Sequence Databases – 21 June 2007 Learning objectives- Be able to describe how information is stored in GenBank. Be able to read a GenBank flat file. Be able to search GenBank for information. Be able to explain the content difference between a header, features and sequence. Be able to say what distinguishes between a primary database and a secondary database. Be able to access and navigate the ENTREZ platform for biological data analysis.

BIOSEQs – entry common to all sequence databases BIOSEQ = Biological sequence Central element in the NCBI database model. Found in both the nucleotide and protein databases Comprises the sequence of a single continuous molecule of nucleic acid or protein. Entry must have At least one sequence identifier (Seq-id) Information on the physical type of molecule (DNA, RNA, or protein) Descriptors, which describe the entire Bioseq Annotations, which provide information regarding specific locations within the Bioseq

What is GenBank? The NIH genetic sequence database, an annotated collection of all publicly available DNA sequences Each record represents a single contiguous stretch of DNA or RNA DNA stretches may have more than one coding region (gene). RNA sequences are presented with T, not U Records are generated from direct submissions to the DNA sequence databases from the investigators (authors). GenBank is part of the International Nucleotide Sequence Database Collaboration.

General Comments on GBFF Three sections: 1) Header-information about the whole record 2) Features-description of annotations-each represented by a key. 3) Nucleotide sequence-each ends with // on last line of record. Nucleic acid (DNA or RNA (cDNA)) sequence translated to amino acid sequence is a “feature” Genbank Flat File (MyoD1 as an example)

Feature Keys Purpose: 1) Indicates biological nature of sequence 2) Supplies information about changes to sequences Feature KeyDescription conflictSeparate determinations of the same seq. differ rep_originOrigin of replication protein_bindProtein binding site on DNA CDSProtein coding sequence

Feature Keys-Terminology Feature Key Location/Qualifiers CDS /product=“alcohol dehydro.” /gene=“adhI” The feature CDS is a coding sequence beginning at base 23 and ending at base 400, has a product called “alcohol dehydrogenase” and corresponds to the gene called “adhI”.

Feature Keys-Terminology (Cont.) Feat. Key Location/Qualifiers CDS join ( , ) /product=“T-cell recep. B-ch.” /partial The feature CDS is a partial coding sequence formed by joining the indicated elements to form one contiguous sequence encoding a product called T-cell receptor beta-chain. (For MyoD1 – Accession number X61655)X61655

Record from GenBank LOCUS SCU bp DNA PLN 21-JUN-1999 DEFINITION Saccharomyces cerevisiae TCP1-beta gene, partial cds, and Axl2p (AXL2) and Rev7p (REV7) genes, complete cds. ACCESSION U49845 VERSION U GI: KEYWORDS. SOURCE baker's yeast. ORGANISM Saccharomyces cerevisiae Eukaryota; Fungi; Ascomycota; Hemiascomycetes; Saccharomycetales; Saccharomycetaceae; Saccharomyces. Modification date GenBank division (plant, fungal and algal) Coding region Unique identifier (never changes) Nucleotide sequence identifier (changes when there is a change in sequence (accession.version)) GeneInfo identifier (changes whenever there is a change) Word or phrase describing the sequence (not based on controlled vocabulary). Not used in newer records. Common name for organism Formal scientific name for the source organism and its lineage based on NCBI Taxonomy Database Locus name

Record from GenBank (cont.1) REFERENCE 1 (bases 1 to 5028) AUTHORS Torpey,L.E., Gibbs,P.E., Nelson,J. and Lawrence,C.W. TITLE Cloning and sequence of REV7, a gene whose function is required for DNA damage-induced mutagenesis in Saccharomyces cerevisiae JOURNAL Yeast 10 (11), (1994) MEDLINE REFERENCE 2 (bases 1 to 5028) AUTHORS Roemer,T., Madden,K., Chang,J. and Snyder,M. TITLE Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein JOURNAL Genes Dev. 10 (7), (1996) MEDLINE Medline UID REFERENCE 3 (bases 1 to 5028) AUTHORS Roemer,T. TITLE Direct Submission JOURNAL Submitted (22-FEB-1996) Terry Roemer, Biology, Yale University, New Haven, CT, USA Submitter of sequence (always the last reference)

Record from GenBank (cont.2) FEATURES Location/Qualifiers source /organism="Saccharomyces cerevisiae" /db_xref="taxon:4932" /chromosome="IX" /map="9" CDS < /codon_start=3 /product="TCP1-beta" /protein_id="AAA " /db_xref="GI: " /translation="SSIYNGISTSGLDLNNGTIADMRQLGIVESYKLKRAVVSSASEA AEVLLRVDNIIRARPRTANRQHM" The 5’ end of the coding sequence begins upstream of the first nucleotide of the sequence. The 3’ end is complete. There are three parts to the feature key: a keyword (indicates functional group), a location (instruction for finding the feature), and a qualifier (auxiliary information about a feature) Keys Location Qualifiers Descriptive free text must be in quotations Start of open reading frame Database cross-refs Protein sequence ID # Note: only a partial sequence Values

Record from GenBank (cont.3) gene /gene="AXL2" CDS /gene="AXL2" /note="plasma membrane glycoprotein" /codon_start=1 /function="required for axial budding pattern of S. cerevisiae" /product="Axl2p" /protein_id="AAA " /db_xref="GI: " /translation="MTQLQISLLLTATISLLHLVVATPYEAYPIGKQYPPVARVN... “ gene complement( ) /gene="REV7" CDS complement( ) /gene="REV7" /codon_start=1 /product="Rev7p" /protein_id="AAA " /db_xref="GI: " /translation="MNRWVEKWLRVYLKCYINLILFYRNVYPPQSFDYTTYQSFNLPQ... “ Cutoff Another location

Record from GenBank (cont.4) BASE COUNT 1510 a 1074 c 835 g 1609 t ORIGIN 1 gatcctccat atacaacggt atctccacct caggtttaga tctcaacaac ggaaccattg 61 ccgacatgag acagttaggt atcgtcgaga gttacaagct aaaacgagca gtagtcagct...//

Primary databases vs. Secondary databases Primary database comprises information submitted directly by the experimenter. is called an archival database. Secondary database comprises information derived from primary database. is a curated database.

Types of primary databases carrying biological infomation GenBank/EMBL/DDBJ PDB-Three-dimensional structure coordinates of biological molecules PROSITE-database of protein domain/function relationships.

Types of secondary databases carrying biological infomation dbSTS-Non-redundant db of sequence-tagged sites (useful for physical mapping) Genome databases-(there are over 20 genome databases that can be searched EPD:eukaryotic promoter database NR-non-redundant GenBank+EMBL+DDBJ+PDB. Entries with 100% sequence identity are merged as one. ProDom PRINTS BLOCKS

RNA cDNA DNA protein DNA databases derived from GenBank containing data for a single gene Non-redundant (nr) dbGSS (genome survey sequences) dbHTGS (high throughput) dbSTS (sequence tagged site) LocusLink RNA (cDNA) databases derived from GenBank containing data for a single gene dbEST (expressed sequence tag) UniGene LocusLink Protein databases derived from GenBank containing data for a single gene Non-redundant (nr) Swissprot PIR (Int’l. protein sequence) LocusLink Secondary Databases

References for understanding the NCBI sequence database model Here is the website for NCBI developer tools. KDOCS/INDEX.HTML KDOCS/INDEX.HTML

Mature mRNA RNA, but NOT mRNA DNA  RNA  PROTEIN RNA processing