# Presentation: 539407

## NCBI Databases

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

**Bldg. 38A, Rm. 6N 603**
**NCBI, NLM, NIH**
**Bethesda MD 20894**

**spouge@ncbi.nlm.nih.gov**

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

## Database Generalities
Outside Database Links on the Web
The General Structure of NCBI Information and Tools
Some Specific NCBI Databases
Their Records 
Their Links
Some Tools for Navigating NCBI Databases
Entrez Genes
Entrez Genomes
Navigating NCBI Sequence Databases with
BLAST
PHI-BLAST
PSI-BLAST
The NCBI Help Desk

**Outside Database Links on the Web**
**The General Structure of NCBI Information and Tools**
**Some Specific NCBI Databases**
**Their Records **
**Their Links**
**Some Tools for Navigating NCBI Databases**
**Entrez Genes**
**Entrez Genomes**
**Navigating NCBI Sequence Databases with**
**BLAST**
**PHI-BLAST**
**PSI-BLAST**
**The NCBI Help Desk**

**Overview**

## 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.)**
**Purpose : UniVec (vectors), UniGene (genes), UniSTS (PCR)**
**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 : Entrez Genes (loci), Homologene (orthologs), OMIM (disease)**

**Types of Databases**

## Other Databases 

**Nucleic Acids Research (Oxford:  IRL Press)**
** ****Database Issue each January 1 (2004)  Vol. 32, No. 1 **
**Web Server Issue each July 1 (2003)  Vol. 31, No. 13 **

## 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 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

## The PubMed Database

## Some NCBI Databases

- ASN.1

- ASN.1

**VAST**

**BLAST**

**BLAST**

**Phylogeny**

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

**Map Viewer**

## NCBI Homepage

- NCBI Homepage

## PubMed Neighbors

**PubMed Neighbors**

- PubMed Homepage

## Format Conversion with ASN.1

**Format Conversion with ASN.1**

- ASN.1

- ASN.1

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

## PubMed Record Fields

**PubMed Record Fields**

**Abstract Format**

## Neighboring within NCBI Databases

**Neighboring within NCBI Databases**

- ASN.1

- ASN.1

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

## PubMed Neighbors

## Links between NCBI Databases

**Links between NCBI Databases**

- ASN.1

- ASN.1

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

## 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 & Genome Map Viewer)
- e.g., Disease to Gene Markers and Genes

## PubMed Links to Other Databases

**PubMed Links to Other Databases**

## The Nucleotide (GenBank) Database

## Nucleotide 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**

**Nucleotide 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**

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

## NCBI Homepage

- NCBI Homepage

## Nucleotide Record Fields

**Nucleotide Record Fields**

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

## Format Conversion with ASN.1

**Format Conversion with ASN.1**

- ASN.1

- ASN.1

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

## GenBank and GenPept Formats

**GenPept (protein)**

## FASTA Format

##  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**

## Derived Databases: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.**

**Accession codes**
**NM_****	****mRNA**
**NP_****	****protein**
**NT_****	****contig**
**NC_****	****chromosome**
**XM_      mRNA**
**XR_      non-coding RNA**

## Derived Databases:The UniGene DatabaseA gene-oriented view of GenBank sequences ESTs and full-length mRNA sequences organized into clusters representing a unique gene in an organism;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/

## The Molecular Modeling DatabaseMMDB(Structure)

## MMDB (Structure) Database

- ASN.1

- ASN.1

**Word Weight**

**BLAST**

**BLAST**

**Phylogeny**

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

**Map Viewer**

## NCBI Homepage

- NCBI Homepage

## Molecular Modeling Database Record

**Molecular Modeling Database Record**

## Structures of Proteins with Cn3D 

**sequence alignment viewer**

**Structures of Proteins with Cn3D **

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

**1UOL**
**mutant**
**p53 tumor suppressor protein complexed with DNA**

**Cn3D**
**viewer**

## BLink (BLAST Link)

**BLink (BLAST Link)**

## The Taxonomy Database

## Taxonomy Record Fields

**Taxonomy Record Fields**

## Other Databases and Navigational Tools

**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**
**CDD : Conserved Domain Database ****a collection of sequence and structure alignments and profiles, with protein domains from Smart, Pfam, and NCBI databases. **

## Using limits and preview/indexto restrict searches 

## PubMedlimits and preview/index

## PubMed Limits and Preview/Index

## PubMed Preview/Index

**Affiliation**
**All Fields**
**Author**
**EC/RN Number**
**Entrez**
**Filter**
**Grant Number**
**Issue**
**Journal**
**Language**
**MeSH Date**
**MeSH Major Topic**
**MeSH Subheading**
**MeSH Terms**
**Pagination**
**Pharmacologic Action**
**Publication Date**
**Publication Type**
**Secondary Source ID**
**Substance Name**
**Text Word**
**Title**
**Title/Abstract**
**Volume**

## PubMed Preview/Index

**PubMed Preview/Index**

## PubMed Limits

**PubMed Limits**

**Affiliation**
**All Fields**
**Author**
**EC/RN Number**
**Entrez**
**Filter**
**Grant Number**
**Issue**
**Journal**
**Language**
**MeSH Date**
**MeSH Major Topic**
**MeSH Subheading**
**MeSH Terms**
**Pagination**
**Pharmacologic Action**
**Publication Date**
**Publication Type**
**Secondary Source ID**
**Substance Name**
**Text Word**
**Title**
**Title/Abstract**
**Volume**

## Entrez Nucleotideslimits (and preview/index)

## Entrez Nucleotides

**Entrez Nucleotides**

## Document Summaries : Mouse [All Fields]

**Not Mouse!**

## Entrez Nucleotides : Limits

**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 **
**Volume**

## Document Summaries: Mouse[Organism]

## Limits on the DNA source 

## Entrez Nucleotides: Limits

**Entrez Nucleotides: Limits**

## Overload of Multiple Copies of the DNA

**Overload of Multiple Copies of the DNA**

**Better to search a genome**
**to find a unique copy!**

## Navigating Entrez Genes to Find Information on anAlzheimer’s Disease Gene

## Entrez: An integrated database search and retrieval system

## Using limits on the organism 

## Entrez Genes : Preview/Index

**Entrez Genes : Preview/Index**

**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 **
**Volume**

## Entrez Genes : Preview/Index

## Entrez Genes : Preview/Index

**Entrez Genes : Preview/Index**

## MapViewer

**View of chromosome 14**

**Gene Name**
**&**
**Links**

## MapViewer

- Online Mendelian Inheritance in Man

- sequence viewer

- protein record

- download

- evidence viewer

- model maker

- homologene

**MapViewer**

**View of chromosome 14**

## MapViewer Links

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

**MapViewer Links**

## BLAST

## BLAST and the NCBI Databases

**Word Weight**

**BLAST and the NCBI Databases**

- ASN.1

- ASN.1

**VAST**

**Phylogeny**

**Genomes**

**Taxonomy**

**Nucleotide Sequences**

**Protein Sequences**

**3 -D Structure**

**MMDB**

**Map Viewer**

## Neighboring with BLink (BLAST Link)

**Neighboring with BLink (BLAST Link)**

## BLAST Page

**BLAST Page**

## Nucleotide BLAST

**Nucleotide BLAST**

**distantly related genomes**

**closely related genomes**

**genomic archives search**

**short sequence search**

**old nucleotide BLAST **
**non-genomic sequences**

- Z Zhang *et al.* (2000) *J Comp Biol ***7:203**
- B Ma *et al.* (2002) *Bioinformatics* **18:440**

## Protein BLAST

**Protein BLAST**

**standard BLAST search**

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

**reverse position-specific BLAST of CDART**

**reverse position-specific BLAST of CDD**

**short sequence search**

- LY Geer *et al.* (2002) *Genome Res* 12**:1619**

## Other Parts of the BLAST Page

**Other Parts of the BLAST Page**

**BLAST 2 sequences, e.g., from a multiple alignment**

**Format and retrieve a previous BLAST search**

## The Basics of the BLAST Algorithms

## 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

- MNPQQQQQQRST = MNPXXXXXXRST

- X **will not match anything in the database.**
**It does preserve position, however.**

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

- “SEG” JC Wootton & S Federhen (1990) *Computers & Chem ***17**:149
- “DUST” RL Tatusov & DJ Lipman *in prep.*

## 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**

**If a diagonal score equals *****S******g****** = *****22 bits, try to extend across gaps, until the score drops to *****X******g***** = -40 bits. **

**gapxdrop Subroutine**

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

## MegaBLAST Algorithms

**gapxdrop Subroutine for very similar sequences**

**MegaBLAST**** ****Algorithms**

- CDEFG = CDE + DEF + EFG

**Contiguous Seeds**

- B Ma *et al.* (2002) *Bioinformatics* **18:440**

- CDEFG = CD_F + DE_G

**Discontiguous Seeds**

- Z Zhang *et al.* (2000) *J Comp Biol ***7:203**

## 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**

**E-value**

**Link to LocusLink**

**Link to MMDB**

**Score**
**Independent of Database**

## Conserved Domain DB BLAST Results

**Conserved Domain DB 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)**

## PHI-BLAST Algorithm

- MNPQQQQQQRST = MNPXXXXXXRST

- X **will not match anything in the database.**
**It does preserve position, however.**

**PHI-BLAST**** ****Algorithm**

## Regular Expression Seed

**PHI-BLAST Algorithm**

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

## Ungapped (Diagonal) Extension

**PHI-BLAST Algorithm**

**Probe along the diagonals containing the regular expression in the input.**

- query

- database

- database

- database

- database

## PHI-BLAST Algorithm

**PHI-BLAST Algorithm**

- query

- database

- database

- database

- database

**Gapped (Off-Diagonal) Extension**

**If a diagonal score equals *****S******g****** = *****22 bits, try to extend across gaps, until the score drops to *****X******g***** = -40 bits. **

**gapxdrop Subroutine**

## 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**

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

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

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

## 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 BLAST**

## Help Desk

**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)**

## Acknowledgements 

**Renata Geer**
**Peter Cooper**

## <footer>

- <date/time>