Submission of Complete Genomic Records with Sequin

Submission of complete genomes or other large sequence records

Sequin

Entrez

BLAST

OMIM

Taxonomy

Structure

Introduction

Sequin can read a five-column, tab-delimited table of feature locations and qualifiers. The table is read into Sequin, along with the DNA sequence (in FASTA or PHRAP/ACE format) and the submitter information, and the record is then ready for submission to GenBank. Even very long chromosomes (e.g., 20 Mb) can be processed in seconds.

The feature table format allows different kinds of features (e.g., gene, mRNA, coding region, tRNA) and qualifiers (e.g., /product, /note) to be indicated. (Systematic sequencing tracking numbers used in genome-level annotation are entered as locus tags.) It also allows multiple intervals per feature to represent the multiple exons in a spliced mRNA, for example. Once the sequence and annotation have been read in, Sequin's validator can be used to check for internal stops in coding regions and use of consensus splice sites. Coding region (CDS), gene, and mRNA feature intervals are also checked for mutual consistency.

The entire process can be automated for multiple chromosomes in a genome with the utility tbl2asn, which produces ASN.1 from pairs of table and sequence files.

FASTA-formatted sequence

Sequin can read nucleotide sequences of any size in FASTA format. FASTA format consists of a single definition line, beginning with a ">" followed by optional text, and subsequent lines of sequence. At minimum, all definition lines must contain an identifier for the sequence, called the SeqId. Other information about the biological source of the organism can also be encoded on the definition line of the sequence and is used to build the record. A sample definition line is

>Sc_16 [organism=Saccharomyces cerevisiae] [strain=S288C] [chromosome=XVI]

Common source modifiers incorporated into the definition line include [strain=yyy] and [chromosome=nnn]. Note that there are no spaces surrounding the equal sign. Complete circular genomes should include the modifier [topology=circular]. Sequences located in the mitochondrion or chloroplast need a modifier of [location=mitochondrion] or [location=chloroplast]. A complete list of modifiers is available from the Sequin help document Source and Organism subpage sections.

An example of a FASTA-formatted sequence is shown in Figure 1.

Table Layout

Sequin reads features from a five-column, tab-delimited table. The feature table specifies the location and type of each feature, and Sequin processes the feature intervals and translates any CDS features into proteins. 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 table and illustrates a number of points about the table format. The GenBank flatfile corresponding to this table is shown in Figure 3, and a graphical overview of the features in this example is in Figure 4.

Features that are on the complementary strand, such as the gene YPR027C and the tRNA tRNA-Phe, are indicated by reversing the interval locations.
Gene features are always a single interval, and their location should cover the intervals of all the relevant features. For example, the gene YIP2 is as long as its mRNA, and is thus longer than its CDS.
If the gene feature spans the intervals of the CDS or mRNA features for that gene, there is no need to include gene qualifiers on those features in the table, because they will be picked up by overlap. For example, in the flatfile, the gene names ATH1 and YPR027C are present as /gene on the overlapping CDS, even though they are not explicitly listed as gene qualifiers on those CDSs in the table. This option can be suppressed by adding a gene qualifier with the value '-' to the feature, as was done in this example for tRNA-Phe. Suppressing the overlapping /gene is important when, for example, a tRNA is encoded within an intron of a housekeeping gene.
If a feature contains multiple intervals, like the spliced tRNA-Phe or the Yip2p CDS, each interval is listed on a separate line by its start and stop position.
Locations of partial (incomplete) features are indicated with a ">" or "<" next to the number. In this example, the first gene, CDS, and mRNA all begin upstream of the start of the nucleotide sequence. The "<" symbol indicates that they are 5' partial features. Furthermore, for the protein to translate correctly, the correct reading frame must be indicated with the qualifier "codon_start" on the first CDS in the table. There is no need to indicate the codon_start on complete CDSs, as it is assumed that the translation starts at the first nucleotide of the interval if no codon_start is provided.
If a protein has more than one name, each can be listed in the table as a separate product qualifier on the CDS in the table . The value of the first product qualifier will become the /product on the CDS in the flatfile, and any additional product qualifie rs will be shown as a /note on the CDS in the flatfile. See the first CDS, which has two product qualifiers, acid trehalase and Ath 1p. All CDS features must have at least one product, even it is the word 'unknown'.
A flatfile /note can be added to any feature using the qualifier note in the table. A note has been added to the second CDS.
Published citations are added using the REFERENCE feature. For most publications, the start and stop of the feature are the first and last nucleotides of the sequence. The qualifier key is PubMed, and the value is the PubMed Identifier (PMID), which can b e found in PubMed.
The [offset] is used to add a specified number to all subsequent nucleotide intervals. In this example, the record was annot ated in two pieces, each piece starting from residue number 1. The sequences themselves were joined together in the FASTA file. The [offset=2000] adds 2000 nt to the location of all features that follow it, sparing the submitter the need to recalculate the locat ion of each feature. This option could be used if the feature intervals for two arms of a chromosome or adjacent contigs are stored separately, but should be joined for the final submission.
Note that when annotating complete genomes, systematic gene names and protein identifiers are required.
In some organisms, like Saccharomyces cerevisiae, genes are identified by a systematic name based on chromosomal position. Genes may also have functional names as assigned in the scientific literature. The systematic gene name should receive the locus_tag qualifier on the gene feature in the table, whereas the functional gene name should receive the gene qualifier. In this example, YPR026W, YPR027C, and YPR028W are the systematic gene names, while ATH1 and YIP2 are the functional gene names. All gene features in genome records must have a locus_tag qualifier.
GenBank prefers that all proteins in a complete genome 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, and string is the identification number. In this example, the protein_id for acid trehalase is gnl|SGD|S0006230. This identifier is saved with the record (in ASN.1 format), but it is not visible in the flatfile. It is also important that a product name or protein description is given.
When submitting a complete bacterial genome, please review the genome guidelines.

Single Record Submissions

Single submissions can be treated like a new Sequin submission. On the starting Sequin page, click on "Start New Submission", and fill out the Submitting Authors form. On the Organism and Sequences form, indicate that this is a Single sequence in FASTA format, choose the Organism and Molecule, indicate that the FASTA definition line starts with a sequence ID, and import the nucleotide sequence. Do not import protein sequence. After you create the initial record containing the sequence, import the table by choosing it with Sequin's File-->Open command. The features listed in the table will be immediately displayed on the flatfile view. Carry out any desired editing, then choose Search-->Validate to check for errors in the record, and fix any mistakes. Use the File-->Save As command to save the record in ASN.1 format. It is this ASN.1 version that should be submitted to GenBank. Additional tips on using Sequin are found in the Sequin Quick Guide.

Multiple Record Submissions

tbl2asn is a command line program that automates parts of the submission process. It is packaged with the Sequin archive (Sequin version 3.30 and above). tbl2asn reads a template, along with the sequence and table files, and outputs ASN.1 for submission to GenBank. Thus, the submitter does not need to read each set of table and sequence files into Sequin. Details about using tbl2asn can be found in this document.

Figure 1: Sample FASTA-formatted sequence:

>Sc_16 [organism=Saccharomyces cerevisiae] [strain=S288C] [chromosome=XVI]
CGACCACAATGGTACGATTGTTCATAAATCAGGAGATGTTCCTATTCATATAAAGATACC
AAACAGATCTCTAATACATGACCAGGATATCAACTTCTATAATGGTTCCGAAAACGAAAG
AAAACCAAATCTAGAGCGTAGAGACGTCGACCGTGTTGGTGATCCAATGAGGATGGATAG
[etc.]

Figure 2: Sequin table format:

>Feature Sc_16
1	7000	REFERENCE
			PubMed		8849441
<1	1050	gene
			gene		ATH1
			locus_tag	YPR026W
<1	1009	CDS
			product		acid trehalase
			product		Ath1p
			codon_start	2
			protein_id	gnl|SGD|S0006230
<1	1050	mRNA
			product		acid trehalase
[offset=2000]
1253	420	gene
			locus_tag	YPR027C
1253	420	CDS
			product		Ypr027cp
			note		hypothetical protein
			protein_id	gnl|SGD|S0006231
1253	420	mRNA
			product		Ypr027cp
2626	2590	tRNA
2570	2535
			gene		-
			product		tRNA-Phe
3450	4536	gene
			gene		YIP2
			locus_tag	YPR028W
3522	3572	CDS
3706	4197
			product		Yip2p
                        prot_desc       similar to human polyposis locus protein 1 (YPD)
			protein_id	gnl|SGD|S0006232
3450	3572	mRNA
3706	4536
			product		Yip2p

Figure 3: GenBank flatfile

LOCUS       Sc_16        7000 bp    DNA             PLN       08-MAY-2000
DEFINITION  Saccharomyces cerevisiae chromosome XVI strain S288C.
ACCESSION   Sc_16
VERSION
KEYWORDS    .
SOURCE      baker's yeast.
  ORGANISM  Saccharomyces cerevisiae
            Eukaryota; Fungi; Ascomycota; Hemiascomycetes; Saccharomycetales;
            Saccharomycetaceae; Saccharomyces.
REFERENCE   1  (bases 1 to 7000)
  AUTHORS   Goffeau,A., Barrell,B.G., Bussey,H., Davis,R.W., Dujon,B.,
            Feldmann,H., Galibert,F., Hoheisel,J.D., Jacq,C., Johnston,M.,
            Louis,E.J., Mewes,H.W., Murakami,Y., Philippsen,P., Tettelin,H. and
            Oliver,S.G.
  TITLE     Life with 6000 genes
  JOURNAL   Science 274 (5287), 546 (1996)
   PUBMED   8849441
REFERENCE   2  (bases 1 to 7000)
  AUTHORS   Ouellette,B.F.F.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-MAY-2000) NCBI/NLM, National Institutes of Health,
            Building 38A, Room 8N805, Bethesda, MD 20894, USA
FEATURES             Location/Qualifiers
     source          1..7000
                     /organism="Saccharomyces cerevisiae"
                     /strain="S288C"
                     /chromosome="XVI"
     mRNA            <1..1050
                     /gene="ATH1"
                     /product="acid trehalase"
     gene            <1..1050
                     /gene="ATH1"
                     /locus_tag="YPR026W"
     CDS             <1..1009
                     /gene="ATH1"
                     /note="Ath1p"
                     /codon_start=2
                     /product="acid trehalase"
                     /translation="DHNGTIVHKSGDVPIHIKIPNRSLIHDQDINFYNGSENERKPNL
                     ERRDVDRVGDPMRMDRYGTYYLLKPKQELTVQLFKPGLNARNNIAENKQITNLTAGVP
                     GDVAFSALDGNNYTHWQPLDKIHRAKLLIDLGEYNEKEITKGMILWGQRPAKNISISI
                     LPHSEKVENLFANVTEIMQNSGNDQLLNETIGQLLDNAGIPVENVIDFDGIEQEDDES
                     LDDVQALLHWKKEDLAKLIEQIPRLNFLKRKFVKILDNVPVSPSEPYYEASRNQSLIE
                     ILPSNRTTFTIDYDKLQVGDKGNTDWRKTRYIVVAVQGVYDDYDDDNKGATIKEIVLN
                     D"
     mRNA            complement(2420..3253)
                     /locus_tag="YPR027C"
                     /product="Ypr027cp"
     gene            complement(2420..3253)
                     /locus_tag="YPR027C"
     CDS             complement(2420..3253)
                     /locus_tag="YPR027C"
                     /note="hypothetical protein"
                     /codon_start=1
                     /product="Ypr027cp"
                     /translation="MVGIYRILASFVPLLGLLFAFHDDDMIDTVTIIKTVYETVTSTS
                     TAPAPAATKSVSEKKLDDTKLTLQVIQTMVSCFSVGENPANMISCGLGVVILMFSLII
                     ELINKLENDGINEPQRLYDLIKPKYVELPSNYVNEKIKTTFEPLDLYLGVNMNTSGSE
                     LNQNCLILKLGEKTALPFPGLAQQICYTKGASNEFTNYKLSDIQGNLNENSQGIANGV
                     FQKISNIRKISGNFKSQLYQISEKITDENWDGSAVGFTAHGREKGPNKSQISVSFYRD
                     N"
     tRNA            complement(join(4535..4570,4590..4626))
                     /product="tRNA-Phe"
     mRNA            join(5450..5572,5706..6536)
                     /gene="YIP2"
                     /product="Yip2p"
     gene            5450..6536
                     /gene="YIP2"
                     /locus_tag="YPR028W"
     CDS             join(5522..5572,5706..6197)
                     /gene="YIP2"
                     /note="similar to human polyposis locus protein 1 (YPD)"
                     /codon_start=1
                     /product="Yip2p"
                     /translation="MSEYASSIHSQMKQFDTKYSGNRILQQLENKTNLPKSYLVAGLG
                     FAYLLLIFINVGGVGEILSNFAGFVLPAYLSLVALKTPTSTDDTQLLTYWIVFSFLSV
                     IEFWSKAILYLIPFYWFLKTVFLIYIALPQTGGARMIYQKIVAPLTDRYILRDVSKTE
                     KDEIRASVNEASKATGASVH"
BASE COUNT     2201 a   1276 c   1255 g   2268 t
ORIGIN
        1 cgaccacaat ggtacgattg ttcataaatc aggagatgtt cctattcata taaagatacc
       61 aaacagatct ctaatacatg accaggatat caacttctat aatggttccg aaaacgaaag
      121 aaaaccaaat ctagagcgta gagacgtcga ccgtgttggt gatccaatga ggatggatag [etc.]

Figure 4: Sequin graphical view

Sequin graphical view displays the nucleotide sequence
as the top bar and lower arrows or bars representing different features.

Questions or Comments?
Write to the NCBI Service Desk

Revised June 14, 2004.