TITLE: SARS-CoV-2 NCBI assembly submission protocol: GenBank
AUTHORS: Ruth Timme, Heather Blankenship, Erin Young, Emma Griffiths, Duncan MacCannell, Stacia Wyman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">PURPOSE: </span></div><div class = "text-block"><span style = "font-weight:bold;">This protocol covers the steps for submitting a SARS-CoV-2 assembly to NCBI's GenBank</span></div><div class = "text-block">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. </div><div class = "text-block">Two protocols cover the PHA4GE guidance for SARS-CoV-2 submission to NCBI (Raw sequence data, metadata, and assemblies).  </div><div class = "text-block"><span>If you need a pipeline for frequent or large volume submissions,  follow Step 1 in the </span><a href="https://www.protocols.io/edit/sars-cov-2-ncbi-submission-protocol-sra-biosample-bf7bjrin" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject</span></a><span style = ":;font-weight:bold;"> </span><span>to get your NCBI submission environment established, then contact </span><a href="mailto:gb-admin@ncbi.nlm.nih.gov" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">gb-admin@ncbi.nlm.nih.gov</span></a><span> to set up an account for submitting through the API. </span></div><div class = "text-block">These protocols cover submission using NCBI's Submission Portal web-interface.</div><div class = "text-block"><span style = "font-weight:bold;">Complete in order (1 then 2):</span></div><div class = "text-block"><span style = "font-weight:bold;">1. </span><a href="https://www.protocols.io/edit/sars-cov-2-ncbi-submission-protocol-sra-biosample-bf7bjrin" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject</span></a></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">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.</li><li style = "counter-reset:ol0;list-style-type:disc;">Submit SARS-CoV-2 raw data to SRA (Sequence Read Archive) and metadata to BioSample.</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">2. SARS-CoV-2 NCBI assembly submission protocol: GenBank (included protocol)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>     </span><span style = "font-style:italic;">Required</span><span>: established BioProject and BioSamples</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Submit SARS-CoV-2 consensus sequences to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data.</li></ul></div><div class = "text-block"><span style = "font-weight:bold;">2. SARS-CoV-2 NCBI assembly submission protocol: GenBank (included protocol)</span></div><div class = "text-block"><span>     </span><span style = "font-style:italic;">Required</span><span>: established BioProject and BioSamples</span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. ["Ingredients" to have in place before starting your submissions]
1.1: Ensure you have a working NCBI user account1.2: Identify your NCBI submission user group or establish a new one if necessary. 1.3: Bookmark the link to your submission portal1.4. BioSample + BioProject assessions in-handAfter these steps are complete you can proceed with data submission in Step 2.
?. ["Ingredients" to have in place before starting your submissions]
Sign in to your NCBI user account: https://www.ncbi.nlm.nih.gov/account/
?. ["Ingredients" to have in place before starting your submissions]
Ensure you have an NCBI user group established and correct permissions are assigned for you to submit.List of submission groups: https://submit.ncbi.nlm.nih.gov/groups/If you don't have a submission group established, please follow this protocol to create one for your laboratory group:https://www.protocols.io/edit/sars-cov-2-ncbi-submission-protocol-sra-biosample-bf7bjrin
?. ["Ingredients" to have in place before starting your submissions]
Bookmark “my submissions” at NCBI: https://submit.ncbi.nlm.nih.gov/subs/. This is your landing page for all new NCBI 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.
?. ["Ingredients" to have in place before starting your submissions]
1. Identify your lab's BioProject accession. Does your laboratory have an established BioProject for this effort? If not please follow instructions in Step 3 of https://www.protocols.io/edit/sars-cov-2-ncbi-submission-protocol-sra-biosample-bf7bjrin for creating a new one.
?. [Data submission (assemblies to GenBank) ]
GenBank assembly submission of SARS-CoV-2:SARS-CoV-2 landing page: https://submit.ncbi.nlm.nih.gov/sarscov2/Click "submit" under GenBank.
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Click the “New submission” box.
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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. **Must be the same person or group that submitted the associated BioSamples and BioProject. Select the appropriate submission group name for your laboratory and check the contact information below.**If you do not have a submission group available to click, see Steps 1.2-1.3 in the SRA submission protocol to establish a new one for your laboratory, or to add your name to a group already established for your lab.Click "Continue" to proceed.
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REVIEW & SUBMIT tab:Check over entire submission, then click submit.
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GenBank accessions:The status of your submission will be updated once it is processed (track the status of your submissions under the "My Submissions" tab: https://submit.ncbi.nlm.nih.gov/subs/).  Sequences with no annotation issues will be listed as Processed and the GenBank accessions will be emailed to you and listed on the submissions page.  Submissions with annotation discrepancies will be marked as Error and a Fix button will appear.  A report is emailed to you and listed on the submissions page with the detailed issues.  If the data is incorrect, click the Fix button and you will return to the sequences page of your submission to upload a corrected file.  If you have evidence that the discrepancy is due to a naturally occurring mutation, send an email to gb-admin@ncbi.nlm.nih.gov with the SUB number and evidence.
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Important data stewardship and curation notes:
For updates to your GenBank records follow the NCBI Curation Protocol hosted by GenomeTrakr: https://www.protocols.io/view/ncbi-data-curation-protocol-bacaiase
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SUBMISSION TYPE tab:Select "SARS-CoV-2" option.
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SEQUENCING TECHNOLOGY tab:This information will get populated as a structured comment on the GenBank record. Pull the sequencing method and assembly information gathered in Step 2.1.Method: sequencing technology or platform.Assembly State: Click "Assembled sequences".Assembly information: Specify program/pipeline AND version.
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SEQUENCES tab:Release date: Click "Release immediately following processing" for all routine surveillance isolates.  Sequences:Sequences can be uploaded one at a time (one per submission), or as a batch upload in a single concatenated FASTA file (https://submit.ncbi.nlm.nih.gov/genbank/help/#fasta) when you are submitting multiple isolates in one submission.  See Step 2.1 for guidance on formating your FASTA file.Click "Choose File" to browse and upload your .fasta file:Click "Continue" and respond to any validation issues.Common validation issues: Ambiguous bases were trimmed warning. Ambiguous bases are non- A, T, G, or Cs. NCBI trims terminal Ns first at the 5' end, then looks to see if 50% of the first 10 bases are ambiguous and trim to last ambiguous. If more than 30% of the first 50 are ambiguous, we trim to the last ambiguous and then recheck the 5' end. If that is fine, we follow the same steps on the 3' end. This procedure is run again if we trimmed vector from an end. NCBI removes sequences that are greater than 50% ambiguous after the trimming. They also remove sequences with internal vector.String of NNNs: If your assembly contains strings of internal NNNs (from mapping to a reference genome), you will get a warning asking for you for more information:Click "A region of estimated length between the sequenced regions based on an alignment to similar sequences or genome" if the NNNs were caused by the reference-based assembly. Click "Continue" again.
Fasta headers must include a unique ID that links the sequence to the source modifiersFor example:FASTA header:>CA-IGI-0042Source modifier templateID from Sequence_ID column in metadata workbook: CA-IGI-0042
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SOURCE MODIFIERS tab:Guidance for populating this metadata outlined in Step 2.2.For a single submission: In the "Other source modifiers" Box, click Add field to add "BioProject" and "BioSample". Then populate these six fields following guidance in SOP for populating the NCBI submission templates, Step 4. For a batch submission.  Upload the csv file created from populating the PHA4GE GenBank source modifiers template in Step 2.2. Upload this file by clicking on the "upload a tab-delimited text file" link.  Ensure that the first column in this spreadsheet, "Sequence_ID" contains an ID that matches exactly the ID used in your FASTA file headers.  Click Continue.  ERRORS:  If you are not listed as an owner on the BioProject/BioSample you will see an error here stating that these are "Unknown".  If you do not have a submission group available to click, see Steps 1.2-1.3 in the SRA submission protocol to establish a new one for your laboratory, or to add your name to a group already established for your lab.
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REFERENCES tab:Sequence Authors: Enter names here from your NCBI submission user group (can be a sub-set of the names or the full submission group list).  Reference: For routine surveillance submissions choose "Unpublished", leave "Reference title" blank, and choose "same as sequence authors".Click Continue.
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Download and populate the PHA4GE GenBank source modifiers template:Populate the metadata spreadsheets for each isolate you intend to submit (you can submit metadata for a single isolate, entire MiSeq run, or for a large collection of isolates you intend to submit in batch). **Ensure that the BioProject and BioSample(s) were registered using the same NCBI user group. If you are not listed as an owner on the BioProject/BioSample(s) you will not be able to properly link the new assembly data to existing records.Save the excel spreadsheet as a tab-delimited text file (.tsv) and ensure that the date field is formatted correctly (e.g. 2020-04-20) in the text file.
Guidance:Follow Step 4 in SOP for populating the NCBI submission templates for populating the source modifiers.Refer to PHA4GE contextual data specification where relevant.
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For all sequences you intend to submit at this time:1. Gather associated BioSample accessions and metadata previously registered in https://www.protocols.io/edit/sars-cov-2-ncbi-submission-protocol-sra-biosample-bf7bjrin along with three pieces of information describing the sequencing method and assembly:Sequencing method. Populate with the PHA4GE field "sequencing instrument"Assembly program/pipeline. Populate with the name from the PHA4GE field "assembly method"Version of the assembly program. Populate with the version from the PHA4GE field "assembly method"  ABCDE1BioSample Accessionsample_nameseq. methodassembly programassembly version or date2SAMN15460792CA-IGI-0042MinIONARTIC-nCoV-bioinformaticsSOP1.1.03SAMN15460793CA-IGI-0031MinIONARTIC-nCoV-bioinformaticsSOP1.1.02. Concatenate all SARS-CoV-2 consensus sequences into a single fasta file, where the fasta headers contain the "sample_name" submitted to the BioSample.Example FASTA file for two sequence submissions:>CA-IGI-0042ATCGATCGGTACCTAAGGATCGATCGGTACCTAAGGATCGATCGGTACCTAAGG....>CA-IGI-0031ATCGATCGGTACCTAAGGATCGATCGGTACCTAAGGATCGATCGGTACCTAAGG....
ABCDE1BioSample Accessionsample_nameseq. methodassembly programassembly version or date2SAMN15460792CA-IGI-0042MinIONARTIC-nCoV-bioinformaticsSOP1.1.03SAMN15460793CA-IGI-0031MinIONARTIC-nCoV-bioinformaticsSOP1.1.0