sequence analysis (i) yuh-shan jou ( 周玉山 ) [email protected] institute of biomedical...

Sequence Analysis (I)

Yuh-Shan Jou ( 周玉山 )[email protected]

Institute of Biomedical Sciences, Academia Sinica

Bioinformatics• Bioinformatics is the application of information

technology to analyze, process, and manage biological data.

• Bioinformatics provides computational tools to facilitate the process of

Data Information Knowledge Discovery

Don’t believe everything you see in DB or even in GenBank!QC is the most important aspect and concern in Bioinformatics!

Roadmap to Genomics

Transcriptional Map of human Genome

Sequencing ofHuman Genome

Human Genome

Human GenomePhysical Maps

Database ESTs (dbEST)

1. Markers: EST: Expressed Sequence Tag. STS: Sequence Tag Site. STR: Short Tandem Repeat.2. genomic DNA contigs: Cosmid contigs YAC contigs

cDNA sequencing

1. BAC or PAC contigs2. Sequencing technologies

Radiation HybridsMapping Panels

Posit ionalCloning

Positional Candidate Approaches

*Diagnosiswith GeneChips

*1. Expression patterns*2. Expression profiles*3. Microarray of genes

Diseases Markersfor diagnosis

Positional Candidate Approaches

Sho

tgun

seq

uen c

ing

Full length cDNAs

Functional Genomics

A Vision for the Future of Genome ResearchFrancis S. Collins (National Human Genome Research Institute, NIH, USA)

Nature 422:835 (2003)

EBI

GenBankGenBank

DDBJDDBJ

EMBLEMBL

EMBLEMBL

Entrez

SRS

getentry

NIGNIGCIB

NCBI

NIHNIH

•Submissions•Updates •Submissions

•Updates

•Submissions•Updates

International Sequence Database Collaboration

Lecture 7.1 6

www.ensembl.org

http://genome.ucsc.edu

Data Bases and Scientific Algorithms Data Bases and Scientific Algorithms

Integration Bioinformatics

Medline(Asn.1)

Medline(Asn.1)

Entrez/NCBI(Asn.1)

Entrez/NCBI(Asn.1)

PDB(Oracle, 3D images)

PDB(Oracle, 3D images)

BLAST(FASTA)

BLAST(FASTA)

IntegrationIntegrationBioInformaticsBioInformatics

IntegrationIntegrationBioInformaticsBioInformatics

ClustalW(FASTA)

KEGG (HTML Text,

Binary Images)

OMIN(Text File)

Microarray Data(RDBMS, Excel)

Web Access: www.ncbi.nlm.nih.gov

NCBI Web Traffic

Christmas and New Year’s Day

User’s per day

The Entrez System: Text Searches

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 49,675,750

Derivative• RefSeq 545,503

• Third Party Annotation 4,544

• PDB 5,561 Total 50,231,358

Entrez Protein: Derivative Databases

GenPept 3,950,968

RefSeq 1,348,072

Third Party Annotation 4,133

Swiss Prot 170,087

PIR 282,821

PRF 12,079

PDB 61,845

Total 5,830,005

BLAST nr total 2,336,522

GenBank Growth

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BasepairsRecords

The Growth of GenBank

Release 148: 45.2 million records 49.4 billion nucleotides

Average doubling time ≈ 14 months*

Organization of GenBank:Traditional Divisions

Records are divided into 17 Divisions.11 Traditional 6 Bulk

Traditional Divisions: Traditional Divisions: • Direct Submissions (Sequin and BankIt)

• Accurate• Well characterized

PRI (28) Primate PLN (13) Plant and FungalBCT (11) Bacterial and Archeal INV (7) InvertebrateROD (15) RodentVRL (4) ViralVRT (7) Other VertebrateMAM (1) Mammalian PHG (1) PhageSYN (1) Synthetic (cloning vectors) UNA (1) Unannotated

Entrez query: gbdiv_xxx[Properties]

Organization of GenBank:Bulk Divisions

Records are divided into 17 Divisions.11 Traditional 6 Bulk

BULK Divisions: BULK Divisions: • Batch Submission (Email and FTP)

• Inaccurate• Poorly characterized

EST (355) Expressed Sequence Tag GSS (132) Genome Survey SequenceHTG (62) High Throughput GenomicSTS (5) Sequence Tagged SiteHTC (6) High Throughput cDNAPAT (17) Patent

Entrez query: gbdiv_xxx[Properties]

File Formats of theSequence Databases

Each sequence is represented bya text record called a flat file.

GenBank/GenPept (useful for scientists) FASTA (the simplest format)

ASN.1 & XML (useful for programmers)

A TraditionalGenBank

Record

LOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA, complete cds.ACCESSION AY182241VERSION AY182241.2 GI:32265057KEYWORDS .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, 84-94 (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, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD 20705, USAREFERENCE 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, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD 20705, USA REMARK Sequence update by submitterCOMMENT On Jun 26, 2003 this sequence version replaced gi:27804758.FEATURES Location/Qualifiers source 1..1931 /organism="Malus x domestica" /mol_type="mRNA" /cultivar="'Law Rome'" /db_xref="taxon:3750" /tissue_type="peel" gene 1..1931 /gene="AFS1" CDS 54..1784 /gene="AFS1" /note="terpene synthase" /codon_start=1 /product="(E,E)-alpha-farnesene synthase" /protein_id="AAO22848.2" /db_xref="GI:32265058" /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

LOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA, complete cds.ACCESSION AY182241VERSION AY182241.2 GI:32265057KEYWORDS .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, 84-94 (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, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD 20705, USAREFERENCE 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, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD 20705, USA REMARK Sequence update by submitterCOMMENT On Jun 26, 2003 this sequence version replaced gi:27804758.

The Header


Header: Locus LineLOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004LOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004

Molecule typeMolecule typeDivisionDivision

Modification DateModification Date

Locus nameLocus name

LengthLength


Header: Database Identifiers

ACCESSION AY182241

VERSION AY182241.2 GI:32265057

ACCESSION AY182241

VERSION AY182241.2 GI:32265057

Accession•Stable•Reportable•Universal

Accession•Stable•Reportable•Universal

VersionTracks changes in sequenceVersionTracks changes in sequence

GI numberNCBI internal useGI numberNCBI internal use


Header: Organism

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.

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.

NCBI-controlled taxonomy

FEATURES Location/Qualifiers source 1..1931 /organism="Malus x domestica" /mol_type="mRNA" /cultivar="'Law Rome'" /db_xref="taxon:3750" /tissue_type="peel" gene 1..1931 /gene="AFS1" CDS 54..1784 /gene="AFS1" /note="terpene synthase" /codon_start=1 /product="(E,E)-alpha-farnesene synthase" /protein_id="AAO22848.2" /db_xref="GI:32265058" /translation="MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSANYKPNIWK NDFLDQSLISKYDGDEYRKLSEKLIEEVKIYISAETMDLVAKLELIDSVRKLGLANLF EKEIKEALDSIAAIESDNLGTRDDLYGTALHFKILRQHGYKVSQDIFGRFMDEKGTLE NHHFAHLKGMLELFEASNLGFEGEDILDEAKASLTLALRDSGHICYPDSNLSRDVVHS LELPSHRRVQWFDVKWQINAYEKDICRVNATLLELAKLNFNVVQAQLQKNLREASRWW ANLGIADNLKFARDRLVECFACAVGVAFEPEHSSFRICLTKVINLVLIIDDVYDIYGS EEELKHFTNAVDRWDSRETEQLPECMKMCFQVLYNTTCEIAREIEEENGWNQVLPQLT KVWADFCKALLVEAEWYNKSHIPTLEEYLRNGCISSSVSVLLVHSFFSITHEGTKEMA DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI LSLLFQPLVN"

The Feature Table

Coding sequenceCoding sequence

start (atg)start (atg) stop (tag)stop (tag)

ImpliedproteinImpliedprotein

GenPept Identifiers

The Sequence: 99.99% Accurate

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

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

1741 ggacccacat cctgtcttta ctattccaac ctcttgtaaa ctagtactca tatagtttga 1801 aataaatagc agcaaaagtt tgcggttcag ttcgtcatgg ataaattaat ctttacagtt 1861 tgtaacgttg ttgccaaaga ttatgaataa aaagttgtag tttgtcgttt aaaaaaaaaa 1921 aaaaaaaaaa a//

1741 ggacccacat cctgtcttta ctattccaac ctcttgtaaa ctagtactca tatagtttga 1801 aataaatagc agcaaaagtt tgcggttcag ttcgtcatgg ataaattaat ctttacagtt 1861 tgtaacgttg ttgccaaaga ttatgaataa aaagttgtag tttgtcgttt aaaaaaaaaa 1921 aaaaaaaaaa a//

>gi|30256|emb|CAA42556.1| c-src-kinase [Homo sapiens] MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREG VKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSI DEEVYFENLMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVM LGDYRGNKVAVKCIKNDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRS RGRSVLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGKLPV KWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIKTHELHL

MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREG VKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSI DEEVYFENLMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVM LGDYRGNKVAVKCIKNDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRS RGRSVLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGKLPV KWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIKTHELHL

FASTA Format

>gi|30256|emb|CAA42556.1| c-src-kinase [Homo sapiens]

>gi number

Database Identifiers:gb GenBankemb EMBLdbj DDBJref RefSeqsp SWISS-PROTpdb Protein Databankpir PIRprf PRFtpg TPA-GenBanktpe TPA-EMBLtpj TPA-DDBJ

Accession.Version Locus Name Organism

Seq-entry ::= set { class nuc-prot , descr { title "Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA, complete cds." , source { org { taxname "Malus x domestica" , common "cultivated apple" , db { { db "taxon" , tag id 3750 } } , orgname { name binomial { genus "Malus" , species "x domestica" } , mod { { subtype cultivar , subname "'Law Rome'" } , { subtype old-name , subname "Malus domestica" , attrib "(10)cultivar='Law Rome'" } } , lineage "Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots; rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus" , gcode 1 ,,

Abstract Syntax Notation: ASN.1

FASTA NucleotideFASTA Nucleotide

FASTAProteinFASTAProtein

GenPeptGenPept GenBankGenBank

ASN.1ASN.1

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

•Batch Submission and htg (email and ftp)•Inaccurate•Poorly Characterized

EST Division: Expressed Sequence Tags

RNA gene products

nucleus30,000 genes

80-100,000 uniquecDNA clones in library

- isolate unique clones -sequence once from each end

make cDNA library

5’

3’

>IMAGE:275615 3', mRNA sequenceNNTCAAGTTTTATGATTTATTTAACTTGTGGAACAAAAATAAACCAGATTAACCACAACCATGCCTTATTATCAAATGTATAAGANGTAAATATGAATCTTATATGACAAAATGTTTCATTCATTATAACAAATTTAATAATCCTGTCAATNATATTTCTAAATTTTCCCCCAAATTCTAAGCAGAGTATGTAAATTGGAAGTTCTTATGCACGCTTAACTATCTTAACAAGCTTTGAGTGCAAGAGATTGANGAGTTCAAATCTGACCAAGGTTGATGTTGGATAAGAGAATTCTCTGCTCCCCACCTCTANGTTGCCAGCCCTC

>IMAGE:275615 3', mRNA sequenceNNTCAAGTTTTATGATTTATTTAACTTGTGGAACAAAAATAAACCAGATTAACCACAACCATGCCTTATTATCAAATGTATAAGANGTAAATATGAATCTTATATGACAAAATGTTTCATTCATTATAACAAATTTAATAATCCTGTCAATNATATTTCTAAATTTTCCCCCAAATTCTAAGCAGAGTATGTAAATTGGAAGTTCTTATGCACGCTTAACTATCTTAACAAGCTTTGAGTGCAAGAGATTGANGAGTTCAAATCTGACCAAGGTTGATGTTGGATAAGAGAATTCTCTGCTCCCCACCTCTANGTTGCCAGCCCTC

>IMAGE:275615 5' mRNA sequenceGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGTGGAGGTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAATTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTTGAACCATGTNGACTTTGTCACAGNCAAGTTNAGTTTAAGTGGGNATCGAGACATGTAAGGCAGGCATCATGGGAGGTTTTGAAGNATGCCGCNTTGGATTGGGATGAATTCCAAATTTCTGGTTTGCTTGNTTTTTTAATATTGGATATGCTTTTG

>IMAGE:275615 5' mRNA sequenceGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGTGGAGGTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAATTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTTGAACCATGTNGACTTTGTCACAGNCAAGTTNAGTTTAAGTGGGNATCGAGACATGTAAGGCAGGCATCATGGGAGGTTTTGAAGNATGCCGCNTTGGATTGGGATGAATTCCAAATTTCTGGTTTGCTTGNTTTTTTAATATTGGATATGCTTTTG

gbdiv_est[Properties]

ESTs in EntrezTotal 26 million recordsHuman 6.0 millionMouse 4.3 millionRat 0.7 millionZebrafish 0.6 millionWheat 0.6 millionBarley 0.3 millionMaize 0.4 million

Total 26 million recordsHuman 6.0 millionMouse 4.3 millionRat 0.7 millionZebrafish 0.6 millionWheat 0.6 millionBarley 0.3 millionMaize 0.4 million

Genome Sequencing - HTG, GSS, (WGS)

Draft Sequence (HTG division)

shredding

Whole BAC insert (or genome)

cloning isolating

assembly

sequencing

GSS divisionor trace archive

whole genome shotgun assemblies (traditional division)

HTG Division: Rice Draft Sequences

•Unfinished sequences of BACs•Gaps and unordered pieces•Finished sequences move to traditional GenBank division

Whole Genome Shotgun Projects• Traditional GenBank Divisions• 200 + projects

– Virus

– Bacteria

– Environmental sequences

– Archaea

– 51 Eukaryotes featuring:• Cow, Chicken, Rat, Mouse, Dog, Chimpanzee, Human

• Pufferfish (2)

• Honeybee, Anopheles, Fruit Flies (3), Silkworm

• Nematode (C. briggsae)

• Yeasts (8), Aspergillus (2)

• Rice

Zebrafish: WGS

wgs_master[Properties]wgs_master[Properties]

Derivative DatabasesUniGeneRefSeq

TPA

Primary vs. DerivativeSequence Databases

GenBankGenBank

SequencingSequencingCentersCenters

GA

GAGA

ATTAT

TC

CGAGA

ATTAT

TC

C

AT

GAGA

ATTC

C GAGA

ATTC

C

TTGACAAT

TGACTA

ACGTGC

TTGACA

CGTGAATTGACTA

TATAGCCG

ACGTGC

ACGTGCACGTGC

TTGACA

TTGACA

CGTGA

CGTGA

CGTGA

ATTGACTA

ATTGACTAATTGACTA

ATTGACTA

TATAGCCG

TATAGCCGTATAGCCGTATAGCCG

TATAGCCG TATAGCCGTATAGCCG TATAGCCGCAT

T

GAGA

ATTC

C GAGA

ATTC

C LabsLabs

AlgorithmsAlgorithms

UniGene

CuratorsCurators

RefSeq

GenomeAssembly

TATAGCCGAGCTCCGATACCGATGACAA

Updated continuall

y by NCBI

Updated ONLY by submitters

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 mRNAAlbumin mRNA

5’ EST hits5’ EST hits

3’ EST hits3’ EST hits

UniGene: Expressed Sequences

Expression Data

RELEASE 11 (May 13, 2005)AVAILABLE ON THE FTP SITE!

• Forming the “best representative” sequence

• Standardizing nomenclature and record structure

• Adding annotation (references, sequence features)

• Stable reference for example, gene identification, polymorphism discovery, comparative analysis

• RefSeq Release 11 includes over 1,425,971 proteins and 2928 organisms.

• The release is available by FTP at: ftp://ftp.ncbi.nih.gov/refseq/release/

• RefSeq number is still not fixed.

srcdb_refseq[Properties]

ftp://ftp.ncbi.nih.gov/refseq/release/



Curated RefSeq Records

COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff. The reference sequence was derived from X66503.1. Summary: Adenylosuccinate synthetase catalyzes the first committed step in the conversion of IMP to AMP.

LOCUS ADSS 1368 bp mRNA linear PRI 27-AUG-2002 DEFINITION Homo sapiens adenylosuccinate synthase (ADSS), mRNA. ACCESSION NM_001126 VERSION NM_001126.1 GI:4557270 RefSeq NucleotideRefSeq Nucleotide

LOCUS ADSS 455 aa linear PRI 27-AUG-2002 DEFINITION adenylosuccinate synthase; Adenylosuccinate synthetase (Ade(-)H-complementing) Homo sapiens . ACCESSION NP_001117 VERSION NP_001117.1 GI:4557271 DBSOURCE REFSEQ: accession NM_001126.1 RefSeq ProteinRefSeq Protein

X records:X records: Genome Annotation & Inferred or PredictedGenome Annotation & Inferred or Predicted vs vs N records:N records: Provisional, Reviewed or ValidatedProvisional, Reviewed or Validated

RefSeq Accession Numbers

mRNAs and Proteins

NM_123456 Curated mRNANP_123456 Curated ProteinNR_123456 Curated non-coding RNAXM_123456 Predicted mRNAXP_123456 Predicted Protein XR_123456 Predicted non-coding RNAGene RecordsNG_123456 Reference Genomic SequenceChromosomeNC_123455 Microbial replicons, organelle genomes, human chromosomesAssembliesNT_123456 Contig NW_123456 WGS Supercontig

Curated genomic DNACurated genomic DNA(NC, NT, NW)(NC, NT, NW)

Curated Model mRNACurated Model mRNA (XM)(XM)(XR)(XR)

Curated mRNACurated mRNA (NM)(NM)(NR)(NR)

Model protein Model protein (XP)(XP)

RefSeq Curation Processes

ProteinProtein (NP)(NP)

Scanning....

http://www.ncbi.nlm.nih.gov/RefSeq/key.html#accessions

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


srcdb_refseq[Properties]

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

Third Party Annotation (TPA) Database

• Annotations of existing GenBank sequences

• Allows for community annotation of genomes

• Direct submissions– BankIt – Sequin

tpa[Properties]

TPA record: WGS Assembly

CDS FeatureTPA protein

Human Nucleotide Sequences

ISDC 8,965,327(GenBank/EMBL/DDBJ)

PRI 916,017(WGS 601,855)EST 6,003,916GSS 905,645HTG 18,364HTC 49,373STS 117,870

PAT 953,269

RefSeq 35,934TPA 893Total 9,002,154

ISDC 8,965,327(GenBank/EMBL/DDBJ)

PRI 916,017(WGS 601,855)EST 6,003,916GSS 905,645HTG 18,364HTC 49,373STS 117,870

PAT 953,269

RefSeq 35,934TPA 893Total 9,002,154

Other NCBI Databases

•dbSNP: nucleotide polymorphism

•Geo: Gene Expression Omnibusmicroarray and other

expression data

•Gene: gene recordsUnifies LocusLink and

Microbial Genomes

•Structure: imported structures (PDB)Cn3D viewer, NCBI

curation

•CDD: conserved domain databaseProtein families

(COGs)

Single domains (PFAM, SMART, CD)

NCBI’s SNP Database

• Primary Database and Derivative (RefSNP)

• Single Nucleotide Polymorphism

• Repeat polymorphisms

• Insertion-Deletion Polymorphisms

• 24 Species

• Over 15 million submissions

Submitted SNP

Hemachromatosis SNP

•Non-redundant•Computational Analysis

•BLAST hits to•genome, mRNA, protein and structure

RefSNP

Sequence Similarity Searching

Basic Local Alignment Search Tool

(BLAST)

BLAST

VAST

Pubmed

Text

Sequence

Structure

• Best score for aligning part of sequences

• Dynamic programming • Algorithm:

Smith-Waterman• Table cells never score

below zero

• Best score for aligning the full length sequences

• Dynamic programming• Algorithm:

Needelman- Wunch• Table cells are allowed

any score

Global Local

Pairwise Alignment Summary

Global vs Local Alignment

Seq 1

Seq 2

Seq 1

Seq 2

Global alignment

Local alignment

Global Alignment

Human: 15 IAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFH 84 +A + + + DL F K D+L I+ T+ W+ GR G IP+NYV + + +++ PW+ Worm: 63 VALFQYDARTDDDLSFKKDDILEILNDTQGDWWFARHKATGRTGYIPSNYVAREKSIES------QPWYF 125

Human: 85 GKITREQAERLLYPP--ETGLFLVRESTNYPGDYTLCVSCDGKVEHYRI-MYHASKLSIDEEVYFENLMQ 151 GK+ R AE+ L E G FLVR+S + D +L V + V+HYRI + H I F L Worm: 126 GKMRRIDAEKCLLHTLNEHGAFLVRDSESRQHDLSLSVRENDSVKHYRIQLDHGGYF-IARRRPFATLHD 194

Human: 152 LVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGN-KVA 220 L+ HY +ADGLC L P Y W ++ + ++L++ IG G+FG+V G + N VAWorm: 195 LIAHYQREADGLCVNLGAPCAKSEAPQTTTFTYDDQWEVDRRSVRLIRQIGAGQFGEVWEGRWNVNVPVA 264

Human: 221 VKCIK-NDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGD 289 VK +K A FLAEA +M +LRH L+ L V ++ + IVTE M + +L+ +L+ RGR Worm: 265 VKKLKAGTADPTDFLAEAQIMKKLRHPKLLSLYAVCTRDE-PILIVTELMQE-NLLTFLQRRGRQCQMPQ 332 Human: 290 CLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLT----KEASSTQDTG-KLPVKWTA 353 L++ S V M YLE NF+HRDLAARN+L++ K++DFGL KE TG + P+KWTA Worm: 333 -LVEISAQVAAGMAYLEEMNFIHRDLAARNILINNSLSVKIADFGLARILMKENEYEARTGARFPIKWTA 401

Human: 354 PEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWH 423 PEA +F+TKSDVWSFGILL EI +FGR+PYP + +V+ +V+ GY+M P GCP +Y++M+ CW Worm: 402 PEAANYNRFTTKSDVWSFGILLTEIVTFGRLPYPGMTNAEVLQQVDAGYRMPCPAGCPVTLYDIMQQCWR 471

Human: 424 LDAAMRPSFLQLREQLEHI 443 D RP+F L+ +LE +Worm: 472 SDPDKRPTFETLQWKLEDL 492

human M--------------SAIQ----------------------AAWPSGT------------ECIAKYNFHG M S .. AA SG. . .A ... .worm MGSCIGKEDPPPGATSPVHTSSTLGRESLPSHPRIPSIGPIAASSSGNTIDKNQNISQSANFVALFQYDA 1 20 40 60

440 450human REQLEHI--------KTHELHL . .:: . : ...worm QWKLEDLFNLDSSEYKEASINF 500

Align program (Lipman and Pearson)

Basic Local Alignment Search Tool

• Widely used similarity search tool

• Heuristic approach based on Smith Waterman algorithm

• Finds best local alignments

• Provides statistical significance

• All combinations (DNA/Protein) query and database.– DNA vs DNA

– DNA translation vs Protein

– Protein vs Protein

– Protein vs DNA translation

– DNA translation vs DNA translation

• www, standalone, and network clients

What BLAST tells you• BLAST reports surprising alignments

– Different than chance

• Assumptions– Random sequences– Constant composition

• Conclusions– Surprising similarities imply evolutionary

homology

Evolutionary Homology: descent from a common ancestorDoes not always imply similar function

BLAST/FASTA variants for different searches

Program Query Database Comparison Searching purpose

blastn/fasta

blastp/fasta

blastx/fastx

tblastn/tfasta

tblastx/tfastx

DNA

DNA

DNA

DNA

DNA

DNA

Protein Protein

Protein

Protein

DNA level

Protein level

Protein level

Protein level

Protein level

homologous DNA

homologous protein

New genes from DNA

New genes from peptide

New genes from DNA

BLAST Web site: http://www.ncbi.nlm.nih.gov/BLASTFASTA Web sites: http://www2.ebi.ac.uk/fasta3/

or http://www.fasta.genome.ad.jp/

BLASTN Databases

nrGenBank, EMBL, DDBJ, PDB and NCBI reference sequences (RefSeq)

htgs High-throughput genomic sequences (draft)

pat Patented nucleotide sequences

mito Mitochondrial sequences

vector Vector subset of GenBank

month GenBank, EMBL, DDBJ, PDB from 30 days

chrom Contigs and chromosomes from RefSeq

BLASTP Databases

nrGenBank CDS translations, RefSeq, PDB, SWISS-PROT, PIR, PRF

swissprot SWISS-PROT

pat Patented protein sequences

pdb Protein Data Bank

monthGenBank CDS translations, PDB, SWISS-PROT, PIR, PRF from 30 days

GTACTGGACATGGACCCTACAGGAACGT

TGGACATGGACCCTACAGGAACGTATAC

CATGGACCCTACAGGAACGTATACGTAA . . .

Nucleotide Words

GTACTGGACAT

TACTGGACATG

ACTGGACATGG

CTGGACATGGA

TGGACATGGAC

GGACATGGACC

GACATGGACCC

ACATGGACCCT . . .

Make a lookuptable of words

GTACTGGACATGGACCCTACAGGAACGTATACGTAAG Query

11-mer

1228megablast

711blastn

Min.Def.WORD SIZE

Protein WordsGTQITVEDLFYNIATRRKALKNQuery:

Neighborhood Words

LTV, MTV, ISV, LSV, etc.

GTQ

TQI

QIT

ITV

TVE

VED

EDL

DLF

...

Make a lookuptable of words

Word size = 3 (default) Word size can only be 2 or 3

Minimum Requirements for a Hit

•Nucleotide BLAST requires one exact match•Protein BLAST requires two neighboring matches within 40 aa

GTQITVEDLFYNI

SEI YYN

ATCGCCATGCTTAATTGGGCTT

CATGCTTAATT

neighborhood words

exact word match

one match

two matches

Query sequenceWords of length W

(1)

(2) Compare the word list to the database and identify exact matches

BLAST Algorithm

W default = 11

(3) For each word match, extend alignment in both directions

(4) Compute E-value

An alignment that BLAST can’t find

1 GAATATATGAAGACCAAGATTGCAGTCCTGCTGGCCTGAACCACGCTATTCTTGCTGTTG || | || || || | || || || || | ||| |||||| | | || | ||| |

1 GAGTGTACGATGAGCCCGAGTGTAGCAGTGAAGATCTGGACCACGGTGTACTCGTTGTCG

61 GTTACGGAACCGAGAATGGTAAAGACTACTGGATCATTAAGAACTCCTGGGGAGCCAGTT

| || || || ||| || | |||||| || | |||||| ||||| | |

61 GCTATGGTGTTAAGGGTGGGAAGAAGTACTGGCTCGTCAAGAACAGCTGGGCTGAATCCT

121 GGGGTGAACAAGGTTATTTCAGGCTTGCTCGTGGTAAAAAC

|||| || ||||| || || | | |||| || |||

121 GGGGAGACCAAGGCTACATCCTTATGTCCCGTGACAACAAC

Here there are no words longer than 6…...for nucleotides

there must be an exact match of at least 7.

An Alignment BLAST Can MakeSolution: compare protein sequences; BLASTX

Score = 290 bits (741), Expect = 7e-77Identities = 147/331 (44%), Positives = 206/331 (61%), Gaps = 8/331 (2%)Frame = +3

Score = 290 bits (741), Expect = 7e-77Identities = 147/331 (44%), Positives = 206/331 (61%), Gaps = 8/331 (2%)Frame = +3

BLAST 2 Sequences (blastx) output:

Nucleotide vs. Protein BLAST

aaccgggtgacggtggtgctcggtgcgcagtggggcgacgaaggcH.sapiens: N R V T V V L G A Q W G D E G + + V + V L G Q W G D E GA.thaliana: S Q V S G V L G C Q W G D E G agtcaagtatctggtgtactcggttgccaatggggagatgaaggt

Comparing ADSS from H. sapiens and A. thaliana

BLASTp finds three matching wordsBLASTn finds no match, because there are no 7 bp words

Protein searches are generallymore sensitive than nucleotide searches.

The Flavors of BLAST

• Standard BLAST– traditional “contiguous” word hit– position independent scoring – nucleotide, protein and translations (blastn, blastp,

blastx, tblastn, tblastx)• Megablast

– optimized for large batch searches– can use discontiguous words

• PSI-BLAST– constructs PSSMs automatically; uses as query– very sensitive protein search

• RPS BLAST– searches a database of PSSMs– tool for conserved domain searches

Megablast: NCBI’s Genome Annotator

• Long alignments for similar DNA sequences

• Concatenation of query sequences

• Faster than blastn

• Contiguous Megablast– exact word match– Word size 28

• Discontiguous Megablast– initial word hit with mismatches– cross-species comparison

MegaBLAST

AI217550AI251192AI254381BE645079

C:\seq\hs.4.fsa

> 1133045 gnl|UG|Hs#S1133045 qd43b11.x1 Homo sapiens cDNA, 3' end CATGTAAGCCATTTATTGGTTTGTTTTAAAAATATGTATTTTATTTATACATGAAGTTTGGTGAGAAGTGCTCGATTAGTTCAGACAACATCTGGCACTTGATGTCTGTCCTTCCCTCCTTTTTCCTACTCTCTTCTCCCCTCCTGCTGGTCATTGTGCAGTTCTGGAAATTAAAAAGGTGACAGCCAGGCTAAAAGCTAAGGGTTGGGTCTAGCTCACCTCCCACCCCCAACCACACCGTCTGCAGCCAGCCCCAGGCACCTGTCTCAAAGCTCCCGGGCTGTCCACACACACAAAAACCACAGTCTCCTTCCGGCCAGCTGGGCTGGCAGCCCGACCTGC> 1141828 gnl|UG|Hs#S1141828 qv37f11.x1 Homo sapiens cDNA, 3' end GAGAAGACGACAGAAGGGGAGAAGAGAGTAGGAAAAAGGAGGGAAGGACAGACATCAAGTGCCAGATGTTGTCTGAACTAATCGAGCACTTCTCACCAAACTTCATGTATAAATAAAATACATATTTTTAAAACAAACCAATAAATGGCTTACATCAAAAAAAAAAAAAAAAAAAAAAAAGTCGTATCGATGT> 1145899 gnl|UG|Hs#S1145899 qv33c06.x1 Homo sapiens cDNA, 3' endGAGAAGACGACAGAAGGGGAGAAGAGAGTAGGAAAAAGGAGGGAAGGACAGACATCAAGTGCCAGATGTTGTCTGAACTAATCGAGCACTTCTCACCAAACTTCATGTATAAATAAAATACATATTTTTAAAACAAACCAATAAATGGCTTACATCAAAAAAAAAAAAAAAAAAAAAAAAGTCGTATCGATGT> 2291670 gnl|UG|Hs#S2291670 7e65f04.x1 Homo sapiens cDNA, 3' end TTTCATGTAAGCCATTTATTGGTTTGTTTTAAAAATATGTATTTTATTTATACATGAAGTTTGGTGAGAAGTGCTCGATTAGTTCAAACAACATCTGGCACTTGATGTCTGTCCTTCCCTCCTTTTTCCTACTCTCTTCTCCCCTCCTGCTGGTCATTGTGCAGTTCTGGAAATTAAAAAGGTGACAGCCAGGCTAAAAGCTAAGGGTTGGGTCTAGCTCACCTCCCACCCCCAACCACACCGTCTGCAGCCAGCCCCAGGCACCTGTCTCAAAGCTCCCGGGCTGTCCACACACACAAAAACCACAGTCTCCTTCCGGCCAGCTGGGCTGGCAGCCCGACCTGCCTCCCAACCGCATTCCTGCCTGTGTAGCAGGCGGTGAGCACCCAGAAGGGGCACATACCTCTCCAAGCCTTGAAAGCAAAGCATGGAGATCTACAAAAATAGGATTTCCACTTGGAGAAATGTCGCTGGGACAGT

Templates for Discontiguous Words

W = 11, t = 16, coding: 1101101101101101W = 11, t = 16, non-coding: 1110010110110111W = 12, t = 16, coding: 1111101101101101W = 12, t = 16, non-coding: 1110110110110111W = 11, t = 18, coding: 101101100101101101W = 11, t = 18, non-coding: 111010010110010111W = 12, t = 18, coding: 101101101101101101W = 12, t = 18, non-coding: 111010110010110111W = 11, t = 21, coding: 100101100101100101101W = 11, t = 21, non-coding: 111010010100010010111W = 12, t = 21, coding: 100101101101100101101W = 12, t = 21, non-coding: 111010010110010010111

Reference: Ma, B, Tromp, J, Li, M. PatternHunter: faster and more sensitive homology search. Bioinformatics March, 2002; 18(3):440-5

W = word size; # matches in template

t = template length (window size within which the word match is evaluated)

Scoring Systems - Nucleotides

A G C T

A +1 –3 –3 -3

G –3 +1 –3 -3

C –3 –3 +1 -3

T –3 –3 –3 +1

Identity matrix

CAGGTAGCAAGCTTGCATGTCA

|| |||||||||||| ||||| raw score = 19-9 = 10

CACGTAGCAAGCTTG-GTGTCA

Scoring Systems - Proteins

Position Independent MatricesPAM 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- and RPS-BLAST

A 4R -1 5 N -2 0 6D -2 -2 1 6C 0 -3 -3 -3 9Q -1 1 0 0 -3 5E -1 0 0 2 -4 2 5G 0 -2 0 -1 -3 -2 -2 6H -2 0 1 -1 -3 0 0 -2 8I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4X 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

Common amino acids have low weights

Rare amino acids have high weights

Negative for less likely substitutions

Positive for more likely substitutions

Gapped Alignments

• Gapping provides more biologically realistic alignments

• Statistical behavior is not completely understood forgapped alignments

• Gapped BLAST parameters must be found by simulations for each matrix

Gap costs: -(a+bk)

a = gap open penalty b = gap extend penalty k= number of residues

For example: A gap of 1 residue receives the score “-(a+b)”.

Scores

V D S – C Y

V E T L C F

BLOSUM62 +4 +2 +1 -12 +9 +3 = 7

PAM30 +7 +2 0 -10 +10 +2 =. 11

Simply add the scoresfor each pair of aligned residues

and (as necessary) factor in the gaps!

Different matrices produce different scores!

Lower BLOSUM series means more divergence

Higher PAM series means more divergence

better for finding local alignments

better for finding global alignments and remote homologs

based on groups of related sequences counted as one

based on minimum replacement or maximum parsimony

Built from vast amout of dataBuilt from small amout of data

Built from local alignmentsBuilt from global alignments

BLOSUMBLOSUMPAMPAM

Matrix differencesMatrix differences

Matrices - Rules of thumb

Need different levels of sensitivity ?– Close relationships (Low PAM number (PAM 1) or

high Blosum number, eg. 80)– Distant relationships (High PAM (e.g. PAM 250),

low Blosum (BLOSUM 45)

Local Alignment StatisticsHigh scores of local alignments between two random sequencesfollow the Extreme Value Distribution

Score

Alig

nm

en

ts

(applies to ungapped alignments)

E = Kmne-S E = mn2-S’

K = scale for search space = scale for scoring system S’ = bitscore = (S - lnK)/ln2

Expect ValueE = number of database hits you expect to find by

chancesize of database

your score

expected number of

random hits

WWW BLAST

The BLAST homepage

Standard databases

Specialized Databases

BLAST Databases: Nucleic Acid

• nr (nt)– Traditional GenBank– NM_ and XM_ RefSeqs

• refseq_rna

• refseq_genomic– NC_ RefSeqs

• dbest – EST Division

• est_human, mouse, others

• htgs – HTG division

• gss – GSS division

• wgs– whole genome shotgun

• env_nt– environmental samples

Options for Advanced Blasting: Nucleotide

Example Entrez Queriesnucleotide all[Filter] NOT mammalia[Organism]green plants[Organism]biomol mrna[Properties]biomol genomic[Properties]

OtherAdvanced-W 7 word size–e 10000 expect value-v 2000 descriptions-b 2000 alignments

BLAST Databases: Non-redundant protein

nr (non-redundant protein sequences)– GenBank CDS translations– NP_ RefSeqs– Outside Protein

• PIR, Swiss-Prot, PRF• PDB (sequences from structures)

pat protein patents

env_nr environmental samples

nr (non-redundant protein sequences)– GenBank CDS translations– NP_ RefSeqs– Outside Protein

• PIR, Swiss-Prot, PRF• PDB (sequences from structures)

pat protein patents

env_nr environmental samples

Advanced Options: Filter

all[Filter] NOT mammals[Organism]

gene_in_mitochondrion[Properties]2003:2005 [Modification Date]tpa[Filter]

Nucleotidebiomol_mrna[Properties]biomol_genomic[Properties]

all[Filter] NOT mammals[Organism]

gene_in_mitochondrion[Properties]2003:2005 [Modification Date]tpa[Filter]

Nucleotidebiomol_mrna[Properties]biomol_genomic[Properties]

Default settingDefault setting

Hides low complexity for initial word hits onlyHides low complexity

for initial word hits only

Masks regions of query in lower case (pre-masked)Masks regions of query in lower case (pre-masked)

BLAST Formatting Page

BLAST Output: Graphic

mouse over

Sort by taxonomy

BLAST Output: Descriptions

link to entrez

Sorted by e values

3 X 10-12

Default e value cutoff 10

Gene Linkout

TaxBLAST: Taxonomy Reports

>gi|127552|sp|P23367|MUTL_ECOLI DNA mismatch repair protein mutL Length = 615

Score = 42.0 bits (97), Expect = 3e-04 Identities = 26/59 (44%), Positives = 33/59 (55%), Gaps = 9/59 (15%)

Query 9 LPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHF-----LHE---ESILEV-QQHIESKL 58 L + P L LEI P VDVNVHP KHEV F +H+ + +L V QQ +E+ LSbjct 280 LGADQQPAFVLYLEIDPHQVDVNVHPAKHEVRFHQSRLVHDFIYQGVLSVLQQQLETPL 338

>gi|127552|sp|P23367|MUTL_ECOLI DNA mismatch repair protein mutL Length = 615

Score = 42.0 bits (97), Expect = 3e-04 Identities = 26/59 (44%), Positives = 33/59 (55%), Gaps = 9/59 (15%)

Query 9 LPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHF-----LHE---ESILEV-QQHIESKL 58 L + P L LEI P VDVNVHP KHEV F +H+ + +L V QQ +E+ LSbjct 280 LGADQQPAFVLYLEIDPHQVDVNVHPAKHEVRFHQSRLVHDFIYQGVLSVLQQQLETPL 338

BLAST Output: Alignments

Identical match

positive score(conservative)

negative substitution

gap

BLAST Output: Alignments

>gi|730028|sp|P40692|MLH1_HUMAN DNA mismatch repair protein Mlh1 1) Length = 756

Score = 233 bits (593), Expect = 8e-62 Identities = 117/131 (89%), Positives = 117/131 (89%)

Query: 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL 60 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLSbjct: 276 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL 335

Query: 61 GSNSSRMYFTQTLLPGLAGPSGEMVKXXXXXXXXXXXXXXDKVYAHQMVRTDSREQKLDA 120 GSNSSRMYFTQTLLPGLAGPSGEMVK DKVYAHQMVRTDSREQKLDASbjct: 336 GSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDA 395

Query: 121 FLQPLSKPLSS 131 FLQPLSKPLSSSbjct: 396 FLQPLSKPLSS 406

>gi|730028|sp|P40692|MLH1_HUMAN DNA mismatch repair protein Mlh1 1) Length = 756

Score = 233 bits (593), Expect = 8e-62 Identities = 117/131 (89%), Positives = 117/131 (89%)

Query: 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL 60 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLSbjct: 276 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL 335

Query: 61 GSNSSRMYFTQTLLPGLAGPSGEMVKXXXXXXXXXXXXXXDKVYAHQMVRTDSREQKLDA 120 GSNSSRMYFTQTLLPGLAGPSGEMVK DKVYAHQMVRTDSREQKLDASbjct: 336 GSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDA 395

Query: 121 FLQPLSKPLSS 131 FLQPLSKPLSSSbjct: 396 FLQPLSKPLSS 406

low complexity sequence filtered

Neighbors: Precomputed BLAST

Nucleotide

Protein

Entrez Related Sequences produces a list of sequences sorted by BLAST score, but with no alignment details.

Blink – Protein BLAST Alignments

• Lists only 200 hits • List is nonredundant

PSI-BLAST

Position-Specific Iterated BLAST

• Mining for protein domains• Confirming relationships among related proteins

Position-Specific Scoring 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 S -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 is scored differently in these two positions.

Active site nucleophile

Position Specific Iterative BLAST:PSI-BLAST

Create your own PSSM:Finding protein families

based on your own sequence.

query BLOSUM62PSSM AlignmentAlignment

>gi|113340|sp|P03958|ADA_MOUSE ADENOSINE DEAMINASE (ADENOSINEMAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGFVIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDEQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAYRTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAVRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKK

PSI-BLAST

e value cutoff for PSSM

RESULTS: Initial BLASTPSame results as protein-protein BLAST

Results of First PSSM Search

Other purine nucleotide metabolizing enzymes not found by ordinary BLAST

Third PSSM Search: Convergence

Just below threshold, another nucleotide metabolism enzyme

Check to add to PSSM

Reverse Position Specific Iterative-BLAST (a.k.a. RPS-BLAST or CDD Search)

A sequence search of the Conserved Domain Database (CDD)

containing curated Position-Specific Scoring Matrices. 10 20 30 40 50 60 . . . . * . . . . | . . . . * . . . . | . . . . * . . . . | . . . . * . . . . | . . . . * . . . . | . . . . * . . . . |consensus 1 KWEIPREDLTLGKKLGEGAFGEVYKGTLKGkgd---nkSIDVAVKTLKEDASEeqIKEFL 571FGI A 1 aWEIPRESLRLEVKLGQGCFGEVWMGTWNG--------TTRVAIKTLKPGTMS--PEAFL 3111BYG A 1 RWELPRDRLVLgkPLGEGAFGQVYLAEAIglgkdkpnrvTKVAVKMLKSDAtedkLSLDI 74gi 125135 1 GWALNMKELKLlqTIGKGEFGDVMLGDYRg---------NKVAVKCIKNDAt---AQAFL 62gi 125702 1 KYEIPRTDLTLkhKLGGGQYGEVYEGVWKky-------sLTVAVKTLKEDTm--eVEEFL 284gi 1174437 1 KWEIPRSELTIlrKLGRGNFGEVFYGKWRn--------sIDVAVKTLREGTm--sTAAFL 325

PSSM Sources

Pfam Sanger 7255SMART EMBL 663COG NCBI 4873KOG NCBI 4825CD NCBI 645

Reverse Position Specific Iterative-BLAST (a.k.a. RPS-BLAST or CD Search)

Query: sequence Database: PSSMs

P03958

Result: TyrKc

Questions:

• Searching for p53 protein homologs with annotation of CDD.

• Can you put codon 72 SNP into 3D protein structure?

Other Areas to Cover

• Genomic Data

• Annotation

• Common Domains prediction WWW

• Other Useful Genome Browsers

sequence analysis (i) yuh-shan jou ( 周玉山 ) [email protected] institute of biomedical...

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