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 prokaryotic 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
-
Prepare annotation table
- Gene features
- locus_tag
- protein_id
- transcript_id
- CDS (coding region) features
- Partial coding regions in incomplete genomes
- Gene fragments
- Transpliced Genes
- Split genes on two contigs
- Ribosomal RNA, tRNA and other RNA features
- mRNA features
- Alternatively spliced genes
- Evidence Qualifiers
- Data base cross references
- Gene Ontology
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 the sequence's SeqID in the FASTA file. 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. The allowed features and their qualifiers are listed in the Feature Table documentation.
Features that are on the complementary strand, such as the genes Ngs_3038 and Ngs_11232 and their corresponding features shown in Figure 2, are indicated by reversing the interval locations.
Please avoid unnecessary capitalization in all text entered in your table.
Additional requirements, as well as suggestions for various types of annotation, are included in the following sections.
Gene features
Gene features are always a single interval, and their location should cover the intervals of all the relevant features such as promoters and polyA binding sites.
Gene names should follow the standard nomenclature rules of the particular organism. For example, mouse gene names begin with an uppercase letter, and the remaining letters are lowercase.
Coding regions (CDS) and RNAs, such as tRNAs and rRNAs, must have a corresponding gene feature. However, other features such as repeat_regions and misc_features do not have a corresponding gene or locus_tag.
locus_tag
All genes should be assigned a systematic gene identifier which should receive the locus_tag qualifier on the gene feature in the table. Genes may also have functional names as assigned in the scientific literature. In this example, KCS_0001 is the systematic gene identifier, while Abc5 is the functional gene name.
Table view of gene with both biological name and locus_tag:
1 1575 gene
gene Abc5
locus_tag KCS_0001
Flatfile view:
gene 1..1575
/gene="Abc5"
/locus_tag="KCS_0001"
Table view of gene with only locus_tag:
1 1575 gene locus_tag KCS_0001
Flatfile view:
gene 1..1575
/locus_tag="KCS_0001"
For consistency the same locus_tag prefix must be used throughout the entire genome. Therefore, all the chromosomes of a genome should have the same locus_tag prefix.
To improve the use of locus_tags we are now requiring that all locus_tag prefixes be registered and that they be unique. The locus_tag prefix should be 3-12 alphanumeric characters and the first character may not be a digit. The locus_tag prefix is 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 special characters can be used in the locus_tag.
The chromosome number can be embedded in the locus_tag, if desired, in the format Prefix_#g#####, where the first # is the chromosome number and ##### is the unique number of the gene. For example, Ajs_4g00123 for a gene on chromosome 4.
Read more about locus_tags and their intended usage.
We have set up a registry to check for the uniqueness of locus_tag prefixes.
Please register your genome project and proposed locus_tag prefix on the Genome Project registration page prior to preparing your submission to GenBank. Each project that is registered here is assigned a project_id, and in the future we intend that the project_id will appear on all entries associated with a particular genome project.
The use of locus_tag is supported in Sequin version 4.35 or later. If you have an older version of Sequin please download the current version. The latest version of tbl2asn is available from the tbl2asn page.
protein_id
All proteins in a WGS or complete genome must be assigned an identification number by the submitter. We use this number to track proteins when sequences are updated. This number is indicated in the table by the CDS qualifier protein_id, and should have the format gnl|dbname|string, where dbname is the username on your ftp account or name assigned by GenBank, and string is the unique protein SeqID assigned by the submitter. 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 as the protein SeqID. In this example, the protein_id for ABC5 is gnl|dbname|KCS_0001.
Example:
<1 >1575 gene gene Abc5 locus_tag KCS_0001 1 1575 CDS product ABC5 protein_id gnl|dbname|KCS_0001
Since the protein_id is used for internal tracking in our database, it is important that the complete protein_id (dbname + SeqID) not be duplicated by a genome center. Thus, if your genome center is submitting more than one complete genome, please be sure to use unique protein_id's for all of the genomes.
The protein_id is also included as a qualifier on the corresponding mRNA feature, to allow the CDS and mRNA to be paired during processing.
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.
transcript_id
The transcript_id is included as a qualifier for both the CDS and its corresponding mRNA. It has the same format as the protein_id, gnl|dbname|identifier. Because each transcript_id and protein_id must be unique, we suggest adding 'mrna' or 't' to the protein_id identifier as a simple way to create the corresponding (unique) transcript_id. However, you can use whatever naming convention you choose, as long as all of the identifiers are unique.
63574 87173 gene locus_tag Ngs_17131 63574 63907 mRNA 75690 75730 84396 85536 85598 85773 85836 86109 86173 86467 86555 86670 86731 87173 product hypothetical protein protein_id gnl|ncbi|Ngs_17131 transcript_id gnl|ncbi|Ngs_mrna17131 84402 85536 CDS 85598 85773 85836 86109 86173 86467 86555 86670 86731 86882 product hypothetical protein protein_id gnl|ncbi|Ngs_17131 transcript_id gnl|ncbi|Ngs_mrna17131
CDS (coding region) features
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 in the correct format for the organism. For example, mouse proteins have the same symbol as the gene name, but the protein name has all capital letters.
- 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
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
Multiple note qualifiers can be included and will be concatenated by tbl2asn or Sequin into a single note with semi-colons as separators.
Bifunctional proteins:
If a protein contains two separate and distinct functions or if it has more than one name, these can be annotated in several ways, as outlined below. Table view:start stop CDS product adenylyltransferase/ADP-heptose synthase note bifunctionalor
start stop CDS product bifunctional adenylyltransferase/ADP-heptose synthase cyclohydrolaseor
start stop CDS
product FolD
function adenylyltransferase
function ADP-heptose synthase cyclohydrolase
note bifunctional
Partial coding regions in incomplete genomes
To annotate an partial coding region, you should use the "<" or ">" in your feature table to designate the feature as either 5' or 3' partial. The coding region should 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.
<1 497 CDS product ABC5 note similar to Bacillus subtilis aldolase codon_start 3 protein_id gnl|dbname|KCS_0001 transcript_id gnl|dbname|KCS_mrna0001 600 >1575 CDS product actin-like protein protein_id gnl|dbname|KCS_0002 transcript_id gnl|dbname|KCS_mrna0002
Partial genes within a sequence should begin or end at consensus splice sites.
The use of partial coding regions is reserved for incomplete genomes only. All coding regions annotated on finished genomes should be complete at both the 5' and 3' ends.
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:
-
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 that this is a
pseudogene. If known, a note qualifier may be added indicating why this gene is
disrupted.
1 200 gene gene Abc5 locus_tag KCS_0001 gene_desc alkaline phosphatase pseudo note frameshift -
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. If known, the reason for the incomplete translation can be included in a
note.
1 200 gene gene Abc5 locus_tag KCS_0001 gene_desc alkaline phosphatase note nonfunctional due to frameshift
-
If the feature is just noting a similarity to genes in the database and is
probably not translated, then it should be annotated as a misc_feature without
a corresponding gene feature.
1 200 misc_feature note similar to Abc5
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 5000 5500 mRNA 6000 >7200 product enolase protein_id gnl|dbname|KCS_2223A transcript_id gnl|dbname|KCS_mrna2223A 5488 5500 CDS 6000 >7200 product enolase protein_id gnl|dbname|KCS_2223A transcript_id gnl|dbname|KCS_mrna2223A note 5' end; 3' end is gene KCS_2223 on contig Cont01.00224
>Feature Cont01.00224 <1 1000 gene locus_tag KCS_2223 <100 1000 mRNA product enolase protein_id gnl|dbname|KCS_2223B transcript_id gnl|dbname|KCS_mrna2223B <100 876 CDS product enolase protein_id gnl|dbname|KCS_2223B transcript_id gnl|dbname|KCS_mrna2223B note 3' end; 5' end is gene KCS_2223 on contig
Ribosomal RNA, tRNA and other RNA features
RNA features (rRNA, tRNA, misc_RNA) need a corresponding gene feature with a locus_tag qualifier. If the amino acid of a tRNA is unknown, use tRNA-Xxx as the product, as in the example.
1 400 gene
locus_tag KCS_00011
1 400 rRNA
product 16S ribosomal RNA
488 560 gene
locus_tag KCS_00012
488 560 tRNA
product tRNA-Phe
700 780 gene
locus_tag KCS_00013
700 780 tRNA
product tRNA-Xxx
900 923 gene
locus_tag KCS_00014
900 923 misc_RNA
product mir-9c
mRNA features
Include an mRNA feature for each translated CDS. Several things to note are:
- Use the same product name for the mRNA and its corresponding CDS.
- If there is no UTR information, then the mRNA's location will agree with its CDS's location, but the mRNA will be partial at its 5' and 3' ends.
- Extend the gene feature to include the entire mRNA.
- If the mRNA is partial, then make the gene partial.
Example
The first example is a complete CDS whose 5' and 3' UTRs are known.
>Feature Cont54 10400 12512 gene locus_tag CCC_03116 10400 10462 mRNA 10533 10577 10651 11098 11182 11642 11716 12512 product hypothetical protein protein_id gnl|dbname|CCC_03116 transcript_id gnl|dbname|CCC_mrna03116 10450 10462 CDS 10533 10577 10651 11098 11182 11642 11716 12233 product hypothetical protein protein_id gnl|dbname|CCC_03116 transcript_id gnl|dbname|CCC_mrna03116
The second example is a CDS that is partial at the 5' end and lacks any 3' UTR information.
>Feature Cont3 <1 >497 gene locus_tag CCC_111011 <1 497 CDS note similar to Bacillus subtilis aldolase product aldolase-like protein codon_start 3 protein_id gnl|dbname|CCC_111011 transcript_id gnl|dbname|CCC_mrna111011 <1 >497 mRNA product aldolase-like protein protein_id gnl|dbname|CCC_111011 transcript_id gnl|dbname|CCC_mrna111011
Alternatively spliced genes
In many cases a gene can be alternatively spliced, yielding alternative transcripts. These transcripts may differ in the coding region and produce different products, or they may differ in the non-translated 5' or 3' UTR and produce the same protein. To annotate alternatively spliced genes, include one mRNA and CDS for each transcript, and include only one gene over all of the features. Give the corresponding mRNA and CDS the same name, and include a note "alternatively spliced" on each. If there are multiple CDS with the same name, then add a note to each mRNA and CDS to refer to each other, eg "transcript variant A" and "encoded by transcript variant A" for one mRNA/CDS pair. If the CDS have different translations, then they should have different product names. Make sure that all the proteins have unique protein_id's.
Example
Example 1 (different products):
>Feature Cont01.00055 10 5000 gene locus_tag CCC_04562 10 500 mRNA 722 1555 2548 3901 4400 5000 product CCC_04562 isoform A note alternatively spliced protein_id gnl|dbname|CCC_04562A transcript_id gnl|dbname|CCC_mrna04562A 102 500 CDS 722 1555 2548 3901 4400 4566 product CCC_04562 isoform A note alternatively spliced protein_id gnl|dbname|CCC_04562A transcript_id gnl|dbname|CCC_mrna04562A 10 500 mRNA 2548 3901 4400 5000 product CCC_04562 isoform B note alternatively spliced protein_id gnl|dbname|CCC_04562B transcript_id gnl|dbname|CCC_mrna04562B 102 500 CDS 2548 3901 4400 4566 product CCC_04562 isoform B note alternatively spliced protein_id gnl|dbname|CCC_04562B transcript_id gnl|dbname|CCC_mrna04562B
Example 2 (same product):
>Feature Cont01.00056 100 1000 gene locus_tag CCC_03222 100 333 mRNA 444 678 800 1000 product hypothetical protein note transcript variant A; alternatively spliced protein_id gnl|dbname|CCC_03222A transcript_id gnl|dbname|CCC_mrna03222A 456 678 CDS 800 865 product hypothetical protein note encoded by transcript variant A; alternatively spliced protein_id gnl|dbname|CCC_03222A transcript_id gnl|dbname|CCC_mrna03222A 100 360 mRNA 444 678 800 1000 product hypothetical protein note transcript variant B; alternatively spliced protein_id gnl|dbname|CCC_03222B transcript_id gnl|dbname|CCC_mrna03222B 456 678 CDS 800 865 product hypothetical protein note encoded by transcript variant B; alternatively spliced protein_id gnl|dbname|CCC_03222B transcript_id gnl|dbname|CCC_mrna03222B
Evidence Qualifiers
The International Nucleotide Sequence Database Collaboration, DDBJ, EMBL and GenBank has adopted a set of new qualifiers to describe the evidence for feature annotation in GenBank records. These are:
/experimental="text"
/inference="TYPE:text", where 'TYPE' is from a select list and
'text' is structured text.
The use of the new evidence qualifiers is supported in Sequin version 6.0 or later, and tbl2asn version 5.1 or later. If you have an older version, please download the current version of Sequin or tbl2asn.
These new qualifiers replace /evidence=experimental and /evidence=non-experimental, respectively, which will no longer be supported.
See more information about the Evidence Qualifiers.
Database cross references
A variety of database 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 databases.
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 helicase go_process chromatin assembly or disassembly|0006333||IEA go_process antimicrobial humoral response|0019730|16163390|IMP go_component nucleus|0005634|14668392|IDA go_component chromatin|0000785||IEA go_function ATP-dependent helicase activity|0008026||ISS go_function nucleic acid binding|0003676||IEA go_function ATP binding|0005524||IEA
The value field is separated by vertical bars '|' into a descriptive string, the GO identifier (leading zeroes are retained), and optionally a PubMed ID and one or more evidence codes. The evidence code is the fourth token, so include blank fields, as necessary (eg the last qualifier has no PubMed ID so the third field is blank).
Revised October 16, 2007