National Center for Biotechnology Information

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National Center for Biotechnology Information

• A Field Guide to GenBank
• and NCBIs Molecular Biology Resources

University of Colorado Health Sciences Center
August 30, 2005
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Topics

• About NCBI
• GenBank overview
• Primary vs derivative databases
• The Reference Sequence (RefSeq) project
• Entrez databases
• Genome resources
• Bookshelf
• -break-
• Entrez text searching
• BLAST sequence searching
• VAST structure searching
• An integrated example

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The National Institutes of Health
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The National Center for Biotechnology Information

• Accepts submissions of primary data
• Develops tools to analyze these data
• Creates derivative databases based on the primary
  data
• Provides free search, link, and retrieval of
  these data, primarily through the Entrez system

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NCBI WWW Users per Day
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Number of Users Per Day
1997 1998 1999 2000 2001 2002
2003
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Homepage - accessing the data
allfilter
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allfilter
1/11/2005
3/15/2005
8/15/2005
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Entrez Nucleotide

• Primary Data
• GenBank / DDBJ / EMBL 57.3 million (97.4 )
• Derivative Data
• RefSeq 1.47 million (2.5 )
• RefSeq reviewed 60,000
• PDB (structures) 5,973
• 
  
• Total
  59 million

records
GenBank
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GenBank NCBIs Primary Sequence Database
Over 100 billion bases!

• full release every two months
• incremental and cumulative updates daily
• available only through internet
• release notes gbrel.txt

ftp//ftp.ncbi.nih.gov/genbank/
ftp//genbank.sdsc.edu/pub ftp//bio-mirror.net/bi
omirror/genbank
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What is GenBank?

• Nucleotide only sequence database
• Archival in nature
• 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

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GenBank Divisions
Organismal
PRI (28) Primate ROD (15) Rodent PLN (13)
Plant and Fungal BCT (11) Bacterial/Archeal INV
(7) Invertebrate VRT (7) Other Vertebrate VRL
(4) Viral MAM (2) Mammalian PHG (1)
Phage SYN (1) Synthetic UNA (1) Unannotated

• Organized by taxonomy (sort of)
• Direct submissions (Sequin/Bankit)
• Accurate (1 error per 10,000 bp)
• Well characterized

Functional
EST (377) Expressed Sequence Tag GSS (138)
Genome Survey Sequence HTG (63) High Throughput
Genomic PAT (17) Patent STS (9) Sequence
Tagged Site CON (1) Contigs, virtual

• Organized by sequence type
• Batch submissions (ftp/email)
• Inaccurate
• Poorly characterized

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GenBank Functional (Bulk) Divisions

• Expressed Sequence Tag
• 1st pass single read cDNA
• Genome Survey Sequence
• 1st pass single read gDNA
• High Throughput Genomic
• incomplete sequences of genomic clones
• Sequence Tagged Site
• PCR-based mapping reagents
• Whole Genome Shotgun

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EST Division Expressed Sequence Tags
gtIMAGE275615 5' mRNA sequence GACAGCATTCGGGCCGAGA
TGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGG
TGGAGGTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAG
CAGAGAATGGAAAGTCAA TTCCTGAATTGCTATGTGTCTGGGTTTCATC
CATCCGACATTGAAGTTGACTTACTGAAGAATGGAGA GAATTGAAAAAG
TGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTG
TACTAC TGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTT
GAACCATGTNGACTTTGTCACAGNC AAGTTNAGTTTAAGTGGGNATCGA
GACATGTAAGGCAGGCATCATGGGAGGTTTTGAAGNATGCCGCN TTGGA
TTGGGATGAATTCCAAATTTCTGGTTTGCTTGNTTTTTTAATATTGGATA
TGCTTTTG
nucleus 30,000 genes
gatccantgccatacg
ctcgccaattcnntcg

• - isolate unique clones
• sequence once from each end

gtIMAGE275615 3', mRNA sequence NNTCAAGTTTTATGATTT
ATTTAACTTGTGGAACAAAAATAAACCAGATTAACCACAACCATGCCTTA
TTATCAAATGTATAAGANGTAAATATGAATCTTATATGACAAAATGTTT
CATTCATTATAACAAATTT AATAATCCTGTCAATNATATTTCTAAATTT
TCCCCCAAATTCTAAGCAGAGTATGTAAATTGGAAGTT CTTATGCACGC
TTAACTATCTTAACAAGCTTTGAGTGCAAGAGATTGANGAGTTCAAATCT
GACCAAG GTTGATGTTGGATAAGAGAATTCTCTGCTCCCCACCTCTANG
TTGCCAGCCCTC
RNA gene products
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GSS, WGS, HTG
Whole BAC insert (or genome)
shred
isolate clones
sequence
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HTG Example Honeybee Draft Sequences

• Unfinished sequences of BACs
• Gaps and unordered pieces
• Finished sequences (Phase 3) move to
  traditional GenBank division

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Whole Genome Shotgun Projects

• 351 projects
• Bacteria (251)
• Environmental sequences (6)
• Archaea (6)
• Eukaryotes (88), including
• Chicken, Rat, Mouse, Dog (2), Chimpanzee, Human
• Pufferfish (2)
• Honeybee, Anopheles, Fruit Flies (3), Silkworm
• Nematode (2)
• Yeasts (8), Aspergillus (2)
• Rice (2)

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Whole Genome Shotgun (WGS) Projects
wgs masterproperties
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Derivative Databases
Sequencing Centers
UniGene
UniSTS
Updated by NCBI
EST
GenBank
STS
Updated ONLY by submitters
RefSeq
RefSeq Entrez Gene and annotation pipelines
HTG
GSS
INV
VRT
PHG
VRL
PRI
ROD
PLN
MAM
BCT
Labs
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Why Make Reference Sequences?
Entrez Nucleotide query humanorganism AND
lipasetitle
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Why Make Reference Sequences?
Entrez Nucleotide query humanorganism AND
lipasetitle
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humanorganism AND lipasetitle AND
endothelialtitle
humanorganism AND lipasetitle AND
endothelialtitle
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RefSeq Benefits
genomes
transcripts
proteins

• non-redundant best representative
• updates to reflect current sequence data and
  biology
• distinct, stable accession series

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Reference Sequence RefSeq
Accession Sequence Type NM_123456789 mRNA NP_1234
56789 protein, from NM_ NR_123456 non-coding
RNA XM_123456 predicted mRNA XP_123456
predicted protein XR_123456 predicted
non-coding RNA ZP_12345678 predicted from
NZ_ NC_123456 genomic, e.g.,
chromosomes NG_123455 genomic, incomplete
region NT_123456 genomic, BAC
assembly NW_123456 genomic, WGS
assembly NZ_ABCD12345678 genomic, WGS
collection bluecurated
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Annotation Process
Genomic DNA (NC, NT, NW)
Scanning....
Model mRNA (XM) (XR)
Model protein (XP)
Curated mRNA (NM) (NR)
Curated Protein (NP)
RefSeq
Genbank Sequences
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Creating NM_ Records
Genome annotation
NMs must have cDNA support
transcript variant 1
transcript variant 2
transcript variant 3
Longest mRNA
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Where is RefSeq?
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The Entrez System
Gene
UniGene
CancerChromosomes
UniSTS
Homologene
SNP
PopSet
Genome
Nucleotide
GEO
Books
Entrez
Taxonomy
PubMed
MeSH
OMIM
Protein
PMC
Journals
Domains
3D Domains
Structure
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A Few Entrez Databases

• UniGene Clusters of ESTs, mRNAs
• dbSNP Single Nucleotide Polymorphisms
• GEO Gene Expression Omnibus
• microarray and other expression data
• CDD Conserved Domain Database
• protein families (COGs and KOGs)
• single domains (PFAM, SMART, CD)

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UniGene
Gene-oriented clusters of expressed sequences

• Automatic clustering using MegaBlast
• Each cluster represents a unique gene
• Informed by genome hits
• Information on tissue types and map locations
• Useful for gene discovery and selection of
  mapping reagents

unique gene
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A Cluster of ESTs
query
5 EST hits
3 EST hits
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UniGene Collections
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Example UniGene Cluster
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Histogram of cluster sizes for UniGene Hs Build
177
(Now at Build 186)
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UniGene Cluster Hs.95351
SELECTED PROTEIN SIMILARITES
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UniGene Cluster Hs.95351
GENE EXPRESSION
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UniGene Cluster Hs.95351 expression
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UniGene Cluster Hs.95351 seqs
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Download sequences
web page
ftp//ftp.ncbi.nih.gov/repository/UniGene/Homo_sap
iens/
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Entrez GEO
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NCBIs SNP Database

• Primary and derivative (RefSNP)
• Single nucleotide polymorphisms
• Repeat polymorphisms
• Insertion-deletion polymorphisms
• Over 19 million refSNPs (rsXXXXXXX)
• (August, 2005)

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Searching dbSNP
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RefSNP
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RefSNP
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RefSNP
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RefSNP
Search Mouse SNP between strains
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RefSNP
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RefSNP
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Entrez GEO
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Submitted by Experimentalists
Curated by NCBI
Submitted by Manufacturer
GDS Grouping of experiments
GPL Platform descriptions
GSE Grouping of slide/chip data a single
experiment
GSM Raw/processed spot intensities from a
single slide/chip
GEO SEries set of related samples
GEO SaMple experimental conditions
Entrez GEO Datasets
Entrez GEO
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Whats a DataSet?
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Gene Expression Omnibus (GEO)
Dataset browser
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GEO Dataset Browser
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GEO Dataset Report
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GEO Profiles
of 12625
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Entrez CDD
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Conserved Domain Database

• Multiple sequence alignments
• Position-specific scoring matrices (PSSM)
• Sources SMART, PFAM, COGs, KOGs, and
• NCBI curated domains (structure-informed
  alignments)

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CDD
gtgi45549418gbAAS67634.1 ATP7A Solenodon
paradoxus IVYQPHLITVEEIKKQIKAVGFPAFIKKQPKYLKLGAID
IERLKNIPVKSSEGSQQMSPSSTNDSKVTLTIDGMHCNSCVSNIESALST
LHYVSSIVVSLQNKSAIIKYNANSVTPEILKKAIEAISPGQYRVSITSEV
ESTSNSPSSSSQKAPLNVVSQPLTQVTVININGMTCNSCVQSIEGVMSKK
AGVKSIQVSLANRNGTVEYDP LLTSPEILRE
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CDD
Click on a colored bar to align your sequence to
the CD
CD
Pfam
COG
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Conserved Domain Database cd00371.1, HMA
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CDD
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CDART Conserved Domain Architecture Retrieval
Tool
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cdd
Linking from Entrez Protein
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Genome Resources
Genomic Biology
Gene database
Homologene
Map Viewer
Trace Archive
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Genomic Biology
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Genome Projects microb
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Genome Resources
Genomic Biology
Gene database
Homologene
Map Viewer
Trace Archive
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Entrez Gene

• A single query interface to
• Sequences
• - RefSeqs
• - GenBank
• - Homologene
• Maps MapViewer
• Entrez links
• Linkouts

• More organisms, 3000
• Entrez integration

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Global Entrez NADH2
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Entrez Gene NADH2
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Gene Record for Pongo NADH2
Not found with nadh2
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A Record With More Data Human HFE
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Human HFE Transcripts
Transcripts with experimental evidence
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Gene Table
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Introns/Exons Gene Table
links to sequence
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Human HFE Links
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Genotype
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Genotype
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Human HFE Links
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GeneView in dbSNP
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SNP in Structure
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SNP in Structure
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SNP in Structure
H41
S43
C260
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Another Variation Source OMIM
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Variants in OMIM
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Genome Resources
Genomic Biology
Gene database
Homologene
Map Viewer
Trace Archive
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The New Homologene
Automated detection of homologs among the
annotated genes of completely sequenced
eukaryotic genomes.

• No longer UniGene based
• Protein similarities first
• Guided by taxonomic tree
• Includes orthologs and paralogs

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The New Homologene
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RAG1 ? Homologene
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RAG1 ? Homolgene
RAG1
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RAG1
RING-finger
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RAG1 ? Homolgene
RAG1
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RAG1
Sugar_tr
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Homologene alignment scores
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BLASTP bl2seq
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Genome Resources
Gene database
Map Viewer
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List View
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Human MapViewer
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MapViewer Human ADAR
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MV Hs ADAR
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Maps Options
Maps Options
--Sequence maps-- Ab initio Assembly Repeats BES_C
lone Clone NCI_Clone Contig Component CpG
island dbSNP haplotype Fosmid GenBank_DNA Gene Phe
notype SAGE_Tag STS TCAG_RNA Transcript
(RNA) Hs_UniGene Hs_EST
--Cytogenetic maps-- Ideogram FISH
Clone Gene_Cytogenetic Mitelman
Breakpoint Morbid/Disease --Genetic
Maps-- deCODE Genethon Marshfield --RH
maps-- GeneMap99-G3 GeneMap99-GB4 NCBI
RH Standford-G3 TNG Whitehead-RH Whitehead-YAC
Mm_UniGene Mm_EST Rn_UniGene Rn_EST Ssc_UniGene Ss
c_EST Bt_UniGene Bt_EST Gga_UniGene Gga_EST Variat
ion
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MapViewer
Component
Gene
UniGene
Repeats
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Phenotype
Variation
Gene
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Maps Options
Maps Options
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Genome Resources
Trace Archive
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Trace Archive Page
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Macaca Mulatta Traces
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(No Transcript)
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Trace Archive BLAST Page
Access to sequences NOT in GenBank
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Literature Links
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BOOKS Database
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BOOKS Database hyperlinked
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BOOKS Database
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BOOKS Database
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BOOKS Database
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Genes Dis
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Genes Dis
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For More Information
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Intermission