Submitting a MAG

The submission of the MAG can be done only using webin-cli.

Again, We will need to create a manifest file, which contains the metadata for our read data and references the flat file we created during the annotation steps.

First, we create another directory:

mkdir /mnt/submission/mags/mags

Then, we gzip the embl file, since it needs to be zipped for submission:

gzip /mnt/WGS-data/megahit_out/metabat/mybin.embl

And change to our submission directory:

cd /mnt/submission/mags/mags

Create the manifest file

We will use this short manifest template for our submission (almost the same as the bin except for the ‘Metagenome-Assembled Genome (MAG)’ and the flat file), fill in YOUR values for STUDY, SAMPLE and RUN accession, be careful to fill in your MAG SAMPLE ACCESSION, not the environmental or bin sample accession:

STUDY   TODO
SAMPLE   TODO
RUN_REF   TODO
ASSEMBLYNAME   TODO
ASSEMBLY_TYPE   Metagenome-Assembled Genome (MAG)
COVERAGE   20
PROGRAM   MEGAHIT
PLATFORM   ILLUMINA
MOLECULETYPE   genomic DNA
DESCRIPTION   TODO
FLATFILE   /mnt/WGS-data/megahit_out/metabat/mybin.embl.gz

Create a file named manifest and fill it with the content above - fill the fields marked with TODO with the appropriate content. Then continue with the next step.

Validating the MAG submission

Before actually submitting, we are validating our manifest file. To do so, we use the option -validate in our call of webin-cli. Also, make sure, to use the -test flag to submit to the ENA test server. We also use -context=genome since we are submitting an assembly. Other options are your -username, -password and the path to the -manifest file:

cd /mnt/submission/mags/mags
java -jar ~/webin-cli-5.2.0.jar -username=$ENA_USER -password=$ENA_PWD -context=genome -manifest=manifest -validate -test

Unfortunately, we get an error:

ERROR: Submission validation failed because of a user error. Please check validation reports for further information: /mnt/submission/mags/mags/genome/mgcourse2022_mag6/validate

Check the file /mnt/submission/mags/mags/genome/mgcourse2022_mag6/validate/mybin.embl.gz.report (it’s different on your system - the assemblyname is in the path):

less /mnt/submission/mags/mags/genome/mgcourse2022_mag6/validate/mybin.embl.gz.report

It should contain many lines like this:

ERROR: Illegal /locus_tag value "LOCUSTAG_MJBABNOI_00673 ". locus_tag prefix "LOCUSTAG" is not registered with the project. [ line: 45893 of mybin.embl.gz]

This is because we didn’t register any locus tag prefixes for our study. Since we are using the test submission server, we cannot proceed here. However, you can still check, if there are any other errors in the validation file - if not, the file is theoretically fine to submit and in a real case you could proceed to submit to the production service.

However… ending the example like this is unsatisfying, so we might at least submit the fasta as MAG, although you wouldn’t do this in a real project, since everything is already submitted as a bin.

Change the line:

FLATFILE   /mnt/WGS-data/megahit_out/metabat/mybin.embl.gz

to (change to your bin-ID):

FASTA   /mnt/WGS-data/megahit_out/metabat/bin.*.fa.gz

make sure, it is zipped:

gzip /mnt/WGS-data/megahit_out/metabat/bin.*.fa.gz

And validate again:

java -jar ~/webin-cli-5.2.0.jar -username=$ENA_USER -password=$ENA_PWD -context=genome -manifest=manifest -validate -test

This should yield a success message.

Submit the MAG

Now, that our MAG submission is validated successfully, we can go on with the submission. Just replace the -validate flag by -submit in the webin-cli call. Do NOT remove the -test flag:

cd /mnt/submission/mags/mags
java -jar ~/webin-cli-5.2.0.jar -username=$ENA_USER -password=$ENA_PWD -context=genome -manifest=manifest -submit -test

If everything works fine, you should receive a message like:

INFO : The TEST submission has been completed successfully. This was a TEST submission and no data was submitted. The following analysis accession was assigned to the submission: ERZ14243535

Now the last thing, we could do, is checking your submission in the webinterface:

https://wwwdev.ebi.ac.uk/ena/submit/webin/

References

ENA - Submitting A Metagenome-Assembled Genome (MAG) https://ena-docs.readthedocs.io/en/latest/submit/assembly/metagenome/binned.html