NCBI Molecular Biology Resources February 2007 Part 1

The National Center for Biotechnology Information Created in 1988 as a part of the National Library of Medicine at NIH –Establish public databases –Research in computational biology –Develop software tools for sequence analysis –Disseminate biomedical information Bethesda,MD

Web Access:

NCBI Databases and Services GenBank largest sequence database Free public access to biomedical literature –PubMed free Medline –PubMed Central full text online access Entrez integrated molecular and literature databases BLAST highest volume sequence search service VAST structure similarity searches Software and Databases

Types of Databases Primary Databases –Original submissions by experimentalists –Content controlled by the submitter Examples: GenBank, SNP, GEO Derivative Databases –Built from primary data –Content controlled by third party (NCBI) Examples: Refseq, TPA, RefSNP, UniGene, NCBI Protein, Structure, Conserved Domain

Entrez Nucleotides Primary GenBank / EMBL / DDBJ 86,011,283 Derivative RefSeq 1,512,656 Third Party Annotation 5,254 PDB 7,261 Total 87,536,454

What is GenBank? NCBI’s Primary Sequence Database Nucleotide only sequence database Archival in nature –Historical –Reflective of submitter point of view (subjective) –Redundant GenBank Data –Direct submissions (traditional records) –Batch submissions (EST, GSS, STS) –ftp accounts (genome data) Three collaborating databases –GenBank –DNA Database of Japan (DDBJ) –European Molecular Biology Laboratory (EMBL) Database

EBI GenBank DDBJ EMBL EMBL Entrez SRS getentry NIG CIB NCBI NIH Submissions Updates Submissions Updates Submissions Updates International Sequence Database Collaboration

GenBank: NCBI’s Primary Sequence Database ftp://ftp.ncbi.nih.gov/genbank/ Release 157December ,434,665Records 150,630,667,561Total Bases 254 Gigabytes (non-WGS) 1072 files (non-WGS) full release every two months incremental updates daily available only via ftp full release every two months incremental updates daily available only via ftp

The Growth of GenBank Non-WGS: 69.0 billion bases WGS: 81.6 billion bases Release 157 Doubling time months

Organization of GenBank: Traditional Divisions Records are divided into 18 Divisions. 12 Traditional 6 Bulk Traditional Divisions: Direct Submissions (Sequin and BankIt) Accurate Well characterized PRI Primate PLN Plant and Fungal BCT Bacterial and Archeal INV Invertebrate ROD Rodent VRL Viral VRT Other Vertebrate MAM Mammalian PHG Phage SYN Synthetic (cloning vectors) ENV Environmental Samples UNA Unannotated Entrez query: gbdiv_xxx[Properties]

Organization of GenBank: Bulk Divisions Records are divided into 18 Divisions. 12 Traditional 6 Bulk BULK Divisions: Batch Submission ( and FTP) Inaccurate Poorly characterized EST Expressed Sequence Tag GSS Genome Survey Sequence HTG High Throughput Genomic STS Sequence Tagged Site HTC High Throughput cDNA PAT Patent Entrez query: gbdiv_xxx[Properties]

A Traditional GenBank Record LOCUS AY bp mRNA linear PLN 04-MAY-2004 DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA, complete cds. ACCESSION AY VERSION AY GI: KEYWORDS. SOURCE Malus x domestica (cultivated apple) ORGANISM Malus x domestica Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots; rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus. REFERENCE 1 (bases 1 to 1931) AUTHORS Pechous,S.W. and Whitaker,B.D. TITLE Cloning and functional expression of an (E,E)-alpha-farnesene synthase cDNA from peel tissue of apple fruit JOURNAL Planta 219, (2004) REFERENCE 2 (bases 1 to 1931) AUTHORS Pechous,S.W. and Whitaker,B.D. TITLE Direct Submission JOURNAL Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab, USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD 20705, USA REFERENCE 3 (bases 1 to 1931) AUTHORS Pechous,S.W. and Whitaker,B.D. TITLE Direct Submission JOURNAL Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab, USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD 20705, USA REMARK Sequence update by submitter COMMENT On Jun 26, 2003 this sequence version replaced gi: FEATURES Location/Qualifiers source /organism="Malus x domestica" /mol_type="mRNA" /cultivar="'Law Rome'" /db_xref="taxon:3750" /tissue_type="peel" gene /gene="AFS1" CDS /gene="AFS1" /note="terpene synthase" /codon_start=1 /product="(E,E)-alpha-farnesene synthase" /protein_id="AAO " /db_xref="GI: " /translation="MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSANYKPNIWK NDFLDQSLISKYDGDEYRKLSEKLIEEVKIYISAETMDLVAKLELIDSVRKLGLANLF EKEIKEALDSIAAIESDNLGTRDDLYGTALHFKILRQHGYKVSQDIFGRFMDEKGTLE DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI LSLLFQPLVN" ORIGIN 1 ttcttgtatc ccaaacatct cgagcttctt gtacaccaaa ttaggtattc actatggaat 61 tcagagttca cttgcaagct gataatgagc agaaaatttt tcaaaaccag atgaaacccg 121 aacctgaagc ctcttacttg attaatcaaa gacggtctgc aaattacaag ccaaatattt 181 ggaagaacga tttcctagat caatctctta tcagcaaata cgatggagat gagtatcgga 241 agctgtctga gaagttaata gaagaagtta agatttatat atctgctgaa acaatggatt // Header Feature Table Sequence The Flatfile Format

Traditional GenBank Record ACCESSION U07418 VERSION U GI: ACCESSION U07418 VERSION U GI: Accession Stable Reportable Universal Accession Stable Reportable Universal Version Tracks changes in sequence Version Tracks changes in sequence GI number NCBI internal use GI number NCBI internal use well annotated the sequence is the data

Bulk Divisions Expressed Sequence Tag –1 st pass single read cDNA Genome Survey Sequence –1 st pass single read gDNA High Throughput Genomic –incomplete sequences of genomic clones Sequence Tagged Site –PCR-based mapping reagents Batch Submission and htg ( and ftp) Inaccurate Poorly Characterized

GenBank Bulk Sequence: EST poorly characterized poorly characterized

ESTs in Entrez Total 41 million records Human 7.9 million Mouse 4.7 million Cow1.3 million Rice1.2 million Zebrafish 1.2 million Maize1.2 million Xenopus tropicalis1.0 million Rat 0.9 million Wheat0.9 million Chicken0.6 million Barley0.4 million Total 41 million records Human 7.9 million Mouse 4.7 million Cow1.3 million Rice1.2 million Zebrafish 1.2 million Maize1.2 million Xenopus tropicalis1.0 million Rat 0.9 million Wheat0.9 million Chicken0.6 million Barley0.4 million

HTG Division: Opossum Draft Sequences Unfinished sequences of BACs Gaps and unordered pieces Finished sequences move to traditional GenBank division Unfinished sequences of BACs Gaps and unordered pieces Finished sequences move to traditional GenBank division

Whole Genome Shotgun Projects ftp://ftp.ncbi.nih.gov/genbank/wgs/ >450 Projects >400 Taxa –302 bacteria –128 eukaryotes 47 fungi 53 animals 3 flowering plants >450 Projects >400 Taxa –302 bacteria –128 eukaryotes 47 fungi 53 animals 3 flowering plants

Mammalian WGS Duck-billed platypus Nine-banded armadillo Northern tree shrew Domestic rabbit Guinea pig Mouse Rat Thirteen-lined ground squirrel Small-eared galago Human Chimpanzee Rhesus macaque Tenrec African elephant Cat Dog European hedgehog Eurasian shrew Cow Little brown bat Gray short-tailed opossum

Derivative Databases

Entrez Protein: Derivative Database Data Source GenPept Sequences 6,749,369 RefSeq 3,261,525 Third Party Annotation 5,079 Swiss Prot 243,887 PIR 30,236 PRF 12,079 PDB 89,953 PAT Division 669,035 Total 10,392,118 BLAST nr total (no patents or env) 4,180,857

FEATURES Location/Qualifiers source /organism="Homo sapiens" /mol_type="mRNA" /db_xref="taxon:9606" /chromosome="3" /map="3p22-p23" gene /gene="MLH1" CDS /gene="MLH1" /note="homolog of S. cerevisiae PMS1 (Swiss-Prot Accession Number P14242), S. cerevisiae MLH1 (GenBank Accession Number U07187), E. coli MUTL (Swiss-Prot Accession Number P23367), Salmonella typhimurium MUTL (Swiss-Prot Accession Number P14161) and Streptococcus pneumoniae (Swiss-Prot Accession Number P14160)" /codon_start=1 /product="DNA mismatch repair protein homolog" /protein_id="AAC " /db_xref="GI:463989" /translation="MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKS TSIQVIVKEGGLKLIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGE ALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIA TRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRS GenPept: GenBank CDS translations >gi|463989|gb|AAC | DNA mismatch repair prote... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... >gi|463989|gb|AAC | DNA mismatch repair prote... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...

Redundant Proteins >gi|741682|prf|| A DNA mismatch repair protei... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... >gi|730028|sp|P40692|MLH1_HUMAN DNA mismatch repair... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... >gi|463989|gb|AAC | DNA mismatch repair prote... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... >gi| |ref|NP_ | MutL protein homolog 1... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... >gi| |gb|AAH | MutL protein homolog 1... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... >gi| |gb|AAA | DNA mismatch repair prot... MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV... EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD... GenPept NCBI RefSeq Swiss-Prot PRF

Protein Sequences from Structures >gi| |pdb|1B63|A Chain A, Mutl Complexed With Adpnp SHMPIQVLPPQLANQIAAGEVVERPASVVKELVENSLDAGATRIDIDIERGGAKLIRIRDNGCGIKKDEL ALALARHATSKIASLDDLEAIISLGFRGEALASISSVSRLTLTSRTAEQQEAWQAYAEGRDMNVTVKPAA HPVGTTLEVLDLFYNTPARRKFLRTEKTEFNHIDEIIRRIALARFDVTINLSHNGKIVRQYRAVPEGGQK ERRLGAICGTAFLEQALAIEWQHGDLTLRGWVADPNHTTPALAEIQYCYVNGRMMRDRLINHAIRQACED KLGADQQPAFVLYLEIDPHQVDVNVHPAKHEVRFHQSRLVHDFIYQGVLSVLQ >gi| |pdb|1B63|A Chain A, Mutl Complexed With Adpnp SHMPIQVLPPQLANQIAAGEVVERPASVVKELVENSLDAGATRIDIDIERGGAKLIRIRDNGCGIKKDEL ALALARHATSKIASLDDLEAIISLGFRGEALASISSVSRLTLTSRTAEQQEAWQAYAEGRDMNVTVKPAA HPVGTTLEVLDLFYNTPARRKFLRTEKTEFNHIDEIIRRIALARFDVTINLSHNGKIVRQYRAVPEGGQK ERRLGAICGTAFLEQALAIEWQHGDLTLRGWVADPNHTTPALAEIQYCYVNGRMMRDRLINHAIRQACED KLGADQQPAFVLYLEIDPHQVDVNVHPAKHEVRFHQSRLVHDFIYQGVLSVLQ

Primary vs. Derivative Sequence DatabasesGenBank SequencingCenters GA ATT C C GA ATT C C AT GA ATT C C GA ATT C C TTGACA ATTGACTA ACGTGC TTGACA CGTGA ATTGACTA TATAGCCG ACGTGC TTGACA CGTGA ATTGACTA TATAGCCG C ATT GA ATT C C GA ATT C C Labs Algorithms UniGene Curators RefSeq Genome Assembly TATAGCCG AGCTCCGATA CCGATGACAA Updated continually by NCBI Updated ONLY by submitters

RefSeq: NCBI’s Derivative Sequence Database Curated transcripts and proteins –reviewed –human, mouse, rat, fruit fly, zebrafish, arabidopsis microbial genomes (proteins), and more Model transcripts and proteins Assembled Genomic Regions (contigs) –human genome –mouse genome –rat genome Chromosome records –Human genome –microbial –organelle ftp://ftp.ncbi.nih.gov/refseq/release / srcdb_refseq[Properties] – chicken – honeybee – sea urchin

RefSeq Benefits non-redundancy explicitly linked nucleotide and protein sequences updates to reflect current sequence data and biology data validation format consistency distinct accession series stewardship by NCBI staff and collaborators

Selected RefSeq Accession Numbers mRNAs and Proteins NM_ Curated mRNA NP_ Curated Protein NR_ Curated non-coding RNA XM_ Predicted mRNA XP_ Predicted Protein XR_ Predicted non-coding RNA Gene Records NG_ Reference Genomic Sequence Chromosome NC_ Microbial replicons, organelle genomes, human chromosomes Assemblies NT_ Contig NW_ WGS Supercontig

GenBank to RefSeq

RefSeq : Genome Annotation Genomic DNA (NC, NT, NW) Model mRNA (XM) (XR) Curated mRNA (NM) (NR) Model protein (XP) Curated Protein (NP) Scanning.... = ? GenBank Sequences RefSeq

Mouse Assembly RefSeq Contig RefSeq Contig BAC WGS Other GenBank Other GenBank RefSeq Transcript RefSeq Transcript UniGene Transcript UniGene Transcript

Expressed Sequences UniGene GEO

A gene-oriented view of sequence entries MegaBlast based automated sequence clustering Now informed by genome hits New! Nonredundant set of gene oriented clusters Each cluster a unique gene Information on tissue types and map locations Includes known genes and uncharacterized ESTs Useful for gene discovery and selection of mapping reagents What is UniGene?

EST hits: Human mRNA Albumin mRNA 5’ EST hits 3’ EST hits

UniGene Chordates Invertebrates Plants Fungi et al.

Xenopus laevis MLH1Cluster Uncharacterized ESTs

UniGene: Expressed Sequences

Expression Data

Other NCBI Databases Structure: imported structures (PDB) Cn3D viewer, NCBI curation CDD: conserved domain database Protein families (COGs and KOGs) Single domains (PFAM, SMART, CD) dbSNP: nucleotide polymorphism Gene: gene records Unifies LocusLink and Microbial Genomes

NCBI Structures and Domains

MM MMDB: Molecular Modeling Data Base Derived from experimentally determined PDB records Value added to PDB records including: –Addition of explicit chemical graph information –Validation (secondary structure elements) –Inclusion of Taxonomy, Citation –Conversion to ASN.1 data description language Structure neighbors determined by Vector Alignment Search Tool (VAST)

Cn3D 4.1: Bacillus thuringiensis Toxin

VAST: Structure Neighbors Vector Alignment Search Tool For each protein chain, locate SSEs (secondary structure elements), and represent them as individual vectors Human IL-4 IL-4 & Leptin align the vectors

Protein Domains Structural Domain –Discrete independently folding unit of a protein Conserved Domain (sequence-based) –Protein region with recognizable position specific pattern of sequence conservation Sequence-based domains often roughly correspond to structural domains Domains often have distinct, identifiable functions

NCBI’s Conserved Domain Database PSI-BLAST –based score matrices Searchable with RPS-BLAST Sources –SMART –PFAM –COGs –NCBI curated domains structure informed alignments

Src Domains

Structure vs Conserved Domain SH2 SH3 TyrKC SH2 Conserved phosphotyrosine binding residues

NCBI Molecular Biology Resources Using Entrez

WWW Access Entrez & BLAST

Genomes Taxonomy Entrez: Database Integration PubMed abstracts Nucleotide sequences Protein sequences 3-D Structure Word weight VAST BLAST Phylogeny Hard Link Neighbors Related Sequences Neighbors Related Sequences BLink Domains Neighbors Related Structures

Database Searching with Entrez uUsing limits and field restriction to find human MutL homolog uLinking and neighboring with MutL uMapping SNPs onto structure and the genome

Global NCBI (Entrez) Search Human hereditary nonpolyposis colon cancer

Global Entrez Search Results

Nucleotide Sequences Nucleotide database now three parts EST expressed sequence tags GSS genome survey sequences CoreNucleotide everything else Nucleotide database now three parts EST expressed sequence tags GSS genome survey sequences CoreNucleotide everything else

Advanced Search Options Tabs

More Precise Nucleotides Search nonpolyposis[All Fields] AND colon cancer[Title] AND human[Organism] AND biomol_mrna[Properties] AND srcdb_refseq[Properties] nonpolyposis[All Fields] AND colon cancer[Title] AND human[Organism] AND biomol_mrna[Properties] AND srcdb_refseq[Properties]

Useful Field Restrictions [Title]: Definition line in GenBank / GenPept format shown in Summary format glyceraldehyde 3 phosphate dehydrogenase[Title] [Organism]: NCBI’s taxonomy. Organizing system for molecular databases mouse[organism]; green plants[organism]; Streptomyces coelicolor[organism] [Properties]: molecule type, location, database source biomol_mrna[properties]; biomol_genomic[properties]; gene_in_mitochondrion[properties]; srcdb pdb[properties] [Filter]: subsets of data, Entrez links all[filter]; nucleotide mapview[filter]; nucleotide omim[filter]

Organism Field: NCBI’s Taxonomy

Useful Properties Field Terms Molecule type biomol_mrna biomol_genomic GenBank division gbdiv_est gbdiv_htg gbdiv_xxx Gene location gene_in_mitochondrion gene_in_chloroplast gene_in_genomic Source Database srcdb_refseq srcdb_pdb srcdb_swiss_prot

Human MutL RefSeq GenBank Records

NM_000249: Links

Literature Links OMIM

OMIM: Human Disease Genes Conserved Domain

Sequence Links Finding Homologs and Structures

Protein Link BLAST Link Conserved Domains

Related Proteins: Homologs and Redundancy Redundant Sequences Bacterial Homologs

BLink: BLAST Link top 200 only Redundant GIs

BLink: non-redundant relatives zebrafish homolog BLAST

Related Proteins: Structure Links

Structures

Short Cut: Related Structures

E. coli MutL Structure Cn3D viewer Conserved Domains 3D Domain Neighbors Structure Neighbors Pubchem compound

MLH1 Domain Structure: CDD ATPase Domain Mismatch Repair Domain

MLH1: ATPase Domain

Mapping Polymorphisms onto Structure

Entrez SNP "coding nonsynon"[Function Class]

GeneView: Variations Human MLH1 ATPase domain

Related Structures

Mapping Variation Onto Structure Conserved Asn Asn Ile Ile – Val

Genome Resources

Higher Genome Resources

Microbial Genomes

NM_000249: Genome Links

The Map Viewer Genome BLAST Previous Builds Available

Map Viewer: Human MLH1 Customizable NCBI Assembly EST Hits Gene Annotations Models Transcripts Download data and sequences

Maps and Options

Mapped Variations

Synteny: Mammalian Genomes

Homologene early globin gene A-chain gene B-chain gene frog A chick A mouse Amouse B chick B frog B paralogs orthologs gene duplication No longer UniGene based Protein similarities first Guided by taxonomic tree Includes orthologs and paralogs No longer UniGene based Protein similarities first Guided by taxonomic tree Includes orthologs and paralogs

Homologene Cluster

Rice Homolog

The Gene Database Gene Centered Information Unifies LocusLink and microbial Genomes 2.4 million records for 3,822 taxa Human38,603Sea Urchin 30,603 Chimpanzee31,502Mosquito 13,763 Mouse60,746Fruit Fly 21,116 Rat38,117C. elegans 20,935 Dog20,154Fungi168,802 Cow23, 677Green Plants 76,847 Chicken18, 469Archea74,627 Zebrafish38, 594Bacteria1,361,390

Genes MLH1: One Stop Shopping

Genes MLH1: One Stop Shopping (cont.)

Genes: Display Options and Links