Evolutionary Analysis

Current Topics in Genome Analysis 2005
Evolutionary Analysis
Fiona Brinkman
Simon Fraser University,
Greater Vancouver, BC, Canada
Why care about Evolutionary Analysis?
What do
• BLAST
• Protein motif searching
• Protein threading
• Multiple sequence alignment
Have in common?
Gene family identification
Gene discovery – inferring gene function, gene

annotation
Origins of a genetic disease, characterization

of polymorphisms
Koski LB, Golding GB

The closest BLAST hit is often not the nearest
neighbor.
J Mol Evol. 2001 Jun;52(6):540-2.
Evolutionary Analysis: Key Concepts

• Foundation of most bioinformatic analyses:
Evolutionary theory
• Unique verses non-unique characters
• Sequence alignments are important!
• Fundamentals of phylogenetics and interpreting

phylogenetic trees (with cautionary notes)
• Overview of some common phylogenetic

methods
• Appreciate the need for new algorithms
18th and 19th centuries: The

evolution of a theory
• Earth erosion, sediment
deposition, strata –
present earth conditions
provide keys to the past
18th and 19th

centuries: The
evolution of a theory
• Discoveries of fossils
accumulated
– Remains of unknown but
still living species that are
elsewhere on the planet?
– Cuvier (circa 1800): the
deeper the strata, the
less similar fossils were
to existing species
• Discoveries of fossils accumulated

– Remains of unknown but still living species that
are elsewhere on the planet?
– Cuvier (circa 1800): the deeper the strata, the
less similar fossils were to existing species
Part of Darwin’s Theory

• The world is not constant, but changing
• All organisms are derived from common

ancestors by a process of branching.
Part of Darwin’s Theory

• This explained…
– Fossil record
– Similarities of organisms classified together
(shared traits inherited from common ancestor)
– Similar species in the same geographic region
– Morphological character-based analysis
What is evolution?
• Think – Pair – Share!
• Come up with a definition of evolution that is

6 words or less. Bonus points for 2-3 words!
Characters
• Heritable changes in features (morphology,
DNA sequence etc…)
• The more similar characters you have, the

more related you are
• However….. characters can be unique and

non-unique
Evolution and characters
time
A Unique Character:
Hair for Mammals
• Hair evolved only once and is “unreversed”
• Presence of hair  strong indication that
organism is a mammal
Homoplasy:
The formation of tails
• Tails evolved independently in the ancestors
of frogs and humans
• Presence of a tail  no useful conclusions
Unique and non-unique characters

Non-unique Unique
bioinformatics
bioinfortatics
bioinfortatios time
oinformatios
informatios
infortation
information
Example: Sequence analysis of functionally similar transporters
All share the same deleted sequence region, which is not found
in any other transporter examined to date
Unique character?
Further investigate for possible functional significance, or use

for classification

Example: Sequence analysis of functionally similar transporters
All have isoleucine at the third position in the sequence,

however some other transporters have isoleucine there too,
while some other transporters have leucine at that position
Non-unique.
Changes from I  L  I are common (see BLOSUM OR

PAM matrices). Not a high priority for further analysis of
significance and not useful for classification.
Classification according to
characters – more characters can
be good
Colour Skin Cost Legs Feathers Hair

Beef red no $$$ four no hair
Duck red yes $$$ two yes no
Chicken most
Pork white no $$ four no often
similar to Tofu?
Chicken white yes $ two yes no
Tofu white sometimes $ none no no
Classification according to
characters

Beef red no $$$ four no hair
Pork white no $$ four no often
Classification according to characters

– increasing the number of characters

Beef red no $$$ four no yes
Pork white no $$ four no yes
Chicken most similar to Duck?

Evolution and characters – the

importance of comparing characters
with common origins (homologous)
bioinformatics
bioinformatics
bioinformatios time
oinformatios
informatios
information
information
Evolution and characters
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bioinformatics
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bioinformatics 㼓㼒㼖㼌㼗㼌㼒㼑㼖㻃㼌㼑㻃㼗㼋㼈
bioinformatios 㼖㼈㼔㼘㼈㼑㼆㼈㻑
time
--oinformatios
---informatios 䇻㻷㼋㼈㼜㻃㼕㼈㼉㼏㼈㼆㼗㻃㼗㼋㼈
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---information 㼌㼑㼖㼈㼕㼗㼌㼒㼑㼖㻒㼇㼈㼏㼈㼗㼌㼒㼑㼖
---information 㼒㼕㻃㼒㼗㼋㼈㼕
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Multiple Sequence Alignment
VTISCTGSSSNIGAG-NHVKWYQQLPG
VTISCTGTSSNIGS--ITVNWYQQLPG The sole purpose
of multiple
LRLSCSSSGFIFSS--YAMYWVRQAPG sequence
LSLTCTVSGTSFDD--YYSTWVRQPPG alignments is to
place homologous
PEVTCVVVDVSHEDPQVKFNWYVDG--
positions of
ATLVCLISDFYPGA--VTVAWKADS-- homologous
AALGCLVKDYFPEP--VTVSWNSG--- sequences into
the same column.
VSLTCLVKGFYPSD--IAVEWESNG--
Multiple sequence alignments and

phylogenetic analysis
• First step in any phylogenetic analysis
• Phylogenetic analysis only as good as the

alignment
in  out!
Clustal: Adding evolutionary theory to

multiple sequence alignment
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994)

CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence
weighting, positions-specific gap penalties and weight
matrix choice. Nucleic Acids Research, 22:4673-4680.
Clustal: Incorporating Biology into

Sequence Alignment Algorithms
• Matrices varied at different alignment stages
according to the divergence of the sequences
• Gap penalties differ for hydrophilic regions to

encourage new gaps in potential loop regions
• Gapped positions in early alignments - reduced gap

penalties to encourage the opening up of new gaps
at these positions
gh
Standard multiple sequence

alignment approach
(first step for phylogenetic analysis)
• Be as sure as possible that the sequences included
are homologous
• Know as much as possible about the gene/protein in

question before trying to create an alignment
(secondary structure etc..)
• Start with an automated alignment: preferably one

that utilizes some evolutionary theory such as Clustal
• Examine alignment:
– Are you confident that aligned residues/bases evolved
from a common ancestor?
– Are domains of the proteins/predicted secondary
structures, etc. aligning correctly?
 No? May need to edit sequences and redo…

_______________________________
_________________ ___ __ ____ _
 Yes? Move on!
• Note indels (insertions and deletions)

– Possible insights into functionally important regions…
• Use alignment as a based for subsequent analyses

(identify consensus or other pattern recognition, for PSSM,
HMM construction, phylogenetic analysis, etc..)
• Remove unreliably aligned regions for phylogenetic

analysis
ILPITSPSKEGYESGKAPDEFSSGG
ILPEH--IKDDGELGAAPHSFSTAG
VLPLD-----S--AGRPADSFSAAG
VLPVDR-------DGQARDEYT-VG
VLPVDN-------KGEARDEYT-VG
LLPYDD-------QGRPQDDYSRAG
GIVSRSG---SNFDGEPKDSYGKVG
Delete?
A phylogenetic tree
A node
Human
A clade
Mouse
Fly
taxon -- Any named group of organisms – evolutionary theory not

necessarily involved.
clade -- A monophyletic taxon (evolutionary theory utilized)

A phylogenetic tree with branch lengths
A node
D
Human
A clade
B
Mouse
C
Fly
A
Branch length can be significant…

In this case the analysis suggests that the mouse
sequence/taxon is slightly more similar to fly
than human is to fly
(i.e. sum of branches A+B+C is less than sum of A+B+D)
Phylogenetic analysis
• Organismal relationships
• Gene/Protein relationships
Organismal relationships
Improving our understanding of organismal

relationships
Realization that rates of change are not constant

Improving our understanding of organismal

relationships
Better appreciation for what sequences may be suitable
for analysis of different degrees of divergence
For the tree of life:
rRNA genes
Multiple genes
“Whole genome” datasets of genes
rRNA genes and multiple suitable genes
Gene/Protein Relationships
Homolog, ortholog, paralog??

Homologs
Have common origins but may or may not have

common activity.
Homologous or not?: Often determined by

arbitrary threshold level of similarity determined
by alignment
Homologs
…have common ancestry, but the way they are related can vary
(i.e. the reasons they have diverged into different sequences can
vary)
• orthologs - Homologs produced only by speciation. They tend to have

similar function.
• paralogs - Homologs produced by gene duplication. They tend to

have differing functions.
• xenologs -- Homologs resulting from horizontal gene transfer

between two organisms.
Orthologous or paralogous homologs
Early globin gene
Gene Duplication
α-chain gene ß-chain gene
mouse α human α cattle α cattle ß human ß mouse ß
Orthologs (α) Orthologs (ß)

Paralogs (cattle)
Homologs
Orthologs – diverged only after speciation – tend to have similar function
Paralogs – diverged after gene duplication – some functional divergence occurs
Therefore, for linking similar genes between species, or performing

“annotation transfer”, identify orthologs
True or False?
A1x is the ortholog in

species x of A1y?
A1x is a paralog of A2x?
A1x is a paralog of A2y?

Identifying Gene/Protein Relationships

from Phylogenetic trees
• orthologs - Homologs produced only by speciation.

ID: Gene phylogeny matches organismal phylogeny.
• paralogs - Homologs produced by gene duplication.

ID: Multiple copies of homologs in a given species, or
genes more/less related than expected by organismal phylogeny.
• xenologs -- Homologs resulting from horizontal gene transfer

between two organisms.
ID: Gene phylogeny does not match organismal phylogeny in a
tree where most genes do match organismal phylogeny well.
What are the probable orthologs and paralogs

of the fly genes BKA and WOOT?
Known organismal phylogeny Chimpanzee
Human
Mouse
Fly
Worm
Chimpanzee gene ABC Human gene SOS
Human gene XYZ Human gene CBA
Mouse gene LMNOP Mouse gene PONML
Fly gene BKA Fly gene WOOT
Fly gene LOTR Worm gene LOTRIII

High Throughput Gene Orthology:

How to detect?
• Most common high throughput computational method: Identify

reciprocal best BLAST hits (EGO, COGs,…)
Example Problem:
• If making comparisons between human and bovine, for example, the

bovine gene dataset is still quite incomplete
• Therefore, current best hit may be a paralog now and the true ortholog
not yet sequenced
human cattle mouse cattle
Can we improve orthology analysis for linking

functionally similar genes?
• One solution: Phylogenetic analysis of all putative human-bovine

orthologs, using mouse as an outgroup
• Assumption:
- Mouse and Human gene datasets are more complete, with more true
orthologs identified
Expect (organismal phylogeny): Reject:
cattle human mouse mouse human

cattle
Bunch
of
Eukaryotes
Two
bacteria
Two
Eukaryotes
Bunch
of
Eukaryotes
A bacteria
Bunch
of
bacteria
2 Forms in 1 Species
+ + ++ +
Slides from Jonathan Eisen

2 Forms in 1 Species - LGT

+ + ++ +
Both forms
maintained
Red and blue forms

diverge
Gene present in
+ common ancestor
2 Forms in 1 Species - Gene Loss

+ + ++ +
Loss
Loss
Gene duplicated in common ancestor

++
Unusual Distribution Pattern

+ +
Unusual Distribution - LGT

+ +
Acquires new
type of gene
Gene originates
here
Unusual Distribution - Gene Loss

+ +
Gene lost
here
Gene present in ancestor
Unusual Distribution -
Evolutionary Rate Variation -?
Gene too diverged to be found
+
Unusual Distribution -
Incomplete Data
+/- +/- + +
Gene present in ancestor
Hope for the future

Better sampling of all the species in our world
2004: Environmental
genomics sampling takes
centre stage
Tyson et al (2004) Community structure

and metabolism through reconstruction
of microbial genomes from the
environment. Nature, 428, 37-43.
Venter et al (2004) Environmental

genome shotgun sequencing of the
Sargasso Sea. Science, 304, 66-74.
“So….. how do we construct a phylogenetic tree??”
Most common methods
• Parsimony
• Neighbor-joining
• Maximum Likelihood
Parsimony
• “Shortest-way-from-A-to-B” method
• The tree implying the least number of changes in
character states (most parsimonious) is the best.
• Note:
– May get more than one tree
– No branch lengths
– Uses all character data
Neighbor-joining
(and other distance matrix methods)
• “speedy-and-popular” method
• distance matrix constructed
• distance estimates the total branch length between
a given two species/genes/proteins
• Neighbor-joining approach: Pairing those
sequences that are the most alike and using that
pair to join to next closest sequence.
Maximum Likelihood
• “Inside-out” approach
• produces trees and then sees if the data could
generate that tree.
• gives an estimation of the likelihood of a
particular tree, given a certain model of
nucleotide substitution.
• Notes:
– All sequence info (including gaps) is used
– Based on a specific model of evolution – gives
probability
– Verrrrrrrrrrrry slow (unless topology of tree is known)
How reliable is a result?

• Non-parametric bootstrapping
– analysis of a sample of (eg. 100 or 1000) randomly
perturbed data sets.
– perturbation: random resampling with replacement,
(some characters are represented more than once, some
appear once, and some are deleted)
– perturbed data analysed like real data
– number of times that each grouping of
species/genes/proteins appears in the resulting profile
of cladograms is taken as an index of relative support
for that grouping
Bootstrapping
The number of times a
particular branch is formed
in the tree (out of the X
times the analysis is done)
can be used to estimate its
probability, which can be
indicated on a consensus tree
High bootstrap values don’t

mean that your tree is the
true tree!
Alignment and evolutionary

assumptions are key
Parametric Bootstrapping
Data are simulated

according to the
hypothesis being tested.
Phylogenetics – More info

Li, Wen-Hsiung. 1997. Molecular evolution Sunderland,
Mass. Sinauer Associates.
- a good starting book, clearly describing the basis of
molecular evolution theory. It is a 1997 book, so is
starting to get a bit out of date.
Nei, Masatoshi & Kumar, Sudhir. 2000. Molecular

evolution and phylogenetics Oxford ; New York. Oxford
University Press.
- a relatively new book, by two very well respected
researchers in the field. A bit more in-depth than the
previous book, but very useful.
Phylogenetic Tree Construction:

Examples of Common Software
PHYLIP
http://evolution.genetics.washington.edu/phylip.html
PAUP
http://paup.csit.fsu.edu/
MEGA 2.1
www.megasoftware.net/
TREEVIEW
http://taxonomy.zoology.gla.ac.uk/rod/treeview.html
Extensive list of software

http://evolution.genetics.washington.edu/phylip/software.html
Challenges
How do we classify?
Computational Challenges
• Need to incorporate more evolutionary theory

into the multiple sequence alignment and
phylogenetic algorithms used in phylogenetic
analysis
• Phylogenetic analyses are computationally

intensive – great way to benchmark your CPU
speed!
More Challenges
• Increasing the sampling of our genetic world
• More accurately differentiating orthologs, paralogs,

and horizontally acquired genes
• How frequent is gene loss, gene duplication, and

horizontal gene transfer in genome evolution?
• To what degree can we predict protein/gene function

using phylogenetic analysis?
Remember:
Evolutionary theory is evolving…

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Current Topics in Genome Analysis 2005

Why care about Evolutionary Analysis?

Why care about Evolutionary Analysis?

Gene family identification

Gene discovery – inferring gene function, gene

Origins of a genetic disease, characterization

Why care about Evolutionary Analysis?

Koski LB, Golding GB

Evolutionary Analysis: Key Concepts

• Unique verses non-unique characters

• Sequence alignments are important!

• Fundamentals of phylogenetics and interpreting

• Overview of some common phylogenetic

• Appreciate the need for new algorithms

18th and 19th centuries: The

18th and 19th

• Discoveries of fossils accumulated

Part of Darwin’s Theory

• All organisms are derived from common

Part of Darwin’s Theory

– Morphological character-based analysis

• Think – Pair – Share!

• Come up with a definition of evolution that is

• The more similar characters you have, the

• However….. characters can be unique and

Evolution and characters

Unique and non-unique characters

Unique and non-unique characters

Example: Sequence analysis of functionally similar transporters

Further investigate for possible functional significance, or use

Unique and non-unique characters

All have isoleucine at the third position in the sequence,

Changes from I  L  I are common (see BLOSUM OR

Colour Skin Cost Legs Feathers Hair

Colour Skin Cost Legs Feathers Hair

Classification according to characters

Colour Skin Cost Legs Feathers Hair

Chicken most similar to Duck?

Evolution and characters – the

Evolution and characters

䇻 㻪㼄㼓 㼖㻃㼕㼈㼓 㼕㼈㼖㼈㼑㼗㻃㼑㼒㼑㻐

Multiple Sequence Alignment

Multiple sequence alignments and

• First step in any phylogenetic analysis

• Phylogenetic analysis only as good as the

Clustal: Adding evolutionary theory to

Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994)

Clustal: Incorporating Biology into

• Gap penalties differ for hydrophilic regions to

• Gapped positions in early alignments - reduced gap

Standard multiple sequence

• Know as much as possible about the gene/protein in

• Start with an automated alignment: preferably one

 No? May need to edit sequences and redo…

 Yes? Move on!

• Note indels (insertions and deletions)

• Use alignment as a based for subsequent analyses

• Remove unreliably aligned regions for phylogenetic

taxon -- Any named group of organisms – evolutionary theory not

䇻㻪㼄㼓㼖㻃㼕㼈㼓㼕㼈㼖㼈㼑㼗㻃㼑㼒㼑㻐