Error on alignment of extracted BUSCO genes

[MattB64]

Dear Edgardo,
I have been using CAPTUS effectively for about a year, but have suddnely encountered an error when trying to align from BUSCO extractions (Basidiomycota, so ~3500 BUSCO genes). The same problem occurs attempting to re-run previous ectracted files that have worked before. I am using a Mac with Apple M3 chip. I tried re-installing with the latest CAPTUS version, but am still seeing the same error reproduced below. Since MUSCLE is mentioned in the error log I tried to use the MAFFT option, but still get the same error message.

I'd appreciate any suggestion as to what the cause could be.

Mant thanks,
Matt

captus align -e 03_extractions

Starting Captus-assembly: ALIGN (2025-05-15 00:54:52)
Welcome to the alignment step of Captus-assembly. In this step, Captus will
collect all the extracted markers across all samples in '03_extractions' and
group them by marker. Then Captus will align each marker using MAFFT or MUSCLE.
If given, the reference loci are also included as alignment guides and removed
after alignment
For more information, please see https://github.com/edgardomortiz/Captus

        Captus version: v1.4.0
               Command: /opt/miniconda3/envs/captus/bin/captus align -e 03_extractions
                    OS: macOS-14.4.1-x86_64-i386-64bit
                  Host: P2LTJX3WR5
              Max. RAM: 15.8GB (out of 16.0GB)
          Max. Threads: 8 (out of 8)

          Dependencies:

Traceback (most recent call last):
File "/opt/miniconda3/envs/captus/bin/captus", line 10, in
sys.exit(main())
^^^^^^
File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/captus_assembly.py", line 1547, in main
CaptusAssembly()
File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/captus_assembly.py", line 86, in init
getattr(self, args.command)()
File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/captus_assembly.py", line 1543, in align
align(full_command, args)
File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/align.py", line 92, in align
muscle_path, muscle_version, muscle_status = muscle_path_version(args.muscle_path)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/misc.py", line 561, in muscle_path_version
version = process.communicate()[0].decode().strip("\n").split()[1]
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~^^^
IndexError: list index out of range

[edgardomortiz]

Hi @MattB64 ,

My guess is that the installed copy of MUSCLE was wrongly compiled at bioconda, the usual solution is to downgrade MUSCLE to immediate previous version. To verify, what happens when you type muscle with the Captus environment activated?

Also, what was the command you used to install Captus?

Edgardo

[MattB64]

“Illegal instruction 4” Thanks Get Outlook for iOS<https://aka.ms/o0ukef>

…

________________________________ From: Edgardo M. Ortiz ***@***.***> Sent: Thursday, May 15, 2025 3:20:40 AM To: edgardomortiz/Captus ***@***.***> Cc: Matt Barrett ***@***.***>; Mention ***@***.***> Subject: Re: [edgardomortiz/Captus] Error on alignment of extracted BUSCO genes (Issue #26) This message was sent from someone external to JCU. Please do not click links or open attachments unless you recognise the source of this email and know the content is safe. [https://avatars.githubusercontent.com/u/5767612?s=20&v=4]edgardomortiz left a comment (edgardomortiz/Captus#26)<#26 (comment)> Hi @MattB64<https://github.com/MattB64> , My guess is that the installed copy of MUSCLE was wrongly compiled at bioconda, the usual solution is to downgrade MUSCLE to immediate previous version. To verify, what happens when you type muscle with the captus environment activated? Edgardo — Reply to this email directly, view it on GitHub<#26 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AI2R5XQKAEVUXEUF4A7LQKD26N3ORAVCNFSM6AAAAAB5DUQASCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDQOBQHE4DKMZXG4>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[edgardomortiz]

Yeah, that is what I thought. In order to downgrade your MUSCLE I need to know the "bad" version that conda installed. With the environment activated, could you tell me what version of MUSCLE is shown when you run conda list?

[MattB64]

muscle 5.3 h8e8ab34_1 bioconda Thanks! From: "Edgardo M. Ortiz" ***@***.***> Reply to: edgardomortiz/Captus ***@***.***> Date: Thursday 15 May 2025 at 10:19 AM To: edgardomortiz/Captus ***@***.***> Cc: Matt Barrett ***@***.***>, Mention ***@***.***> Subject: Re: [edgardomortiz/Captus] Error on alignment of extracted BUSCO genes (Issue #26) This message was sent from someone external to JCU. Please do not click links or open attachments unless you recognise the source of this email and know the content is safe. [Image removed by sender.]edgardomortiz left a comment (edgardomortiz/Captus#26)<#26 (comment)> Yeah, that is what I thought. In order to downgrade your MUSCLE I need to know the "bad" version that conda installed. With the environment activated, could you tell me what version of MUSCLE is shown when you run conda list? — Reply to this email directly, view it on GitHub<#26 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AI2R5XQ45ZNI7FPVSVB2H2D26PMO5AVCNFSM6AAAAAB5DUQASCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDQOBRHA3TQNZRHE>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[edgardomortiz]

Try downgrading like this:

conda install -c bioconda muscle=5.3=h28ef24b_1

Did you install Captus using the instructions for Macs with Apple Silicon?

[MattB64]

I did install the Apple Silicon version last year, and re-installed this year (without uninstalling the previous version). I downgraded to the version you suggested, closed and re-opened terminal and captus, but the problem persists, and “conda list” still returns the old version: muscle 5.3 h8e8ab34_1 bioconda Matt From: "Edgardo M. Ortiz" ***@***.***> Reply to: edgardomortiz/Captus ***@***.***> Date: Thursday 15 May 2025 at 11:25 AM To: edgardomortiz/Captus ***@***.***> Cc: Matt Barrett ***@***.***>, Mention ***@***.***> Subject: Re: [edgardomortiz/Captus] Error on alignment of extracted BUSCO genes (Issue #26) This message was sent from someone external to JCU. Please do not click links or open attachments unless you recognise the source of this email and know the content is safe. [Image removed by sender.]edgardomortiz left a comment (edgardomortiz/Captus#26)<#26 (comment)> Try downgrading like this: conda install -c bioconda muscle=5.3=h28ef24b_1 Did you install Captus using the instructions for Macs with Apple Silicon? — Reply to this email directly, view it on GitHub<#26 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AI2R5XUIEVEMKPOWRQXSOT326PUGTAVCNFSM6AAAAAB5DUQASCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDQOBRHE2TKMJVGU>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[edgardomortiz]

What was the message that conda showed when you executed conda install -c bioconda muscle=5.3=h28ef24b_1?

[MattB64]

Apologies, I thought it had downloaded and didn’t notice the error message below. Going to the anaconda site, the second-most recent file (and the only other build of v5.3) is: osx-64/muscle-5.3-h79b9f80_0.tar.bz2<https://anaconda.org/bioconda/muscle/5.3/download/osx-64/muscle-5.3-h79b9f80_0.tar.bz2> Alternately there are versions of 5.2? Matt conda install -c bioconda muscle=5.3=h28ef24b_1 Channels: - bioconda - conda-forge - defaults Platform: osx-64 Collecting package metadata (repodata.json): done Solving environment: failed PackagesNotFoundError: The following packages are not available from current channels: - muscle==5.3=h28ef24b_1 Current channels: - https://conda.anaconda.org/bioconda - https://conda.anaconda.org/conda-forge - defaults To search for alternate channels that may provide the conda package you're looking for, navigate to https://anaconda.org<https://anaconda.org/> and use the search bar at the top of the page. From: "Edgardo M. Ortiz" ***@***.***> Reply to: edgardomortiz/Captus ***@***.***> Date: Thursday 15 May 2025 at 12:27 PM To: edgardomortiz/Captus ***@***.***> Cc: Matt Barrett ***@***.***>, Mention ***@***.***> Subject: Re: [edgardomortiz/Captus] Error on alignment of extracted BUSCO genes (Issue #26) This message was sent from someone external to JCU. Please do not click links or open attachments unless you recognise the source of this email and know the content is safe. [Image removed by sender.]edgardomortiz left a comment (edgardomortiz/Captus#26)<#26 (comment)> What was the message that conda showed when you executed conda install -c bioconda muscle=5.3=h28ef24b_1? — Reply to this email directly, view it on GitHub<#26 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AI2R5XUSRZDG2RWYLWBHBRT26P3OTAVCNFSM6AAAAAB5DUQASCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDQOBSGAZTCNJRHE>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[edgardomortiz]

And trying conda install -c bioconda muscle=5.3=h79b9f80_0?

[edgardomortiz]

Weird, my search command looks like this (I am using an M4 Pro):

$ conda search -c bioconda muscle
Loading channels: done
# Name                       Version           Build  Channel
muscle                      3.8.1551      h4675bf2_8  bioconda
muscle                         5.1.0      h025cef0_1  bioconda
muscle                           5.2      h025cef0_0  bioconda
muscle                           5.3      h025cef0_0  bioconda
muscle                           5.3      h28ef24b_1  bioconda

I don't have the build h79b9f80_0

[MattB64]

Weird, I get: muscle 3.8.31 0 bioconda muscle 3.8.1551 1 bioconda muscle 3.8.1551 2 bioconda muscle 3.8.1551 h04f5b5a_4 bioconda muscle 3.8.1551 h470a237_3 bioconda muscle 3.8.1551 h5fa12a8_7 bioconda muscle 3.8.1551 h5fa12a8_8 bioconda muscle 3.8.1551 h770b8ee_5 bioconda muscle 3.8.1551 hb280591_6 bioconda muscle 5.1 h09c63de_2 bioconda muscle 5.1.0 h79b9f80_1 bioconda muscle 5.1.0 h7d747a2_0 bioconda muscle 5.1 h7d747a2_3 bioconda muscle 5.1 hb339e23_1 bioconda muscle 5.1 he1e422e_0 bioconda muscle 5.2 h79b9f80_0 bioconda muscle 5.3 h79b9f80_0 bioconda muscle 5.3 h8e8ab34_1 bioconda The build h79b9f80_0 downgraded ok , and “captus list” gives the new version: muscle 5.3 h79b9f80_0 bioconda However the problem still persists: captus align -e 03_extractions Starting Captus-assembly: ALIGN (2025-05-15 12:48:33) Welcome to the alignment step of Captus-assembly. In this step, Captus will collect all the extracted markers across all samples in '03_extractions' and group them by marker. Then Captus will align each marker using MAFFT or MUSCLE. If given, the reference loci are also included as alignment guides and removed after alignment For more information, please see https://github.com/edgardomortiz/Captus Captus version: v1.4.0 Command: /opt/miniconda3/envs/captus/bin/captus align -e 03_extractions OS: macOS-14.4.1-x86_64-i386-64bit Host: P2LTJX3WR5 Max. RAM: 15.8GB (out of 16.0GB) Max. Threads: 8 (out of 8) Dependencies: Traceback (most recent call last): File "/opt/miniconda3/envs/captus/bin/captus", line 10, in <module> sys.exit(main()) ^^^^^^ File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/captus_assembly.py", line 1547, in main CaptusAssembly() File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/captus_assembly.py", line 86, in __init__ getattr(self, args.command)() File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/captus_assembly.py", line 1543, in align align(full_command, args) File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/align.py", line 92, in align muscle_path, muscle_version, muscle_status = muscle_path_version(args.muscle_path) ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ File "/opt/miniconda3/envs/captus/lib/python3.12/site-packages/captus/misc.py", line 561, in muscle_path_version version = process.communicate()[0].decode().strip("\n").split()[1] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~^^^ IndexError: list index out of range From: "Edgardo M. Ortiz" ***@***.***> Reply to: edgardomortiz/Captus ***@***.***> Date: Thursday 15 May 2025 at 12:44 PM To: edgardomortiz/Captus ***@***.***> Cc: Matt Barrett ***@***.***>, Mention ***@***.***> Subject: Re: [edgardomortiz/Captus] Error on alignment of extracted BUSCO genes (Issue #26) This message was sent from someone external to JCU. Please do not click links or open attachments unless you recognise the source of this email and know the content is safe. [Image removed by sender.]edgardomortiz left a comment (edgardomortiz/Captus#26)<#26 (comment)> Weird, my search command looks like this: $ conda search -c bioconda muscle Loading channels: done # Name Version Build Channel muscle 3.8.1551 h4675bf2_8 bioconda muscle 5.1.0 h025cef0_1 bioconda muscle 5.2 h025cef0_0 bioconda muscle 5.3 h025cef0_0 bioconda muscle 5.3 h28ef24b_1 bioconda I don't have the build h79b9f80_0 — Reply to this email directly, view it on GitHub<#26 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AI2R5XUZUZ67RBAN3NTRHNT26P5R5AVCNFSM6AAAAAB5DUQASCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDQOBSGA2TANRWGU>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[edgardomortiz]

I noticed your environment didn't even installed the latest captus version (1.4.2), it is not recommended to reinstall on top of a previous environment, something got broken there. Deactivate the captus environment and remove it:

conda deactivate your_captus_env

conda remove -n your_captus_env

conda clean --all

conda create --platform osx-64 -n captus142 -c bioconda -c conda-forge captus megahit=1.2.9=hfbae3c0_0 mmseqs2=16.747c6

conda activate captus142

Execute the steps one by one, and let me know if the problem persists. We updated the installation instructions in April so the old instructions might have something to do with the errors.

[MattB64]

Thanks for the instructions, I’ve removed and reinstalled captus -with a slight change to the deactivation instructions you gave which didn’t work, I had to use: conda deactivate conda remove --name captus --all However, I’m still getting the same error at the same stage: (captus142) P2LTJX3WR5:_CAPTUS_BUSCOattemts_FAIL jc660591$ captus align -e 03_extractions Starting Captus-assembly: ALIGN (2025-05-15 13:58:17) Welcome to the alignment step of Captus-assembly. In this step, Captus will collect all the extracted markers across all samples in '03_extractions' and group them by marker. Then Captus will align each marker using MAFFT or MUSCLE. If given, the reference loci are also included as alignment guides and removed after alignment For more information, please see https://github.com/edgardomortiz/Captus Captus version: v1.4.2 Command: /opt/miniconda3/envs/captus142/bin/captus align -e 03_extractions OS: macOS-14.4.1-x86_64-i386-64bit Host: P2LTJX3WR5 Max. RAM: 15.8GB (out of 16.0GB) Max. Threads: 8 (out of 8) Dependencies: Traceback (most recent call last): File "/opt/miniconda3/envs/captus142/bin/captus", line 10, in <module> sys.exit(main()) ^^^^^^ File "/opt/miniconda3/envs/captus142/lib/python3.12/site-packages/captus/captus_assembly.py", line 1547, in main CaptusAssembly() File "/opt/miniconda3/envs/captus142/lib/python3.12/site-packages/captus/captus_assembly.py", line 86, in __init__ getattr(self, args.command)() File "/opt/miniconda3/envs/captus142/lib/python3.12/site-packages/captus/captus_assembly.py", line 1543, in align align(full_command, args) File "/opt/miniconda3/envs/captus142/lib/python3.12/site-packages/captus/align.py", line 92, in align muscle_path, muscle_version, muscle_status = muscle_path_version(args.muscle_path) ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ File "/opt/miniconda3/envs/captus142/lib/python3.12/site-packages/captus/misc.py", line 561, in muscle_path_version version = process.communicate()[0].decode().strip("\n").split()[1] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~^^^ IndexError: list index out of range From: "Edgardo M. Ortiz" ***@***.***> Reply to: edgardomortiz/Captus ***@***.***> Date: Thursday 15 May 2025 at 12:56 PM To: edgardomortiz/Captus ***@***.***> Cc: Matt Barrett ***@***.***>, Mention ***@***.***> Subject: Re: [edgardomortiz/Captus] Error on alignment of extracted BUSCO genes (Issue #26) This message was sent from someone external to JCU. Please do not click links or open attachments unless you recognise the source of this email and know the content is safe. [Image removed by sender.]edgardomortiz left a comment (edgardomortiz/Captus#26)<#26 (comment)> I noticed your environment didn't even installed the latest captus version (1.4.2), it is not recommended to reinstall on top of a previous environment, something got broken there. Deactivate the captus environment and remove it: conda deactivate your_captus_env conda remove -n your_captus_env conda clean --all conda create --platform osx-64 -n captus142 -c bioconda -c conda-forge captus megahit=1.2.9=hfbae3c0_0 mmseqs2=16.747c6 conda activate captus142 Execute the steps one by one, and let me know if the problem persists. We updated the installation instructions in April so the old instructions might have something to do with the errors. — Reply to this email directly, view it on GitHub<#26 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AI2R5XSKKS2U5O4CJTMXW6T26P64PAVCNFSM6AAAAAB5DUQASCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDQOBSGA3DIOJXHA>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[edgardomortiz]

I am using Mac 15.4 (yours is still 14.4), so my guess is that you have to downgrade MUSCLE a couple of versions. Run conda search -c bioconda muscle inside the active environment, and install the previous available version as I showed you above, if that doesn't work keep falling back until a MUSCLE version works.

[edgardomortiz]

You could also install manually a MUSCLE somewhere else in your computer and provide the path to Captus with --muscle_path. I'm sorry, I know how frustrating conda can be sometimes, I have had to completely remove it several times, especially when they update it!