National Center for Biotechnology Information Web Tools Introduction

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National Center for Biotechnology Information Web
Tools (Introduction)

• Craig R. Street, M. Biot.
• Biomedical Informatics Facility
• 20 April 2004

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Types of Databases

• Primary Databases
• Original submissions by experimentalists
• Content controlled by the submitter
• Examples GenBank, SNP
• Derivative Databases
• Built from primary data
• Content controlled by third party (NCBI)
• Examples Refseq, UniGene, NCBI Protein,
  Structure, Conserved Domain

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A Traditional GenBank Record
LOCUS AF062069 3808 bp
mRNA linear INV 23-OCT-2002 DEFINITION
Limulus polyphemus myosin III mRNA, complete
cds. ACCESSION AF062069 VERSION AF062069.2
GI7144484 KEYWORDS . SOURCE Limulus
polyphemus (Atlantic horseshoe crab) ORGANISM
Limulus polyphemus Eukaryota
Metazoa Arthropoda Chelicerata Merostomata
Xiphosura Limulidae Limulus. REFERENCE
1 (bases 1 to 3808) AUTHORS
Battelle,B.-A., Andrews,A.W., Calman,B.G.,
Sellers,J.R., Greenberg,R.M. and
Smith,W.C. TITLE A myosin III from Limulus
eyes is a clock-regulated phosphoprotein
JOURNAL J. Neurosci. 18 (12), 4548-4559 (1998)
MEDLINE 98279067 PUBMED 9614231 REFERENCE
2 (bases 1 to 3808) AUTHORS
Battelle,B.-A., Andrews,A.W., Calman,B.G.,
Sellers,J.R., Greenberg,R.M. and
Smith,W.C. TITLE Direct Submission
JOURNAL Submitted (29-APR-1998) Whitney
Laboratory, University of Florida,
9505 Ocean Shore Blvd., St. Augustine, FL 32086,
USA
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GenBank Overview

• Comprehensive Public Database of Nucleotide
  Sequences
• Composed Primarily of Submission of Sequence Data
  from Authors and Bulk Submission of ESTs, GSSs,
  and Other High-Throughput Data
• Continues to Grow at an Exponential Rate with 9
  Million New Sequences Added in 2003

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GenBank Overview

• Complete Genomes Represent a Growing Portion of
  the Database
• Over a Dozen Eukaryote Genomes Are Now Available
• Each Entry Contains a Concise Description of the
  Sequence, the Scientific Name Taxonomy of the
  Source Organism, Bibliographic References and a
  Table of Features

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GenBank Overview

• Record Features Include Source, Gene, Variations
  (SNPs, etc.), Coding Sequence, etc.
• Each Record (Consisting of a Sequence and its
  Annotations) is Assigned a Stable and Unique
  Accession Number
• ESTs Continue to be Major Source of New Sequence
  Records

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GenBank Overview

• Sequence Records Are Accessible via NCBIs
  Retrieval System Entrez
• Sequence-Similarity Searches are Performed on
  GenBank Data Using the BLAST Family of Tools

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LOCUS AF062069 3808 bp mRNA
linear INV 23-OCT-2002
GenBank Record Locus
LOCUS AF062069 3808 bp
mRNA linear INV 23-OCT-2002 DEFINITION
Limulus polyphemus myosin III mRNA, complete
cds. ACCESSION AF062069 VERSION AF062069.2
GI7144484 KEYWORDS . SOURCE Limulus
polyphemus (Atlantic horseshoe crab) ORGANISM
Limulus polyphemus Eukaryota
Metazoa Arthropoda Chelicerata Merostomata
Xiphosura Limulidae Limulus. REFERENCE
1 (bases 1 to 3808) AUTHORS
Battelle,B.-A., Andrews,A.W., Calman,B.G.,
Sellers,J.R., Greenberg,R.M. and
Smith,W.C. TITLE A myosin III from Limulus
eyes is a clock-regulated phosphoprotein
JOURNAL J. Neurosci. 18 (12), 4548-4559 (1998)
MEDLINE 98279067 PUBMED 9614231 REFERENCE
2 (bases 1 to 3808) AUTHORS
Battelle,B.-A., Andrews,A.W., Calman,B.G.,
Sellers,J.R., Greenberg,R.M. and
Smith,W.C. TITLE Direct Submission
JOURNAL Submitted (29-APR-1998) Whitney
Laboratory, University of Florida,
9505 Ocean Shore Blvd., St. Augustine, FL 32086,
USA COMMENT On Mar 2, 2000 this sequence
version replaced gi3132700.
Length
Division
Molecule type
Locus name
Modification Date
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Actin Record (FASTA Format)
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RefSeq NCBIs Derivative Sequence Database

• Curated transcripts and proteins
• reviewed
• human, mouse, rat, fruit fly, zebrafish,
  arabidopsis
• Model transcripts and proteins
• Assembled Genomic Regions (contigs)
• human genome
• mouse genome
• Chromosome records
• Human genome
• microbial
• organelle

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RefSeq Benefits

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

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RefSeq Accession Numbers
mRNAs and Proteins NM_123456 Curated
mRNA NP_123456 Curated Protein NR_123456 Curated
non-coding RNA XM_123456 Predicted
mRNA XP_123456 Predicted Protein
XR_123456 Predicted non-coding RNA Gene
Records NG_123456 Reference Genomic
Sequence Chromosome NC_123455 Microbial
replicons, organelle genomes, human
chromosomes Assemblies NT_123456 Contig
NW_123456 WGS Supercontig
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Text
Entrez
Sequence
BLAST
Structure
VAST
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Entrez Data Retrieval System

• Provides Integrated Access to a Wide Range of
  Data Domains
• Entrez is Grouped into Nodes. Each Node is a
  Collections of Data that is Grouped and Indexed
  Together and Often Referred to as an Entrez
  Database

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Entrez Data Retrieval System

• Popular Nodes Include
• Nucleotide sequence database (GenBank)
• Protein sequence database
• Genome whole genome sequences
• SNP single nucleotide polymorphism
• PopSet population study data sets

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Entrez Data Retrieval System
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Entrez Database Integration
Word weight
PubMed abstracts
Phylogeny
VAST
3-D Structure
3 -D Structure
Taxonomy
Genomes
Protein sequences
Nucleotide sequences
BLAST
BLAST
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Global Entrez Search
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Entrez Nucleotides Limits Preview/Index
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Entrez Nucleotides Limits
Accession All Fields Author Name EC/RN
Number Feature key Filter Gene Name Issue Journal
Name Keyword Modification Date Organism Page
Number Primary Accession Properties Protein
Name Publication Date SeqID String Sequence
Length Substance Name Text Word Title Uid Volume
Field Restriction
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BLAST Basic Local Alignment Search Tool

• Why align sequences ?
• - because it is the best way to infer
  structure-function relationships for the
  unknown biomolecules
• Global vs Local Alignments
• BLAST Basics
• Interpretation of Results
• PSI-BLAST

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BLAST 2.0 (a.k.a. Gapped BLAST)

• Calculates Similarity for Biological Sequences
• Finds Best LOCAL Alignments
• Heuristic Approach Based on Smith-Waterman
  Algorithm
• Searches for Matching Words and then Extends
  the Hits
• Uses Statistical Theory to Determine if a Match
  Might have Occurred by Chance

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BLAST 2.0 (a.k.a. Gapped BLAST)

• blastp Compares an amino acid query sequence
  against a protein sequence database.
• blastn Compares a nucleotide query sequence
  against a nucleotide sequence database.
• blastx Compares a nucleotide query sequence
  translated in all reading frames against a
  protein sequence database.
• tblastn Compares a protein query sequence against
  a nucleotide sequence database dynamically
  translated in all reading frames.
• tblastx Compares the six-frame translations of a
  nucleotide query sequence against the
  six- frame translations of a nucleotide sequence
  database.
• Please note that the tblastx program cannot be
  used with the nr database on the BLAST Web page
  because it is computationally intensive.

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BLAST Databases Nucleic Acid

• nr (nt)
• Traditional GenBank Divisions
• NM_ and XM_ RefSeqs
• dbest
• EST Division
• htgs
• HTG division
• gss
• GSS division
• chromosome
• NC_ RefSeqs
• wgs
• whole genome shotgun

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BLAST Databases Amino Acid

• nr (non-redundant protein sequences)
• GenBank CDS translations
• NP_ RefSeqs
• Outside Protein
• PIR, Swiss-Prot, PRF
• PDB (sequences from structures)

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How BLAST Works

• Make a lookup table of all words in the query
• Scan the database for matching words
• Initiate extensions from these matches

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Words
GTQ TQI QIT ITV TVE
VED EDL DLF
LFY
Make a lookup table of words
Word Size 3
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Scan DatabaseInitiate Extensions
Protein BLAST requires two hits
GTQITVEDLFYNI lt------ TVE FYN ------gt
two words (threshold score)
Nucleotide BLAST requires exact matches
ATCGCCATGCTTAATTGGGCTT
lt------ CATGCTTAATT ------gt
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An Alignment that BLAST Cant Find
1 GAATATATGAAGACCAAGATTGCAGTCCTGCTGGCCTGAACC
ACGCTATTCTTGCTGTTG
1
GAGTGTACGATGAGCCCGAGTGTAGCAGTGAAGATCTGGACCACGGTGTA
CTCGTTGTCG 61 GTTACGGAACCGAGAATGGTAAAGACTACTGG
ATCATTAAGAACTCCTGGGGAGCCAGTT

61 GCTATGGTGTTAAGGGTGGGAAGAAGTACTGGCTCGTCAAGAACAGC
TGGGCTGAATCCT 121 GGGGTGAACAAGGTTATTTCAGGCTTGCT
CGTGGTAAAAAC
121 GGGGAGACCAAGGCTACATCCTTATGTCCCG
TGACAACAAC
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Local Alignment Statistics
High scores of local alignments between two
random sequences follow the Extreme Value
Distribution
Expect Value E number of database hits you
expect to find by chance
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Scoring Systems - Nucleotides
Identity matrix
A G C T A 1 3 3 -3 G 3 1 3 -3 C 3 3
1 -3 T 3 3 3 1
CAGGTAGCAAGCTTGCATGTCA
raw score 19-9 10 CACGTAGCAAGCTTG-GTGTCA
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Scoring Systems - Proteins

• Position Independent Matrices
• PAM Matrices (Percent Accepted Mutation)
• Derived from observation small dataset of
  alignments
• Implicit model of evolution
• All calculated from PAM1 (PAM250 widely used)
• BLOSUM Matrices (BLOck SUbstitution Matrices)
• Derived from observation large dataset of
  highly conserved blocks
• Each matrix derived separately from blocks with
  a defined percent identity cutoff
• BLOSUM62 - default matrix for BLAST
• Position Specific Score Matrices (PSSMs)
• PSI-BLAST

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A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3
-3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2
5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0
0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2
-3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1
-2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2
-1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0
6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4
7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1
4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1
1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1
-4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2
-1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3
3 1 -2 1 -1 -2 -2 0 -3 -1 4 X 0 -1 -1 -1 -2
-1 -1 -1 -1 -1 -1 -1 -1 -1 -2 0 0 -2 -1 -1 -1
A R N D C Q E G H I L K M F P S
T W Y V X
BLOSUM62
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Options for Advanced Blasting Nucleotide
Example Entrez Queries nucleotide allFilter NOT
mammaliaOrganism green plantsOrganism biomol
mrnaProperties biomol genomicProperties Other
Advanced -Word size Expect value
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Options for Advanced Blast Protein
Example Entrez queries proteins allFilter NOT
mammaliaOrganism green plantsOrganism srcdb
refseqProperties Other advanced -Word
size Expect value
Limit by taxon Mus musculusOrganism MammaliaOrg
anism ViridiplantaeOrganism

• Matrix Selection
• PAM30 -- most stringent
• BLOSUM45 -- least stringent

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BLAST Graphical Overview
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BLAST Alignments
gi7160701embCAB04427.2 C. elegans KIN-22
protein (corresponding sequence F49B2.5)
Caenorhabditis elegans gi17508235refNP_49350
2.1 Tyrosine kinase with SH2, SH3 and N
myristoylation domains, Drosophila suppressor of
pole hole homolog (57.5 kD) (kin-22)
Caenorhabditis elegans Length 507 Score
290 bits (742), Expect 1e-78 Identities
170/440 (38), Positives 245/440 (55), Gaps
21/440 (4)
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PSI-BLASTPosition-Specific Iterated BLAST

• Mining for protein domains
• Confirming relationships among related proteins

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Position Specific Substitution Rates
Weakly conserved serine
Active site serine
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Position Specific Score Matrix (PSSM)
A R N D C Q E G H I L K M
F P S T W Y V 206 D 0 -2 0 2 -4 2 4
-4 -3 -5 -4 0 -2 -6 1 0 -1 -6 -4 -1 207 G
-2 -1 0 -2 -4 -3 -3 6 -4 -5 -5 0 -2 -3 -2 -2
-1 0 -6 -5 208 V -1 1 -3 -3 -5 -1 -2 6 -1
-4 -5 1 -5 -6 -4 0 -2 -6 -4 -2 209 I -3 3
-3 -4 -6 0 -1 -4 -1 2 -4 6 -2 -5 -5 -3 0 -1
-4 0 210 D -2 -5 0 8 -5 -3 -2 -1 -4 -7 -6
-4 -6 -7 -5 1 -3 -7 -5 -6 211 S 4 -4 -4 -4
-4 -1 -4 -2 -3 -3 -5 -4 -4 -5 -1 4 3 -6 -5 -3
212 C -4 -7 -6 -7 12 -7 -7 -5 -6 -5 -5 -7 -5 0
-7 -4 -4 -5 0 -4 213 N -2 0 2 -1 -6 7 0
-2 0 -6 -4 2 0 -2 -5 -1 -3 -3 -4 -3 214 G
-2 -3 -3 -4 -4 -4 -5 7 -4 -7 -7 -5 -4 -4 -6 -3
-5 -6 -6 -6 215 D -5 -5 -2 9 -7 -4 -1 -5 -5
-7 -7 -4 -7 -7 -5 -4 -4 -8 -7 -7 216 S -2 -4
-2 -4 -4 -3 -3 -3 -4 -6 -6 -3 -5 -6 -4 7 -2 -6
-5 -5 217 G -3 -6 -4 -5 -6 -5 -6 8 -6 -8 -7
-5 -6 -7 -6 -4 -5 -6 -7 -7 218 G -3 -6 -4 -5
-6 -5 -6 8 -6 -7 -7 -5 -6 -7 -6 -2 -4 -6 -7 -7
219 P -2 -6 -6 -5 -6 -5 -5 -6 -6 -6 -7 -4 -6 -7
9 -4 -4 -7 -7 -6 220 L -4 -6 -7 -7 -5 -5 -6
-7 0 -1 6 -6 1 0 -6 -6 -5 -5 -4 0 221 N
-1 -6 0 -6 -4 -4 -6 -6 -1 3 0 -5 4 -3 -6 -2
-1 -6 -1 6 222 C 0 -4 -5 -5 10 -2 -5 -5 1
-1 -1 -5 0 -1 -4 -1 0 -5 0 0 223 Q 0 1
4 2 -5 2 0 0 0 -4 -2 1 0 0 0 -1 -1 -3 -3
-4 224 A -1 -1 1 3 -4 -1 1 4 -3 -4 -3 -1
-2 -2 -3 0 -2 -2 -2 -3
Serine scored differently in these two positions
Active site nucleophile
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PSI-BLAST
gtgi113340spP03958ADA_MOUSE ADENOSINE
DEAMINASE (ADENOSINE MAQTPAFNKPKVELHVHLDGAIKPETILY
FGKKRGIALPADTVEELRNIIGMDKPLSLPGF VIAGCREAIKRIAYEFV
EMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVD EQAFG
IKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFP
GHVEAY RTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLL
KENMHFEVCPWSSYLTGA VRFKNDKANYSLNTDDPLIFKSTLDTDYQMT
KKDMGFTEEEFKRLNINAAKSSFLPEEEKK
e value cutoff for PSSM
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LocusLink

• Provides a Single Query Interface to Curated
  Sequence and Descriptive Information about
  Genetic Loci
• LocusLink will be Replaced by Entrez Gene
• Model Organisms Include C. elegans, chicken, cow,
  dog, human, human immunodeficiency virus type 1,
  mouse, pig, rat, S. purpuratus, X. laevis, X.
  tropicalis, and zebrafish.

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LocusLink

• Each Locuslink Record Consists of a Collection of
  Links to More Information About a Locus
• A Unique Integer is Assigned to Each Locus

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LocusLink Query Results Page
Links field small, color-coded boxes indicate
when links are available for PubMed, OMIM,
RefSeq, GenBank Nucleotide, Protein, HomoloGene,
UniGene, and Variation data
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Genome Resources Pages
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Online Tutorials
http//www.ncbi.nlm.nih.gov/Education/index.html
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Future Courses

• Data Mining
• Protein Structure
• SQL (Structured Query Language)