# Humann3 running indefinitely! **URL:** \ **Category:** HUMAnN\ **Created:** [July 25, 2024, 5:24pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317 "2024-07-25T17:24:27Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![mashuk-utoledo](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mashuk-utoledo/32/1645_2.png) [@mashuk-utoledo](https://forum.biobakery.org/u/mashuk-utoledo)\ **Post date:** [July 25, 2024, 5:24pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/1 "2024-07-25T17:24:27Z")
Hi, I am using Hunmann3 on Linux computer for analyzing metagenomic sequence and it keeps running metaphlan indefinitely and no output is coming I am not sure if there was something wrong with the settings or database folder. I would appreciate your suggestions on this issue. ## Thanks in advance Mashuk I have copied the command, database folder detail, human configuration, and log of the running commands below: 1. Here is the command: (mpa)msiddiq7@enggpz1p23:/media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq$ humann --input 86-ELS\_SME\_L001\_R1\_001.fastq.gz --output MG24\_ur50/USACE\_86-ELS\_R1\_uniref50 --bypass-translated-search Creating output directory: /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50 Output files will be written to: /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50 Decompressing gzipped file … Removing spaces from identifiers in input file … Running metaphlan … * * * 1. Database folder details: (base) msiddiq7@enggpz1p23:~/humann\_dbs$ ls chocophlan uniref50\_201901b\_ec\_filtered.dmnd uniref uniref90\_201901b\_full.dmnd (base) msiddiq7@enggpz1p23:~/humann\_dbs/uniref$ ls uniref50\_201901b\_full.dmnd (base) msiddiq7@enggpz1p23:~/humann\_dbs/uniref$ ## (base) msiddiq7@enggpz1p23:~/utility\_mapping$ ls map\_ec\_name.txt.gz map\_level4ec\_uniref50.txt.gz map\_eggnog\_name.txt.gz map\_level4ec\_uniref90.txt.gz map\_eggnog\_uniref50.txt.gz map\_pfam\_name.txt.gz map\_eggnog\_uniref90.txt.gz map\_pfam\_uniref50.txt.gz map\_go\_name.txt.gz map\_pfam\_uniref90.txt.gz map\_go\_uniref50.txt.gz map\_uniref50\_name.txt.bz2 map\_go\_uniref90.txt.gz map\_uniref50\_uniref90.txt.gz map\_ko\_name.txt.gz map\_uniref90\_name.txt.bz2 map\_ko\_uniref50.txt.gz uniref50-tol-lca.dat.bz2 map\_ko\_uniref90.txt.gz uniref90-tol-lca.dat.bz2 (base) msiddiq7@enggpz1p23:~/utility\_mapping$ 1. Humann3 settings (configuration) detail: (mpa) msiddiq7@enggpz1p23:~$ humann\_config HUMAnN Configuration ( Section : Name = Value ) database\_folders : nucleotide = /home/utad/msiddiq7/humann\_dbs/chocophlan database\_folders : protein = /home/utad/msiddiq7/humann\_dbs/uniref database\_folders : utility\_mapping = /home/utad/msiddiq7/utility\_mapping run\_modes : resume = False run\_modes : verbose = False run\_modes : bypass\_prescreen = False run\_modes : bypass\_nucleotide\_index = False run\_modes : bypass\_nucleotide\_search = False run\_modes : bypass\_translated\_search = False run\_modes : threads = 1 alignment\_settings : evalue\_threshold = 1.0 alignment\_settings : prescreen\_threshold = 0.01 alignment\_settings : translated\_subject\_coverage\_threshold = 50.0 alignment\_settings : translated\_query\_coverage\_threshold = 90.0 alignment\_settings : nucleotide\_subject\_coverage\_threshold = 50.0 alignment\_settings : nucleotide\_query\_coverage\_threshold = 90.0 output\_format : output\_max\_decimals = 10 output\_format : remove\_stratified\_output = False output\_format : remove\_column\_description\_output = False ## (mpa) msiddiq7@enggpz1p23:~$ 1. Log file of the running sequence: 06/23/2024 11:29:33 PM - humann.humann - INFO: Running humann v3.0.1 06/23/2024 11:29:33 PM - humann.humann - INFO: Output files will be written to: /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50 06/23/2024 11:29:33 PM - humann.humann - INFO: Writing temp files to directory: /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50/86-ELS\_SME\_L001\_R1\_001\_humann\_temp 06/23/2024 11:29:33 PM - humann.utilities - INFO: File ( /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/86-ELS\_SME\_L001\_R1\_001.fastq.gz ) is of format: fastq.gz 06/23/2024 11:29:33 PM - humann.utilities - INFO: Decompressing gzipped file … 06/23/2024 11:37:46 PM - humann.humann - INFO: Removing spaces from identifiers in input file 06/23/2024 11:46:33 PM - humann.utilities - DEBUG: Check software, metaphlan, for required version, 3.0 06/23/2024 11:46:35 PM - humann.utilities - INFO: Using metaphlan version 3.0 06/23/2024 11:46:35 PM - humann.utilities - DEBUG: Check software, bowtie2, for required version, 2.2 06/23/2024 11:46:35 PM - humann.utilities - INFO: Using bowtie2 version 2.3 06/23/2024 11:46:35 PM - humann.config - INFO: Run config settings: DATABASE SETTINGS nucleotide database folder = /home/utad/msiddiq7/humann\_dbs/chocophlan protein database folder = /home/utad/msiddiq7/humann\_dbs/uniref pathways database file 1 = /home/utad/msiddiq7/.local/lib/python3.7/site-packages/humann/data/pathways/metacyc\_reactions\_level4ec\_only.uniref.bz2 pathways database file 2 = /home/utad/msiddiq7/.local/lib/python3.7/site-packages/humann/data/pathways/metacyc\_pathways\_structured\_filtered\_v24 utility mapping database folder = /home/utad/msiddiq7/utility\_mapping RUN MODES resume = False verbose = False bypass prescreen = False bypass nucleotide index = False bypass nucleotide search = False bypass translated search = True translated search = diamond threads = 1 SEARCH MODE search mode = uniref90 nucleotide identity threshold = 0.0 translated identity threshold = 80.0 ALIGNMENT SETTINGS bowtie2 options = --very-sensitive diamond options = --top 1 --outfmt 6 evalue threshold = 1.0 prescreen threshold = 0.01 translated subject coverage threshold = 50.0 translated query coverage threshold = 90.0 nucleotide subject coverage threshold = 50.0 nucleotide query coverage threshold = 90.0 PATHWAYS SETTINGS minpath = on xipe = off gap fill = on INPUT AND OUTPUT FORMATS input file format = fastq.gz output file format = tsv output max decimals = 10 remove stratified output = False remove column description output = False log level = DEBUG 06/23/2024 11:46:35 PM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use 06/23/2024 11:46:35 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use 06/23/2024 11:46:53 PM - humann.humann - INFO: Load pathways database part 1: /home/utad/msiddiq7/.local/lib/python3.7/site-packages/humann/data/pathways/metacyc\_reactions\_level4ec\_only.uniref.bz2 06/23/2024 11:46:53 PM - humann.humann - INFO: Load pathways database part 2: /home/utad/msiddiq7/.local/lib/python3.7/site-packages/humann/data/pathways/metacyc\_pathways\_structured\_filtered\_v24 06/23/2024 11:46:53 PM - humann.search.prescreen - INFO: Running metaphlan … 06/23/2024 11:46:53 PM - humann.utilities - DEBUG: Using software: /opt/miniconda3/envs/mpa/bin/metaphlan 06/23/2024 11:46:53 PM - humann.utilities - INFO: Execute command: /opt/miniconda3/envs/mpa/bin/metaphlan /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50/86-ELS\_SME\_L001\_R1\_001\_humann\_temp/tmpo6zw2o4m/tmp884uw5gi -t rel\_ab -o /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50/86-ELS\_SME\_L001\_R1\_001\_humann\_temp/86-ELS\_SME\_L001\_R1\_001\_metaphlan\_bugs\_list.tsv --input\_type fastq --bowtie2out /media/msiddiq7/KangLab1/ASU\_FTP\_Server\_Kang/MyKL2023/fastq/MG24\_ur50/USACE\_86-ELS\_R1\_uniref50/86-ELS\_SME\_L001\_R1\_001\_humann\_temp/86-ELS\_SME\_L001\_R1\_001\_metaphlan\_bowtie2.txt * * * ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [August 2, 2024, 7:59pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/2 "2024-08-02T19:59:39Z")
First, I would definitely upgrade to a newer HUMAnN 3.x, as we’ve made some important bugfixes along the 3.0 branch. Second, if it feels like you’re getting stuck running MetaPhlAn, I would try a MetaPhlAn run on a sample outside of HUMAnN and see how that goes. The first time you run MetaPhlAn it does some downloading / indexing of its database. It’s possible that something is getting stuck in that process. ---
**Author:** ![mashuk-utoledo](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mashuk-utoledo/32/1645_2.png) [@mashuk-utoledo](https://forum.biobakery.org/u/mashuk-utoledo)\ **Post date:** [August 3, 2024, 4:16pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/3 "2024-08-03T16:16:29Z")
Thank you a lot! Is it possible to suggest some commands that could work? ---
**Author:** ![Alex\_Grier](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/alex_grier/32/1922_2.png) [@Alex\_Grier](https://forum.biobakery.org/u/Alex_Grier)\ **Post date:** [August 27, 2024, 10:59pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/4 "2024-08-27T22:59:59Z")
I’ve been dealing with this problem for the past week or so and found the solution. The problem is that metaphlan checks online to see if you have the most recent version of the database installed. If you do not, it asks if you want to install it. When running within humann3, it does not prompt the user to respond to this question, so it just sits there unanswered and nothing happens. In your case, the solution is to run metaphlan by itself, using basically the same command found in your log: /opt/miniconda3/envs/mpa/bin/metaphlan /path/to/my/input.fastq -t rel\_ab -o /path/to/my/output/metaphlan\_bugs\_list.tsv --input\_type fastq --bowtie2out /path/to/my/output/metaphlan\_bowtie2.txt You will be prompted to download the new database. Respond Y. Then retry running humann3 and it should work fine. If you want to continue using this version of the database (even if new ones come out), you want to include the following argument in the humann command: –metaphlan-options “-x mpa\_vJun23\_CHOCOPhlAnSGB\_202403” For everyone else, you can repeat the commands above to get the new database. If you want to intentionally use an older database version, add the following argument to humann: –metaphlan-options “–bowtie2db /path/to/metaphlan/db -x MY-INDEX” where MY-INDEX will be something like mpa\_vOct22\_CHOCOPhlAnSGB\_202212. That should do the trick. ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [August 29, 2024, 3:22pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/5 "2024-08-29T15:22:42Z")
I checked in with the MetaPhlAn developers about this and they informed me that MetaPhlAn has an `--offline` flag that will prevent this behavior when running in a grid context (for example). Adding that to your `--metaphlan-options` should solve this problem when MetaPhlAn is called inside of HUMAnN. We’ll likely make this a default in a future HUMAnN release to avoid users needing to know about it ahead of time. ---
**Author:** ![mashuk-utoledo](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mashuk-utoledo/32/1645_2.png) [@mashuk-utoledo](https://forum.biobakery.org/u/mashuk-utoledo)\ **Post date:** [August 29, 2024, 4:48pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/6 "2024-08-29T16:48:19Z")
Thank you both @Alex_Grier and @franzosa ! ---
**Author:** ![Alex\_Grier](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/alex_grier/32/1922_2.png) [@Alex\_Grier](https://forum.biobakery.org/u/Alex_Grier)\ **Post date:** [August 29, 2024, 7:31pm UTC](https://forum.biobakery.org/t/humann3-running-indefinitely/7317/7 "2024-08-29T19:31:32Z")
I’m not sure if this is universally the case, but I did try passing `"--offline"` to `--metaphlan-options` and I got an error that humann couldn’t find the metaphlan\_bugs\_list.tsv file after the metaphlan step. Specifying the location of my installed metaphlan database and the index with `--metaphlan-options` `--bowtie2db` and `-x` fixed everything though.