Sequence Based Analysis Tutorial

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Sequence Based Analysis Tutorial

• NIH Proteomics Workshop
• Lai-Su L. Yeh, Ph.D.
• Protein Science Team Lead
• Protein Information Resource at
• Georgetown University Medical Center

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Retrieval, Sequence Search Classification
Methods

• Retrieve protein info by text / UID
• Sequence Similarity Search
• BLAST, FASTA, Dynamic Programming
• Family Classification
• Patterns, Profiles, Hidden Markov Models,
  Sequence Alignments, Neural Networks
• Integrated Search and Classification System

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Sequence Similarity Search

• Based on Pair-Wise Comparisons
• Dynamic Programming Algorithms
• Global Similarity Needleman-Wunch
• Local Similarity Smith-Waterman
• Heuristic Algorithms
• FASTA Based on K-Tuples (2-Amino Acid)
• BLAST Triples of Conserved Amino Acids
• Gapped-BLAST Allow Gaps in Segment Pairs
• PHI-BLAST Pattern-Hit Initiated Search
• PSI-BLAST Position-Specific Iterated Search

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Sequence Similarity Search

• Similarity Search Parameters
• Scoring Matrices Based on Conserved Amino Acid
  Substitution
• Dayhoff Mutation Matrix, e.g., PAM250 (20
  Identity)
• Henikoff Matrix from Ungapped Alignments, e.g.,
  BLOSUM 62
• Gap Penalty
• Search Time Comparisons
• Smith-Waterman 10 Min
• FASTA 2 Min
• BLAST 20 Sec

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Feature Representation

• Features Residue Physicochemical Properties,
  Context (Local Global) Features, Evolutionary
  Features
• Alternative Alphabets Classification of Amino
  Acids To Capture Different Features of Amino Acid
  Residues

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Substitution Matrix

• Likelihood of One Amino Acid Mutated into Another
  Over Evolutionary Time
• Negative Score Unlikely to Happen (e.g.,
  Gly/Trp, -7)
• Positive Score Conservative Substitution (e.g.,
  Lys/Arg, 3)
• High Score for Identical Matches Rare Amino
  Acids (e.g., Trp, Cys)

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BLAST

• BALST (Basic Local Alignment Search Tool)
• Extremely fast
• Robust
• Most frequently used
• It finds very short segment pairs (seeds)
  between the query and the database sequence
• These seeds are then extended in both directions
  until the maximum possible score for extensions
  of this particular seed is reached

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BLAST Search

• From BLAST Search Interface
• Table-Format Result with BLAST Output and SSEARCH
  (Smith-Waterman) Pair-Wise Alignment

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BLAST/SSEARCH Results
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Family Classification Methods

• Based on Family Information
• ClustalW Multiple Sequence Alignment
• ProSite Pattern Search
• Profile Search
• Hidden Markov Models (HMMs)
• Neural Networks
• Integrated Analysis

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Multiple Sequence Alignment

• ClustalW
• Progressive Pairwise Approach
• Base on Exhaustive Pairwise Alignments
• Neighbor Joining
• Joining Order Corresponding to a Tree
• Alignment Varies
• Dependent on Joining Order

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How do you build a tree?

• Pick sequences to align
• Align them
• Verify the alignment
• Keep the parts that are aligned correctly
• Build and evaluate a phylogenetic tree

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Multiple Alignment and Tree

• From Text/Sequence Search Result or ClustalW
  Alignment Interface

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Motif Patterns (Regular Expressions)

• Signature Patterns for Functional Motifs

ProClass Motif Alignments
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PIR Pattern Search

• From Text/Sequence Search Result or Pattern
  Search Interface
• One Query Sequence Against PROSITE Pattern
  Database
• One Query Pattern (PROSITE or User-Defined)
  Against Sequence DB

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Pattern Search Result (I)

• One Query Sequence Against PROSITE Pattern
  Database

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Pattern Search Result (II)

• One Query Pattern Against Sequence Database

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Profile Method

• Profile A Table of Scores to Express Family
  Consensus Derived from Multiple Sequence
  Alignments
• Num of Rows Num of Aligned Positions
• Each row contains a score for the alignment with
  each possible residue.
• Profile Searching
• Summation of Scores for Each Amino Acid Residue
  along Query Sequence
• Higher Match Values at Conserved Positions

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PIR HMM Domain/Motif Search

• From Text/Sequence Search Result or HMM Search
  Interface
• HMMER Model Building Sequence Search
• Search One Query Protein Against All HMMs
• Search One HMM Against Sequence DB

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HMM Search Result (I)

• One Query Protein Against All Pfam HMMs

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HMM Search Result (II)

• Search User-Built HMM Against Protein Sequence DB
• Input Sequences (Optional Residue Ranges) -gt
  Multiple Sequence Alignment -gt Model Building -gt
  HMM Search

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Secondary Structure Features

• a Helix Patterns of Hydrophobic Residue
  Conservation Showing I, I3, I4, I7 Pattern Are
  Highly Indicative of an a Helix (Amphipathic)
• b Strands That Are Half Buried in the Protein
  Core Will Tend to Have Hydrophobic Residues at
  Positions I, I2, I4, I6

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Integrated Bioinformatics System for Function and
Pathway Discovery

• Data Integration
• Associative Analysis

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Analytical Pipeline
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Integrated Bioinformatics System

• Global Bioinformatics Analysis of 1000s of Genes
  and Proteins
• Pathway Discovery, Target Identification

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Lab Section
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Peptide Search Results
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Blast Similarity Search
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Blast Search Results
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Pair-Wise Alignment
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Multiple Sequence Alignment
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Pattern Search Results
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HMM Domain Search Result
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Building HMM Profile
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Using HMM Profile for Searching
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Rabbit Alpha Crystallin A Chain An iProClass View
of the entry
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alpha-Crystallin and Related Proteins