TITLE: SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject
AUTHORS: Ruth  Timme, Emma  Griffiths, Duncan  MacCannell, Lee  Katz, Michael  Weigand, Technical  Outreach and Assistance for States Team

[DESCRIPTION]
PURPOSE: 
This is a SARS-CoV-2 specific protocol that covers the steps needed to establish a new NCBI submission environment for your laboratory, including the creation of new BioProject(s) and submission groups. Once these are step up, the protocol then walks through the process for submitting raw reads to SRA and sample metadata to BioSample through the Submission portal.  

For new submitters, there's quite a bit of groundwork that needs to be established before a laboratory can start its first data submission.  We recommend that one person in the laboratory take a few days to get everything set up in advance of when you expect to do your first data submission. 
 
If you need a pipeline for frequent or large volume submissions,  follow Step 1 in the SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject to get your NCBI submission environment established, then contact gb-admin@ncbi.nlm.nih.gov to set up an account for submitting through the API. 

These protocols cover submission using NCBI's Submission Portal web-interface.

Complete in order::

1. Populate your templates first.

2. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject (included protocol)
Step-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort.
SARS-CoV-2 raw data submission to SRA (Sequence Read Archive) and metadata to BioSample.  Users can modify this protocol to just create a BioSample with no linked raw data. 

3. SARS-CoV-2 NCBI consensus submission protocol: GenBank
     Required: established BioProject and BioSamples
Submit SARS-CoV-2 assemblies to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data.

Version history:
V4: Direct links provided to download metadata templates (instead of hosting duplicate files). Other minor edits throughout the protocol. 

[BEFORE_START]
This protocol has three sections:

Section 1: Setting up NCBI accounts (for new users)
Section 2: Single-step data submission to SRA for raw reads and associated sequence metadata and to BioSample for sample metadata
Section 3: Detailed steps for creating a BioProject (usually done once during the account set-up)

Associated protocols:
SOP for populating the NCBI submission templates for SARS-CoV-2 (e.g. BioSample and SRA metadata)
GenBank submission protocol for submitting SARS-CoV-2 assemblies or consensus sequences.
NCBI Data Curation protocol for making updates, corrections, or retractions to your data.

Link to PHA4GE contextual data specification

[STEPS]
SECTION: &quot;Ingredients&quot; to have in place before starting your submissions
1. Set up a new NCBI submission environment for your lab
1.1: Create an NCBI user account
1.2: Set up an NCBI submission user group for your lab
1.4: Bookmark the link to your Submission Portal
1.5. Identify or establish new BioProjects (detailed in Step 3)

Ready for data submission:
After these steps are complete you can proceed with BioSample + SRA data submission in Step 2.

   
SECTION: &quot;Ingredients&quot; to have in place before starting your submissions
1.1. Create an NCBI user account at NCBI: https://www.ncbi.nlm.nih.gov/account 


   



SECTION: &quot;Ingredients&quot; to have in place before starting your submissions
1.2. Establish an NCBI submission user group for your laboratory. 

We recommend using this user group for all NCBI submissions related to microbial genome surveillance. This will link your laboratory's NCBI data ownership to the user group and not to individuals, allowing anyone in the current group to perform updates or retractions and answer inquiries from the NCBI staff, even if there's been a complete turnover of staff since the original data submission. 

User groups also ensure consistent data ownership across BioProjects, BioSamples, and sequence data.  If your laboratory has non-overlapping research groups submitting and managing data at NCBI, multiple user groups can be established to track these efforts separately. 

Your laboratory might already have a submission group established!  Check the "Group" tab in the Submission Portal, https://submit.ncbi.nlm.nih.gov/groups/.  Ask your colleagues to do the same thing, to ensure your laboratory doesn't already have one in place.  

  


Creating a new submission group: 

1. Submit an email request to submit-help@ncbi.nlm.nih.gov containing the following information: 

 

2. Look for an email reply entitled "NCBI Submission Portal Group invitation" and click on the enclosed link to accept the invitation. 
SECTION: &quot;Ingredients&quot; to have in place before starting your submissions
1.3. Managing your NCBI submission user group.  

After a user group has been established it can be edited for membership and permissions by clicking in the “group” tab of the Submission Portal (https://submit.ncbi.nlm.nih.gov/groups/), then on the Group Id hyperlink, e.g 'fda_ny' in the above example. 

Users with admin privileges can update contact information in the "profile" tab and membership in the "Members" tab. New members can be invited by clicking on the "Invite members" link.  

  
This user group should be kept up-to-date as members enter and leave the laboratory.

Permissions levels:
READ: primarily for collaborators who can see the submissions, but not edit them.
MODIFY, SUBMIT, DELETE: Permissions to submit, modify, or retract data (members usually have all or none of these permissions)
ADMIN: Can invite or remove members of the submission group. Ensure that at least one (or more) members of your group have ADMIN privileges.

SECTION: &quot;Ingredients&quot; to have in place before starting your submissions
1.4. Bookmark “my submissions” at NCBI: https://submit.ncbi.nlm.nih.gov/subs/. This is the page where you view and track all of your past submissions. 

If you see a blank page with a yellow box in the upper right corner saying “please login”, click this link and login using the credentials created in Step 1.1.  

   

SECTION: &quot;Ingredients&quot; to have in place before starting your submissions
1.5. Identify or establish a new BioProject

Umbrella BioProjects. If you are already part of a surveillance network, (e.g. SPHERES, COG-UK, CanCOGeN, etc) you should use one of their established umbrella BioProjects. For reference, here are some of the umbrella projects established for SARS-CoV-2 surveillance:

SPHERES (US): PRJNA615625
CanCOGeN (Canada), PRJNA623807

Data BioProjects. Does your laboratory have an established data BioProject for this effort? If not please follow instructions in Step 3 for creating a new one. 

Countries with single data projects (not exhaustive):
COG-UK (United Kingdom): PRJEB37886
Turkey: PRJNA636004
Switzerland: PRJEB38472
South Africa: PRJNA624358
 

SECTION: Data submission (BioSample and SRA)
2. Data submission (source metadata and sequence data):

This protocol follows a one-step data submission process where the source metadata is submitted at the same time as the sequence data.

SARS-CoV-2 landing page: https://submit.ncbi.nlm.nih.gov/sarscov2/

    

Click "Submit" under the Sequence Read Archive (SRA) option

SECTION: Data submission (BioSample and SRA)
2.1. Download and populate the sample (BioSample) and sequence (SRA) metadata templates:

Current excel template versions can be accessed in the Overview of NCBI's SARS-CoV-2 submission process and the metadata required (Step 2 and Step 3).

Ensure that the BioSample and SRA templates are populated before proceeding to the next step.
SECTION: Data submission (BioSample and SRA)
2.2. Click the “New submission” box.  

   

SECTION: Data submission (BioSample and SRA)
2.3. Submitter tab: 

Populate with submitter info. The “submitter” is the name of the person, or user group, who is physically doing the submissions, not a supervisor or PI. 

Select the appropriate submission group name (see Step 1.2 for creating a new submission group), and describe the submitting organization or laboratory name.  This will be auto-populated from the contact info you included in your NCBI user account. 

  
Click "Continue" to proceed.
SECTION: Data submission (BioSample and SRA)
2.4. GENERAL INFO tab:

1. BioProject:  Did you already register a BioProject for this effort?  If not please follow instructions in Step 3 for creating a new BioProject and return back to this step with accession in hand.

   Click "Yes" and paste in your data BioProject accession, e.g. PRJNA614995. 
 
2. BioSample: Click "NO" here. You will be registering BioSamples within this current submission

3. Release date:  
**BioProjects established for wastewater data can be flagged for automated  human-read scrubbing (preformed prior to public release. tool here: https://github.com/ncbi/sra-human-scrubber).  This flag needs to be set along side the first data submission for that BioProject. One the flag is set, subsequent data submissions will get automatically scrubbed.  


First submission?  Choose "Release on specified date", then enter a date 1-week in the future.  This will give you some time to establish the flag prior to data release.


Send the following email to sra@ncbi.nlm.nih.gov asap:
 




Otherwise, choose "Release immediately following processing" (for all subsequent submissions). 

  

4. Click Continue.
SECTION: Data submission (BioSample and SRA)
2.5. BIOSAMPLE TYPE tab:

You are choosing the appropriate metadata package here (i.e. what kind of samples are you submitting?).

Select "SARS-CoV-2: clinical or host-associated"

  
 



SECTION: Data submission (BioSample and SRA)
2.6. BIOSAMPLE ATTRIBUTES tab:

Choose "Upload a file using Excel or text format (tab-delimited) that includes the attributes for each of your BioSamples".


  

Then click "Choose File" and browse to your populated PHA4GE BioSample_template Excel file.

If you have not populated your PHA4GE BioSample metadata template yet, download and follow the guidance in Step  2.1.

**Skip antibiogram sections (not relevant for SARS-CoV-2)

Click "Continue".

NCBI will do a validation check on your metadata.  Resolve any red "errors" reported back by editing the spreadsheet and replacing the uploaded file.  Review any yellow "Warnings" and proceed if everything looks ok.

Click "Continue".
SECTION: Data submission (BioSample and SRA)
2.7. SRA metadata tab:

Choose: "Upload a file using Excel or text format (tab-delimited)"

   
Upload the SRA metadata template populated in Step 2.1 (Excel file works here). 

Click "Continue".

NCBI will do a validation check on your metadata.  Resolve any red "errors" reported back by editing the spreadsheet and replacing the uploaded file.  Review any yellow "Warnings" and proceed if everything looks ok.

Click "Continue".
SECTION: Data submission (BioSample and SRA)
2.8. Files tab:

Each laboratory will establish its own path for transferring files.  

In general, selecting the web browser option should work for uploading ~48 sequences at a time. For a more stable internet connection, your laboratory can use FTP or Aspera. Directions for doing so pop up after clicking the FTP radio button

   

SECTION: Data submission (BioSample and SRA)
2.9. REVIEW & SUBMIT tab:

Check over your entire submission, then click submit.

If corrections are needed, you can go back and select individual tabs to edit your submission.
 

SECTION: Data submission (BioSample and SRA)
2.10. BioSample accessions will be automatically created upon submission and will be available on the “my submissions” page of the Submission Portal by clicking on “## objects” within the submission record (usually within 2 hours). You can also download by clicking the “Download attributes file with BioSample accessions”. Accessions will start with SAMNxxxxxxxx.  You will also receive an email containing these same accessions.  


   

SECTION: Data submission (BioSample and SRA)
2.11. SRA Accessions:

SRA run accessions will be available on the “my submissions” page of the Submission Portal by clicking on “## objects” within the submission record (usually within 2 hours). You can also download by clicking the “Download metadata file with SRA accession”. Accessions will start with SRRxxxxxxx.” You will also receive an email containing these same accessions.

 
SECTION: Data submission (BioSample and SRA)
2.12. Important data stewardship and curation notes:

Develop an internal method for storing and tracking your BioSample and SRR accessions! They are required for making future updates to your records.

For updates, corrections, or retractions to your BioSample and SRA records, follow the guidance provided in the NCBI Curation Protocol: https://www.protocols.io/view/ncbi-data-curation-protocol-bacaiase. Some edits can be made within the Submission Portal and others need to be done via email. 
 



SECTION: BioProject Creation
3. Create a new BioProject  

BioProjects are an organizing tool at NCBI that pulls together different kinds of data submitted across multiple NCBI databases. Each BioProject has a unique URL, providing a home page with a title, description, links to lab websites, publications, funding resources associated with a particular project, along with links to the deposited data. A basic data BioProject holds actual sequence data, assemblies, and their associated metadata. An umbrella BioProject is a way to group two or more data BioProjects together, which is useful for coordinating disease surveillance and for looking across the grouped BioProjects in a single view.

This protocol describes the steps for creating a new data BioProject linked to an existing umbrella BioProject (usually established by a coordinating group, e.g. SPHERES or CanCOGen). 
 


SECTION: BioProject Creation
3.1. 
Navigate to the “My Submissions” page, https://submit.ncbi.nlm.nih.gov/subs/, and click “BioProject” in the “Start a new submission” box.

    

SECTION: BioProject Creation
3.2. Click the “New submission” button:

    

SECTION: BioProject Creation
3.3. SUBMITTER tab: 

Populate with submitter info. An NCBI "submitter” is the name of the person or submission group who is managing the submissions, not a supervisor or PI. 

**Select the appropriate submission group name (see Step 1.2 for creating a new submission group), and describe the submitting organization or laboratory name.  This will be auto-populated from the contact info you included in your NCBI user account.  

  

SECTION: BioProject Creation
3.4. PROJECT TYPE tab: 

*Project data type: 
Choose: "Genome sequencing and assembly"

*Sample scope: 

For a Data BioProject: select "multi-isolate:. This will allow you to submit multiple isolates or strains of the same species. 

  

SECTION: BioProject Creation
3.5. TARGET tab:

For a Data BioProject: 

Populate ONLY the Organism name here: "Severe acute respiratory syndrome coronavirus 2", or higher level taxonomic classification for broader surveilliance.

Leave the strain info and Description fields blank. 

  
SECTION: BioProject Creation
3.6. GENERAL INFO tab: 

Click “Release immediately following processing”.

Include a brief title describing the effort. 
Data BioProject Title: e.g., “SARS-CoV-2 WGS: Utah Public Health Laboratory”. 

Public Description: e.g., “Whole-genome sequencing of XXX pathogens as part of XXXX surveillance effort.” 

Relevance: medical. 

Is your project part of a larger initiative that is already registered at NCBI? 
Data BioProjects. Click “Yes” and include a brief description and umbrella BioProject accession number (e.g. for SPHERES use PRJNA615625, or refer to Step 1.5 for full list). This will properly link your data project to the umbrella. 

External links: Include a link to your laboratory’s website here.  

    

SECTION: BioProject Creation
3.7. BioSample tab: 

Leave blank!!  You will create biosamples separately. 
SECTION: BioProject Creation
3.8. Publications tab: 

If relevant, include publications from your laboratory.
SECTION: BioProject Creation
3.9. Review and Submit tab: 

Check if everything looks correct and edit if necessary, then click “submit.”  

  

SECTION: BioProject Creation
3.10. The BioProject accession will be available within a few minutes on the “my submissions” page of the Submission Portal, “PRJNAxxxxxx.”  You will also receive an email containing the new accession.  

  
SECTION: BioProject Creation
3.11. If you are part of a coordinated surveillance effort, like SPHERES, please alert the coordinating body that a new BioProject was created under an existing umbrella.

For SPHERES, contact oamd@cdc.gov.
SECTION: BioProject Creation
3.12. Important data stewardship and curation notes:

Develop an internal method for storing and tracking your BioProject accessions! They are required for every BioSample and sequence data submission to ensure proper linkage.

Bookmark URLs for each of your BioProjects to monitor the public-facing view of your submissions.
        e.g. Virgina DCLS's SARS-CoV-2 BioProject: https://www.ncbi.nlm.nih.gov/bioproject/625551

For updates to your BioProjects, follow the guidance provided in the NCBI Curation Protocol: https://www.protocols.io/view/ncbi-data-curation-protocol-bacaiase. Most edits can be made within the Submission Portal by clicking the "Manage Data" button. Others need to be done via email.