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NCBI Molecular Biology Resources

January 2008 Peter Cooper

Using NCBI BLAST

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eBasic 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 client

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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

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BLAST and BLAST-like programs• Traditional BLAST (blastall) nucleotide, protein, translations

– blastn nucleotide query vs. nucleotide database

– blastp protein query vs. protein database

– blastx nucleotide query vs. protein database

– tblastn protein query vs. translated nucleotide database

– tblastx translated query vs. translated database

• Megablast nucleotide only

– Contiguous megablast• Nearly identical sequences

– Discontiguous megablast • Cross-species comparison

• Position Specific BLAST Programs protein only

– Position Specific Iterative BLAST (PSI-BLAST)• Automatically generates a position specific score matrix (PSSM)

– Reverse PSI-BLAST (RPS-BLAST)• Searches a database of PSI-BLAST PSSMs

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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 or E = mn2-S’

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

Expect Value

E = number of database hits you expect to find by chance

size of database

your score

expected number of random hits

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Scoring Systems

•Position Independent MatricesPosition Independent Matrices•Nucleic Acids – identity matrix•Proteins

•PAM Matrices (Percent Accepted Mutation)•Implicit model of evolution•Higher PAM number all calculated from PAM1•PAM250 widely used

•BLOSUM Matrices (BLOck SUbstitution Matrices)•Empirically determined from alignment of conserved blocks•Each includes information up to a certain level of identity•BLOSUM62 widely used

•Position Specific Score Matrices (PSSMs)Position Specific Score Matrices (PSSMs)•PSI and RPS BLAST

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WWW BLAST Interface

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eThe BLAST homepage

www.ncbi.nlm.nih.gov/blastwww.ncbi.nlm.nih.gov/blast

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Basic BLAST: Databases

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BLAST Databases: Non-redundant protein

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

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

pat protein patents

env_nr environmental samples

Servicesblastpblastx

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Nucleotide Databases: Human and Mouse

• Human and mouse genomic and transcript now default• Separate sections in output for mRNA and genomic• Direct links to Map Viewer for genomic sequences

Megablast, blastn service

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Nucleotide Databases: Traditional

Servicesblastntblastntblastx

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Nucleotide Databases: Traditional

• 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

Databases are mostly non-overlapping

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Basic BLAST: Protein Searches

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Universal Form: Protein

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3000 Myr3000 Myr

1000 Myr1000 Myr

540 Myr540 Myr

Alzheimer’sDisease

Ataxiatelangiectasia

Colon cancer

Pancreaticcarcinoma

Yeast BacteriaWormFlyHuman

BLAST and Molecular Evolution

MLH1 MutL

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Protein BLAST Page

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Limiting Database: Organism

Organism autocompleteOrganism autocomplete

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Limiting Database: Entrez Query

all[filter] NOT mammals[organism]

gene_in_mitochondrion[Properties]2006:2007 [Modification Date]

Nucleotidebiomol_mrna[Properties]biomol_genomic[Properties]

all[filter] NOT mammals[organism]

gene_in_mitochondrion[Properties]2006:2007 [Modification Date]

Nucleotidebiomol_mrna[Properties]biomol_genomic[Properties]

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Run Search

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BLAST Formatting Page

Conserved Domain ResultsConserved Domain Results

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eBLAST Output: Graphical

Overview

mouse overmouse over

Sort by taxonomySort by taxonomy

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BLAST Output: Descriptions

Link to entrezLink to entrez

Sorted by e valuesSorted by e values

5 X 10-145 X 10-14

Default e value cutoff 10Default e value cutoff 10

Gene LinkoutGene Linkout

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TaxBLAST: Taxonomy Reports

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BLAST Output: Alignments

Identical matchIdentical match

positive score(conservative)positive score(conservative)

Negative or zeroNegative or zero

gapgap

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Position Specific Iterative BLAST

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MLH1 and ETR1>gi|4557757|ref|NP_000240.1| MutL protein homolog 1 [Homo sapiens] MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRK EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPK PCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNA STVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVY AAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLP GLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDIS SGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRKEM TAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELF YQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEI DEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC

>gi|22095656|sp|O81122.1|ETR1_MALDO Ethylene receptorMLACNCIEPQWPADELLMKYQYISDFFIALAYFSIPLELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFSIHSRTVAMVMTTAKVLTAVVSCATALMLVHIIPDLLSVKTRELFLKNKAAELDREMGLIRTQEETGRHVRMLTHEIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLRQQNPVGYTVPIHLPVINQVFSSNRAVKISANSPVAKLRQLAGRHIPGEVVAVRVPLLHLSNFQINDWPELSTKRYALMVLMLPSDSARQWHVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNIALDLARREAETAIRARNDFLAVMNHEMRTPMHAIIALSSLLQETELTAEQRLMVETILRSSNLLATLINDVLDLSRLEDGSLQLEIATFNLHSVFREVHNMIKPVASIKRLSVTLNIAADLPMYAIGDEKRLMQTILNVVGNAVKFSKEGSISITAFVAKSESLRDFRAPDFFPVQSDNHFYLRVQVKDSGSGINPQDIPKLFTKFAQTQALATRNSGGSGLGLAICKRFVNLMEGHIWIESEGLGKGCTATFIVKLGFPERSNESKLPFAPKLQANHVQTNFPGLKVLVMDDNGVSRSVTKGLLAHLGCDVTAVSLIDELLHVISQEHKVVFMDVSMPGIDGYELAVRIHEKFTKRHERPVLVALTGSIDKITKENCMRVGVDGVILKPVSVDKMRSVLSELLEHRVLFEAM

Human Mismatch Repair ProteinHuman Mismatch Repair Protein

Apple ethylene receptorApple ethylene receptor

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PSI-BLAST: Iteration 1

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PSI-BLAST:Iteration 4

Plant ethylene receptors, bacterial two-component regulatory system kinasesPlant ethylene receptors, bacterial two-component regulatory system kinases

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RPS-BLAST: Conserved Domains

Histidine Kinase-like ATPase DomainHistidine Kinase-like ATPase Domain

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eAlgorithm parameters: Protein

Adjust to set stringencyAdjust to set stringency

May limit resultsMay limit results

Default statistics adjustmentfor compositional bias

Default statistics adjustmentfor compositional bias

Off now by default. Conflicts withcomp-based statsOff now by default. Conflicts withcomp-based stats

ExpandExpand

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Automatic Short Sequence Adjustment

e-value 20000Word Size 2Matrix PAM30Comp Stats OffLow Comp Filter Off

Nucleotide and Protein

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Basic BLAST: Nucleotide

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Universal Form: Nucleotide

SpeedSpeedSensitivitySensitivity

More

Less

Less

More

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Nucleotide Results: ALB mRNA

megablastmegablast

disco. megablastdisco. megablast

blastnblastn

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eNucleotide BLAST: Human

Genome

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Sortable Results

Pseudogene on Chromosome 9Pseudogene on Chromosome 9

Separate Sections for

Transcript and Genome

Separate Sections for

Transcript and Genome

Direct links to Entrez DatabasesDirect links to Entrez Databases

Functional Gene on Chromosome 1Functional Gene on Chromosome 1

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Total Score: All Segments

Functional Gene Now FirstFunctional Gene Now First

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Alignments: Sorting in Exon Order

Default Sorting Order: ScoreLongest exon usually firstDefault Sorting Order: ScoreLongest exon usually first

Query start positionExon orderQuery start positionExon order

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Links to Map Viewer

Chromosome 1 Chromosome 9

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Algorithm parameters: Nucleotideblastnblastn

•Masks species-specific interspersed repeats•Essential for genomic query sequences•Masks species-specific interspersed repeats•Essential for genomic query sequences

•Prevents starting alignment in masked region•Allows extensions through masked regions•Prevents starting alignment in masked region•Allows extensions through masked regions

Masks LC sequence (simple repeats)Masks LC sequence (simple repeats)

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BLAST Formatting Options

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Protein Formatting Page

ShowAlignmentPSSMPssmWithParametersBioseq

ShowAlignmentPSSMPssmWithParametersBioseq

asHTMLPlain TextASN.1XML

asHTMLPlain TextASN.1XML

Alignment ViewPairwisePairwise with dots for identitiesQuery-anchored with dots for identitiesQuery-anchored with letters for identitiesFlat query-anchored with dots for identitiesFlat-query anchored with letters for identitiesHit table

Alignment ViewPairwisePairwise with dots for identitiesQuery-anchored with dots for identitiesQuery-anchored with letters for identitiesFlat query-anchored with dots for identitiesFlat-query anchored with letters for identitiesHit table

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Structured formats: XML and ASN.1

<Iteration_hits>−<Hit><Hit_num>1</Hit_num><Hit_id>gi|730028|sp|P40692|MLH1_HUMAN</Hit_id>−<Hit_def>DNA mismatch repair protein Mlh1 (MutL protein homolog 1)</Hit_def><Hit_accession>P40692</Hit_accession><Hit_len>756</Hit_len>−<Hit_hsps>−<Hsp><Hsp_num>1</Hsp_num><Hsp_bit-score>1568.9</Hsp_bit-score><Hsp_score>4061</Hsp_score><Hsp_evalue>0</Hsp_evalue><Hsp_query-from>1</Hsp_query-from><Hsp_query-to>756</Hsp_query-to><Hsp_hit-from>1</Hsp_hit-from><Hsp_hit-to>756</Hsp_hit-to><Hsp_query-frame>0</Hsp_query-frame><Hsp_hit-frame>0</Hsp_hit-frame><Hsp_identity>0</Hsp_identity><Hsp_positive>0</Hsp_positive><Hsp_gaps>0</Hsp_gaps><Hsp_align-len>756</Hsp_align-len>

Seq-annot ::= { desc { user { type str "Hist Seqalign" , data { { label str "Hist Seqalign" , data bool TRUE } } } , user { type str "Blast Type" , data { { label id 0 , data int 0 } } } , user { type str "BLAST database title" , data { { label str "Non-redundant SwissProt

Seq-annot ::= { desc { user { type str "Hist Seqalign" , data { { label str "Hist Seqalign" , data bool TRUE } } } , user { type str "Blast Type" , data { { label id 0 , data int 0 } } } , user { type str "BLAST database title" , data { { label str "Non-redundant SwissProt

XMLXML

ASN.1ASN.1

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The Hit Table# BLASTP 2.2.17 (Aug-26-2007)# Query: gi|4557757|ref|NP_000240.1| MutL protein homolog 1 [Homo sapiens]# Database: swissprot# Fields: query id, subject ids, % identity, % positives, alignment length, mismatches, gap opens, q. start, q. end, s. start, s. end, evalue, bit score# 80 hits foundref|NP_000240.1||gi|4557757 gi|1709056|sp|P38920|MLH1_YEAST 36.68 56.91 796 426 18 8 756 5 769 7e-138 491ref|NP_000240.1||gi|4557757 gi|48474996|sp|Q9P7W6|MLH1_SCHPO 37.24 54.04 768 371 16 8 756 9 684 8e-122 437ref|NP_000240.1||gi|4557757 gi|25090753|sp|Q8RA70|MUTL_THETN 37.44 54.62 390 231 7 8 394 4 383 5e-59 229ref|NP_000240.1||gi|4557757 gi|25090732|sp|Q8KAX3|MUTL_CHLTE 35.95 54.05 370 229 5 8 375 4 367 5e-55 215ref|NP_000240.1||gi|4557757 gi|127552|sp|P23367.2|MUTL_ECOLI 35.99 58.11 339 202 7 8 334 3 338 8e-55 214ref|NP_000240.1||gi|4557757 gi|29427778|sp|Q8FAK9|MUTL_ECOL6 35.99 58.11 339 202 7 8 334 3 338 1e-54 214ref|NP_000240.1||gi|4557757 gi|20455084|sp|Q8XDN4|MUTL_ECO57 35.99 58.11 339 202 7 8 334 3 338 1e-54 214ref|NP_000240.1||gi|4557757 gi|59798328|sp|Q72PF7|MUTL_LEPIC 36.27 55.20 375 221 8 6 375 2 363 3e-54 213ref|NP_000240.1||gi|4557757 gi|13431695|sp|P57886|MUTL_PASMU 35.48 58.94 341 213 6 8 345 3 339 4e-54 212ref|NP_000240.1||gi|4557757 gi|1171080|sp|P44494|MUTL_HAEIN 35.74 59.87 319 198 6 8 323 3 317 5e-54 212ref|NP_000240.1||gi|4557757 gi|20455102|sp|Q8ZIW4|MUTL_YERPE 36.01 58.63 336 207 6 8 339 3 334 6e-54 212ref|NP_000240.1||gi|4557757 gi|20455152|sp|Q9JYT2|MUTL_NEIMB 33.96 55.35 374 224 8 8 376 4 359 2e-53 210ref|NP_000240.1||gi|4557757 gi|20139217|sp|Q9KAC1|MUTL_BACHD 35.39 55.90 356 214 6 8 362 4 344 2e-53 209ref|NP_000240.1||gi|4557757 gi|31076794|sp|Q87L05|MUTL_VIBPA 35.33 58.38 334 210 5 8 338 3 333 3e-53 209ref|NP_000240.1||gi|4557757 gi|20455150|sp|Q9JTS2|MUTL_NEIMA 36.94 58.28 314 183 5 8 316 4 307 5e-53 209ref|NP_000240.1||gi|4557757 gi|56749233|sp|Q6GHD9|MUTL_STAAR 38.28 58.46 337 193 7 6 335 2 330 1e-52 207ref|NP_000240.1||gi|4557757 gi|25090739|sp|Q8NWX9|MUTL_STAAW 38.28 58.46 337 193 7 6 335 2 330 1e-52 207ref|NP_000240.1||gi|4557757 gi|71151979|sp|Q5HGD5|MUTL_STAAC 38.28 58.46 337 193 7 6 335 2 330 1e-52 207ref|NP_000240.1||gi|4557757 gi|54037875|sp|P65492|MUTL_STAAN 38.28 58.46 337 193 7 6 335 2 330 2e-52 207ref|NP_000240.1||gi|4557757 gi|20043258|sp|Q9KV13|MUTL_VIBCH 35.74 58.56 333 204 6 8 335 3 330 2e-52 207ref|NP_000240.1||gi|4557757 gi|127553|sp|P14161|MUTL_SALTY 35.10 56.93 339 205 7 8 334 3 338 3e-52 206ref|NP_000240.1||gi|4557757 gi|20455140|sp|Q9CDL1|MUTL_LACLA 36.31 56.55 336 196 5 6 334 2 326 4e-52 206ref|NP_000240.1||gi|4557757 gi|61214242|sp|Q7MH01|MUTL_VIBVY 34.63 58.51 335 213 5 8 339 3 334 4e-52 206ref|NP_000240.1||gi|4557757 gi|20455099|sp|Q8Z187|MUTL_SALTI 35.10 56.93 339 205 7 8 334 3 338 4e-52 206ref|NP_000240.1||gi|4557757 gi|31076809|sp|Q8DCV0|MUTL_VIBVU 34.63 58.51 335 213 5 8 339 3 334 6e-52 205ref|NP_000240.1||gi|4557757 gi|71648717|sp|Q5E2C6|MUTL_VIBF1 36.71 59.81 316 186 6 8 316 3 311 1e-51 204ref|NP_000240.1||gi|4557757 gi|37999611|sp|Q88DD1|MUTL_PSEPK 30.34 48.97 435 278 7 8 419 7 439 2e-51 203

Importable into spreadsheetsImportable into spreadsheets

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PSSMs: Restart PSI-BLAST

ASCII encoded, Web onlyASCII encoded, Web only

ASN.1 ScoreMat, PortableASN.1 ScoreMat, Portable

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BLAST TreeView

Black bear mt genome vs. RefSeq GenomicBlack bear mt genome vs. RefSeq Genomic

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Distance Tree Carnivore Mitochondrial Genome

bearswalrus fur seal

sea lions

true seals

dogsmongooses

catsred pandaweasels

raccoon

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Managing Searches

Recent Results

Saved Strategies

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Recent Results

Login to My NCBI to save search strategiesLogin to My NCBI to save search strategies

Results available for 36 hoursResults available for 36 hours

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Saved Strategies

Re-run searches to keep up to dateRe-run searches to keep up to date

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Genome and Specialized BLAST

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Genome BLAST pages

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Map Viewer Homepage

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Poplar Genome BLAST

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tblastn Genome BLAST Results

Protein-nucleotide alignmentsProtein-nucleotide alignments

Exons and genes mixedExons and genes mixed

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eGenomic Context of BLAST Hits

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Hits in Map Viewer

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Specialized BLAST Pages

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Service Addresses

•General Help info@ncbi.nlm.nih.gov•BLAST blast-help@ncbi.nlm.nih.gov

Telephone support: 301- 496- 2475