Evaluate bins with BUSCO in MOSHPIT tutorial 4.1.6 section

Technical Support
1

MANIFEST is not a(n) MultiMAGManifestFormat file

Dear all

I having issues to move to the next step in this MOSHPIT tutorial (https://moshpit.qiime2.org/_/downloads/en/2025.7.0/pdf/), I am running this code:
mosh annotate evaluate-busco
--i-mags ./cache:mags
--i-db ./cache:busco_db
--p-lineage-dataset bacteria_odb12
--p-cpu 32
--o-visualization /mags.qzv
--o-results ./cache:busco_results
--verbose

I got this error message:
MANIFEST is not a(n) MultiMAGManifestFormat file:

No sample records found in manifest, only observed comments, blank lines, and/or a header row.

Plugin error from annotate:

/MANIFEST is not a(n) MultiMAGManifestFormat file:

No sample records found in manifest, only observed comments, blank lines, and/or a header row.

I was tinking to modify the MANIFEST file and adapt the MANIFEST file to the MIltiMAGManifestFormat file format. Unfortunatelly I didnt find nothing helpful to move forward.

If its helpful, this is an example of my current MANIFEST header file:

sample-id,mag-id,filename
1163,0453259e-8e3b-4f09-a8cc-71307561ebe6,1163/0453259e-8e3b-4f09-a8cc-71307561ebe6.fasta
1163,178f605e-bce8-40a2-acba-70045480966f,1163/178f605e-bce8-40a2-acba-70045480966f.fasta
1163,218dc332-3ac1-4539-87e5-6ac6146fcd63,1163/218dc332-3ac1-4539-87e5-6ac6146fcd63.fasta
1163,244ba0b5-ae16-48c0-b00e-6c8c6d97d682,1163/244ba0b5-ae16-48c0-b00e-6c8c6d97d682.fasta

Everything is stored in a cache directory, as recommended at the beginning of the tutorial.
I appreciate any suggestions or comments.

3

Hey @ferflei,

Sorry to hear you experienced this issue! From the documentation link you included I can see that you used the 2025.7 version of the docs - was your environment also 2025.7? If yes, would you mind trying the tutorial using the newest MOSHPIT version (2026.1)? We have patched up a few things here and there so perhaps that can help. If you still get an error, let us know and we'll do some more troubleshooting.

Thanks!

Cheers,
Michal

6

Hi @misialq thanks for your answer.

Yes, the enviroment is 2025.7

I will update the version, then I will let you knwon if works or not.

Thanks!

Cheers.

Fernando.

7

Dear @jakabaesj10

Thanks for your comments, I verified both Unix-style line endings and UTF-8 encoding. The manifest file looks is fine or what do you think? here the outputs that I ran:
file MANIFEST
MANIFEST: ASCII text

cat -A MANIFEST | head
sample-id,mag-id,filename$
1163,0453259e-8e3b-4f09-a8cc-71307561ebe6,1163/0453259e-8e3b-4f09-a8cc-71307561ebe6.fasta$
1163,178f605e-bce8-40a2-acba-70045480966f,1163/178f605e-bce8-40a2-acba-70045480966f.fasta$
1163,218dc332-3ac1-4539-87e5-6ac6146fcd63,1163/218dc332-3ac1-4539-87e5-6ac6146fcd63.fasta$

If you have another option ro veryfy Unix-style line endings and UTF-8 encoding please let me know.

Thanks.

Best.

Fernando.

8

Hi @misialq

I asked the technical team at the institution where I work to update the qiime-mospit version as you suggested. They are having issues installing the latest version (2026.4). It seems that version 2026.4 is not yet finalized, so they installed version 2026.1 instead. I ran my script again, but I got the same error. Do you think that using the latest version (2026.4) would make a difference?

Thaks for your comments.

Fernando.

9

Hey @ferflei,

Thanks for trying out the 2026.1 version. You are correct - the 2026.4 release is not finalized yet and should be available later this month but I am not sure this one would fix the issue. I will look into it a bit more and get back to you - sorry for the inconveniance!

Cheers,
Michal

16

Hi @ferflei,
I am so sorry for our delayed response here.
We have tried to recreate this error but have not successfully recreated the issue.

Just to clarify, Is this your own data that you are running through the tutorial or are you using tutorial data? If you are using tutorial data is it from the PDF linked above or Whole metagenome analysis with MOSHPIT - MOSHPIT documentation. I am just trying to make sure we are recreating your issue with the right data.

If it is your own data would you be willing to send it to us so we could try to debug on our end?

Again, I apologize for the delay on this response.

18

Hello MOSHPIT Team / @cherman2 ,

I am running into the same problem with 2026.4 version too.

Some background info:

I am running on a local machine. They are my own samples - 4-sample co-assembly (mosh assembly assemble-megahit), and reads were back-mapped using Bowtie2. MetaBAT2 completed successfully, reporting that 3 bins were formed (3 bins formed, 84.02% of large contigs binned).

However, hen passing the resulting mags.qza into evaluate-busco, the internal MultiMAGManifestFormat validator fails, stating that no sample records were found in the manifest.

bash:

mosh annotate evaluate-busco
--i-mags ../mags.qza
--i-db busco_db.qza
--p-lineage-dataset bacteria_odb12
--p-cpu 12
--o-visualization busco_report.qzv
--o-results busco_raw.qza
--verbose

Error code:

rachis.core.exceptions.ValidationError: /tmp/qiime2/user/tmp/rachis-OutPath-ih9wwzbg/MANIFEST is not a(n) MultiMAGManifestFormat file: No sample records found in manifest, only observed comments, blank lines, and/or a header row. Plugin error from annotate: No sample records found in manifest, only observed comments, blank lines, and/or a header row.

19

Hi @pollyhere,

hmmm, this is really weird.

Is it possible for you to send over that mag.qza to me? I am sure that its big but maybe you could upload it to google drive and send me a download link?

We haven't been able to reproduce this issue so having data with this issue would really help us narrow down the issue.

20

Hi @cherman2 , sure, how can I reach you?

I am now trying to use CheckM2 for the evaluation, or do you have other tools to recommend that is working well within the moshpit env?

Thank you!

21

Hi @pollyhere,
If you click on my profile there should be a button to message me!

@misialq or @colinvwood, do yall have any suggestions for evaluation tools?

22

Dear Chloe.

Sorry for my delay to answer your message!

Regarding your question, I was running the pipeline using my own data. I think the MANIFEST file might be useful for you. I cannot attach it here, but you can access it using the following link:https://drive.google.com/drive/folders/17F_jelUuL-i7nRdSrcS3f_5FNd0MK__Y?usp=drive_link

Please let me know if you need something else,

Thank you for your help

All the best.

Fernando.

24

Hi @ferflei

Thank you for send that. Can you update who has access to that link? It says I don't have access

26

Can you please send an email account.

Thanks.

27

Hi @ferflei,
Thanks for sharing your manifest.

After looking at your file there should be no reason for that error. This leads me to believe that even though you have a manifest MOSHPIT is not finding it.

If either of you could share the mag data that this manifest comes from that would help me identify where we are hitting this error.

28

Hi @cherman2

Thanks for your help and attention to this issue

You should acces to this folder: data - Google Drive

This is another subset with more mags, I had the same issue so this should be helpful.

Please let me know if you need something else.

Thank you.

Fernando.