# Humann can not run with metaphlan together **URL:** \ **Category:** HUMAnN\ **Created:** [July 26, 2020, 1:08pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774 "2020-07-26T13:08:33Z")\ **Posts on this page:** 20\ **Page:** 1
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 26, 2020, 1:08pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/1 "2020-07-26T13:08:33Z")
hi, I installed metaphlan whose database in /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan\_databases/. When I run humann like following: (py37) [zhangwenping@localhost try\_humann]$ humann --input /data/liying\_metagenome/clean\_data\_liying/SRR5130527\_paired.1.fastq --output try\_SRR5130527\_paired.1 --metaphlan /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan Output files will be written to: /data/try\_humann/try\_SRR5130527\_paired.1 Removing spaces from identifiers in input file … WARNING: Can not call software version for bowtie2 Running metaphlan … CRITICAL ERROR: Error executing: /data/xmjd/miniconda2/envs/py37/bin/metaphlan /data/try\_humann/try\_SRR5130527\_paired.1/SRR5130527\_paired.1\_humann\_temp/tmpbs8jnhgf/tmpf8cqxgux -t rel\_ab -o /data/try\_humann/try\_SRR5130527\_paired.1/SRR5130527\_paired.1\_humann\_temp/SRR5130527\_paired.1\_metaphlan\_bugs\_list.tsv --input\_type fastq --bowtie2out /data/try\_humann/try\_SRR5130527\_paired.1/SRR5130527\_paired.1\_humann\_temp/SRR5130527\_paired.1\_metaphlan\_bowtie2.txt Error message returned from metaphlan : Downloading [https://www.dropbox.com/sh/7qze7m7g9fe2xjg/AAA4XDP85WHon\_eHvztxkamTa/file\_list.txt?dl=1](https://www.dropbox.com/sh/7qze7m7g9fe2xjg/AAA4XDP85WHon_eHvztxkamTa/file_list.txt?dl=1) Warning: Unable to download [https://www.dropbox.com/sh/7qze7m7g9fe2xjg/AAA4XDP85WHon\_eHvztxkamTa/file\_list.txt?dl=1](https://www.dropbox.com/sh/7qze7m7g9fe2xjg/AAA4XDP85WHon_eHvztxkamTa/file_list.txt?dl=1) Traceback (most recent call last): File “/data/xmjd/miniconda2/envs/py37/bin/metaphlan”, line 10, in sys.exit(main()) File “/data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan.py”, line 916, in main pars[‘index’] = check\_and\_install\_database(pars[‘index’], pars[‘bowtie2db’], pars[‘bowtie2\_build’], pars[‘nproc’], pars[‘force\_download’]) File “/data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/ **init**.py”, line 269, in check\_and\_install\_database index = resolve\_latest\_database(bowtie2\_db, ls\_f[‘mpa\_latest’], force\_redownload\_latest) UnboundLocalError: local variable ‘ls\_f’ referenced before assignment Can you tell me which options for humann will show the database of metaphlan locate in /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan\_databases/? Thank you very much! wenping ---
**Author:** ![lauren.j.mciver](https://avatars.discourse-cdn.com/v4/letter/l/f05b48/32.png) [@lauren.j.mciver](https://forum.biobakery.org/u/lauren.j.mciver)\ **Post date:** [July 28, 2020, 7:51pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/2 "2020-07-28T19:51:39Z")
Hi Wenping, Thanks for all the info! The MetaPhlAn databases are usually installed by default in a subfolder of the MetaPhlAn executable. Sorry we don’t have an option in HUMAnN to provide the location of the MetaPhlAn database. Based on your info I think the databases would be installed in the directory you listed `/data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan_databases/` . It looks like the issue with installing the database might be resolved if you upgrade to a newer version of MetaPhlAn. Would you try upgrading the MetaPhlAn software and then rerun? If you continue to see errors please let us know. Thanks! Lauren ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 29, 2020, 4:45am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/3 "2020-07-29T04:45:29Z")
Hi Lauren, Thank you for your reply. I upgraded the metaphlan and the version is MetaPhlAn version 3.0.1 (25 Jun 2020). However, it doesn’t work and metaphlan stiil want to download the database from dropbox that is not availble for us. ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [July 29, 2020, 7:35am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/4 "2020-07-29T07:35:38Z")
If Dropbox is not accessible you can try to download the database from Zenodo, here’s the link [https://zenodo.org/record/3957592](https://zenodo.org/record/3957592). I’ve pushed a new release (3.0.2) in which the database automatically is retrieved from Zenodo if Dropbox is not reachable. ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 29, 2020, 7:54am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/5 "2020-07-29T07:54:56Z")
Hi Francesco, Thank you very much for your reply. I downloaded the database of metaphlan and metaphlan can run succesfully when showing the pathway of the database. However, no options for running humann to show the pathway of database of metaphlan. ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [July 29, 2020, 8:08am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/6 "2020-07-29T08:08:54Z")
You can use the HUMAnN `--metaphlan-options` parameter to provide additional MetaPhlAn parameters for the internal exection (e.g. `-x --bowtie2db `) ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 29, 2020, 8:27am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/7 "2020-07-29T08:27:55Z")
Hi Francesco, I run the following code, but it doesn’t work: (py37) [zhangwenping@localhost try\_humann]$ humann --input /data/liying\_metagenome/clean\_data\_liying/SRR5130527\_paired.1.fastq --output try\_SRR5130527\_paired.1 --metaphlan-options -x mpa\_v30\_CHOCOPhlAn\_201901 --bowtie2db /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan\_databases/ usage: humann [-h] [–version] [-v] [-r] [–bypass-prescreen] [–bypass-nucleotide-index] [–bypass-translated-search] [–bypass-nucleotide-search] -i \ -o [–nucleotide-database \] [–annotation-gene-index \<3\>] [–protein-database \] [–evalue \<1.0\>] [–search-mode {uniref50,uniref90}] [–metaphlan] [–metaphlan-options \] [–diamond-options \] [–bowtie-options \] [–o-log \] [–log-level {DEBUG,INFO,WARNING,ERROR,CRITICAL}] [–remove-temp-output] [–threads \<1\>] [–prescreen-threshold \<0.01\>] [–nucleotide-identity-threshold \<0.0\>] [–translated-identity-threshold \] [–translated-subject-coverage-threshold \<50.0\>] [–nucleotide-subject-coverage-threshold \<50.0\>] [–translated-query-coverage-threshold \<90.0\>] [–nucleotide-query-coverage-threshold \<90.0\>] [–bowtie2] [–usearch] [–rapsearch] [–diamond] [–taxonomic-profile \] [–id-mapping \] [–translated-alignment {usearch,rapsearch,diamond}] [–xipe {on,off}] [–minpath {on,off}] [–pick-frames {on,off}] [–gap-fill {on,off}] [–output-format {tsv,biom}] [–output-max-decimals \<10\>] [–output-basename \] [–remove-stratified-output] [–remove-column-description-output] [–input-format {fastq,fastq.gz,fasta,fasta.gz,sam,bam,blastm8,genetable,biom}] [–pathways-database \] [–pathways {metacyc,unipathway}] [–memory-use {minimum,maximum}] humann: error: argument --metaphlan-options: expected one argument ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [July 29, 2020, 8:40am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/8 "2020-07-29T08:40:47Z")
You are missing the quotes, it should be ` --metaphlan-options "-x mpa_v30_CHOCOPhlAn_201901 --bowtie2db /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan_databases/"` ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 29, 2020, 9:23am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/9 "2020-07-29T09:23:28Z")
thank you very much. It works, but another error shows as following: (py37) [zhangwenping@localhost try\_humann]$ humann --input /data/liying\_metagenome/clean\_data\_liying/SRR5130527\_paired.1.fastq --output try\_SRR5130527\_paired.1 --metaphlan-options ‘-x mpa\_v30\_CHOCOPhlAn\_201901 --bowtie2db /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan\_databases/’ Output files will be written to: /data/try\_humann/try\_SRR5130527\_paired.1 Removing spaces from identifiers in input file … WARNING: Can not call software version for bowtie2 Running metaphlan … CRITICAL ERROR: Error executing: /data/xmjd/miniconda2/envs/py37/bin/metaphlan /data/try\_humann/try\_SRR5130527\_paired.1/SRR5130527\_paired.1\_humann\_temp/tmpkcc2\_rom/tmpw83r1n2g -x mpa\_v30\_CHOCOPhlAn\_201901 --bowtie2db /data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan\_databases/ -o /data/try\_humann/try\_SRR5130527\_paired.1/SRR5130527\_paired.1\_humann\_temp/SRR5130527\_paired.1\_metaphlan\_bugs\_list.tsv --input\_type fastq --bowtie2out /data/try\_humann/try\_SRR5130527\_paired.1/SRR5130527\_paired.1\_humann\_temp/SRR5130527\_paired.1\_metaphlan\_bowtie2.txt Error message returned from metaphlan : Use of uninitialized value $bt2\_args[2] in join or string at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 423. Use of uninitialized value bt2\_args[3] in join or string at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 423. Use of uninitialized value _[2] in string eq at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 360. Use of uninitialized value $_[3] in string eq at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 360. Use of uninitialized value in exists at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 81. Use of uninitialized value in exists at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 81. Use of uninitialized value $bt2\_args[2] in join or string at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 459. Use of uninitialized value $bt2\_args[3] in join or string at /data/xmjd/miniconda2/envs/py37/bin/bowtie2 line 459. Traceback (most recent call last): File “/data/xmjd/miniconda2/envs/py37/bin/metaphlan”, line 10, in sys.exit(main()) File “/data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan.py”, line 991, in main min\_alignment\_len=pars[‘min\_alignment\_len’], read\_min\_len=pars[‘read\_min\_len’], min\_mapq\_val=pars[‘min\_mapq\_val’]) File “/data/xmjd/miniconda2/envs/py37/lib/python3.7/site-packages/metaphlan/metaphlan.py”, line 429, in run\_bowtie2 nreads = int(read\_fastx\_stderr[0]) IndexError: list index out of range ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [July 29, 2020, 3:25pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/10 "2020-07-29T15:25:17Z")
Could you run `read_fastx.py -l 70 > /dev/null` on the input fastq and see if the total number of reads is printed on stderr? ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 30, 2020, 2:44am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/11 "2020-07-30T02:44:18Z")
I run the code and no message was shown as the following: (py37) [zhangwenping@localhost clean\_data\_liying] read\_fastx.py -l 70 SRR5130527\_paired.1.fastq \> /dev/null (py37) [zhangwenping@localhost clean\_data\_liying] ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [July 30, 2020, 8:54am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/12 "2020-07-30T08:54:53Z")
Are you sure the input file is not empty and well-formed? ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 30, 2020, 9:05am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/13 "2020-07-30T09:05:04Z")
The input file is as the following: (py37) [zhangwenping@localhost clean\_data\_wuqi] read\_fastx.py -l 70 CRR055205\_paired.1.fastq \> /dev/null (py37) [zhangwenping@localhost clean\_data\_wuqi] more CRR055205\_paired.1.fastq @HWI-ST531R:260:C4GKMACXX:3:1101:1954:1955 1:N:0:GTCTG TAAAGTATACATTTATAATTAGTTAGGAGAAGAATTTATGTCGTTTTCAGC + CCCFFEDEHHHHGJJJIJJJJJHIJJJGGJIJIJJJJIIJHIJHIIJJIJI @HWI-ST531R:260:C4GKMACXX:3:1101:2131:1942 1:N:0:GTCTG TCANCAGTCATTATTGCTTCACCATGAACTTCAAAAAAATCACCCGTTG + @@@#4ADDHHHHHEIHEIJIIIJJJJJIJIJJIIJJIJJGBDFFHHAEH @HWI-ST531R:260:C4GKMACXX:3:1101:2108:1942 1:N:0:GTCTG CATNTTTATCATTAACTGGTCGAACAAGAATGTCAAATAATTGGGGATCGG + CCC#4ADFHHHHHJJJJJJHIJJJIJJJHIJJJJJJJJJJJJJJJIFGIIJ @HWI-ST531R:260:C4GKMACXX:3:1101:2487:1994 1:N:0:GTCTG GTCATAGATTAAGGCATGAAGAAAACTATTGTGAAGAAATTAATCTCAA + ?@@DFDD?CFFFFHIIIGEHIIIIGEC@HICFDGHGEGHGHIICDG\>DH @HWI-ST531R:260:C4GKMACXX:3:1101:2647:1943 1:N:0:GTCTG AGTNGTGTCGTGTGTTGAGGGTGTTATGGAACCTGTATTTATTGACATCTG + @@?#4=BDFFFFBGFFEEGII1CCFEGEIDFFIIFDEGIICGIIFFIEFII @HWI-ST531R:260:C4GKMACXX:3:1101:2676:1948 1:N:0:GTCTG CCGNTTGGTACAATAATTCATACAGGAGATTTTAAAATAGACTATACTCCA + @@C#4ADDBFHHHJJJJJIJDHJJJJIIFHGIJIIIJEEGHIHIJIJJIJJ @HWI-ST531R:260:C4GKMACXX:3:1101:2694:1962 1:N:0:GTCTG TTTAAGACAAATTCAAGATGGGATAGAGCTATTTATACTTGGACTAGGAAG + CCCFFFFFHHHHHJJIJIJJJJIJIJJJJJJJJJIGJJJJJJJJJIJJIII @HWI-ST531R:260:C4GKMACXX:3:1101:2570:1982 1:N:0:GTCTG CTATATCATTAAATCCATCCTTATTGAGAGTTTCTATGATTGCTGCAAAAACTTC + CCCFDFDFHHFHHIIHIIIGIJIHHJHIHIAHHGIGGIJIJIJJJJJIIHJJIJJ (py37) [zhangwenping@localhost clean\_data\_wuqi]$ ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [July 30, 2020, 9:27am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/14 "2020-07-30T09:27:24Z")
It looks like that all the reads may be filtered since the length is below 70bp. You should try adding `--read_min_len 49 ` to `--metaphlan-options` ---
**Author:** ![wenping](https://avatars.discourse-cdn.com/v4/letter/w/e56c9b/32.png) [@wenping](https://forum.biobakery.org/u/wenping)\ **Post date:** [July 31, 2020, 4:56am UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/15 "2020-07-31T04:56:15Z")
Hi, Francesco Thank you very much, it works. ---
**Author:** ![mghanbari](https://avatars.discourse-cdn.com/v4/letter/m/3d9bf3/32.png) [@mghanbari](https://forum.biobakery.org/u/mghanbari)\ **Post date:** [December 11, 2020, 12:58pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/16 "2020-12-11T12:58:18Z")
Hi I faced a similar issue with humann and metaphlan. `humann3 --input ./MASTER_FASTQ_BC/${1}.fastq --output humann3/${1} --metaphlan-options "-unknown_estimation"` humann: error: argument --metaphlan-options: expected one argument Any idea? Regards Mehdi ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [December 11, 2020, 1:15pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/17 "2020-12-11T13:15:31Z")
Have you tried using `--metaphlan-options="-unknown_estimation"`? Also, it seems you are using ` “` instead of `"` ---
**Author:** ![mghanbari](https://avatars.discourse-cdn.com/v4/letter/m/3d9bf3/32.png) [@mghanbari](https://forum.biobakery.org/u/mghanbari)\ **Post date:** [December 11, 2020, 1:29pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/18 "2020-12-11T13:29:28Z")
Thank you for the fast reply. Your suggestion almost worked :), instead of `--metaphlan-options="-unknown_estimation"`, I used `--metaphlan-options="--unknown_estimation"` and it is working now. Regards Mehdi ---
**Author:** ![haoteng](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/haoteng/32/697_2.png) [@haoteng](https://forum.biobakery.org/u/haoteng)\ **Post date:** [March 26, 2021, 5:04pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/19 "2021-03-26T17:04:49Z")
Hello, I use the command below. However, it didn’t work. human … --metaphlan-options="-t=rel\_ab\_w\_read\_stats" --metaphlan-options="–read\_min\_len=30" Are there any solutions? Thank you! ---
**Author:** ![lauren.j.mciver](https://avatars.discourse-cdn.com/v4/letter/l/f05b48/32.png) [@lauren.j.mciver](https://forum.biobakery.org/u/lauren.j.mciver)\ **Post date:** [March 26, 2021, 8:37pm UTC](https://forum.biobakery.org/t/humann-can-not-run-with-metaphlan-together/774/20 "2021-03-26T20:37:26Z")
Hello, The MetaPhlAn options flag is not appending. Sorry for any confusion about this. So with your example only the final flag would be used for the run. If you would like two options for MetaPhlAn include them with a single flag `--metaphlan-options="-t rel_ab_w_read_stats --read_min_len 30"` . Please let me know if this does not resolve the issue you are seeing. Thank you, Lauren