# Presentation: 526957

## NCBI Databases

**Their Organization**
**And**
**Practical Use**

**Bldg. 45, Rm. 6AS 47J**
**NCBI, NLM, NIH**
**Bethesda MD 20894**

**spouge@nih.gov**

**John L. Spouge**
**National Center for Biotechnology Information**

## Personal Research

**Physical chemistry of viral blocking ****	**JL Spouge (1994) *J. Virology ***68**:1782-1789
**Quantitation of viral reproduction****	**JL Spouge & SP Layne (1999) *PNAS ***96**:7017-7022
**Algorithmic problems in bioinformatics**
**BLAST search strategies****	**** **S Rajasekaran *et al.* (2001) *J. Comput. Biol. ***to appear**
**Statistical problems in Bioinformatics**
**Protein structure comparison statistics****	**** **JF Gibrat *et al.* (1997) *Biophys. J. ***72**:MP298
**Protein threading statistics****	**** **JL Spouge *et al.* (1999) *Ann. Combinatorics ***3**:81-93
**DNA-word search statistics****	**** **TG Wolfsberg *et al.* (1999) *Genome Res. ***9**:775-792
**Statistical evaluation of retrieval ****	**** **AA Schaffer *et al.* (2001) *Nucleic Acids Res. ***29**:2994-3005
**Rigorous ungapped BLAST statistics****	**** **JL Spouge (2001) *J. Appl. Prob. ***38**:1-16
**Heuristic gapped BLAST statistics **
**Genomic clustering statistics****	**** **IK Jordan *et al.* (2001) *Genome Res*. **11**:556-556

**Personal Research**

## Database Generalities

## Some NCBI Databases and Their Links 

**Some NCBI Databases and Their Links**** **

## Archival or Primary Data 
Text : PubMed
DNA Sequence : GenBank/EMBL/DDBJ
Protein Structures : Brookhaven PDB

**Text : PubMed**
**DNA Sequence : GenBank/EMBL/DDBJ**
**Protein Structures : Brookhaven PDB**

**Specialized Databases **
**Organism : Maps in Entrez Genomes (human, mouse, yeast, etc.)**
**Function : Sequences in UniVec (vectors), UniGene (genes) **
**Sequencing Methods : Sequences in dbEST, dbGSS, , dbSTS, HTG**

**Curated or Processed Data**
**Sequences : RefSeq (curated, non-redundant DNA, mRNA, protein, etc.)**
**Protein Sequences and Structures : MMDB, nr **
**Organism Maps : Entrez Genomes (human, mouse, yeast, etc.) **
**Genes : LocusLink (loci), Homologene (orthologs), OMIM (disease)**

**Types of Databases**

## Links within and between the Databases

- Same Article (Expert Authorship)
- e.g., Text to Sequence
- Similar Vocabulary (PubMed neighboring)
- e.g., Article to Article
- Similar Sequence (BLAST neighboring)
- Sequence to Sequence
- Similar Structure (VAST neighboring)
- Structure to Structure
- Physical Proximity on Chromosome (Genome Map Viewer)
- e.g., STS Marker to Gene
- Clinical and Laboratory Correlates of Heredity (OMIM and LocusLink)
- e.g., Disease to Gene Markers and Genes

## Other Databases 

**Nucleic Acids Research (Oxford:  IRL Press)**
** ****Database Issue each January 1 (2001)  Vol. 29, No. 1 **
**Includes articles on about 100 different databases**

## Web Portals for Biological Databases 

- Amos’ Links
- http://www.expasy.ch/alinks.html
- EBI’s Biocatalog
- http://www.ebi.ac.uk/biocat/
- Infobiogen’s DBCat
- http://www.infobiogen.fr/services/dbcat/
- Indiana University IUBio Archive:
- http://iubio.bio.indiana.edu/
- Pedro's BioMolecular Research Tools
- http://www.public.iastate.edu/~pedro/research_tools.html
- NCBI
- http://www.ncbi.nlm.nih.gov/

## NCBI Homepage

- NCBI Homepage

## NCBI Site Map

- NCBI Site Map

## NCBI Homepage

- NCBI Homepage

## NCBI Home Page : Title Bar 

**Sequence**
**Similarity**
**Searching**

**NCBI Home Page : Title Bar**

## Entrez: An integrated database search and retrieval system

- All of MEDLINE plus others
- Abstracts
- Links to online Journals

- GenBank, EMBL, DDBJ
- RefSeq, PDB

- GenBank, DDBJ, EMBL translations
- PDB, PIR, SWISS-PROT, PRF, RefSeq

- NCBI’s MMDB - derived from PDB

- Graphical views
- Assembled sequence and mapping data

- NCBI’s Taxonomy
- Hierarchical tree structure
- access to sequences

- MIM
- Now in Entrez

- Population and phylogenetic studies

**Entrez: ****An integrated database search and retrieval system**

## Some NCBI Databases

- ASN.1

- ASN.1

**Map Viewer**

**Word Weight**

**VAST**

**BLAST**

**BLAST**

**Phylogeny**

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**Article Abstracts**

**MedLine**

**3 -D Structure**

**MMDB**

## The PubMed Database

## NCBI Homepage

- NCBI Homepage

## PubMed Homepage

- PubMed Homepage

## PubMed Record Fields

**PubMed Record Fields**

**Abstract Format**

## Neighboring within NCBI Databases 

**Neighboring within NCBI Databases**** **

## PubMed Neighbors

## PubMed Neighbors

## Links between NCBI Databases 

**Links between NCBI Databases**** **

## PubMed Links to Other Databases

**Specialty Non-NCBI Links**

**PubMed Links to Other Databases**

## The GenBank Database

## NCBI Homepage

- NCBI Homepage

## GenBank Record Fields

**GenBank Record Fields**

**New Sequence**
**New gi # **
**New Accession #**
**Modification **
**Fixed gi #**
**New Version #**
**Exception: HTG **
**Fixed accession #**
**New gi #****	**

## GenBank Database Divisions

**Traditional Divisions**
**BCT,INV,MAM,PHG,PLN,PRI**
**ROD,SYN,UNA,VRL,VRT**
**Special Sequence Divisions**
**EST****	****	****Expressed Sequence Tags **
**STS****	****	****Sequence Tagged Site**
**GSS****	****	****Genome Survey Sequence**
**HTG****	****	****High Throughput Genome**
**PAT****	****	****Patent**

**GenBank Database Divisions**

## ESTs (Expressed Sequence Tags)

**ESTs (Expressed Sequence Tags)**

- dbEST http://www.ncbi.nlm.nih.gov/dbEST/

## Segment of known gene, EST, mRNA or genomic DNA of known position (e.g., microsatellite)
PCR with STS primers gives unique product (one per genome)
Basis of Radiation Hybrid Mapping
Related resource: Electronic PCR
     http://www.ncbi.nlm.nih.gov/genome/sts/epcr.cgi

- PCR with STS primers gives unique product (one per genome)
- Basis of Radiation Hybrid Mapping
- Related resource: Electronic PCR
-      **http://www.ncbi.nlm.nih.gov/genome/sts/epcr.cgi**

**STSs (Sequence Tagged Sites)**

- dbSTS http://www.ncbi.nlm.nih.gov/dbSTS/

## GSSs (Genome Survey Sequences)

- E.g., BAC end sequences
- BAC and other first pass surveys

**GSSs (Genome Survey Sequences)**

- dbGSS http://www.ncbi.nlm.nih.gov/dbGSS/

## HTGS: High Throughput Genome Sequences

-   have gaps in the nucleotide sequence
-   low accuracy
-   no annotations

**HTGS: High Throughput Genome Sequences**

- HTGS http://www.ncbi.nlm.nih.gov/HTGS/

**40 kb**
**to**
** ****> 350 kb**

**New! Phase 0. First pass surveys.**
**acc = AC021876****	****    gi = 6729081**

## Other Sequence Formats

## GenBank and GenPept Formats

## Creating New NCBI Databases 

**Creating New NCBI Databases**** **

- MedLine

- MMDB

- Nucleotide Sequences

##  FASTA Format

**GGCAGCCAGGCCATGAGATCTTAGGCCATGTCGAGACGTGACGTGGTCCTTACCAATGTTACTGTTGTCC**
**AGCTACGGCGGGACCGATGCCCATGCCCATGCCCATGCCCATGTCCATGCCCATGCCCTGTGATCAGACC**
**ACCTCCACCCAAGCTTGAGGATCCACCACCCACGGTTGAAGAACAGCCACCGCCACCGCCGCCGCCACCT**
**CCACCTCCACCACCACCTCCTCCTCCTCCTCCACCCCAGATAGAGCCAGACAAGTTTGAAGAGGCTCCCC**
**CTCCCCCTCCCCCTCCTCCTCCTCCTCCCCCTCCCCCTCCTCCACCACTCCAAAAGCCAGCTAGAGAGCT**
**GACAGTGGGTATCAATGGATTTGGACGCATTGGTCGTCTGGTGCTGCGAGTCTGCATGGAGAAGGGCATT**
**AGGGTGGTAGCAGTGAATGACCCATTCATTGATCCAGAATACATGGTTTACATGTTCAAATATGACTCCA**
**CACATGGTAGATACAAAGGAAACGTGGAACATAAGAATGGACAACTAGTTGTGGACAACCTTGAGATCAA**
**CACGTACCAGTGCAAAGACCCTAAAGAAATCCCCTGGAGCTCTATAGGGAATCCCTACGTGGTGGAGTGT**
**ACAGGCGTCTATCTGTCCATCGAGGCAGCTTCGGCACATATTTCATCTGGTGCCAGGCGTGTGGTGGTCA**
**CTGCACCCTCCCCCGATGCACCCATGTTTGTCATGGGAGTGAACGAGAAGGACTATAACCCTGGCTCTAT**
**GACCATTGTCAGCAATGCATCCTGTACCACCAACTGCCTGGCTCCTCTCGCCAAGGTTATTCATGAAAAC**
**TTCGGGATCGTGGAAGGGCTAATGACCACAGTCCATTCCTACACAGCCACTCAGAAGACAGTGGATGGGC**
**CATCAAAGAAGGACTGGCGAGGTGGCCGCGGCGCTCACCAAAACATCATCCCATCGTCCACTGGGGCTGC**
**CAAGGCTGTAGGCAAAGTCATCCCAGAGCTCAAAGGGAAGCTAACAGGAATGGCATTCCGGGTGCCAACC**
**CCAAACGTGTCAGTTGTGGACCTGACCTGCCGCCTGGCCAAGCCTGCTTCTTACTCGGCTATCACGGAGG**
**CTGTGAAAGCTGCAGCCAAGGGACCTTTGGCTGGCATCCTTGCTTACACAGAGGACCAGGTGGTCTCCAC**
**GGACTTTAACGGCAATCCCCATTCTTCCATCTTTGATGCTAAGGCTGGAATTGCCCTCAATGACAACTTC**
**GTGAAGCTTGTTGCCTGGTACGACAACGAATATGGCTACAGTAACCGAGTGGTCGACCTCCTCCGCTACA**
**TGTTTAGCCGAGAGAAGTAACACAAAAGGCCCCTCCTTGCTCCCCTGCGCACCTCGCGTTCCTGACTTCG**
**GCTTCCACTCAAAGGCGCCGCCACCGGGTCAACAATGAAATAAAAACGAGAATGCGC**

- >

** ****FASTA Format**

**FASTA Definition Line**
**>gi|193425|gb|M60978.1|MUSGAPDS**

**gi number**

**Database Identifiers**
**gb****	****GenBank**
**emb****	****EMBL**
**dbj****	****DDBJ**
**sp****	****SWISS-PROT**
**pdb****	****Protein Databank**
**pir****	****PIR**
**ref****	****RefSeq**

**Accession number**

**Locus Name**

## The RefSeq DatabaseNCBI database of Reference SequencesCurated, non-redundant set (unlike nr) includinggenomic DNA contigs, mRNAs and genes for proteins

## RefSeq Record Fields

**LOCUS       NM_000492    6159 bp    mRNA            PRI      26-JUL-1999**
**DEFINITION  Homo sapiens cystic fibrosis transmembrane conductance**
**            ****regulator(CFTR) mRNA.**
**ACCESSION   NM_000492**

**COMMENT  **_**REFSEQ:**_** This reference sequence was derived from M55131.**
**         ****PROVISIONAL RefSeq: This is a provisional reference sequence**
**         ****record that has not yet been subject to human review. The final**
**         ****curated reference sequence record may be somewhat different from**
**         ****this one.**

**LOCUS       NP_000483    1480 aa                    PRI      26-JUL-1999**
**DEFINITION  cystic fibrosis transmembrane conductance regulator.**
**ACCESSION   NP_000483**
**PID         g4502785**
**VERSION     NP_000483.1  GI:4502785**
**DBSOURCE    REFSEQ: accession **_**NM_000492.1**_

- RefSeq Protein

- RefSeq Nucleotide

_**REFSEQ**_**: This reference sequence was derived from M28668.1,**
**M55131.1.**
**On Feb 17, 2000 this sequence version replaced gi:4502784.**
**Summary: Cystic fibrosis transmembrane conductance regulator is**
**member 7 of the ATP-binding cassete sub-family C. The protein**
**functions as a chloride channel and controls the regulation of**
**other transport pathways. Mutations in this gene cause the**
**autosomal recessive disorder, cystic fibrosis (CF) and congenital**
**bilateral aplasia of the vas deferens (CBAVD). Alternative splice**
**variants have been described, many of which result from mutations**
**in the CFTR gene.**
**COMPLETENESS: full length.**

- New style accession numbers
- NM_	mRNA
- NP_	protein
- NT_	contig
- NC_	chromosome

## The UniGene DatabaseA gene-oriented view of GenBank sequences ESTs and full-length mRNA sequences organized into clusters representing a unique human gene;annotated with mapping and expression information; and with cross-references to other NCBI resources

## UniGene Database

**Nonredundant set of gene oriented clusters**
**Each cluster a unique gene**
**Information on tissue types and map locations**
**Includes well-characterized genes and novel ESTs**
**Useful for discovering genes**
**Useful for selecting mapping reagents**

**UniGene Database**

- UniGene http://www.ncbi.nlm.nih.gov/UniGene/

## OMIM: Online Mendelian Inheritance in Man

## OMIM record for Presenilin 1 (PSEN1)

**OMIM record for Presenilin 1 (PSEN1)**

**Each record provides a state of the art summary of current knowledge of genetic disease**

## The Structure Database

**Molecular Modeling Database (MMDB)**
**   **

## 3-Dimensional Protein Structures

**x-ray crystallography**
**NMR**

**1TUP**
**p53 tumor suppressor protein complexed with DNA**
**Viewed with Cn3D**

## MMDB Record Fields

- COMPND    Alzheimer's Disease Amyloid Beta-Peptide (Residues 1 - 28)
- COMPND   2 (E.C. Number Not Assigned) (Nmr, 5 Structures)
- SOURCE    ORGANISM_SCIENTIFIC: Homo sapiens; ORGANISM_COMMON: human
- AUTHOR    J.Talafous, K.J.Marcinowski, G.Klopman & M.G.Zagorski
- REMARK  00 NCBI PDB FORMAT VERSION 5.0
- REMARK  00 NOTE:  NCBI-MMDB PDB-Format File derived from ASN.1
- REMARK  00 Refer to original ASN.1 file or PDB file for data records
- REMARK  00 not included.  Changes from original PDB files include:
- REMARK  00 - residues are derived by graph matching algorithms.
- REMARK  00 - residue numbers always increase numerically (e.g. no 8A).
- REMARK  00 - taxonomic information reannotated using NCBI Taxonomy.
- REMARK  00 - element names are made consistent.
- REMARK  00 - CONECT records are derived algorithmically.
- REMARK  00 - Secondary structure is derived algorithmically.
- SEQRES   1     28  ASP ALA GLU PHE ARG HIS ASP SER GLY TYR GLU VAL HIS
- SEQRES   2     28  HIS GLN LYS LEU VAL PHE PHE ALA GLU ASP VAL GLY SER
- SEQRES   3     28  ASN LYS
- HELIX    1 hel ALA      2  ASP     23
- ATOM      1  C   ASP     1      17.590  -5.142  -0.280  1.00  0.00           C
- ATOM      2  CA  ASP     1      18.832  -4.846  -1.120  1.00  0.00           C
- ATOM      3  CB  ASP     1      18.639  -5.405  -2.532  1.00  0.00           C
- ATOM      4  CG  ASP     1      19.661  -4.773  -3.477  1.00  0.00           C
- ATOM      5  HA  ASP     1      18.985  -3.778  -1.174  1.00  0.00           H
- ATOM      6  N   ASP     1      20.022  -5.487  -0.492  1.00  0.00           N
- Etc.

**MMDB Record Fields**

## The Corresponding GenPept Record

**The Corresponding GenPept Record**

## The Taxonomy Database

## Taxonomy Record Fields

## COGs : comparison of complete microbial genomes natural system of gene families from complete genomesHomoloGene : a gene homology tool that compares nucleotide sequences between pairs of organisms, including human, mouse, rat, zebrafish, and fruit fly, in order to identify putative orthologsCD-Search : Conserved Domain Search can be used to identify the conserved domains present in a protein sequenceDART : Domain Architecture Retrieval Tool displays the functional domains that make up the protein and lists proteins with similar domain architectures (not yet in site map)

**Other Databases and Navigational Tools**

## Links between NCBI Databases 

**Links between NCBI Databases**** **

**Word Weight**

**VAST**

**BLAST**

**BLAST**

**Phylogeny**

**Map Viewer**

## Text Searching	Entrez	OMIM	LocusLinkSequence Similarity Searching	BLAST (nucleotide and protein sequences)Structure Similarity Searching	VAST (protein structures)

**Searching the Databases**

## Using Entrez to Search the GenBank Database

## Searching the NCBI GenBank Database 

## Using limits and restricting the GenBank division to find mouse GAPD

## Entrez Nucleotides

**Entrez Nucleotides**

## Document Summaries : Mouse [All Fields]

**Document Summaries : ****Mouse [All Fields]**

**Not Mouse!**

**Mouse**

**Entrez Nucleotides : Limits & Preview/Index**

## Entrez Nucleotides : Limits

**Only From**
**RefSeq**
**GenBank**
**EMBL**
**DDBJ**

**Exclude unwanted categories of sequences**

**Molecule**
**Genomic DNA/RNA**
**mRNA**
**rRNA**

**Gene Location**
**Genomic DNA/RNA**
**Mitochondrion**
**Chloroplast**

**Mouse**

**Entrez Nucleotides : Limits**

## Entrez Nucleotides: Limits: Organism

## Document Summaries: Mouse[Organism]

**Document Summaries: Mouse[Organism]**

## Adding Terms: Preview/Index

**glyceraldehyde 3 phosphate dehydrogenase**

**Adding Terms: Preview/Index**

- Search History

**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 Word**
**Uid**
**Volume**

## EST Overload

## Entrez Nucleotides: Limits: Organism

**Entrez Nucleotides: Limits: Organism**

## Using field restriction instead to eliminate ESTs andto find mouse GAPD mRNA

## Properties Field: Molecule Type

**Properties Field: Molecule Type**

## Properties Field: GenBank Division

**Properties Field: GenBank Division**

## Mouse GAPD Records

## Displaying Mouse GAPD Records

**Summary**
**Brief**
**GenBank**
**ASN.1**
**FASTA**
**LinkOut**
**PubMed Links**
**Protein Links**
**Nucleotide Neighbors**
**PopSet Links**
**Structure Links**
**Genome Links**

**Links and neighbors (related records)**

**Displaying Mouse GAPD Records**

**Formats**

## Protein Neighbors-Structure Links

**Protein Neighbors-Structure Links**

## Neighboring with Blink (BLAST link)

**Neighboring with Blink (BLAST link)**

## Blink Hits for gi 567204

## CD Search (RPS-BLAST) for gi 567204

**Reverse Position-Specific BLAST**
**Sequence against Protein Families**

## Displaying Mouse GAPD Records

**Displaying Mouse GAPD Records**

## GAPD PopSet Links

## GAPD PopSets

## PopSet Alignment Display

## Navigating OMIM(Online Mendelian Inheritance in Man)

## OMIMHomepage

**Catalog of human genes and genetic disorders**

- OMIMHomepage

## OMIM : Limits

**Mouse**

**OMIM : Limits**

## OMIM Limits Options

**OMIM Limits Options**

## OMIM Search for Alzheimer’s Disease

**OMIM Search for Alzheimer’s Disease**

## OMIM Search Results by Titles

**OMIM Search Results by Titles**

## OMIM Links

**OMIM Links**

## Allelic Variants of PSEN1

**Allelic Variants of PSEN1**

**Allelic Variants**

**Allelic Variants**

**Allelic Variants**

**Alzheimer Disease, Familial Type 3**
**Also known as:**
**AD3                   Presenilin 1        PSEN1              PS1**

## Navigating Databases with LocusLink

## LocusLink

**Text querying**

**Multiple Organisms**

**Alphabetical listings**

**Stable Locus ID**

**Approved symbol**

**Description**

**Genome Position**

**External Links**

**Curated Resource **
**Central hub of information for human, mouse, rat, zebrafish, and fruit fly loci**

## LocusLink Links

## LocusLink : LocusID 5663   PSEN1

## Navigating the Genomes Database with Entrez

## NCBI Genomes Database 

**NCBI Genomes Database**** **

**Map Viewer**

## Entrez Genome 

**Entrez Genome**** **

## Entrez Genome : Gene Location 

**View of chromosome 14**

## Integrated View : 3 Maps of Chromosome 7

**Chromosome 7 **
**GenBank Map Contig Map     STS Map**

**Integrated View : 3 Maps of Chromosome 7**

**Multiple Maps**
**STSs, ESTs, etc.**

## Entrez Genome : Gene Location 

**View of chromosome 14**

## Entrez Genome : Gene Location 

**Entrez Genome : Gene Location**** **

**Entrez Genomes   Map Viewer**
**Chromosome 14 Cytogenetic map**

## BLAST

## BLAST and the NCBI Databases 

**BLAST and the NCBI Databases**** **

**BLAST**

**BLAST**

**Map Viewer**

- Genomes

- Nucleotide Sequences

## Neighboring with Blink (BLAST link)

**Neighboring with Blink (BLAST link)**

## New QBLAST Page

**Standard BLAST Search**

**Genomic BLAST Search**

**Standard BLAST Search**

**Position-Specific Iterated Pattern-Hit Initiated BLAST Search**

**Short Sequence Search**

## New QBLAST Page (continued)

## Protein-Protein BLAST

## BLAST (Basic Local Alignment Search Tool)

**a query sequence **
**a database of biological sequences **

**Output **
**a ranked list of “hits”, database sequences that are **
**locally similar to the query sequence (from which the **
**unknown function of the query sequence might be**
**inferred)**
**the statistical significance of each hit**

- SF Altschul *et al.* (1990) *J Mol Biol* **215**:403-410
- SF Altschul *et al.* (1997) *Nucleic Acids Res. ***17**:3389-3402

**BLAST**** ****(Basic Local Alignment Search Tool)**

## BLAST Algorithm

**BLAST**** ****Algorithm**

## BLAST Algorithm

**BLAST**** ****Algorithm**

## BLAST Algorithm

**BLAST**** ****Algorithm**

**Probe along a diagonal, if it contains**
**Two**** *****T**** *= 11 **hits within 40 positions of each other**
**A single**** *****T**** *= 13 **hit**

## BLAST Algorithm

**Gapped (Off-Diagonal) Extension**

**BLAST**** ****Algorithm**

## Protein-Protein BLAST Page

**Protein-Protein BLAST Page**

**Useful options for short queries**

## Immediate Protein-Protein BLAST Results

**Immediate Protein-Protein BLAST Results**

## Formatted Protein-Protein BLAST Results

**Formatted Protein-Protein BLAST Results**

## Large Nucleotide Sequences

**GAATTCAAGTGTCTTATTTCTTCAGGTAATACGGCAATTGTACTAGTTGGGAAATGAAATATCAAA**
**GGCTTCCAGTTTTAACTAACGAGTCAATTTATATGTATTAAAGCTGTCCTGGGCTTGTTTAAAAGA**
**AGCTTTATTTTTCAAGCATATGTGCAAAGTTCAGGACCAAAAGAAAGTTTAGTTATTTATCTCTGG**
**TGCAGATACAATTAAATGTAAGTAAATGCTAACTATTCTTTAATGAAAATTACAAACATAATTGAA**
**ATATTTAACTAAAGGCAGAGTTGTTAGTTAAACAAATGATGTCAGAGCTCCAGTTTTCTTATTCAC**
**TTTAATTATGCTTAGTCTTTTAACACAGGGATTTCCACACAGGTCTCCATTTCATGTACTCTGTAG**
**TATCTTTGCAAACCATTTAAGAGTTTTCTGACTGACAAAATAAAGTAGGCTGGAAGTTTAAAAGAA**
**ATGTCTGGCTTCTGACAGGATCCTAGCTTTAGATGTTTCTATACGGTTTGTATAAATGCCAGCCCT**
**AAGCTATTCAATCTTTTTTCTTTTTCTTTTTTTTTCTTTTTAAAACTTTCTGACTAGATCAAGACA**
**AGGACTGTAGAAGTATGACGGGGAATAAAAAGGTCAAATTACACACAAAAGCATTAAGGTTCACTT**
**CAGCATCAGCCAAGGTGTGCATTGACAAGAAAATGTAATCTGACAGAAATTAACAGCAATGATTCA**
**ACTGTGTTCACTACAAAATGGGGGTGGGTGGGGTGGGTATTATGCCACAAATGTGGAAACATATGG**
**ATAAAAATTTTTGGCTGGTAAATATATAAAGAAATCTTGAATAATTAAAAGTAATATGGGGAACAC**
**ATTCAAGAATGATCTCATTCTCAAACCGTTTGTTTCAAAGATATGTTAAGACATTTAAAGGACTCC**
**AGTGATGAATTAATTCATTAAAAATGACATGAGCAATATTAAATGATAGAATTATAATAACAGGAA**
**ATGTTGGTAATTCTATGGGTAGTTCATTTACTTGGAAAAGTATAGCATCCCTGGCTGGGCACAGTG**
**ATACCTGTAATCCCAGTGCTTTGGGAGGCTGAGTTGGGAGGATAGCTCGAGGCCAGAAGTTTGAGA**
**...**

**Large Nucleotide Sequences**

**ERROR: Blast: CPU limit exceeded**

**use MEGABLAST!**

**Mask ALU repeats**

**Decrease the E-value to decrease the false positives**

**Decrease the number of seeds by increasing the length of the word matches required**

## PSI-BLAST & PHI-BLAST

**Position-Specific Iterated BLAST**
**&**
**Pattern-Hit Initiated BLAST**

## PHI-BLAST (Pattern-Hit Initiated BLAST)

**a query sequence**
**a regular expression (pattern) in the query sequence**** **
**a database of biological sequences **

**Output **
**a ranked list of “hits”, database sequences that are **
**locally similar to the query sequence, ****which contain the pattern**
**the statistical significance of each hit**

- SF Altschul *et al.* (1997) *Nucleic Acids Res. ***17**:3389-3402

**PHI-BLAST**** ****(Pattern-Hit Initiated BLAST)**

## Slide 112

## Regular Expression Seed

**PHI-BLAST Algorithm**

**Match the query’s regular expression in the database**

## Slide 114

## Slide 115

## PSI-BLAST (Position-Specific Iterated BLAST)

**a query sequence**
**a database of biological sequences**
**an E-value threshold for inclusion into a position-specific scoring matrix (PSSM) **

**Output **
**a ranked list of “hits”, database sequences that are **
**locally similar to the query sequence**
**the statistical significance of each hit**
**a PSSM describing the hits closest to the query**

- SF Altschul *et al.* (1997) *Nucleic Acids Res. ***17**:3389-3402

**PSI-BLAST**** ****(Position-Specific Iterated BLAST)**

**Input**** **
**a PSSM**

## PSI-BLAST Algorithm

**PSI-BLAST**** ****Algorithm**

## PSI-BLAST Algorithm

**PSI-BLAST Iteration**

**PSI-BLAST**** ****Algorithm**

**New Position-Specific Scoring Matrix**

**convergence occurs when the set of closely related sequences does not change**

## PSI-BLAST Algorithm

**PSI-BLAST**** ****Algorithm**

**corruption occurs when the threshold permits unrelated sequences to continue to enter the PSSM on each iteration**

## PSI-BLAST Page

**PSI-BLAST Page**

**Matrix from multiple alignment**

**PROSITE Pattern**

## PSI-BLAST : First Iteration

**PSI-BLAST : First Iteration**

## PSI-BLAST : Later Iterations

**BLAST did not find this sequence**

**To prevent corruption, new but unwanted sequences can be examined and excluded from the PSSM by hand on biological grounds**

**PSI-BLAST : Later Iterations**

## PSI-BLAST : Convergence

**PSI-BLAST : Convergence**

**PSI-BLAST **
**PSSM matrix provides a picture of a whole protein family**
**Useful for repeated searches on a specific family**
**Usually more sensitive than QBLAST**

## NCBI Homepage

- NCBI Homepage

**Help Desk**

## Please Send Help

**Subject:  Help Desk**
**Dear sirs, **
**I am working in the field of cancer molecular biology, could you please send me a help desk.**
**	****	****	****	****	****(Nov. 5, 1999)**

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