NCBI resources II: web-based tools and ftp

resourcesYanbinYin

Mostmaterialsaredownloaded fromftp://ftp.ncbi.nih.gov/pub/education/

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Outline

• Tools– BLAST– SpecializedBLAST– GEO

• ftpdownload• Handsonexercise

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References

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http://homepages.ulb.ac.be/~dgonze/TEACHING/stat_scores.pdf

http://www.bioinformatics.wsu.edu/bioinfo_course/notes/lecture6.pdf

NCBIdiscoveryworkshopsftp://ftp.ncbi.nih.gov/pub/education/discovery_workshops/NLM/2012/Sept2012/

Evolutionofpairwisealignmenttools

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Smith-Watermanalgorithm

FASTA

BLAST

Fasterbut lessaccurate

Needleman-Wunsch algorithm1970

1981

1985

1990

BasicLocalAlignmentSearchTool

• Widelyusedsimilaritysearchtool• HeuristicapproachbasedonSmithWatermanalgorithm• Findsbestlocalalignments• Providesstatisticalsignificance• Allcombinations(DNA/Protein)queryanddatabase

– DNAvs DNA– DNAtranslationvs Protein– Proteinvs Protein– Proteinvs DNAtranslation– DNAtranslationvs DNAtranslation

• www,standalone,andnetworkclient

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6http://www.bioinformatics.wsu.edu/bioinfo_course/notes/lecture6.pdf

7http://homepages.ulb.ac.be/~dgonze/TEACHING/stat_scores.pdf

8http://www.bioinformatics.wsu.edu/bioinfo_course/notes/lecture6.pdf

9http://www.bioinformatics.wsu.edu/bioinfo_course/notes/lecture6.pdf

LocalAlignmentStatisticsHigh scores of local alignments between two random sequencesfollow the Extreme Value Distribution

Score

Alig

nmen

ts

(applies to ungapped alignments)

E = Kmne-lS or E = mn2-S’

K = scale for search spacel= scale for scoring systemS’ = bitscore = (lS - lnK)/ln2

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

sizeofdatabase

yourscore

expectednumberofrandomhits

10http://www.youtube.com/ncbinlm

LocalAlignmentScoring:Protein

KK +5

KE +1

QF -3

Gap-(11 + 4(1))= -14

Number of Chance Alignments = 4 X 10-50

Scores from BLOSUM62, a position independent matrix

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LocalAlignmentScoring:Nucleotide

Gap-(5 + 4(2))= -13

Number of Chance Alignments = 2 X 10-73

Match=+2 Mismatch=-3

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BLASTandBLAST-likeprograms

• TraditionalBLAST(formerlyblastall)nucleotide,protein,translations– blastn nucleotidequeryvs.nucleotidedatabase– blastp proteinqueryvs.proteindatabase– blastx nucleotidequeryvs.proteindatabase– tblastn proteinqueryvs.translatednucleotidedatabase– tblastx translatedqueryvs.translateddatabase

• Megablastnucleotideonly– Contiguousmegablast

• Nearlyidenticalsequences

– Discontiguousmegablast• Cross-speciescomparison

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Position-specificBLASTPrograms(proteinonly)

• PositionSpecificIterativeBLAST(PSI-BLAST)Automaticallygeneratesapositionspecificscorematrix(PSSM)

• Position-HitInitiatedBLAST(PHI-BLAST)Focusessearcharoundpattern(motif)

• DomainEnhancedLookupTimeAccelerated(DELTA)BLASTUsesdomainPSSM infirstroundofsearch

• ReversePSI-BLAST(RPS-BLAST)Searchesadatabaseof PSI-BLASTPSSMsConservedDomainDatabaseSearch

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http://www.ch.embnet.org/CourseAthens/slides/intro_hmm_profile.pdf

Non-redundantprotein

nr (non-redundant proteinsequences)– GenBank CDS

translations– NP_,XP_refseq_protein– OutsideProtein

• PIR,Swiss-Prot,PRF• PDB (sequencesfromstructures)

pat proteinpatentsenv_nrmetagenomes

(environmental samples)

Servicesblastpblastx

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NucleotideDatabases:Traditional

Servicesblastntblastntblastx

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NucleotideDatabases:Traditional

• nr(nt)– TraditionalGenBank– NM_andXM_RefSeqs

• refseq_rna

• NCBIGenomes– NC_RefSeqs– GenBankChromosomes

• dbest– ESTDivision

• non-human,non-mouseests

• htgs– HTGdivision

• gss– GSSdivision

• wgs– wholegenomeshotgun

contigs

• tsa– transcriptomeshotgun

assembly

• 16Smicrobial– Selected16Ssequences

(targetedloci)

Databases are mostly non-overlapping

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SpecializedBLASTPages

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Handsonexercise1

blastn andmegablast

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Searchagainsthumandatabase

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Alotofthingsyoumayexplore

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Changehereto1000

Uploadatextfilewithhumantp53mRNAfasta sequenceDownload fromcoursewebpage

Question:howmanyESTsmatchtp53genes?

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Ittook~1minutetofinish

Alotofthingsyoumayexplore!!!

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Searchagainstotherrefseq genomes

Handsonexercise2

Proteinblast(blastp andtblastn)

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Ifnotselectorganisms…

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Youcanstillspecifyorganisms…

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Uploadatextfilewithtwo arabidopsis protein fastasequenceDownload fromcoursewebpage

Typeinpopulus tochoosepopulus trichocarpa

Youmaysubmitmanysequences,butexpectittakestime

Question:whatarethehomologs inpoplartree?

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Ittook~1minute(smallerdatabase)

Clickheretochoosetoviewwhichqueryprotein

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Howtodeterminewhatisagoode-valuecutofftoselecthomologs?

http://www.youtube.com/watch?v=nO0wJgZRZJs&list=PL8FD4CC12DABD6B39&index=6

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Typeincharoph tochoosecharophytes

Question:whataretheESThomologs incharophytic algae?

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Handsonexercise3

PHI-BLASTQueryprotein+shortmotif/pattern

&PSI-BLAST(iteratedBLAST)

Multi-roundBLASTP

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Example:plantglycosyltransferase family8(GT8)hassignaturemotif

WewanttosearchArabidopsisGAUT1protein(gi #:86611465)andtheHXXGXXKPWmotif

ProSite stylepattern:H-x(2)-G-x(2)-K-P-W

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Handsonexercise4

RPS-BLASTGivenproteinsequences,findconservedfunctionaldomains

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Nextclass:NCBIGEOandftpresource(withalittlebitintroto

Linuxskills)andpractice

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