Comparing Two
Sequences
© Wiley Publishing. 2007. All Rights Reserved.
Learning Objectives
Get the basics about dot plots
Know how to interpret the most common
patterns in a dot plot
Use Dotlet
Use Lalign to extract local alignments
Outline
Some reasons for comparing two sequences
Basic principles of dot-plot comparisons
Using Dotlet
Making local alignments with Lalign
Why Compare Two
Sequences?
Database searches are useful for finding homologues
Database searches don’t provide precise comparisons
More precise tools are needed to analyze the sequences in
detail including
• Dot plots for graphic analysis
• Local or global alignments for residue/residue analysis
The alignment of two sequences is called a pairwise
alignment
Using The Right Tool
Some Applications of Pairwise
Alignments
Convince yourself two sequences are homologous
Identify a shared domain
Identify a duplicated region
Locate important features such as
• Catalytic domains
• Disulphide bridges
Compare a gene and its product
What Is a Dot Plot ?
A dot plot is a graphic representation of pairwise similarity
The simplicity of dot plots prevents artifacts
Ideal for looking for features that may come in different orders
Reveal complex patterns
Benefit from the most sophisticated statistical-analysis tool in the
universe . . . your brain
Choosing Your Two
Sequences
Making pairwise comparisons takes time
Use BLAST to rapidly select your sequences
• More than 70% identity for DNA
• More than 25% identity for proteins
If your sequences are too similar, comparing them
yields no useful information
Self-comparisons
Start comparing your sequence with itself
You can discover
• Repeated domains
• Motifs repeated many times (low complexity)
• Mirror regions (palindromes) in nucleic acids
What Can You Analyze
with a Dot Plot ?
Any pair of sequences
• DNA
• Proteins
• RNA
DNA with proteins
• Dotlet is an appropriate tool
• To compare full genomes, install the program locally
Sequences longer than 1000 symbols are hard to analyze online
Some Typical Dot-plot
Comparisons
Divergent sequences where only a segment is homologous
Long insertions and deletions
Tandem repeats
• The square shape of the pattern is characteristic of these repeats
Using Dotlet
Dotlet is one of the handiest tools for making dot
plots
Dotlet is a Java applet
Open and download the applet at the following site:
• [Link]/java/dotlet
Use Firefox or IE (if one doesn’t work, use the other)
Set Dotlet Parameters
Dotlet slides a window along
each sequence
If the windows are more similar
than the threshold, Dotlet prints Window
Window size
Size
a dot at their intersection
You can control the similarity
threshold with the little window Threshold
Threshold
on the left
The Dotlet Threshold
Every dot has a score given by the window
comparison
When the score is
• Below threshold 1 black dot Log curve
• Between thresholds 1 and 2 grey dot
• Above threshold 2 white dot
The blue curve is the distribution of scores in
the sequences
The peak most common score,
• Most common less informative
Getting Your Dot Plot Right
Window size and the stringency control the aspect of your
dot plot
• Very stringent = clean dot plot, little signal
• Not stringent enough = noisy dot plot, too much signal
Play with the threshold until a usable signal appears
Which Size for the
Window?
Long window
• Clean dot plots
• Little sensitivity
Short window
• Noisy dot plots
• Very sensitive
The size of the window should be in the range of the
elements you are looking for
• Conserved domains: 50 amino acids
• Transmembrane segments: 20 amino acids
Shorten the window to compare distantly related sequences
Looking at Repeated
Domains with Dotlet
The square shape is typical
of tandem repeats
The repeats are not perfect
because the sequences
have diverged after their
duplication
Comparing a Gene and
Its Product
Eukaryotic genes are transcribed
into RNA
The RNA is then spliced to
remove the introns’ sequences
It may be necessary to compare
the gene and its product
Dotlet makes this comparative
analysis easy
Aligning Sequences
Dotlet dot plots are a good way to provide an
overview
Dot plots don’t provide residue/residue
analysis
For this analysis you need an alignment
The most convenient tool for making precise
local alignments is Lalign
Lalign and BLAST
Lalign is like a very precise BLAST
It works on only two sequences at a time
You must provide both sequences
Lalign Output
Lalign produces an output similar
to the alignment section of BLAST
The E-value indicates the
significance of each alignment
Low E-value good alignment
Going Farther
If you need to align coding DNA with a protein, try these
sites:
• [Link] => protogene
• [Link]/pal2nal
If you need to align very large sequences, try this site:
• [Link]/blast/bl2seq/[Link]
If you need a precise estimate of your alignment’s statistical
significance, use PRSS
• The program is available at [Link]