1. Sequence Based Analysis Tutorial
NIH Proteomics Workshop
Lai-Su L. Yeh, Ph.D.
Protein Information Resource at
Georgetown University Medical Center

2. 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

3. 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

4. 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 10

5. 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

6. 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) 10

7. 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

8. BLAST Search From BLAST Search Interface
Table-Format Result with BLAST Output and SSEARCH (Smith-Waterman) Pair-Wise Alignment
Links to iProClass and
UniProtKB reports
Link to NCBI
taxonomy
Link to PIRSF
report
Click to see
SSearch alignment
Click to see
alignment

9. Blast Result & Pairwise Alignment
BLAST Aligment

10. 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?

11. Classification Databases
C - x(2,4) - C - x(3) - [LIVMFYWC] - x(8) - H - x(3,5) - H The 2 C's and the 2 H's are zinc ligands
Group proteins according to the presence of a common domain
Protein motif
Protein domain
3-D structure
Whole-protein
Group proteins according to common 3D structure
Group proteins according to common domain architecture and length
Protein Domain: Structurally compact, independently folded unit that forms a stable 3D-structure and shows a certain level of evolutionary conservation
Protein motif: A set of conserved amino acid residues that are important for protein function and located within a certain distance from one another

12. 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

13. 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

14. 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

15. Multiple Alignment and Tree
From Text/Sequence Search Result or ClustalW Alignment Interface

16. Here is an example of two different functions easily separated on a phylogenetic tree. Each functional group is used to build an HMM.

17. Motif Patterns (Regular Expressions)
Signature Patterns for Functional Motifs
ProClass Motif Alignments

18. 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

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

20. Pattern Search Result (II)
One Query Pattern Against Sequence Database
Links to iProClass and
UniProtKB reports
Link to NCBI
taxonomy
Link to PIRSF
report 2 1 3
Sorting arrows
Display the query
pattern

21. 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

22. 1
PIRSF scan
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

23. Secondary Structure Features
a Helix Patterns of Hydrophobic Residue Conservation Showing I, I+3, I+4, I+7 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, I+2, I+4, I+6

24. Proteins share the same fold suggesting homology
3D Structure
Proteins share the same fold suggesting homology
Gamma Crystallin C
Beta B1 Crystallin

25. Creation and Curation of PIRSFs

26. Integrated Bioinformatics System for Function and Pathway Discovery
Data Integration
Associative Analysis

27. Analytical Pipeline Family Classification & Functional Analysis
Query Sequence
UniProt
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
Analytical Pipeline

28. Integrated Bioinformatics System
Global Bioinformatics Analysis of 1000’s of Genes and Proteins
Pathway Discovery, Target Identification

29. Lab Section

30. Text Search

31. Text Search Result (I) Pre-computed 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

32. Text Search Result (III)
Number of Related
Seq. at 3 different
E-value cut-offs

33. 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

34. Peptide Search

35. Peptide Search & Results
Links to iProClass and
UniProtKB reports
Link to NCBI
taxonomy
Link to PIRSF
report
Matching peptide
highlighted in
the sequence
Sorting arrows

36. Choose columns to be displayed
Batch Retrieval Results (I)
Choose columns to be displayed 3 4 5 2 1 6
Links to iProClass and
UniProtKB reports
Retrieve more sequences

37. Curated domain architecture (N- to C- termini) with links
Batch Retrieval Results (II)
Choose columns to be displayed
Retrieve more
families 3 4 5 2 1 6
Links PIRSF reports
Curated domain architecture
(N- to C- termini) with links
to Pfam database

38. Blast Similarity Search

39. Blast / Related Sequences Results

40. Blast Result & Pairwise Alignment
BLAST Aligment

41. Pairwise Alignment

42. Multiple Alignment Interactive Phylogenetic Tree and Alignment

43. Phylogenetic Tree and Alignment View

44. Pattern Search (I)

45. Pattern Search (II) Links to iProClass and UniProtKB reports
Link to NCBI
taxonomy
Link to PIRSF
report
Sorting arrows
Display the query
pattern

46. PIRSF scan

47. PIRSF Report

48. PIRSF Family Hierarchy

49. Taxonomic Distribution & Phylogenetic Pattern

50. Rabbit Alpha Crystallin A Chain An iProClass View of the entry
Pre-computed
BLAST results
See protein
synonyms
See IDs
from different
databases

51. alpha-Crystallin and Related Proteins