Literature Databases Online databases available at CSU – Galileo Galileo – JSTOR JSTOR Online databases at other sites – PubMed. If you find a useful article, you can check PubMed Central to see if it is available online for free. PubMed PubMed Central Where to get articles – PubMed Central PubMed Central – GIL GIL – Interlibrary loan Interlibrary loan
DNARNA cDNA *ESTs UniGene phenotype genomic DNA databases protein sequence databases protein Sources of Molecular Data *Expressed Sequence Tags
Molecular Databases Primary Database – Archival - sequences submitted directly from experimental sequencing results Very little interpretation Anyone can submit; accuracy not checked Examples – Nucleic Acid: EMBL, DDJB, GenBANK EMBLDDJBGenBANK – Protein: Swiss-Prot, PIR, PDB Swiss-ProtPIRPDB Secondary Databases – Curated – sequences are validated/checked and may be annotated Refseq (nucleic acids and proteins, but limited to certain organisms) Refseq TrEMBL, GenPept, Uniprot
Nucleic Acid Databases Contain: – Nucleic acid sequences Chain termination method (Sanger sequencing)Sanger sequencing – Used for sequences 100-1000 bp Whole Genome Shotgun (WGS) SequencingWGS – Used for sequences >1000 bp – DNA chopped into little chunks – Sequenced using chain termination method (reads) – Numerous, overlapping reads are collected and assembled into sequence (computational methods) – Annotations for each sequence Putative identification of open reading frames (ORFs = parts of gene that encode protein) in sequence Putative intron(excised)/exon(retained) locations Authors, dates, publication, etc.
GenBankEMBL DDBJ International Nucleotide Sequence Database Collaboration (Public nucleotide and protein sequence databases) Name: European Molecular Biology Laboratory (EMBL) Location: European Bioinformatics Institute (EBI) Name: DNA Database of Japan (DDBJ) Location: National Institute of Genetics, Mishima Daily Info sharing Name: GenBank Location: National Institutes of Health, National Center for Biotechnology Information Daily Info sharing
GenBank As of April 2011, There were approximately 126,551,501,141 bases in 135,440,924 sequence records in the traditional GenBank divisions. Read the following paper: http://www.ncbi.nlm.nih.gov/pubmed/21071399http://www.ncbi.nlm.nih.gov/pubmed/21071399 Home Page: http://www.ncbi.nlm.nih.gov/genbank/http://www.ncbi.nlm.nih.gov/genbank/ Homo sapiens 14.9 billion bases Mus musculus 8.9b Rattus norvegicus 6.5b Bos taurus5.4b Zea mays 5.0b Sus scrofa4.8b Danio rerio 3.1b Strongylocentrotus purpurata1.4b Oryza sativa (japonica) 1.2b Nicotiana tabacum 1.2b Most sequenced organisms
NCBI key features: PubMed National Library of Medicine's search service 21 million citations from MEDLINE & others (as of 2011) Links to other online journals http://www.ncbi.nlm.nih.gov/pubmed Starting point for most research
Use “Advanced search” to limit by author, year, language, etc.
PubMed search strategies Try the tutorial Use boolean queries (capitalize AND, OR, NOT) lipocalin AND disease Try using limits (see Advanced search) There are links to find Entrez entries and external resources
lipocalin AND disease (504 results) lipocalin OR disease (2,500,000 results) lipocalin NOT disease (2,370 results) 1 AND 2 1 OR 2 1 NOT 2 1 1 1 2 2 2
Scholar Google Search http://scholar.google.com/ Includes references that may not be found in PubMed
A search from NCBI main page will search: the scientific literature; DNA and protein sequence databases; 3D protein structure data; population study data sets; assemblies of complete genomes String search Search by author, date, keyword, publication, etc. NCBI key features Classroom exercise: Author searches Paper searches Protein searches
BLAST is… Basic Local Alignment Search Tool NCBI's sequence similarity search tool supports analysis of DNA and protein databases NCBI key features: BLAST 3CLN
Online Mendelian Inheritance in Man Catalog of human genes and genetic disorders NCBI key features: OMIM
Browser for the major divisions of living organisms (archaea, bacteria, eukaryota, viruses) Taxonomy information such as genetic codes Molecular data on extinct organisms Useful to find a protein or gene from a species NCBI key features: Taxonomy Browser
Molecular Modelling Database (MMDB) biopolymer structures obtained from the Protein Data Bank (PDB) Cn3D (a 3D-structure viewer) vector alignment search tool (VAST) NCBI key features: Structure
Cn3D A 3D-structure viewer Must download ( ftp://ftp.ncbi.nlm.nih.gov/cn3d/Cn3D-4.3.msi) ftp://ftp.ncbi.nlm.nih.gov/cn3d/Cn3D-4.3.msi Use to align structures identified as similar by VAST
Example: Researching beta globin Beta globin is protein, so it will be found in 3 different types of databases DNA*RNAProteins GenBank dbGSS GenBank dbHTGS GenBank dbSTS GenBank Entrez Gene GenBank dbEST UniGene Gene Expression Omnibus Entrez Protein UniProt PDB SCOP CATH *Because RNA is unstable, it can be transcribed into complementary DNA (cDNA)
Necessary (yet annoying) Definitions Sequence Tagged Site (STS): Small DNA fragments with both DNA sequence data and mapping data (genes assigned to chromosomes) Expressed Sequence Tags (EST): Partial DNA sequence of a complementary (cDNA) clone – Typically these are randomly-selected cDNA clones sequenced on a single strand (300-800 bp) – Useful for identifying novel genes – Higher rate of error http://genome.wellcome.ac.uk/doc_WTD020755.html
Unigene Unique Gene (Unigene) Project to create gene-oriented clusters by partitioning ESTs into non-redundant sets – http://www.ncbi.nlm.nih.gov/unigene – Ultimately there should be only 1 cluster per gene – Usually more than 1 due to errors – Types of errors 2 or more clusters may represent different parts of the same gene Sequence errors Cloning artifacts (DNA transcribed during creation of cDNA that doesn’t correspond to authentic transcript) EST’s Unigene Cluster
GenBank Flatfile A format for organizing genomic sequence data. Includes the following: Sequence and annotations Header – Locus name or accession number: unique to sequence description – Size: number of nucleotide bases or amino acid residues – Molecule: DNA, RNA, strandedness (ds, ss), and type of RNA or DNA – Genbank division code: 18 divisions (PRI = primate, PLN = plant, BAC = bacterial, etc.) – Date of last modification Definition Line: brief description of sequence (e.g. source organism, protein/gene name, function) Accession: unique identifier for a record Version – May be more than one accession – Record modification (accession.1; accession.2) – GI: is specific to version; may be more than one Keywords Source: organism or clone description Reference: publications that discuss data reported Authors and Journal publication info PubMed identifier: link to sequence record (abstract) Features: vary (chromosomal info., coding info, protein id, % of each nucleotide) Sequence Data Jump to example
What is an accession number? An accession number is label that is used to identify a sequence. It is a (unique) string of letters and/or numbers that corresponds to a molecular sequence. Examples (all for retinol-binding protein, RBP4): X02775GenBank genomic DNA sequence NT_030059Genomic contig (overlapping DNA fragments) Rs7079946dbSNP (single nucleotide polymorphism) N91759.1An expressed sequence tag (1 of 170) NM_006744RefSeq DNA sequence (from a transcript) NP_007635RefSeq protein AAC02945GenBank protein Q28369SwissProt protein 1KT7Protein Data Bank structure record protein DNA RNA
NCBI’s important RefSeq project: best representative sequences RefSeq (accessible via the main page of NCBI) provides an expertly curated accession number that corresponds to the most stable, agreed-upon “reference” version of a sequence. RefSeq identifiers include the following formats: Complete genomeNC_###### Complete chromosomeNC_###### Genomic contigNT_###### mRNA (DNA format)NM_###### e.g. NM_006744 ProteinNP_###### e.g. NP_006735
UniGene Name Search: Oncomodulin All results listed Allows filtering
UniGene Name Search: Select Human Oncomodulin 4 Expressed Sequence Tags from 1 complementary DNA library Identifies chromosome and map position on chromosome Compares cluster transcripts with refseq proteins
UniGene Name Search: Select Human Oncomodulin Click on link for menu of other links: Conserved domains Gene summary Protein sequence Clicking on Protein sequence link then takes you to predicted protein sequence file (NP_006179.2)
UniGene Name Search: Select Human Oncomodulin 1 2 3 4 Once here, you can: 1.Open FASTA file 2.Run BLAST 3.Identify and view conserved domains 4.See related proteins
Access to sequences: Gene at NCBI Gene is a great starting point: it collects key information on each gene/protein from major databases. It covers all major organisms. Example: RefSeq provides a curated, optimal accession number for each DNA (NM_000518 for beta globin DNA corresponding to mRNA) or protein (NP_000509) These references should be more reliable data
Gene Name Search: Oncomodulin Returns list of gene entries for oncomodulin for different organisms Click on a highlighted link to see details
Gene Name Search: Select Human Oncomodulin Summary of all gene information, including mapping (when available). Note that this sequence has been validated as a RefSeq. Scrolling down, you can find link to protein data through UniProt.
FASTA format: versatile, compact with one header line followed by a string of nucleotides or amino acids in the single letter code
Comparison of Gene to other resources Gene: collects key information on each gene/protein from major databases. It covers all major organisms. UniGene: Database with information on where in a body, when in development, and how abundantly a transcript is expressed HomoloGene: Gathers information on sets of related proteins based on common genetic ancestry.
Homologene Name Search: Oncomodulin Provides list of homologous (related) genes
Homologene Name Search: Oncomodulin Shows conserved domains of protein sequences. If you click on graphic, takes you to summary of domain/family information.
ExPASy to access protein and DNA sequences ExPASy (Expert Protein Analysis System) sequence retrieval system Visit http://www.expasy.ch/ Similar to Entrez for NCBI Example: Search for calmodulin Jump to PrositeProsite
UniProt: a centralized protein database (uniprot.org) This is separate from NCBI, and interlinked.
UniProt: Calmodulin Search Results for bovine calmodulin (P62157)
Protein Secondary Structure: PDBSum (EMBL-EBI) http://www.ebi.ac.uk/pdbsum/ Either enter PDB file or can load new/existing sequence
Genome Browsers Genomic DNA is organized in chromosomes. Genome browsers display ideograms (pictures) of chromosomes, with user-selected “annotation tracks” that display many kinds of information. The two most essential human genome browsers are at Ensembl and UCSC. We will focus on UCSC (but the two are equally important). The browser at NCBI is not commonly used.
click human Ensembl genome browser (www.ensembl.org)
Ensembl output for beta globin includes views of chromosome 11 (top), the region (middle), and a detailed view (bottom). There are various horizontal annotation tracks.
The UCSC Genome Browser This browser’s focus is on humans and other eukaryotes you can select which tracks to display (and how much information for each track) tracks are based on data generated by the UCSC team and by the broad research community you can create “custom tracks” of your own data! Just format a spreadsheet properly and upload it The Table Browser is equally important as the more visual Genome Browser, and you can move between the two
 Visit http://genome.ucsc.edu/, click Genome Browser  Choose organisms, enter query (beta globin), hit submit
 On the UCSC Genome Browser: --choose which tracks to display
Protein Databases What do they contain? – Amino acid sequences Primary sequence – Direct submissions - protein sequencing – SWISS-PROT, PIR Secondary sequence – Translations - putative proteins resulting from modifying (i.e. intron splicing) nucleic acid sequence – GenPept, TrEMBL Structure – Protein Data Bank Protein Data Bank – Annotations Function, domains, etc.
SWISS-PROT Created by Amos Bairoch in 1986 at the Department of Medical Biochemistry in Geneva Maintained by the Swiss Institute of Bio-informatics (SIB) and funded by GeneBio Few redundancies Direct submission (from sequencing, not translation) PIR (The Protein Information Resource) was created by M.O. Dayhoff in 1965 Maintained by many In 2004, joined with other databases (Swiss-Prot and TrEMBL) to become part of the UniProt consortiumUniProt consortium PIR
Protein Data Bank Protein Data Bank Archive of 3-D structural data of biological macromolecules Based on experimental data Managed by the Research Collaboratory for Structural Bioinformatics (RCSB) – Rutgers & UCSD As of January 11, 2012 contained 78477 structures ~ 5000 membrane proteins http://www.rcsb.org/pdb/statistics/contentGrowthChart.do?content=total&seqid=100
PDB: Source of protein sequence and structure data