Bacterial Genome Submission Guide

Annotation

Sequin and tbl2asn use a simple five-column tab-delimited table of feature locations and qualifiers in order to generate annotation.

The format of this feature table allows diferent kinds of features (e.g. gene, coding region, tRNA, repeat_region) and qualifiers (e.g. /product, /note) to be indicated. The validator will check for errors such as internal stops in coding regions.

Guidelines for eukaryotic genome submissions.

If you do not understand any of the instructions presented here or you have questions, please contact us by email at genomes@ncbi.nlm.nih.gov prior to creating your submission. This will save us both a lot of time.

Table of Contents

1. Prepare annotation table
   • Gene features
   • locus_tag is required
   • protein_id
   • CDS (coding region) features and protein names
   • Intein containing coding region
   • Partial coding regions in incomplete genomes
   • Gene fragments
   • Intron Containing Genes
   • Transpliced Genes
   • Split genes on two contigs
   • Ribosomal RNA, tRNA and other RNA features
   • Evidence Qualifiers
   • Functional bacteriophage
   • Insertion sequences and transposons
   • Data base cross references
   • Gene Ontology
   • Other Annotation

Prepare annotation table

The features must be in a simple five-column tab-delimited table, called the feature table. The feature table specifies the location and type of each feature for tbl2asn or Sequin to include in the GenBank submission that is created. The first line of the table contains the following basic information:

>Features SeqId table_name

The SeqId must be the same as that used on the sequence. The table_name is optional. Subsequent lines of the table list the features. Columns are separated by tabs.

• Column 1: Start location of feature
• Column 2: Stop location of feature
• Column 3: Feature key
• Column 4: Qualifier key
• Column 5: Qualifier value

Figure 2 shows a sample feature table and illustrates a number of points about the feature table format. The GenBank flatfile corresponding to this table is shown in Figure 3

Features that are on the complementary strand (such as the gene abrB in the examples below) and its corresponding CDS, are indicated by reversing the interval locations. Please avoid unnecessary capitalization in all text entered in your table.

Gene features

Gene features are usually a single interval, and their location should cover the intervals of all the relevant features such as promoters and operator binding sites. Gene names must follow the standard bacterial nomenclature rules of three lower case letters. Different loci are distinguished by a suffix of uppercase letters.

Example

  correct cytB
  incorrect CYTB
  incorrect cytochrome B
  incorrect orf1
  incorrect putative gene fragment

If a gene is a pseudogene, please do not add the word "pseudo" to the gene name or protein name. Instead use the /pseudo qualifier on the gene feature. Please see Gene fragments for more details.

locus_tag

All genes must be assigned a systematic gene identifier which must receive the locus_tag qualifier on the gene feature in the annotation table. Genes may also have functional names as assigned in the scientific literature. In this example, OBB_0001 is the systematic gene identifier, while abcD is the functional gene name. The locus_tag prefix must be 3-12 alphanumeric characters and the first character may not be a digit. Additionally locus_tag prefixes are case sensitive. All chromosomes and plasmids of an individual genome must use the exactly same locus_tag prefix followed by an underscore and then an alphanumeric identification number that is unique within the given genome. Other than the single underscore used to separate the prefix from the identification number no other special characters can be used in the locus_tag. Locus_tags must only be used in combination with a gene feature. Read more about locus_tags and their intended usage.

The use of locus_tag is supported in Sequin version 4.35 or newer. If you have an older version of Sequin please download the current version.

You may search our locus_tag data base to determine if the prefix you prefer is available.

Table view of gene with both biological name and locus_tag

1 1575   gene
		gene	abcD
		locus_tag     OBB_0001

Flatfile view

gene     1..1575
	/gene="abcD"
	/locus_tag="OBB_0001"

Table view of gene with only locus_tag:

1 1575   gene
		locus_tag     OBB_0001

Flatfile view:

gene     1..157
	/locus_tag="OBB_0001"

protein_id

The submitter must assign an identification number to all proteins. NCBI uses this number to track proteins when sequences are updated. This number is indicated in the table by the CDS qualifier protein_id, and must have the format gnl|dbname|string, where dbname is the username on your ftp account or name assigned by GenBank, and string is the identification number.

Example

The protein_id for abcD is gnl|dbname|OBB_0001. This identifier is saved with the record (in ASN.1 format), but it is not visible in the flatfile. We recommend using the locus_tag number as the protein identification number.

1	1575	gene
			gene	abcD
			locus_tag     OBB_0001
1	1575	CDS
			product	AbcD
			protein_id	gnl|dbname|OBB_0001

The protein_id is used for internal tracking in our database, it is important that the complete protein_id (dbname + string) not be duplicated by a genome center. Note that when WGS submissions are processed, the dbname in the protein_id is automatically changed to 'WGS:XXXX', where XXXX is the project's accession number prefix. After your genome is released into GenBank, the proteins are assigned accession numbers. We will provide a table of the protein SeqIDs and accession numbers for you to use in future updates.

protein names

All CDS features must have a product qualifier (protein name). NCBI protein naming conventions are adopted in part from the UniProt-Swiss-Prot Protein Knowledgebase

Consistent nomenclature is indispensable for communication, literature searching and data retrieval. Many species-specific communities have established gene nomenclature committees that try to assign consistent and, if possible, meaningful gene symbols. Other scientific communities have established protein nomenclatures for a set of proteins based on sequence similarity and/or function. But there is no established organization involved in the standardization of protein names, nor are there any efforts to establish naming rules that are valid across the largest spectrum of species possible.

Ambiguities regarding gene/protein names are a major problem in the literature and it is even worse in the sequence databases which tend to propagate the confusion. For this reason, we ask that you follow some basic guidelines in naming your proteins. The protein naming guidelines are based on the premise that a good and stable recommended name for a protein is a name that is as neutral as possible.

Guidelines for naming proteins:

• If it exists, use the approved nomenclature.
• Use a concise name, not a description or phrase.
• Ideally the name should be unique and attributed to all orthologs.
• The protein name should not contain specific characteristics of the protein, and it should not reflect the function of the protein, its subcellular location, its domain structure, its molecular weight or its species of origin. This information can be included in the note.
• In cases where the protein name is not known use "unknown" or "hypothetical protein" as the product name. We recommend the use of "hypothetical protein" as this will allow the locus_tag identifier to be appended to the product name in BLAST and Entrez summary lines.
• Protein names may be denoted by the same symbol as the corresponding gene, but the symbol begins with a capital letter.
• Avoid the use of molecular weights in protein names "unicornase subunit A" is preferred to "unicornase 52 kDa subunit"
• Do not use the term "homolog" in a protein as this infers an evolutionary relationship that has generally not been determined.
• Where possible, avoid the use of commas in protein names
• Use lowercase letters, except when uppercase are required (for example, in acronyms such as DNA or ATP).
• Wherever appropriate, the name should use American spelling conventions.
• Avoid the use of Roman numerals where possible. Use instead Arabic numbers.
• Do not build molecular weights into abbreviations
• For proteins that belong to a multigene family, it is recommended that you choose a coherent nomenclature with numbers to specify the different members of the family.
• When naming proteins which can be grouped into a family based on homology or according to a notion of shared function, the different members should be enumerated with a dash "-" followed by an Arabic number. e.g. "desmoglein-1", "desmoglein-2", etc.
• Greek letters must be written in full e.g. "alpha", and written entirely in lower case with the exception of "Delta" in the context of steroid/fatty acid metabolism nomenclature. Additionally the Greek letters that are followed by a number should be preceded or followed by a dash "-" e.g. "unicornase alpha-1".
• Do not use diacritics, such as accents, umlauts. Many computer sytems (ours included) can only understand ASCII characters.
• Do not use plurals in a protein name. e.g. "ankyrin repeats-containing protein 8" is wrong.
• Proteins of unknown function which contain a defined domain or motif, can be named according to the domain present. The name should be of the following type: "<domain|repeat>-containing protein". e.g. "PAS domain-containing protein 5".

Here are some examples of good protein names:

cytochrome b

CytB

aconitate hydrase B

hypothetical protein

cytochrome b-like protein

4Fe-4S cluster binding protein

adenylyltransferase/ADP-heptose synthase

2-hydroxyhepta-2,4-diene-1,7-dioate isomerase

short-chain specific acyl-CoA dehydrogenase

formylmethanofuran--tetrahydromethanopterin formyltransferase

serine/threonine-protein kinase

translation initiation factor 1

triphosphoribosyl-dephospho-CoA synthetase

thiamine biosynthesis protein ThiC

PAS domain-containing protein 5

ABC transporter ATP-binding protein AlbC

stage 0 sporulation protein J

These names all concisely describe the function of the protein, where known, and avoid references to structure, homology and species.

Here are some examples of bad protein names:

required for the efficient incorporation of molybdate into molybdoproteins

This describes the protein's role in a biosynthetic process but is not a protein name.

chaperone Hsp70; DNA biosynthesis; autoregulated heat shock proteins

The name "chaperone Hsp70" is fine however the remaining comments would be best fielded as a note or in the function qualifier.

putative carbonic anhdrase (EC 4.2.1.1)

The EC number should not be part of the protein name but instead fielded in the EC_number qualifier

similar to aconitrate hydrase B

This statement is fine as a note, however as a protein name aconitrate hydrase B-like protein is preferred

related to protein of unknown function

Does this tell you anything?

cytochrome b-like

cytochrome b-like protein is preferred

ABC transporter related

vague name, there are many ABC transporters and subunits, be more specific

pirin, N-terminal:pirin, C-terminal

uniformative name noting similarity in N and C terminus

helix-turn-helix motif

Describes a motif or structural domain but is not an appropriate protein name.

PP-loop

Describes a motif or structural domain but is not an appropriate protein name.

alpha/beta hydrolast fold

Describes a motif or structural domain but is not an appropriate protein name.

pentapeptide repeat

Describes a motif or structural domain but is not an appropriate protein name.

phosphopantetheine-binding domain

Describes a motif or structural domain but is not an appropriate protein name.

protein of unknown function:conserved

Does this tell you anything?

hypothetical 32.5 kDa protein homologous to phytoene and squalene synthethases

Hypothetical protein alone is appropriate. The remaining comments should be fielded as a note.

ribosomal protein L3 (E. coli)

Protein names should not contain references to organism names. Ribosomal protein L3 is an appropriate name by itself.

saccharopine dehydrogenase or related protein

"saccharopine dehydrogenase" or "saccharopine dehydrogenase-like protein" would be more appropriate

tyrosine-protein kinase (capsular polysaccharide biosynthesis)

tyrosine-protein kinase is fine as a protein name but capsular polysaccharide biosynthesis would be more appropriate as a function.

RimM protein, required for 16S rRNA processing

RimM is fine as a protein name but descriptive comments should be placed in the note.

involved in flagellar biosynthesis

This is a functional comment and not a protein name.

Notes

Please avoid including notes indicating a specific percentage of similarity to other entries in the database, since the corresponding record that you have pointed to may change and make your current note inaccurate, incorrect and obsolete. Descriptions, notes describing similarity to other proteins, and functional comments must be placed in the appropriate CDS qualifiers such as note, or prot_desc, as they are descriptors of the product. E.C. numbers must be fielded in an EC_number qualifier.

start	stop	CDS
			product	DNA gyrase subunit B
			EC_number     5.99.1.3
			note	required for the gyration of DNA

Qualifiers that can be used on the CDS feature are:

start	stop	CDS
			product
			prot_desc
			function
			EC_number
			note
			experiment
			inference
			go_component
			go_process
			go_function
			db_xref
			pseudo
			exception
			transl_except

Bifunctional Proteins

If a protein contains two separate and distinct functions or if it has more than one name, it can be annotated in several ways as outlined below.

Table view:

start	stop	CDS
			product	adenylyltransferase/ADP-heptose synthase
			note	bifunctional
			EC_number     2.7.7.2
			EC_number     1.4.1.13

or

start	stop	CDS
			product	bifunctional adenylyltransferase/ADP-heptose synthase cyclohydrolase
			EC_number     2.7.7.2
			EC_number     1.4.1.13

or

start	stop	CDS
			product	FolD 
			function	adenylyltransferase
			function	ADP-heptose synthase cyclohydrolase
			note	bifunctional
			EC_number     2.7.7.2
			EC_number     1.4.1.13

Intein-containing coding regions

Intein-containing coding regions must be represented as follows:

946506	950039	gene
			gene	recA
			locus_tag     OBB_0010
946506	950039	CDS
			product	DNA recombination protein precursor
			protein_id	gnl|dbname|OBB_0010
946506	946790	misc_feature
948057	950036
			note	DNA recombination protein
946791	948056	misc_feature
			note	intein	

Inteins should be annotated with two mat_peptide features, one for the intein and one for the final protein. We also add "precursor" to the product name on the CDS feature. Unfortunately, you can not add a mat_peptide feature in a table. Instead, you can add a misc_feature and we can convert them for you. Please see accession number AY847267 for an example of an intein containing protein.

Partial coding regions in incomplete genomes

Annotate a partial coding region using the "<" or ">" in your feature table to designate the feature as either 5' or 3' partial. The coding region must begin at the first nucleotide present, however the translation will start at the first complete codon.

Example

In the first example below, the "<" designates this coding region as 5' partial and "codon_start 3" tells the software to start translation with the third nucleotide of the CDS. Note that if the codon_start is not specified, then the software assumes a codon_start of 1. The second coding region below is partial at the 3' end so ">" is used to indicate a 3' partial feature. The third example is of a 3' partial coding region on the complementary or minus strand.

<1	497	gene
			gene	abcD
			locus_tag     OBB_0001
<1	497	CDS
			product	AbcD
			note	similar to Bacillus subtilis aldolase
			codon_start	3
			protein_id	gnl|dbname|OBB_0001
200	>1575	gene
			gene	xyzA
			locus_tag     OBB_0002
200	>1575	CDS
			product	actin-like protein
			protein_id	gnl|dbname|OBB_0002
436	>1	gene
			gene	nirK
			locus_tag     OBB_0003
436	>1	CDS
			product	NirK
			protein_id	gnl|dbname|OBB_0003

Partial coding regions may be used for incomplete genomes only. All coding regions annotated on finished genomes must be complete at both the 5' and 3' ends.

Disrupted genes and gene fragments

Sometimes a genome will have adjacent or nearby genes that seem to be only part of a protein. In many cases these indicate a possible problem with the sequence and/or annotation. A related issue is the presence of internal stop codons in the conceptual translation of a CDS that looks like it should be a real CDS. These problems may be due to a variety of reasons, including mutations or sequencing artifacts. They can be annotated in a number of ways:

1. Annotate the gene as a pseudogene. If multiple gene fragments were present initially, then add a single gene feature which covers all of the potential coding regions and add the pseudo qualifier indicating this is a pseudogene. If known, a note qualifier may be added indicating why this gene is disrupted.

   1	200	gene
   			gene	phoA
   			gene_desc	alkaline phosphatase
   			locus_tag     OBB_0001
   			pseudo
   			note	frameshift
   

2. Alternatively, if you are not sure if the disrupted gene is a "pseudogene" you can just use a gene feature without the /pseudo. Please use the complete nucleotide spans of the frameshifted gene. A note can be added to indicate the reason for the incomplete translation.

   1	200	gene
   			gene	phoA
   			gene_desc	alkaline phosphatase
   			locus_tag     OBB_0001
   			note	nonfunctional due to frameshift
   

3. A coding region containing a frameshift that is thought to be corrected by ribosomal slippage can be annotated using joined feature spans. Joined spans on a feature are used to combine two non-contiguous regions of sequence that are joined together to encode a protein, for example. This is typically used to combine eukaryotic exons to translate the coding region. To create a join CDS you must specify the spans of each contiguous region of sequence that encodes the protein. The use of the joined feature spans is rare in bacteria.

   333255	333181	CDS
   333179	332157
   			product	AbcD
   			protein_id     gnl|dbname|OBB_0001
   			exception      ribosomal slippage
   

   In this case the CDS must also include an exception qualifier with the exact text "ribosomal slippage". If you include a join feature for a different reason, please include a note qualifier indicating why the two nucleotide spans are joined.

4. If a gene is localized but the translation is unknown, it is possible to simply annotate the gene feature without a corresponding coding region. The gene_desc qualifier can be used to annotate both the gene symbol and the gene description.

   1	200	gene
   			gene	phoA
   			gene_desc	alkaline phosphatase
   			locus_tag     OBB_0001
                       

5. Gene containing an authentic frameshift induced by phase variation can be represented by a gene feature with a note.

   1	200	gene
   			gene	phoA
   			gene_desc	alkaline phosphatase
   			locus_tag     OBB_0001
   			note    authentic frameshift induced by phase variation; This region contains 
   			an authentic frameshift or in-frame stop in the coding sequence and is not the 
   			result of a sequencing error
                       

Intron-Containing Genes

While rare, there are some examples of bacterial genes containing introns. Annotate the gene feature of any intron-containing gene such that the gene feature spans are a single span covering all exons and introns. The actual feature (CDS, tRNA, etc.) should then be annotated with sets of nucleotide spans showing how the exons are joined to create the correct product. In this example there are two exons transcribed to create a tRNA. The first exon is from 1456 to 1419 and the second is from 1400 to 1361. Note how the gene feature spans encompass both exons and the intron.

1456	1361	gene
			locus_tag     APO_t01
1456	1419	tRNA
1400	1361
			product	tRNA-Cys

Transpliced Genes

Transpliced genes are the exception to the rule for annotating gene feature spans. Transpliced genes are similar to intron containing genes except the two pieces of the gene are found on different regions of the chromosome. These genes are transcribed as two or more separate RNA products that are transpliced into a single mRNA or tRNA. To annotate this using a table, enter the nucleotide spans so that the complementary (minus strand) spans are arranged from high to low and vice versa for the plus strand.

36700	36618	gene
86988	87064
			locus_tag    NEQ_t38
			exception    trans-splicing
36631	36618	misc_feature
			note	sequence cleaved during processing of trans-spliced tRNAs
36673	36635
87030	87064	tRNA
			product	tRNA-Glu
			exception    trans-splicing
			note	this trans-spliced tRNA consists of two halves on mixed strands; it shares 
			a 3' half with another tRNA

     gene            join(complement(36618..36700),86988..87064)
                     /locus_tag="NEQ_t38"
                     /trans_splicing
     misc_feature    complement(36618..36631)
                     /locus_tag="NEQ_t38"
                     /note="sequence cleaved during processing of trans-spliced tRNAs"
     tRNA            join(complement(36635..36673),87030..87064)
                     /locus_tag="NEQ_t38"
                     /product="tRNA-Glu"
                     /trans_splicing
                     /note="this trans-spliced tRNA consists of two halves on
                     mixed strands; it shares a 3' half with another tRNA"

Split genes on two contigs

Sometimes in incomplete genomes the ends of a gene may be on different contigs. Annotate these as separate genes with the same locus_tag, plus separate CDS with different protein_id's. In addition, link the features together with notes that refer to the other part of the gene.

Example

>Feature Cont01.00111
5000	>7500	gene
			locus_tag     KCS_2223
5488	5500	CDS
6000	>7200
			product	enolase
			protein_id	gnl|dbname|KCS_2223A
			note	5' end; 3' end is gene KCS_2223 on contig Cont01.00224

>Feature Cont01.00224
<1	1000	gene	
			locus_tag     KCS_2223
<100	876	CDS
			product	enolase
			protein_id	gnl|dbname|KCS_2223B
			note	3' end; 5' end is gene KCS_2223 on contig Cont01.00111

Ribosomal RNA, tRNA and other RNA features

RNA features (rRNA, tRNA, RNA) must include a corresponding gene feature with a locus_tag qualifier. Please be sure to specify which amino acid the tRNA gene corresponds to. If the amino acid of a tRNA is unknown, use tRNA-Xxx as the product, as in the example. Many submitters like to label the tRNAs such as tRNA-Gly1, etc. If you wish to do this please include "tRNA-Gly1" as a note and not in /gene. The use of /gene is reserved for the actual biological gene symbol such as "trnG". If a tRNA is a pseudogene, please use the /pseudo qualifier.

1	400	gene
			locus_tag     OBB_0001
1	400	rRNA
			product	16S ribosomal RNA
401	500	gene
			gene	trnG
			note	tRNA-Gly1
			locus_tag     OBB_0002
401	500	tRNA
			product	tRNA-Gly
501	600	gene
			locus_tag     OBB_0003
501	600	misc_RNA
			product	tmRNA
601	700	gene
			locus_tag     OBB_0004
601	700	tRNA
			product	tRNA-Xxx
701	800	gene
			locus_tag     OBB_0005
			pseudo
701	800	tRNA	
			product	tRNA-Phe
			pseudo

Evidence Qualifiers

At the 2005 annual meeting of the International Nucleotide Sequence Databases (INSD), DDBJ, EMBL and GenBank agreed to adopt two qualifiers to describe the evidence for features in sequence records. These are "/experimental=text" and "/inference=TYPE:text", where 'TYPE' is from a select list and 'text' is structured text. These new qualifiers replace "evidence=experimental" and "evidence=non-experimental", respectively, which are no longer supported. Read more about Evidence Qualifiers

1     100   gene  
		locus_tag   Test_0001
1     100   CDS
		product     RecA
		protein_id  gnl|center_name|Test_0001
		inference   ab initio prediction:Genscan:2.0
200   300   gene
		locus_tag   Test_0002
200   300   CDS
		product     SecA
		protein_id  gnl|center_name|Test_0002
		inference   similar to DNA sequence, (same species):INSD:DQ060639.1
400   500   gene  
		locus_tag   Test_0003
400   500   CDS
		product     ribonuclease R
		protein_id  gnl|center_name|Test_0003
		inference   protein motif:InterPro:IPR001900
		db_xref	InterPro:IPR001900
600   700   gene  
		locus_tag   Test_0004
600   700   CDS
		product     nitroreductase A
		protein_id  gnl|center_name|Test_0004
		experiment  expression of GST fusion protein    

Functional bacteriophage

If a bacterial genome contains a functional phage, an additional source feature must be included with the spans covering the complete phage sequence. However, if the phage is not functional or if you are not sure, annotate it as a misc_feature.

Example

361	4200	source
			organism	Bacteriophage xyz

Insertion sequences and transposons

Insertion sequences and transposons must be annotated as repeat_region features. The name of the insertion sequence or transposon must be added in a insertion_seq or transposon qualifier. Note that transposons and insertion sequences should not be given locus_tags.

1	100	repeat_region
			mobile_element	insertion sequence:IS1363

500	600	repeat_region
			mobile_element	transposon:Athena-Av1

Data base cross references

A variety of data base cross references can be added to a feature. These appear as /db_xref on the features. This qualifier serves as a vehicle for linking of sequence records to other external databases. See the full list of db_xref.

1     100   gene  
		locus_tag   Test_0001
1     100   CDS
		product     RecA
		protein_id  gnl|center_name|Test_0001
		db_xref	InterPro:IPR000111

Gene Ontology

GO (Gene Ontology) terms can be included in genomes in order to describe protein functionality. Gene Ontology (GO) terms can be indicated with the following qualifiers

1	100	CDS
			product	AbcD
			go_component    exocyst|0000145
			go_process      regulation of transcription, DNA-dependent|0006355
			go_process      exocytosis|0006887
			go_function     DNA binding|0003677

Other Annotation

Riboswitches should be annotated using the misc_binding feature if the bound moiety is known.

1	100	misc_binding
			note    Cobalamin riboswitch as predicted by Rfam (RF00174), score 19.56
			bound_moiety    adenosylcobalamin

If the bound moiety is unknown annotate riboswitches using a misc_feature instead of misc_binding.

1	100	misc_feature
			note    yybP-ykoY element as predicted by Rfam (RF00080), score 62.42		
			

Revised May 7, 2007

Submission Instructions

• Introduction
• Annotation Instructions
• Annotation Examples

Links

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• about Sequin
• about tbl2asn
• Sequin MacroSend

Genomes

• Genome Project List
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• Project Instructions
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• Evidence Qualifiers