output out_dir --threads 16 --nucleotide-database databases/chocophlan/ --protein-database databases/uniref

I am working in a conda environment with HUMAnN and MetaPhlAn installed in it.

Thank you very much again for your help!

Hi - Thank you for the follow up! Do you see any other errors when you run? More specifically do you see any errors from MetaPhlAn? It looks like there might be an issue generating the taxonomic profile.

Thank you,
Lauren

Hello again. Yes, indeed looking at a log file, seems that the problem is that our cluster nodes don’t have network connection so MetaPhlAn is not able to download a database. As I have already downloaded the MetaPhlAn database, I was wondering if there is some way to tell HUMAnN where this database is and see if that resolves the problem. Here are the last lines of the log file, I have edited only the paths because they were very long:

02/16/2021 02:00:52 PM - humann.utilities - INFO: Execute command: /path/miniconda3/envs/metabiome-taxonomic-profiling/bin/metaphlan /path/humann3-out/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_humann_temp/tmp2t5huxit/tmpm8b2_apb -t rel_ab -o /path/humann3-out/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_humann_temp/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /path/humann3-out/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_humann_temp/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_metaphlan_bowtie2.txt
02/16/2021 02:00:53 PM - humann.utilities - DEBUG: b’WARNING: It seems that you do not have Internet access.\nERROR: Cannot find a local database. Please run MetaPhlAn using option “-x <database_name>”.\n You can download the MetaPhlAn database from \n http://cmprod1.cibio.unitn.it/biobakery3/metaphlan_databases \n https://zenodo.org/record/3957592 \n Dropbox - MetaPhlAn_databases - Simplify your life \n \n’
02/16/2021 02:00:53 PM - humann.utilities - CRITICAL: Can not find file /path/humann3-out/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_humann_temp/A102_FDSW202351806-1r_H375TDSXY_L3_paired_bt2_metaphlan_bugs_list.tsv

Thank you again for your help!

Hello, You can run MetaPhlAn with the following options to indicate the database has already been downloaded (and its location): --bowtie2db=<full/path/to/folder> --index mpa_v30_CHOCOPhlAn_201901. Then provide the taxonomic profile to your HUMAnN run with the option --taxonomic-profile <file>. Alternatively you could run HUMAnN and MetaPhlAn providing the MetaPhlAn options with --metaphlan-options=<options>.

Thanks!
Lauren

Hi, I tried to run HUMAnN with the option --metaphlan-options="--bowtie2db /path/to/database/", and I think this resolved my previous problem. However, now I am getting a different error:

Error message returned from metaphlan :
perl: warning: Setting locale failed.
perl: warning: Please check that your locale settings:
LANGUAGE = (unset),
LC_ALL = (unset),
LANG = “en_US.utf8”
are supported and installed on your system.
perl: warning: Falling back to the standard locale (“C”).
/home/juan.arboleda2/7_12_Metagenomics_training/miniconda3/envs/metabiome-humann/bin/bowtie2-align-s: error while loading shared libraries: libtbb.so.2: cannot open shared object file: No such file or directory
(ERR): Description of arguments failed!
Exiting now …
OSError: “Command ‘[‘bowtie2’, ‘-h’]’ returned non-zero exit status 127.”
Fatal error running BowTie2. Is BowTie2 in the system path?
WARNING: It seems that you do not have Internet access.
WARNING: Cannot connect to the database server. The latest available local database will be used.

What could be happening now? Thanks for your help!

Hi - I think MetaPhlAn also needs the database name to use the database you provided. Try adding the option --index mpa_v30_CHOCOPhlAn_201901 and I think MetaPhlAn will not try to download and install a new database.

Thank you,
Lauren

Hi, I’ve just tried your suggestion but it seems that now there’s some problem with Bowtie2:

Error message returned from metaphlan :
perl: warning: Setting locale failed.
perl: warning: Please check that your locale settings:
LANGUAGE = (unset),
LC_ALL = (unset),
LANG = “en_US.utf8”
are supported and installed on your system.
perl: warning: Falling back to the standard locale (“C”).
/home/juan.arboleda2/7_12_Metagenomics_training/miniconda3/envs/metabiome-humann/bin/bowtie2-align-s: error while loading shared libraries: libtbb.so.2: cannot open shared object file: No such file or directory
(ERR): Description of arguments failed!
Exiting now …
OSError: “Command ‘[‘bowtie2’, ‘-h’]’ returned non-zero exit status 127.”
Fatal error running BowTie2. Is BowTie2 in the system path?

Moreover, if I try to run bowtie2 --help inside the conda environment I get a similar error message:

/home/juan.arboleda2/7_12_Metagenomics_training/miniconda3/envs/metabiome-humann/bin/bowtie2-align-s: error while loading shared libraries: libtbb.so.2: cannot open shared object file: No such file or directory
(ERR): Description of arguments failed!
Exiting now …

I found here that installing the Tbb library could solve the problem, however since I am not sudoer I installed it with conda install tbb. After that it seems that Bowtie2 is working properly. I ran HUMAnN again and now it seems to be working fine, however the job has not ended yet, when it ends I will tell you if it was successful.

Thanks a lot for your help!