Sequence Based Analysis Tutorial

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Sequence Based Analysis Tutorial

• NIH Proteomics Workshop
• Lai-Su L. Yeh, Ph.D.
• 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 (I)

• 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 (II)

• 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 of Amino Acids Physicochemical
  Properties, Context (Local Global) Features,
  Evolutionary Features
• Alternative Amino Acids 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

Link to NCBI taxonomy
Link to PIRSF report
Click to see alignment
Links to iProClass and UniProtKB reports
Click to see SSearch alignment
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Blast Result Pairwise Alignment
BLAST Aligment
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Classification

• What is classification?
• Why do we need protein classification?
• Different levels of classification
• Basis for functional protein classification
• How to classify a protein of unknown function?

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Classification Databases

• Protein motif
• Protein domain
• 3-D structure
• Whole-protein

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Family Classification Methods

• Based on Other Classification Information
• Multiple Sequence Alignment (ClustalW)
• ProSite Pattern Search
• Profile Search
• Hidden Markov Models (HMMs)
• Domain (Pfam) Whole protein (PIRSF)
• Neural Networks

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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
• 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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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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PIRSF scan
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Shows PIRSF that the query belongs to

• Search One Query Protein Against all the
  Full-length and Domain HMM models for the fully
  curated PIRSFs by HAMMER
• The matched regions and statistics will be
  displayed.

Statistical data for all domains
Statistical data per domain
Alignment with consensus sequence
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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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3D Structure
Proteins share the same fold suggesting homology
Beta B1 Crystallin
Gamma Crystallin C
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Creation and Curation of PIRSFs
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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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Text Search
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Text Search Result (I)
Extend your search or start over
Choose columns to be displayed
Expand view
Pre-computed BLAST Results
Links to iProClass and UniProtKB reports
Link to NCBI taxonomy
Link to PIRSF report
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Text Search Result (III)
Number of Related Seq. at 3 different E-value
cut-offs
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Text Search Result (II)
Extend your search or start over
Choose columns to be displayed
Curated domain architecture with links to
Pfam database
Link to PIRSF report
Extent of family curation
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Peptide Search
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Peptide Search Results
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Batch Retrieval Results (I)
Retrieve more sequences
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Batch Retrieval Results (II)
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Blast Similarity Search
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Blast Search Results
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Blast / Related Sequences Results
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Blast Result Pairwise Alignment
BLAST Aligment
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Pairwise Alignment
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Multiple Alignment Interactive Phylogenetic Tree
and Alignment
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Phylogenetic Tree and Alignment View
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Pattern Search (I)
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Pattern Search (II)
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PIRSF scan
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PIRSF Report
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PIRSF Family Hierarchy
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Taxonomic Distribution Phylogenetic Pattern
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Rabbit Alpha Crystallin A Chain An iProClass View
of the entry
Pre-computed BLAST results
See protein synonyms
See IDs from different databases
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alpha-Crystallin and Related Proteins