Submitting an environment sample
==================

There are two ways to submit a sample to ENA (the same as for studies):

1) Using the Webinterface
-----------------------
The Webinterface for the dev submission database can be found at: 

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

You could follow this guide: 

https://ena-docs.readthedocs.io/en/latest/submit/samples/interactive.html

Download the following file: 

https://github.com/metagenomics/metagenomics-workshop/blob/denbi_nfdi_simplevm/files/environmental_sample.tsv

and submit it in the webinterface as described above. At the end of the process, you can download a file containing your submission details, you will need the sample accession later on, so make sure to save the file, for example in::

  /mnt/submission/sample.txt

Samples can be submitted programmatically was well as described below (for reference), we will do that for the bin samples later. Continue with the next step now: read submission.

2) Registering a sample programmatically
-----------------------
... using the linux command line tool ``curl``.

As for the study submission, we will also use this approach in this short course. 

First, create a work folder for submissions and a subfolder for the sample files::

  mkdir -p /mnt/submission/assembly/sample/
  cd /mnt/submission/assembly/sample

Create submission.xml
^^^^^^^^^^
The next thing you need to to is to create a file submission.xml with the following content::
  
  <SUBMISSION>
     <ACTIONS>
        <ACTION>
           <ADD/>
        </ACTION>
     </ACTIONS>
  </SUBMISSION>

You can use an editor like ``gedit`` to do so. It is actually the same file as the submission.xml for the study (except the HOLD part) so you could just copy that as well.

Get the correct ENA checklist
^^^^^^^^^^

The next thing, you need to do, is to identify the correct checklist for your sample. Checklists can be browsed here, look for example for "environmental":
https://www.ebi.ac.uk/ena/browser/checklists

Since we have an artifical dataset let's choose this checklist for example:
https://www.ebi.ac.uk/ena/browser/view/ERC000025


Create sample.xml
^^^^^^^^^^

There are a number of fields to be filled. You can download the XML file to see what can be filled out. Since this is a test submission, 
we will reduce that to the mandatory fields only::

	<SAMPLE_SET>
	  <SAMPLE alias="course_test_environmental sample">
	    <TITLE>Environmental sample for the metagenomic course 2022</TITLE>
	    <SAMPLE_NAME>
	      <TAXON_ID>1839947</TAXON_ID>
	      <SCIENTIFIC_NAME>outdoor metagenome</SCIENTIFIC_NAME>
	      <COMMON_NAME>outdoor metagenome</COMMON_NAME>
	    </SAMPLE_NAME>
	    <DESCRIPTION>DESCRIPTION</DESCRIPTION>
	    <SAMPLE_ATTRIBUTES>
	      <SAMPLE_ATTRIBUTE>
		<TAG>ENA-CHECKLIST</TAG>
		<VALUE>ERC000025</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>project name</TAG>
		<VALUE>MGCourse 2022</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
	       <TAG>sequencing method</TAG>
		<VALUE>MiSeq</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>collection date</TAG>
		<VALUE>2022-11-03</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>geographic location (country and/or sea)</TAG>
		<VALUE>Germany</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>geographic location (latitude)</TAG>
		<VALUE>52.019101</VALUE>
		<UNITS>DD</UNITS>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		 <TAG>geographic location (longitude)</TAG>
		 <VALUE>8.531007</VALUE>
		 <UNITS>DD</UNITS>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>broad-scale environmental context</TAG>
		<VALUE>temperate woodland</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>local environmental context</TAG>
		<VALUE>temperate woodland</VALUE>
	      </SAMPLE_ATTRIBUTE>
	      <SAMPLE_ATTRIBUTE>
		<TAG>environmental medium</TAG>
		<VALUE>forest soil</VALUE>
	      </SAMPLE_ATTRIBUTE>
	    </SAMPLE_ATTRIBUTES>
	  </SAMPLE>
	</SAMPLE_SET>


Some notes on the selected values:

The sequencing method is choosen from the following recommended vocabulary:
https://ontobee.org/ontology/OBI?iri=http://purl.obolibrary.org/obo/OBI_0400103

The broad/local scale environmental context is choosen from the following recommended vocabulary:
http://purl.obolibrary.org/obo/ENVO_00000428

The environmental medium is choosen from the following recommended vocabulary:
http://purl.obolibrary.org/obo/ENVO_00010483

There are several designated taxids in the NCBI taxonomy for metagenomes. Here is a list:
https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=408169

The chosen values in our example are of course fictitious.

Submit the sample
^^^^^^^^^^^^^^^^

Now, it is time to submit::
  
  cd /mnt/submission/assembly/sample
  curl -u $ENA_USER:$ENA_PWD -F "SUBMISSION=@submission.xml" -F "SAMPLE=@sample.xml" "https://wwwdev.ebi.ac.uk/ena/submit/drop-box/submit/" > receipt.xml

Make sure to use wwwdev to submit to the ENA test server.

Get the sample accession number
^^^^^^^^^^^^^^^

The response is stored in the file "receipt.xml". You can find the accession number for your sample in this line::

  <SAMPLE accession="ERS13654528" alias="course_test_environmental sample" status="PRIVATE">
  
Also note, that this number is only valid for today (as for the study accession), since it is discarded after 24 hours::

     <MESSAGES>
          <INFO>This submission is a TEST submission and will be discarded within 24 hours</INFO>
     </MESSAGES>

Note your accession number somewhere, you will need it for the next steps.

Now let's submit our reads for this study.



References
^^^^^^^^^^
**ENA - Registering a Sample** https://ena-docs.readthedocs.io/en/latest/submit/samples.html