# PhyloPhlAn3: muscle returned non-zero exit status 1

**URL:** <https://forum.biobakery.org/t/phylophlan3-muscle-returned-non-zero-exit-status-1/5270>\
**Category:** PhyloPhlAn\
**Created:** [May 12, 2023, 2:36pm UTC](https://forum.biobakery.org/t/phylophlan3-muscle-returned-non-zero-exit-status-1/5270 "2023-05-12T14:36:03Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![Anderson1](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/anderson1/32/3041_2.png) [@Anderson1](https://forum.biobakery.org/u/Anderson1)\
**Post date:** [May 12, 2023, 2:36pm UTC](https://forum.biobakery.org/t/phylophlan3-muscle-returned-non-zero-exit-status-1/5270/1 "2023-05-12T14:36:03Z")

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When I ran the program, the following error occurred.

Here is my log file：  
##################################################################################  
PhyloPhlAn version 3.0.67 (24 August 2022)

Command line: /share/home/wengenz/anaconda3/envs/phylophlan/bin/phylophlan  
-i /share/home/wengenz/GIWMD/phylo/phoD\_genome/  
–genome\_extension fa  
-d phylophlan\_databases/phylophlan/  
-f phoD\_tol\_nt.cfg  
–diversity high  
–fast  
–force\_nucleotides  
-o phoD\_output  
–nproc 20  
–verbose

Automatically setting “database=phylophlan” and “databases\_folder=/share/home/wengenz/GIWMD/phylo/phylophlan\_databases”  
Automatically setting “input=phoD\_genome” and “input\_folder=/share/home/wengenz/GIWMD/phylo”  
**[w] “/share/home/wengenz/anaconda3/envs/phylophlan/lib/python3.11/site-packages/phylophlan/phylophlan\_configs/” folder does not exists**  
Creating folder “phoD\_output”  
Creating folder “phoD\_output/tmp”  
“high-fast” preset  
Setting “sort=True” because “database=phylophlan”  
Setting “min\_num\_markers=100” since no value has been specified and the “database=phylophlan”

Arguments: {‘input’: ‘phoD\_genome’, ‘clean’: None, ‘output’: ‘phoD\_output’, ‘database’: ‘phylophlan’, ‘db\_type’: None, ‘config\_file’: ‘phoD\_tol\_nt.cfg’, ‘diversity’: ‘high’, ‘accurate’: False, ‘fast’: True, ‘clean\_all’: False, ‘database\_list’: False, ‘submat’: ‘pfasum60’, ‘submat\_list’: False, ‘submod\_list’: False, ‘nproc’: 20, ‘min\_num\_proteins’: 1, ‘min\_len\_protein’: 50, ‘min\_num\_markers’: 100, ‘trim’: ‘greedy’, ‘gap\_perc\_threshold’: 0.67, ‘not\_variant\_threshold’: 0.9, ‘subsample’: \<function phylophlan at 0x7fc0d4f2fa60\>, ‘unknown\_fraction’: 0.3, ‘scoring\_function’: \<function trident at 0x7fc0d4f30180\>, ‘sort’: True, ‘remove\_fragmentary\_entries’: False, ‘fragmentary\_threshold’: 0.67, ‘min\_num\_entries’: 4, ‘maas’: None, ‘remove\_only\_gaps\_entries’: False, ‘mutation\_rates’: False, ‘force\_nucleotides’: True, ‘convert\_N2gap’: False, ‘input\_folder’: ‘/share/home/wengenz/GIWMD/phylo/phoD\_genome’, ‘data\_folder’: ‘phoD\_output/tmp’, ‘databases\_folder’: ‘/share/home/wengenz/GIWMD/phylo/phylophlan\_databases’, ‘submat\_folder’: ‘/share/home/wengenz/anaconda3/envs/phylophlan/lib/python3.11/site-packages/phylophlan/phylophlan\_substitution\_matrices/’, ‘submod\_folder’: ‘/share/home/wengenz/anaconda3/envs/phylophlan/lib/python3.11/site-packages/phylophlan/phylophlan\_substitution\_models/’, ‘configs\_folder’: None, ‘output\_folder’: ‘’, ‘genome\_extension’: ‘.fa’, ‘proteome\_extension’: ‘.faa’, ‘update’: False, ‘verbose’: True}

Loading configuration file “phoD\_tol\_nt.cfg”  
Checking configuration file  
Checking “/share/home/wengenz/anaconda3/envs/phylophlan/bin/diamond”  
Checking “/share/home/wengenz/anaconda3/envs/phylophlan/bin/muscle”  
Checking “/share/home/wengenz/anaconda3/envs/phylophlan/bin/trimal”  
Checking “/share/home/wengenz/anaconda3/envs/phylophlan/bin/iqtree”  
Checking “/share/home/wengenz/anaconda3/envs/phylophlan/bin/raxmlHPC-PTHREADS-SSE3”  
“db\_aa” database “/share/home/wengenz/GIWMD/phylo/phylophlan\_databases/phylophlan/phylophlan.dmnd” present  
Loading files from “/share/home/wengenz/GIWMD/phylo/phoD\_genome”  
Creating folder “phoD\_output/tmp/clean\_dna”

**[e] Command ‘[’/share/home/wengenz/anaconda3/envs/phylophlan/bin/muscle’, ‘-quiet’, ‘-maxiters’, ‘2’, ‘-in’, ‘phoD\_output/tmp/markers/p0276.fa’, ‘-out’, ‘/share/home/wengenz/GIWMD/phylo/phoD\_output/tmp/msas/p0276.aln’]’ returned non-zero  
\*\*\*\* exit status 1**.\*\*  
##################################################################################

This bug has been bothering me for a long time. Would someone kindly help to answer it? Thank you!

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**Author:** ![f.asnicar](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/f.asnicar/32/196_2.png) [@f.asnicar](https://forum.biobakery.org/u/f.asnicar)\
**Post date:** [August 28, 2023, 9:07am UTC](https://forum.biobakery.org/t/phylophlan3-muscle-returned-non-zero-exit-status-1/5270/2 "2023-08-28T09:07:53Z")

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Hi there and thanks for using PhyloPhlAn!

Have you tried running the single MUSCLE command without the `-quite` param? This will allow us to better understand what’s causing the problem.

```auto
/share/home/wengenz/anaconda3/envs/phylophlan/bin/muscle -maxiters 2 -in phoD_output/tmp/markers/p0276.fa -out /share/home/wengenz/GIWMD/phylo/phoD_output/tmp/msas/p0276.aln

```

Thanks a lot,  
Francesco