Bio Tools

Bioinformatics Tools
Byoung-Tak Zhang and Chul Joo Kang

School of Computer Science and Engineering
Seoul National University
Contents
1. Sequence Alignment
2. Multiple Sequence Alignment
3. Pattern Finding
4. Structure Prediction
5. DNA Microarray
6. Major Tools for Proteomics
(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)
Bioinformatics Tools
Problems
Tools or Database
Sequence Alignment
BLAST, FASTA
Multiple Sequence
Alignment
Clustal W, Macaw
Pattern Finding
GRAIL, FGENEH, tRNAscan-SE, NNPP,

eMOTIF, PROSITE, ChloroP
Structure Prediction
Bend.it, RNA Draw, NNPREDICT, SWISSMODEL
DNA Microarray
GeneX, GOE, MAT, GeNet
Hardwares for
Proteomics
2D Gel, MALDI-TOF
Bioinformatics Based on Sequence

!
Genome sequencing project: get huge sequence

data
Rretrieving useful information from sequence data

- Find genes and other elements
- Classification
- Predict its function
- Others
Read the articles on Nature and Science

1. Sequence Alignment
Pairwise Sequence Alignment

!
!
!
!
!
!
!
Basic job handling sequences

Alignment between two sequences
Sequence database search
Finding similar DNA or protein sequences
Smith-Waterman algorithm
BLAST
FASTA
Smith-Waterman Algorithm (1)

!
!
Finding local alignments

Using dynamic programming
TCAT*G
*CATTG
Smith-Waterman Algorithm (2)

!
!
!
!
Best results and slow performance

Can grasp results missed by BLAST or FASTA.
Available on web:
spiral.genes.nig.ac.jp/homolgy/ssearch-e.shtml
Ref : Smith, T. F. and Watermann, M. S. (1981)
Identification of common molecular subsequence.
Journal of Molecular Biology, 147: 196-197.
BLAST & FASTA

!
!
!
!
Using heuristic algorithms

Word based match
Faster than Smith &Waterman
BLAST: www.ncbi.nlm.nih.gov/BLAST
Ref: Altschul, S. F., et al. (1990) Basic local alignment
search tool. Journal of Molecular Biology, 215, 403410.
FASTA: www.ebi.ac.uk/fasta3
Ref: Wilbur, W. J. and Lipman, D. J. (1983) Rapid
similarity searches of nucleic acid and protein data
banks. Proceedings of the National Academy of
Sciences USA 80, 726-30.
The BLAST Search

For the query, find the list
of high scoring words of
length W.
Compares the word list to
the database and identifies
exact matches.
For each word match,
extends the alignment in
both directions to find
alignments that score
greater than a threshold of
value S.
10
BLAST Result
Program: BLASTP
Query: human MYB
binding protein
Database: Swissprot
11
2. Multiple Sequence Alignment
12
Multiple Sequence Alignment

!
!
Align multiple sequences

Finding well conserved regions
4Finding motifs or other elements
!
!
!
Building gene families

Constructing a phylogenetic tree
Using whole dynamic programming ?
4O(n^k) problem (n = sequence length, k = number of
sequences)
!
!
Clustal W
Macaw
13
Clustal W
!
!
!
!
Step 1: Pairwise alignment between two sequences

Step 2: Making sequence weights and clusters
Step 3: Alignment between most similar sequences or
clusters
Step 4: Making one optimal output sequence
Ref: Higgins, D. G. and Sharp, P. H. (1988) CLUSTAL: A
package for performing multiple sequence alignment
on a microcomputer. Gene 73, 237-244.
14
Multiple Alignment by Clustal W
15
Phylogenetic Tree Construction Based

on Clustal W Results
16
3. Pattern Finding
17
Pattern Finding
! DNA
sequences have all information about

their products
4Gene finding
4Other sequence element finding
4Motif finding
4Localization prediction
4And others
18
Gene Structure
19
Gene Finding
! From
an unannotated DNA sequence, find

putative expressive regions
! Distinguish exon and intron regions (in
eukaryote).
! Solutions
4Compare known genes (alignments)
4Simply find conserved region
4Using statistical models like hidden Markov
models and others
20
GRAIL: Find Exon and Intron

!
!
!
!
!
!
!
In eukaryotes, RNA is processed.

Intron: splice out
Exon: join together
GU AG rule
GRAIL: exon prediction from a
genomic sequence with
RepeatMasker filtering
Neural networks which combine a
series of coding prediction
algorithms
http://compbio.ornl.gov/Grail-1.3
Ref: Einstein et al. (1992) Computerbased construction of gene using
the GRAIL gene assembly program.
Oak Ridge Natl. Lab. Report TM12174.
21
GRAIL Results
22
FGENEH: Prediction of Multiple Genes

in Human DNA Sequences
!
!
!
Finding genes from DNA sequences

HMM based human gene structure prediction
Available on web:
http://searchlauncher.bcm.tmc.edu/seq-search/genesearch.html
Ref: Algorithm is trained on our data and based on HMM
similar with Genescan (Burge, C. and Karlin, S. (J.
Mol. Biol. 1997, 268, 78-94.) and Genie (Kulp, D.,
Haussler, D., Reese, M.G., and Eeckman, F. H. (1996),
Proc. Conf. on Intelligent Systems in Molecular
Biology '96, 134-142).
23
FGENEH: Prediction of Multiple Genes

in Human DNA Sequences
24
tRNAscan-SE: Find tRNA Genes

!
!
!
!
!
Finding tRNA from genomic sequence

Finding polII promoter sites, searching conserved
secondary structures - easier problem than finding protein
coding genes
Combining several earlier programs and algorithms
Http://www.genetics.wustl.edu/eddy/tRNAscan-SE/
Ref: Lowe, T. M. and Eddy. S. R. (1997) tRNAscan-SE:
A program for improved detection of transfer RNA
genes in genomic sequence. Nucl. Acids Res. 25: 955964.
25
tRNAscan-SE Results
26
Sequence Element Finding
* Promoter region of E.coli lacZ operon
Promoter and other elements: regulatory and other protein binding

sites
27
NNPP: Neural Network Promoter

Prediction
!
!
!
Finding promoter sequences on DNA sequences

Time-delay neural networks
Two feature layers
4Recognizing TATA boxes
4Recognizing initiators
http://www.fruitfly.org/seq_tools/promoter.html
28
Neural Network Promoter Prediction
29
Motif Finding
!
!
Find conserved residues from DNA or protein sequences.

Commonly, conserved residues are related to protein functions.
Solutions:
Finding consensus sequences
Alignment
Position specific weight matrix
Hidden Markov models
Motif of one class of zinc finger protein
(C2H2)
Cx{2,4}Cx{12}Hx{3,5}H
30
Motif Databases: eMOTIF, PROSITE

!
Prosite, eMotif: protein motif databases
eMotif (motif.stanford.edu/emotif)
eMotif Maker: building new motif from multiple alignment
eMotif scan: find motif pattern
eMotif search: find motif from protein sequence
Ref: Huang, J. Y. and Brutlag, D. L. (2001) The eMOTIF Database.

Nucleic Acids Research, 29(1), 202-204
Craig et al. (1998) Highly specific protein sequence motif for
genomic analysis. Proc. Natl. Acad. Sci. 95: 5865-5871
31
eMOTIF (1)
32
eMOTIF (2)
33
eMOTIF (3)
34
PROSITE (1)
!
!
PROSITE: Database of protein families and

domains (http://www.expasy.ch/prosite/)
Consists of biologically significant sites, patterns
and profiles that help to reliably identify to which
known protein family (if any) a new sequence
belongs.
35
PROSITE (2)
e.g.) C2H2 zinc finger
protein
36
ScanPROSITE
!
Compares query sequences

(protein) to Prosite.
! Find patterns
37
Others: Find Gene Destination

!
!
After translation, proteins are located to proper places

(nuclear, mitochondria, outside of cell)
That process determined by amino acid sequences on
proteins
4 Finding signal peptide
4 Predicting protein targets
38
ChloroP: Prediction of Chloroplast

Importing Protein (1)
!
!
!
!
Predicting chloroplast targeting transit peptides

Predicting cleavage sites
Neural network based method
Ref: Emanuelsson, O., Nilesen, H., and von
Heijne, G. (1999) A neural network-based
method for predicting chloroplast transit
peptides and their cleavage sites. Protein
Science 8, 978-984.
Service site:
http://www.cbs.dtu.dk/services/ChloroP/
39
ChloroP: Prediction of Chloroplast

Importing Protein (2)
40
4. Structure Prediction
41
!
!
!
DNA, RNA and protein structure prediction

These structures affect their function, stability, etc.
Protein structure prediction by calculation of
biochemical properties of each amino acid
4In most cases of protein secondary or tertiary structure
prediction, it is a terribly huge computing job (almost
impossible)
Prediction based on known structures
42
DNA & RNA Structure

!
!
DNA & RNA strands: dynamic one, not linear form

DNA: regionally denatured (breathing), bending
4 Affects its expressions and others.
RNA: forming intra strand pairing

4 Affects its stability, function (RNase), and others.
43
DNA Bending
!
!
!
DNA structure is flexible.

DNA double strand is
bended by protein binding.
The bending of the DNA
structure will influence
other proteins to bind to it.
44
Bend.it: Predict DNA Bendability

!
!
!
!
Predicting DNA curvatures from DNA sequences

The curvature is calculated as a vector sum of
dinucleotide geometries (roll, tilt and twist angles).
Expressed as degrees per helical turn.
Ref: Munteanu, M. G., et al. (1998) Rod models of
DNA: sequence-dependent anisotropic elastic
modelling of local bending phenomena.
Trends Biochem. Sci. 23(9), 341-346
Service site:
http://www2.icgeb.trieste.it/~dna/bend_it.html
45
RNA Draw: Predict RNA Secondary

Structure on PC (1)
!
!
Predicting RNA secondary conformation under given E

state
Using dynamic programming, base pair probability and E
parameters
Clover leaf structure of tRNA and its 3D structure

46
RNA Draw: Predict RNA Secondary

Structure on PC (2)
E.coli tRNA-Ile
predicted
structure on 37 C
47
Protein Structure
Primary structure (amino acid

sequence) determine its
secondary structure (partial
structure: helix, -sheet) and
tertiary structure
Protein function: determined by its structure

(e.g.: enzyme inactivated by heat - because its structure
was changed by heat.)
48
Secondary Structure Prediction

!
Prediction of secondary
structure
4 -helix
4 -sheet
!
!
Can be helpful for its 3D

conformation study.
Solutions
4 Calculate propensity for each
amino acid to be in helix, sheet
4 Calculate information content
4 Neural network based method
4 Nearest neighbor
49
NNPREDICT: Protein Secondary

!
!
Two-layer, feed-forward neural network

Finding secondary structure from amino acid sequences
http://www.cmpharm.ucsf.edu/~nomi/nnpredict.html
50
Tertiary Structure Prediction
!
!
Using E state?: only select lowest E state - impossible

Using homology modeling: similar sequence = similar
structure
51
SWISS-MODEL
!
Predicting protein 3D structure
52
Swiss-Pdb Viewer
53
SWISS-MODEL
!
Ref: Guex, N. and Peitsch, M. C. (1997) SWISSMODEL and the Swiss-PdbViewer: An

environment for comparative protein modelling.
Electrophoresis 18:2714-2723.
Guex, N. and Peitsch, M. C. (1999) Molecular
modelling of proteins. Immunology News 6:132-134.
Available on web:
http://www.expasy.ch/swissmod/SWISS-MODEL.html
54
5. DNA Microarray
55
DNA Microarray (1)
!
!
!
Monitoring of gene expression

cDNA chip: spotting cDNAs
Oligonucleotide chip: spotting
short DNA sequences
56
DNA Microarray: cDNA chip (2)

!
!
DNAs are spotted on glass surfaces.

Applications: drug effects, drug metabolism, disease diagnosis, finding
gene pathway, finding disease genes, etc.
57
DNA Microarray: oligonucleotide chip (3)

!
!
Short (10~25 nt) oligonucleotides on chip

Applications: find sNPs, detect mutation, sequencing by chip,
genotyping
58
DNA Microarray (4)

!
Processing DNA chip images to obtain qualified data
Data mining from DNA chip images like clustering (hierarchical

algorithms, partitioning algorithms)
59
DNA Microarray (4)

!
Ref: Schena, M., et al. (1996) Parallel human

genome analysis: Microarray-based expression
monitoring of 1000 genes. Proc Natl. Acad. Sci.
USA. 93(20):10614-9.
Useful websites:
4www.affimatrix.com
4www.genechip.co.kr
4inkage.rockefeller.edu/wli/microarray
4cmgm.stanford.edu/~plf58/microArray
60
6. Major Tools for Proteomics
61
Major Tools for Proteomics: 2D Gel &

MALDI-TOF
!
Proteomics:
4 Protein expression profile
4 Protein interaction
4 Protein structure
4 Protein variation
Why proteomics?
4 Post-Genomics Era: Understanding functions
4 Applications in target discovery
4 Novel proteins and genes
4 Disease associated proteins
4 Elucidate pathways and processes in disease/cell biology
62
2-D Gel Electrophoresis

!
!
!
Separates proteins by their netcharge and

molecular weights.
Displays whole proteins
Like DNA chip, compares expression under
different treatments, and detects tissues or cell
specific expressions.
4Finding specific proteins for further study
Poor reproduction problem
63
2-D Gel Image of Human Liver
64
2-D Electrophoretic Gel Images
65
MALDI-TOF: Matrix-Assisted Laser

Desorption Ionization
!
!
Detection of protein molecular weights (mass

spectrophotometer)
Can determine protein sequence.
66
Other bioinformatic work

!
Sequence assembly
4 From scattered DNA sequences, construct full sequence
Specific database warehousing

4 Structural databases, protein block databases, drug databases
sNPs
4 Find single nucleotide polymorphisms in population
Construct gene to gene, protein to protein and gene to

protein interaction pathway
67

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Bioinformatics Tools

Byoung-Tak Zhang and Chul Joo Kang

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

GRAIL, FGENEH, tRNAscan-SE, NNPP,

Bend.it, RNA Draw, NNPREDICT, SWISSMODEL

GeneX, GOE, MAT, GeNet

Bioinformatics Based on Sequence

Genome sequencing project: get huge sequence

Rretrieving useful information from sequence data

Read the articles on Nature and Science

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Pairwise Sequence Alignment

Basic job handling sequences

Smith-Waterman Algorithm (1)

Finding local alignments

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Smith-Waterman Algorithm (2)

Best results and slow performance

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

BLAST & FASTA

Using heuristic algorithms

The BLAST Search

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

2. Multiple Sequence Alignment

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Multiple Sequence Alignment

Align multiple sequences

Building gene families

Step 1: Pairwise alignment between two sequences

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Multiple Alignment by Clustal W

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Phylogenetic Tree Construction Based

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

sequences have all information about

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

an unannotated DNA sequence, find

GRAIL: Find Exon and Intron

In eukaryotes, RNA is processed.

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

FGENEH: Prediction of Multiple Genes

Finding genes from DNA sequences

FGENEH: Prediction of Multiple Genes

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

tRNAscan-SE: Find tRNA Genes

Finding tRNA from genomic sequence

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Sequence Element Finding

* Promoter region of E.coli lacZ operon

Promoter and other elements: regulatory and other protein binding

NNPP: Neural Network Promoter

Finding promoter sequences on DNA sequences

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Neural Network Promoter Prediction

(C) 2001 SNU CSE Artificial Intelligence Lab (SCAI)

Find conserved residues from DNA or protein sequences.

Motif Databases: eMOTIF, PROSITE

Prosite, eMotif: protein motif databases