Bioinformatics and Functional Genomics Student ID: 21485094
Lecturer: Dr. Obed Brew Beatriz Manso
1
PHYLOGENETIC ANALYSIS
Introduction
Phylogeny explains how sequences evolved, their genealogical relationships, and how they became
today.
In this exercise, we will explore how to build and interpret a phylogenetic tree using the
morphological characteristics of the organism.
This is an introduction to modern taxonomy that classifies organisms according to their evolutionary
relationships.
Activity 1 - Basics of Phylogenetic Analysis
1. Look at the organisms shown in Figure 1 and identify seven features that differ between them. List
those features in the first row (header row) of the Table 1. Fill in the table, recording data for each of
organisms, A - F.
2. Now compare each pair of organisms and, in the small table, record the total number of
differences between each pair. This is the data matrix that will be used to build the phylogenetic tree
in the next step:
Dotted
body
Body
colour
Body
shape
Head
colour
ge
Antennae
✓
✓
✓
Oval
Oval
Oval
Round
Round
Round
Yellow
Yellow
Orange
Pink
Pink
Orange
White
White
Black
Black
Black
Black
Body line
Orange
Orange
Black
Black
Claws
Grey
Grey
Black
Black
Black
White
Dots
colour
✓
✓
✓
✓
✓
✓
✓
Bioinformatics and Functional Genomics Student ID: 21485094
Lecturer: Dr. Obed Brew Beatriz Manso
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We’ve learned how to create a matrix in R.
3. The numbers in the data matrix can now be used to draw a phylogenetic tree showing the
relatedness of all six organisms. The next steps demonstrate how to do this.
4. Using the records from the first table, a data matrix, recording the number of differences between
pairs of organisms, has also been produced:
a. Currently living (extant) organisms will be placed at the top of the tree - all of the
organisms analysed live in the present.
b. Coming down the tree, we are moving into the past. The last common ancestor of the
organisms will be shown on the very bottom of the tree.
To create the tree, proceed as follows:
1. Start by finding the closest relatives - the pair or pairs of organisms that have the fewest
differences between them. In this example,
a. Beetles A and B have only one difference, so they are the closest relatives.
b. C and F also have just one difference, so they are the closest relatives as well.
c. Place these pairs at the top of the tree.
d. The last common ancestor of A and B lived relatively recently.
e. Mark a dot under A and B, showing the separation of evolutionary lines leading to
the contemporary forms of A and B.
f. Repeat this for C and F (Figure 1
2. Next, find organisms that have more than one difference.
a. B differs from C by two features, so the last common ancestor of this pair lived
before the common ancestor of A and B and C and F. Draw a dot to represent the
common ancestor of both of these pairs and link them together.
b. Organisms D and E also differ by just two features, so they are closely related to
each other, but not to the other organisms.
c. D and E are paired up by linking them with a common ancestor.
3. Establish which organisms differ by three or four characteristics. In this way, we can
confirm the earlier assumption of the relationship between A, B, C and F.
4. Next, look for organisms that differ by five features. These are beetles A and D, C and E
and E and F. When there groups are linked by a common ancestor, the tree is complete
2
4
4
6
4
2
5
5
4
7
4
2
2
6
4
Bioinformatics and Functional Genomics Student ID: 21485094
Lecturer: Dr. Obed Brew Beatriz Manso
3
Bioinformatics and Functional Genomics Student ID: 21485094
Lecturer: Dr. Obed Brew Beatriz Manso
4
Activity 2 - Phylogenetic Analysis with SeaView
A gastroenterologist was convicted of attempted second-degree murder by injecting his former
girlfriend with blood or blood products obtained from an HIV type 1 (HIV-1)-infected patient under
his care.
Phylogenetic analyses of HIV-1 sequences were admitted and used as evidence in this case,
representing the first use of phylogenetic analyses in a criminal court case in the United States.
Phylogenetic analysis of HIV env/gp120 sequences was used as evidence in the trial of a Louisiana
gastroenterologist accused of deliberately infecting someone (the victim) with HIV-infected blood,
from one of the gastroenterologist's patients.
PNAS _ October 29, 2002 _ vol. 99 _ no. 22
The aim of this exercise is to determine how well the data supports the hypothesis that the victim
(whose sequence identifiers all begin with a "V") was directly infected by blood taken from the
patient (whose sequence identifiers all begin with a "P"). The other sequences in the file are from
other people in Louisiana who are neither the patient, nor the victim.
1. Open the SeaView program
2. Go to file, click open and Load in ThirtySeqsLouisianaGastroUnaligned.fasta
3. Click align ➔ align all (the sequences will be aligned with clustal omega by default)
4. Click ‘ok’ to view the alignment
5. Click Trees > Distance Methods> Run, click ‘ok’
6. Interpret your results
Fig.1 – Sequences before alignment
Bioinformatics and Functional Genomics Student ID: 21485094
Lecturer: Dr. Obed Brew Beatriz Manso
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FIG.2 – sequences after alignment
Fig.3 – Phylogeny tree
1. What is the main difference visible in the sequences before and after alignment?
Why do you think it is important to align the sequences before you draw a phylogeny?
Before the aligning the sequences, it is very hard to see which ones have similar sections. After the
sequences have been aligned, we can observe the similarity between each sequence.
2. Do you think the gastroenterologist committed the crime? How confident are you
in this verdict?
The tree we obtained from the alignment supports the hypothesis that the doctor indeed has
infected the victim, because all of the sequences taken from the patients (P) have a common
ancestor with the victim (V), compared to the random people from Louisiana.
Bioinformatics and Functional Genomics Student ID: 21485094
Lecturer: Dr. Obed Brew Beatriz Manso
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