Sequence Based Analysis Sequence Based Analysis TutorialTutorial

March 26, 2004 March 26, 2004 NIH Proteomics Workshop NIH Proteomics Workshop

Lai-Su L. Yeh, Ph.D.Lai-Su L. Yeh, Ph.D.Protein Science Team LeadProtein Science Team LeadProtein Information Resource at Protein Information Resource at Georgetown University Medical CenterGeorgetown University Medical Center

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Retrieval, Sequence Search & Retrieval, Sequence Search & Classification MethodsClassification Methods

Retrieve protein info by text / UIDRetrieve protein info by text / UID Sequence Similarity SearchSequence Similarity Search

BLAST, FASTA, Dynamic ProgrammingBLAST, FASTA, Dynamic Programming Family Classification Family Classification

Patterns, Profiles, Hidden Markov Models, Patterns, Profiles, Hidden Markov Models, Sequence Alignments, Neural NetworksSequence Alignments, Neural Networks

Integrated Search and Classification Integrated Search and Classification SystemSystem

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Sequence Similarity SearchSequence Similarity Search

Based on Based on Pair-Wise ComparisonsPair-Wise Comparisons Dynamic Programming AlgorithmsDynamic Programming Algorithms

Global Similarity: Needleman-WunchGlobal Similarity: Needleman-Wunch Local Similarity: Smith-WatermanLocal Similarity: Smith-Waterman

Heuristic AlgorithmsHeuristic Algorithms FASTA: Based on K-Tuples (2-Amino Acid)FASTA: Based on K-Tuples (2-Amino Acid) BLAST: Triples of Conserved Amino AcidsBLAST: Triples of Conserved Amino Acids Gapped-BLAST: Allow Gaps in Segment PairsGapped-BLAST: Allow Gaps in Segment Pairs PHI-BLAST: Pattern-Hit Initiated SearchPHI-BLAST: Pattern-Hit Initiated Search PSI-BLAST: Position-Specific Iterated SearchPSI-BLAST: Position-Specific Iterated Search

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Sequence Similarity SearchSequence Similarity Search

Similarity Search ParametersSimilarity Search Parameters Scoring Matrices – Based on Conserved Amino Scoring Matrices – Based on Conserved Amino

Acid Substitution Acid Substitution • Dayhoff Mutation Matrix, e.g., PAM250 (~20% Dayhoff Mutation Matrix, e.g., PAM250 (~20%

Identity)Identity)• Henikoff Matrix from Ungapped Alignments, Henikoff Matrix from Ungapped Alignments,

e.g., BLOSUM 62 e.g., BLOSUM 62 Gap PenaltyGap Penalty

Search Time ComparisonsSearch Time Comparisons Smith-Waterman: 10 MinSmith-Waterman: 10 Min FASTA: 2 MinFASTA: 2 Min BLAST: 20 SecBLAST: 20 Sec

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Feature RepresentationFeature Representation

Features:Features: Residue Physicochemical Properties, Context Residue Physicochemical Properties, Context (Local & Global) Features, Evolutionary Features(Local & Global) Features, Evolutionary Features

Alternative Alphabets:Alternative Alphabets: Classification of Amino Acids To Classification of Amino Acids To Capture Different Features of Amino Acid ResiduesCapture Different Features of Amino Acid Residues

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Substitution MatrixSubstitution Matrix Likelihood of One Amino Acid Mutated into Another Over Evolutionary Likelihood of One Amino Acid Mutated into Another Over Evolutionary

TimeTime Negative Score: Unlikely to Happen (e.g., Gly/Trp, -7)Negative Score: Unlikely to Happen (e.g., Gly/Trp, -7) Positive Score: Conservative Substitution (e.g., Lys/Arg, +3)Positive Score: Conservative Substitution (e.g., Lys/Arg, +3) High Score for Identical Matches: Rare Amino Acids (e.g., Trp, Cys)High Score for Identical Matches: Rare Amino Acids (e.g., Trp, Cys)

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BLASTBLAST

BLASTBLAST (Basic Local Alignment Search Tool) (Basic Local Alignment Search Tool) To search a sequence against the databaseTo search a sequence against the database Extremely fastExtremely fast Robust Robust Most widely usedMost widely usedIt finds very short segment pairs between the query It finds very short segment pairs between the query

and sequence in the databaseand sequence in the databaseThese segments are then extended in both directions These segments are then extended in both directions

until the maximum possible score of this particular until the maximum possible score of this particular segment is reached segment is reached

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BLAST SearchBLAST Search From BLAST Search InterfaceFrom BLAST Search Interface Table-Format Result with BLAST Output and SSEARCH Table-Format Result with BLAST Output and SSEARCH

(Smith-Waterman) Pair-Wise Alignment(Smith-Waterman) Pair-Wise Alignment

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BLAST/SSEARCH ResultsBLAST/SSEARCH Results

SSEARCH Alignment

BLAST Alignment

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Family Classification MethodsFamily Classification Methods

Based on Based on Family InformationFamily Information ClustalW Multiple Sequence AlignmentClustalW Multiple Sequence Alignment ProSite Pattern SearchProSite Pattern Search Profile Search Profile Search Hidden Markov Models (HMMs)Hidden Markov Models (HMMs) Neural NetworksNeural Networks Integrated AnalysisIntegrated Analysis

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Multiple Sequence AlignmentMultiple Sequence Alignment

ClustalWClustalW Progressive Pairwise ApproachProgressive Pairwise Approach

Base on Exhaustive Pairwise AlignmentsBase on Exhaustive Pairwise Alignments Neighbor JoiningNeighbor Joining

Joining Order Corresponding to a Tree Joining Order Corresponding to a Tree Alignment VariesAlignment Varies

Dependent on Joining OrderDependent on Joining Order

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How do you build a tree?How do you build a tree?

Pick sequences to alignPick sequences to align Align themAlign them Verify the alignmentVerify the alignment Keep the parts that are aligned correctlyKeep the parts that are aligned correctly Build and evaluate a phylogenetic treeBuild and evaluate a phylogenetic tree

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Multiple Alignment and TreeMultiple Alignment and Tree From Text/Sequence Search Result or ClustalW Alignment InterfaceFrom Text/Sequence Search Result or ClustalW Alignment Interface

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Motif Patterns (Regular Expressions)Motif Patterns (Regular Expressions) Signature Patterns for Functional MotifsSignature Patterns for Functional Motifs

ProClass Motif Alignments

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PIR Pattern SearchPIR Pattern Search From Text/Sequence Search Result or Pattern Search InterfaceFrom Text/Sequence Search Result or Pattern Search Interface One Query Sequence Against PROSITE Pattern DatabaseOne Query Sequence Against PROSITE Pattern Database One Query Pattern (PROSITE or User-Defined) Against Sequence DBOne Query Pattern (PROSITE or User-Defined) Against Sequence DB

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Pattern Search Result (I)Pattern Search Result (I) One Query Sequence Against PROSITE Pattern DatabaseOne Query Sequence Against PROSITE Pattern Database

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Pattern Search Result (II)Pattern Search Result (II) One Query Pattern Against Sequence DatabaseOne Query Pattern Against Sequence Database

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Profile MethodProfile Method

Profile: A Table of Scores to Express Family Consensus Derived from Multiple Profile: A Table of Scores to Express Family Consensus Derived from Multiple Sequence AlignmentsSequence Alignments Num of Rows = Num of Aligned PositionsNum of Rows = Num of Aligned Positions Each row contains a score for the alignment with each possible residue.Each row contains a score for the alignment with each possible residue.

Profile SearchingProfile Searching Summation of Scores for Each Amino Acid Residue along Query SequenceSummation of Scores for Each Amino Acid Residue along Query Sequence Higher Match Values at Conserved PositionsHigher Match Values at Conserved Positions

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PIR HMM Domain/Motif SearchPIR HMM Domain/Motif Search

From Text/Sequence From Text/Sequence Search Result or HMM Search Result or HMM Search InterfaceSearch Interface

HMMER Model Building HMMER Model Building & Sequence Search & Sequence Search

Search One Query Search One Query Protein Against All HMMs Protein Against All HMMs

Search One HMM Search One HMM Against Sequence DBAgainst Sequence DB

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HMM Search Result (I)HMM Search Result (I) One Query Protein Against All Pfam HMMsOne Query Protein Against All Pfam HMMs

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HMM Search Result (II)HMM Search Result (II) Search User-Built HMM Against Protein Sequence DBSearch User-Built HMM Against Protein Sequence DB Input Sequences (Optional Residue Ranges) -> Multiple Input Sequences (Optional Residue Ranges) -> Multiple

Sequence Alignment -> Model Building -> HMM SearchSequence Alignment -> Model Building -> HMM Search

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Secondary Structure FeaturesSecondary Structure Features HelixHelix Patterns of Hydrophobic Residue Conservation Showing I, Patterns of Hydrophobic Residue Conservation Showing I,

I+3, I+4, I+7 Pattern Are Highly Indicative of an I+3, I+4, I+7 Pattern Are Highly Indicative of an Helix (Amphipathic)Helix (Amphipathic) StrandsStrands That Are Half Buried in the Protein Core Will Tend to Have That Are Half Buried in the Protein Core Will Tend to Have

Hydrophobic Residues at Positions I, I+2, I+4, I+6Hydrophobic Residues at Positions I, I+2, I+4, I+6

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Integrated Bioinformatics System for Integrated Bioinformatics System for Function and Pathway DiscoveryFunction and Pathway Discovery

Data IntegrationData Integration Associative AnalysisAssociative Analysis

Sequence Analysis Pipeline

(Family Classification & Feature Identification)

Data Mining Tools

(Retrieval, Visualization, Analysis, Correlation)

Data Warehouse

(Gene, Protein, Family, Function, Structure, Pathway, Interaction)

Graphical User Interface

(Browsing, Querying, Navigation)

Input

(Gene/Protein Expression Data)

Output

(Analysis Results, Biological Interpretation)

Integrated Bioinformatics System

User

Input

(Local Data, Search Criteria, Report Format)

Sequence Analysis Pipeline

(Family Classification & Feature Identification)

Data Mining Tools

(Retrieval, Visualization, Analysis, Correlation)

Data Warehouse

(Gene, Protein, Family, Function, Structure, Pathway, Interaction)

Graphical User Interface

(Browsing, Querying, Navigation)

Input

(Gene/Protein Expression Data)

Output

(Analysis Results, Biological Interpretation)

Integrated Bioinformatics System

User

Input

(Local Data, Search Criteria, Report Format)

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Analytical Analytical PipelinePipeline

Query SequencePIR-NREFiProClass

Top-Matched Superfamilies/Domains

BLAST Search HMM Domain Search

Predicated Superfamilies/Domains/Motifs/Sites/SignalPeptides/TMHs

SSEARCH CLUSTALW

Superfamily/Domain/Motif Alignments

Family Relationships & Functional Features

Family Classification & Functional Analysis

HMM Motif Search Pattern Search SignalP/TMHMM

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Integrated Bioinformatics SystemIntegrated Bioinformatics System

Global Bioinformatics Global Bioinformatics Analysis of 1000’s of Analysis of 1000’s of Genes and ProteinsGenes and Proteins

Pathway Discovery, Pathway Discovery,

Target IdentificationTarget Identification

Gene Expression Data Proteomic Data

Clustering

Expression Pattern

Visualization & Statistical Analysis

Clustered Matrix Pathway Map Process HierarchyClustered Graph

Gene/Peptide-Protein Mapping

Pathway Discovery (Browsing, Sorting, Visualization & Statistical Analysis)

Functional Analysis (Sequence Analysis & Information Retrieval)

Integrated Protein Knowledge System

Comprehensive Protein

Information Matrix

Protein List

Gene Expression Data Proteomic Data

Clustering

Expression Pattern

Visualization & Statistical Analysis

Clustered Matrix Pathway Map Process HierarchyClustered GraphClustered Matrix Pathway Map Process HierarchyClustered Graph

Gene/Peptide-Protein Mapping

Pathway Discovery (Browsing, Sorting, Visualization & Statistical Analysis)

Functional Analysis (Sequence Analysis & Information Retrieval)

Integrated Protein Knowledge System

Comprehensive Protein

Information Matrix

Protein List

Gene/Peptide-Protein Mapping

Pathway Discovery (Browsing, Sorting, Visualization & Statistical Analysis)

Functional Analysis (Sequence Analysis & Information Retrieval)

Integrated Protein Knowledge System

Comprehensive Protein

Information Matrix

Protein List

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Lab SectionLab Section

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Peptide Search & ResultsPeptide Search & Results

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Blast Similarity SearchBlast Similarity Search

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Blast Search ResultsBlast Search Results

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Pair-Wise AlignmentPair-Wise Alignment

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Multiple Sequence AlignmentMultiple Sequence Alignment

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Pattern Search Results Pattern Search Results

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HMM Domain Search ResultHMM Domain Search Result

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Building HMM ProfileBuilding HMM Profile

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Using HMM Profile for Using HMM Profile for SearchingSearching

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Rabbit Alpha Crystallin A Chain Rabbit Alpha Crystallin A Chain An An iiProClass View of the entryProClass View of the entry