Prokaryotic and Eukaryotic Genomes Submission Guide

Both WGS and non-WGS genomes, including gapless complete bacterial chromosomes, can be submitted via the Submission Portal. You will be asked to choose whether the genome being submitted is considered WGS or not. The differences for GenBank purposes are:

non-WGS

• Each chromosome is in a single sequence and there are no extra sequences
• Each sequence in the genome must be assigned to a chromosome or plasmid or organelle
• Plasmids and organelles can still be in multiple pieces.

WGS

• One or more chromosomes are in multiple pieces and/or some sequences are not assembled into chromosomes

In both cases

• There can still be gaps within the sequences; you will supply that information in the submission
• Plasmids and organelles can still be in multiple pieces.
• Internal sequences must be arranged in the correct order and orientation.
• Sequences concatenated in unknown order are not allowed.

Table of Contents

• Type of submission
• Events
• Submission files : fasta , .sqn , AGP , Genome Info
• Common metadata for all genomes
• Submitting genomes
• Requesting Prokaryotic Genome Annotation Pipeline (PGAP) annotation of prokaryotic genomes
• Running PGAP yourself

Type of submission

• No annotation or requesting PGAP annotation? Submit the fasta sequences in single or Batch mode
• Annotated genome? Create a .sqn file in ASN format and submit it in single or Batch mode
• Lots of genomes in the same BioProject? Use the Batch mode
• Individual assemblies of the haplotypes of a diploid or polyploid genome? See Submitting Multiple Haplotype Assemblies

Submit a single genome

This is the simplest submission route because you just fill in a web form in the Submission Portal and upload fasta (or sqn) files of the genome sequences. You will need to:

• Provide the BioProject created for this research effort, e.g., during submission of the reads to SRA OR register a new BioProject during the genome submission.
• Provide the BioSample created during submission of the reads to SRA OR register a BioSample during the genome submission
• Assert whether this is a WGS or non-wgs genome assembly
• Upload fasta sequences of the genome (or .sqn, file if the genome is annotated)
• Upload optional AGP file(s) to assemble scaffolds (unplaced or unlocalized) and/or chromosomes from the submitted sequences. This is for WGS only. Remember that you can submit the gapped scaffolds themselves instead of submitting contigs plus an AGP file
• Provide information in response to prompts during the genome submission (see the common metada section):
  • Genome Assembly Data and other information about this genome assembly
  • Gap Information (What the Ns represent)
  • Chromosome and plasmid assignments. Every sequence in a non-wgs genome must have a chromosome or plasmid assignment and every chromosome must be submitted as a single sequence.
  • Authors and a title (for fasta submissions)
  • Release date (immediately after processing OR a specific date. Release will be on that date or upon publication, whichever is first)
  • Optional request for annotation of prokaryotic genomes by PGAP

Submit a batch of genomes

This submission route allows you to submit as many as 400 WGS or non-wgs genomes in a single batch submission. In this route you choose Batch/multiple in the Genome Submission Portal , fill in the web form, upload a Genome Info file with genome metadata, and upload or preload fasta files (or sqn files if there is annotation) of the genome sequences. All the genomes within a batch must:

• Be part of the same BioProject
• Be either WGS or non-wgs, not a mix of both types
• Have the same (initial) release date
• Have the same gap/Ns information
• Contain either fasta files or ASN ( .sqn ) files, not a mix of file types. We recommend submitting fasta files unless the submission needs to include annotation or the Genome-Assembly-Data structured comment
• Have a single file for each genome, including any plasmid or organelle sequences
• Have a separate file for each genome, not all the genomes together
• Request PGAP annotation or not (only relevant for prokaryotic genomes)
• Be just a single layer (= no AGP file(s))

You will need to:

• Provide the BioProject created for this research effort, e.g., during submission of the reads to SRA OR register a new BioProject during the genome submission.
• Provide the BioSamples that were preregistered, eg during submission of the reads to SRA OR register BioSamples during the genome submission
• Include assignment (ie, chromosome, plasmid or organelle) information about the sequence in the fasta files (see the Additional requirements for batch submissions section)
• Upload or preload fasta sequences (or sqn files for annotated genomes) of the genomes. Each genome is in a separate single file, uniquely named, but the files can be archived together
  • An option for batch submission is to preload the files of genome assemblies before beginning the submission, rather than uploading them in the browser during the submission. You can preload using Aspera, the FTP protocol or Filezilla. Detailed instructions for using the preload option for genome submissions are at How to preload files.
• Upload a Genome Info table with information specific to each genome
• Provide this information in response to prompts on the web pages during the genome submission (see the common metadata section):
  • Gap Information (What the Ns represent)
  • Authors and a title (for fasta submissions)
  • Release date (immediately after processing OR a specific date. Release will be on that date or upon publication, whichever is first)
  • Optional request for annotation of prokaryotic genomes by PGAP

Events

1. Only if you will be submitting a genome with annotation and you have not yet registered a BioProject and BioSample for this genome, then you will register the genome sequencing project with the BioProject and BioSample databases so that a locus_tag prefix will be assigned to the BioProject:BioSample pair. If you have already registered a BioProject and BioSample for this genome, eg when submitting the reads to SRA, then a locus_tag prefix should have already been assigned. A file of the locus_tag prefix(es) for the BioSamples within a BioProject is linked to the BioProject submission. Write to genomes@ncbi.nlm.nih.gov if you did not receive a locus_tag prefix. Do not register a duplicate BioProject or BioSample for the same genome. Provide these preregistered BioProject and BioSample accessions in the genome submission. Remember that annotation is optional for genome submissions. If you are submitting a genome without annotation, even if you will be requesting PGAP annotation, then you'll create the BioSample (and BioProject, if necessary) during the genome submission. Genomes sequenced as part of the same research effort can belong to a single BioProject, so it's common to create a BioProject during the submission of one genome and then include that BioProject during the submission of additional genomes.

2. Make the genome assembly data files.

   • Unannotated genomes just need fasta files
   • Annotated genomes need to make .sqn file submissions by running the command line program table2asn (the replacement of tbl2asn), and then fixing Errors and Fatals that are indicated in the .val and .dr files. Failure to do this will cause serious delays in processing.

3. If you have higher-level assembly information, scaffolds and/or chromosomes, then generate an AGP file to build those objects from the wgs-contigs.

4. If you are submitting a batch of genomes (maximum of 400 per batch), then create a Genome Info file. Note that for batch submissions all chromosome and plasmid assignment information must be included in the header of the relevant fasta sequence, as described in the 'see details' section of the Additional requirements for batch submissions
5. Submit via the Genome Submission Portal.
6. What happens after submission

Submission Files

Fasta files

Put the sequences file into fasta format

• These files have the suffix .fsa.
• Each sequence has a definition line beginning with a '>' and a unique identifier (SeqID), eg contig001, contig002. The SeqIDs must:
  • Be <50 characters
  • Can only include letters, digits, hyphens (-), underscores (_), periods (.), colons (:), asterisks (*), and number signs (#).
  • Be unique within a genome
• Include in the definition line the organism and the relevant strain, breed, cultivar or isolate, if one exists for the sequenced organism. Any additional source qualifiers will be added from the registered BioSample to the genome during processing.
• Remove any Ns from the beginning or end of each sequence.
• Contigs should be >199nt, unless they are part of multi-component scaffolds in an AGP file

IMPORTANT Additional requirements for batch submissions

[1] All the sequences of single genome must be in one file

[2] The chromosome, plasmid, and organelle assignment information must be encoded in the input files of a batch submission, as described in these details:

• To indicate that a single gapped or ungapped sequence represents the chromosome, include [location=chromosome] in the fasta definition line. This must be true for at least one sequence when "option 1" (non-wgs genome) is selected.
• Sequences that are a complete circular chromosome or plasmid need to have the circular topology and the completeness included. If a gapped or nongapped circular chromosome has a gap at the end or has not been circularized, please also include that information unbracketed. Examples:
  • [topology=circular] [completeness=complete]
  • [topology=circular] gap at end, not circularized
• Sequences that are part of a plasmid, or an organellar chromosome, or specific nuclear chromosomes need to have that information included in the fasta definition line, in these formats:
  • [plasmid-name=pBR322]
  • [plasmid-name=unnamed] (when the plasmid name is not known. However, be sure that each plasmid has a unique name, eg unnamed1 and unnamed2. )
  • [location=mitochondrion]
  • [location=chloroplast]
  • [chromosome=2]
• Follow the Plasmid and chromosome names rules

• Here is an example of the definition line for the complete plasmid of a bacterial submission (all the text must be in a single line):

  >contig02 [organism=Clostridium difficile] [strain=ABDC] [plasmid-name=pABDC1] [topology=circular] [completeness=complete]

• Here is an example of a gapped sequence that represents chromosome 2 of a eukaryotic genome (all the text must be in a single line), so both the chromosome location and chromosome name are included:

  >Seq001 [organism=Puma concolor] [isolate=ABDC] [location=chromosome] [chromosome=2]

• Here is an example of sequences that belong to chromosome 5 so only the chromosome name is included (all the text must be in a single line):

  >Seq001 [organism=Puma concolor] [isolate=ABDC] [chromosome=5]

  >Seq002 [organism=Puma concolor] [isolate=ABDC] [chromosome=5]

.sqn files

These are generally required only when the submitter wants to include annotation. Annotation is optional for GenBank genome submissions.

see details

AGP file (optional)

AGP files provide the ordering and orientation information to construct scaffolds from contigs, or to construct chromosomes from scaffolds and/or contigs. However, remember that we do accept the gapped scaffolds themselves as the basic sequences of the genome. If you choose to submit a multi-layer submission with and AGP file, then know that the AGP file defines these genome assemblies, so be sure to include all wgs-contigs that are considered to be part of the genome in the AGP file. However, if the sequences in the fasta (or .sqn) files are already the scaffolds or chromosomes, then do not make an AGP file.

see details

Genome Info table

The Genome Info table is required for batch submissions and is used to provide the Genome Assembly Data of each. You can either fill in the table during the genome submission or prepare the file ahead of time and upload it during the submission. To prepare it ahead of time, download the Genome Info file template. The instructions are on the first tab of this file and the template is on the second tab. Complete the second tab (Genome_Data), then save the worksheet as a Text (Tab-delimited) file -- (use 'File, Save as, Save as type: Text (Tab-delimited)' ).

see details of the required and optional information

Metadata required for all genome submissions

BioProject

The BioProject contains the description of the research effort, relevant grant(s), and has links to the public data for the proejct. Each genome must belong to a BioProject, and genomes sequenced as part of the same research effort can belong to a single BioProject. Use the same BioProject for the sequence reads and genome assembly made from those reads; do not create duplicate BioProjects. If a new BioProject is necessary for unannotated (or PGAP-annotated) genomes, then registering during the genome submission process is simplest. However, genomes submitted with annotation will need to be pre-registered so that a locus_tag prefix can be assigned to the BioProject/BioSample pair and used to identify each gene within that genome uniquely. A file of the locus_tag prefix(es) for the BioSamples within a BioProject is linked to the BioProject submission. Write to genomes@ncbi.nlm.nih.gov if you did not receive a locus_tag prefix after preregistering a BioSample for your BioProject.

BioSample

The BioSample contains the source information of the sample that was sequenced. Use the same BioSample for the sequence reads and genome assembly made from those reads; do not create duplicate BioSamples. Registering a new BioSample can be done during the genome submission process for unannotated (or PGAP-annotated) genomes; however, genomes submitted with annotation will need to be pre-registered to get a locus_tag prefix. Include the registered BioProject when you register the BioSample so that a locus_tag prefix is assigned to the pair. You'll find the locus_tag assignment(s) in a file linked to the BioProject submission.

During processing of the genome the relevant information from the genome and BioSample will be merged so that they are in agreement. If the genome and BioSample have a conflict in the value of an attribute, we will stop and ask the submitter to clarify what the correct value is. There is some extra validation in GenBank compared to BioSample, eg ‘altitude’ is defined as being in meters in BioSample but it must have ‘m’ present in the GenBank genome. We will fix simple issues like this.

Genome Assembly Data and other information about a genome assembly

• Assembly method : Name of the assembly algorithm(s)
• Assembly method version or date : version of the algorithm or date it was run
• Genome coverage : The estimated base coverage across the genome, eg 12x.
• Sequencing technology : sequencing platform(s) used
• Assembly date : Optional. Year, month or day the assembly was made. Date formats: YYYY-MM-DD; YYYY-MM; YYYY
• Assembly name : Optional and not usually relevant for prokaryotes. This is a short name suitable for display that does not include the organism name eg, LoxAfr_3.0 for a Loxodonta africana assembly, version 3.0
• Full or Partial Genome in the sample : the answer is nearly always "yes, Full". Choose "no, partial" only if a subset of the sample was deliberately selected, eg just exomes or a single chromosome of a eukaryote or only the non-repetitive regions of the genome
• Reference genome : If this is NOT a de novo assembly, you will need to provide the accession.version and/or the assembly name of the genome assembly that was used as the reference guide for this assembly
• Update : accession of the genome being updated, when appropriate
• bacteria_available_from : Optional. For prokaryotes provide a name and physical address (not email) of the lab or PI, or a culture collection identifier where scientists could obtain this bacterial culture

Gap Information: What the Ns represent

• The minimum number of consecutive Ns that represents a gap (must be 10 or less). Be aware that the assembly statistics are always calculated using 10 or more Ns as a gap, regardless of the presence/absence of gaps in the final genome sequence.
• The number of Ns that represents a gap of completely unknown length (usually 0; sometimes 100 or another value)
• The evidence used to assert that the sequence on either side of the gap is linked (usually paired-ends)
• This information is collected in the submission form for individual and batch submissions. Default answers are those that have been most commonly submitted. Be sure to select the correct answer when the defaults are incorrect for the genome(s) being submitted.

Chromosome and plasmid assignments

• Indicate any sequences that are chromosomes or plasmids, or that belong to chromosomes or plasmids.
• Follow the chromosome and plasmid names rules
• Every sequence in a non-wgs genome must have a chromosome or plasmid assignment and every chromosome must be submitted as a single sequence.

Plasmid and chromosome names rules

see details

Submit the genomes to the Genome Submission Portal

All files must be submitted via the Genome Submission Portal. Choose "Single genome" or "Batch/multiple genomes". Answer the questions and upload the necessary files Review the summary page and click the "Submit" button. The submission will be given a 'SUB' temporary identifier which you can use in correspondence before an accession number is assigned to the genome submission.

What happens next

Once we receive your genome submission, several automated validations are run and a member of our staff conducts an initial review. If no significant issues are found, the genome will be assigned an accession number.

If there are problems

Submission statuses in the submission portal

• Queued : the submission is waiting for initial review
• Error : one or more genomes has errors in its files, so needs to be resubmitted. Use the same file name when resubmitting batch submissions.
• Processing, and no accession number : all of the genomes have passed the initial automated validations and are waiting for additional review.
• Processing, and accession number : genome accessions have been assigned and the genomes will be processed by NCBI staff. Genomes will remain at this status until they are released. We will contact you during processing if the submission has issues that require additional information.
• Processed : the genome has been publicly released.

If you elected to hold your genome until a particular date (or publication, whichever is first), we ask that you provide us with the expected publication date and also notify us in a timely manner of the upcoming publication and the relevant citation details. This will allow us to coordinate the release of your genome with the appearance of the paper. Please provide at least two weeks' notice of any upcoming publication.

NOTE: As of January 2017, genomes will be released on their release date without additional communication, as is the normal GenBank policy. Be sure to request an extension of the release date if the genome is not yet published and you wish to continue to keep it confidential.

Requesting PGAP annotation of prokaryotic genomes

Requests for annotation by the Prokaryotic Genomes Annotation Pipeline is a step during submission of the genome to GenBank. Prepare a regular GenBank genome submission and request PGAP annotation during the submission process by clicking on the box "Annotate this prokaryotic genome in the NCBI Prokaryotic Annotation Pipeline before being released". The annotated genome will be posted back to the Submission Portal for your review. You may edit the file and resubmit that to GenBank; however, this is not required and is generally not recommended, as it will slow processing and may introduce problems that you would need to fix.

Running PGAP yourself

If you would like to annotate your prokaryotic genome with the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) before or without submitting your data to GenBank, NCBI has made an external version available for you to download and run. It will generate a GenBank-compliant annotated genome that is submission-ready. If you are interested in running PGAP yourself, please see the NCBI Insights announcement and find more details at github, or see this short video.

After your genome is annotated using external PGAP, you may choose to submit it to GenBank:

• Submit the ASN output file of your PGAP run to GenBank via the usual Genome Submission Portal.
  • Information for single submission.
  • Information for batch submission.
• As usual during the genome submission, you will be asked to provide the BioProject and BioSample that you may have already created for that genome or its sequencing reads, or to create them if they do not already exist. Please do not create duplicates.
• Any locus_tag prefix can be used to run PGAP. However, during GenBank processing that value will be automatically changed to the officially registered locus_tag prefix for the BioProject:BioSample pair of that genome. Be sure to include the official locus_tags in your publication.
  • You have the option to obtain the official locus_tag prefix before running PGAP, but that is not necessary. To do that, you would register the BioProject, then register the BioSample(s), then look for the locus_tag_prefix.txt file posted to the BioProject submission. A separate BioSample is required for each strain, but multiple genomes can, and usually do, belong to a single BioProject.
• The submitted genomes will undergo the standard validations, as described above including being screened for foreign contaminants and vector sequences and also being analyzed to check the organism identification. Any annotated assemblies that do not pass these validations may need to be modified.
• Genomes that pass the validations will be assigned accession numbers and made public on the release date you have selected during submission. Be sure to use the official GenBank-assigned accessions when you publish.