Whole Genome Shotgun Sequence Submissions

DDBJ/EMBL/GenBank accepts both complete and incomplete genomes. Whole Genome Shotgun (WGS) sequencing projects are incomplete genomes or incomplete chromosomes that are being sequenced by a whole genome shotgun strategy. WGS projects may be annotated, but annotation is not required. The pieces of a WGS project are the contigs (overlapping reads), and they do not include any gaps. An AGP file can be submitted to indicate how the contig sequences are assembled together into scaffolds (contig sequences separated by gaps) and/or chromosomes. We must have the contig sequences without gaps as the basic units for all WGS projects.

WGS projects without annotation require at least two weeks to be processed. Projects with annotation require at least one month for processing. Please submit your project with enough lead time.

Below are detailed instructions for preparing a WGS submission. We recommend sending us a test file via GenomesMacroSend to see if there are problems before committing to generating the entire project.

See the list of WGS projects. The Annotation column indicates whether a project is annotated on the contigs (Y-c), on the scaffolds (Y-s) or is not annotated (-).

Table of Contents

• Introduction
• What To Do
• WGS Project Info
• How to Create a WGS Submission
• AGP Files to Build Scaffolds and/or Chromosomes
• Updating a WGS Project

Introduction

Each WGS project is assigned a stable 4-letter WGS accession prefix, which does not change as the project is updated. In addition to the WGS accession prefix, the contig identifiers have a version number corresponding to a particular WGS project update. Finally, each individual contig within the assembly is assigned a unique accession number prefixed by the WGS accession prefix and version number. For instance, if a WGS project's assigned accession number is XXXX00000000, then that project's first assembly version would be XXXX01000000, and the first contig of that version would be XXXX01000001. (The last six digits of this ID identify each individual contig).

The nucleotide data from most WGS projects go into the BLAST wgs database, whereas proteins go into the BLAST nr database. Nucleotides from environmental projects are present in either the BLAST wgs or env_nt database, depending upon whether that sequence has been identified as a particular organism (wgs), or if the organism is not yet known (env_nt). Similarly, the proteins from those projects are in the nr or env_nr BLAST database.

See the Metagenome Submission Guide for information about how to submit the various elements of a metagenome project.

What To Do

The table below shows three examples of WGS projects that have both contigs and scaffolds. One is unannotated and the others have annotation on either the contigs or the scaffolds. You can see that when the contigs are annotated, that annotation is displayed up on the corresponding scaffold in Entrez. Annotated records are shown as GenBank(Full) view. The accession number of each WGS project is included in the table:

Annotated Contigs	Annotated Scaffolds	No Annotation
ACZS00000000	ABXC00000000	AAGU00000000
WGS contig	WGS contig	WGS contig
Scaffold CON	Scaffold CON	Scaffold CON

How to Create a WGS Submission

File Format

Sequence

Other information about the biological source of the organism, including the organism name, should also be included in the definition line of the sequence. In addition to organism name, include other source modifiers that are known, eg [strain=yyy] [chromosome=nnn]. Note that there are no spaces surrounding the equal sign. A complete list of modifiers is available from the Sequin FAQ page.

If there is annotation and the organism does not use the standard genetic code, include the correct genetic code in the defline. For example, include [gcode=11] for bacteria.

The definition line must be on a single line with no line break.

A sample definition line is
>SeqID [organism=Mus musculus] [strain=BALB/c] [tech=wgs] [chromosome=2]

Annotation

Annotation can be included by creating a 5-column table in a .tbl file for each .fsa file. Go to the appropriate page for information about the format of the table and the desired annotation for eukaryotic or prokaryotic genomes. Three required fields are

• locus_tag for genes
• The locus_tag is the systematic name of the gene and is used for tracking individual genes. It therefore must be unique across all the genes in a project. If a gene's biological name is known, then it is included as the gene qualifier in the table.


• protein_id for proteins
• The protein_id is the SeqID of the protein (analogous to the nucleotide SeqID) and is used to track the protein. All of the SeqIDs, both nucleotide and protein, must be unique within a project. For WGS projects you can use type general protein_id's (format: gnl|dbname|SeqID) or local protein_id's (format: lcl|SeqID). Note that during our processing both forms of protein_id's are converted to type general id's in the format gnl|WGS:XXXX|SeqID, where XXXX is the project_ID. You can use your name or your lab name as the dbname, if you choose to create these as type general protein_id's.


• product for proteins
• The product is free text, chosen by the submitter. Protein names should be concise names, not descriptions or phrases. BLAST similarity results can be included as a note, or can be modified to be used as the product name. For example, if BLAST results indicate that the translation is similar to XYZ protein, then the product name could be "XYZ-like protein". If the protein is predicted and the product name is not known, use "hypothetical protein" as the product name.

Note that the nucleotide SeqID appears in the DEFINITION line in the flatfile view of the record. Although the protein SeqIDs are not displayed in the final flatfile view, they are present in the ASN.1.

See example *.fsa and *.tbl files for various situations, such as partial CDS or features on the minus strand.

Quality Scores

Use tbl2asn to include the Phrap/Consed quality scores of a sequence. The scores must be in files named *.qvl that are in the same directory and have the same nucleotide SeqIDs as the corresponding *.fsa files.

Template File

If there is a published reference, it can be sent separately with the submission, to be added to the records by GenBank staff during WGS processing.

tbl2asn does not include a release date in the output file, so include that information in your email message to us when you submit.

Using tbl2asn

In a specified directory, tbl2asn looks for .fsa files and any .tbl and .qvl files with the same basename, for example file1.fsa, file1.tbl and file1.qvl, and it builds ASN.1 records from them. The ASN.1 record for this example would be called file1.sqn. The results of the validation would be in a file named file1.val. Most validation errors must be fixed before the .sqn files can be submitted to GenBank; however, taxonomy-related errors can generally be ignored. Upload the .sqn files to us with GenomesMacroSend. Be sure to include in the comment box the Project ID that was assigned when you registered your project with the Genome Project database.

Go to the tbl2asn page for more detailed information about tbl2asn, its command line arguments, and file formats.

Sample command:

tbl2asn -t template.sbt -p path_to_files -V v

Multiple Sequences in a Single .fsa File

If there are more than 1000 contigs, then we recommend that you combine the fasta (and other) files, putting 10,000 or fewer into each file. Combining multiple fasta sequences into a single file can be useful for smaller projects too.

The corresponding .tbl and .qvl files must have the information for all of the sequences that are in the .fsa file. Run tbl2asn with the "-a s" argument so that each definition line is recognized as the beginning of a new sequence. A single .sqn file will then be generated for the multiple sequences of each .fsa file.

Sample command:

tbl2asn -t template.sbt -p path_to_files -a s -V v

See example .fsa and .tbl files.

AGP Files to Build Scaffolds and/or Chromosomes

If there is assembly information, of how the contigs are assembled into scaffolds (supercontigs) or chromosomes, then submit an AGP file with that information. AGP files provide the ordering and orientation information to construct supercontigs or scaffolds from contigs, or to construct chromosomes from supercontigs and/or contigs. More information about genome assemblies is here. See this page for the AGP format.

Some specific requests are: