Comparative Genomics
Todd Castoe
Biochemistry and Molecular Genetics
The First Genomes
Figure 18.6 Genomes 3 (© Garland Science 2007)
Tree of life from David Hillis’ lab (based on ~3000 rRNAs)
animals
plants
you are here
protists
fungi
http://www.zo.utexas.edu/faculty/antisense/Download.html
bacteria
archaea
Tree of life from David Hillis’ lab (based on ~3000 rRNAs)
you are here
http://www.zo.utexas.edu/faculty/antisense/Download.html
Hedges, Nat Rev Genet 2003
An argument for model species
and the need for comparative genomics
Most human proteins are ancient
>90%
Timescale of eukaryote
evolution
~75%
HUMAN PROTEINS…
~50%
~30%
Divergences within 749 gene families in the Human Genome
Gu X. et al. Nature Genetics (2002) 31 205-209
Genomes have been recycling for Billions of years
What is comparative genomics
There are many ways that genomes can be compared
• Whole genome
– Genome size
– Genome alignments
– Synteny (gene order conservation)
– Gene number
– Anomalous regions
• Gene-centric
– Gene families and unique genes
– Gene clustering by function
• Gene sequence variations
– Codon usage, SNPs, inDels, pseudogenes
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Why Comparative Genomics?
1. Conservation over long evolutionary distances suggests functional constraints
2. Lack of conservation over short distances may be indicative of adaptive
evolution
3. Helps us identify both coding and non-coding genes and regulatory elements
4. Characterizing the differences between organisms reveals mechanisms of change
5. Allows us to achieve a greater understanding of vertebrate evolution
6. Leveraging knowledge between species for annotation and inference of function
7. Tells us what is common and what is unique between different species at the
genome level
8. The function of human genes and other regions may be revealed by studying
their counterparts in simpler model organisms
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Comparing Genome Size
The ‘C-value paradox’
Genome size does NOT correlate with organismal
complexity
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Why Are Some Genomes So Large?
• There is no clear correlation between genome size and genetic
complexity.
• C-value – The total amount
of DNA in the genome (per
haploid set of chromosomes)
• C-value paradox – The
lack of relationship
between the DNA content
(C-value) of an organism
and its coding potential.
Haploid Genome Size (log scale)
Contrasted Genome Landscapes
Transposable Element
The amount of TE correlate positively with genome size
Mb
Genomic DNA
3000
2500
TE DNA
2000
Protein-coding
DNA
1500
1000
500
0
Feschotte & Pritham 2006
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Transposable Elements…
• Variation in gene numbers cannot explain variation in genome size among
eukaryotes
• Most of variation in genome size is due to variation in the amount of repetitive
DNA (mostly derived from TEs)
• TEs accumulate in intergenic and intronic regions
•CONCLUSIONS…
•TEs have played an important role in genome evolution and
diversification
•Facilitate expansion and contraction of genomes AND gene
families
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Coarse Comparisons of Genomes
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Fugu Genome
Science 2002
365 Mb
(1/10 the human)
Tiny vertebrate
genome
Humans and Fish
shared common
ancestor 450Mya!
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Among the Smallest
Vertebrate Genome
• Genome is < 1/6 repetitive DNA
– Vs. ~50% in us
• ¾ of human proteins have a strong
match to Fugu (pretty good for 450My)
• ¼ of human proteins had highly
diverged from, or had no pufferfish
homologs
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Shadows of the Ancient
Vertebrate Genome…
• Conserved linkages between Fugu and human
– Preservation of chromosomal chunks from the
common vertebrate ancestor (synteny)
• BUT, lots of cut/copy-paste…. And some
general scrambling of gene order
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Shadows of the Ancient
Vertebrate Genome…
• Conserved linkages between Fugu and human
– Preservation of chromosomal chunks from the
common vertebrate ancestor
• BUT, lots of cut/copy-paste…. And some
general scrambling of gene order
What a little genome…
…with little introns
• The Fugu genome is compact partly because introns
are shorter compared with the human genome
• The Fugu mode of intron size is 79 bp
– 75% of introns 425 bp in length
• The human mode is 87 bp
– 75% of introns 2609 bp
• Fugu: 500 introns > 10Kb --- Human: 12,000 > 10Kb
• The total numbers of introns are roughly the same
– 161,536 introns in Fugu
– 152,490 introns in human
What a little genome…
…with little introns
GC Content Differences
Probably related to the relative complexity of the chromatin
structure in humans versus the Fugu.
Fugu-Human Synteny
http://blast.fugu-sg.org/fugu-synteny/viewer_newServer.php
I think their maps, however, are confusing and not that informative,
-scaffolds were not physically mapped to chromosomes…
Let’s look instead at the other pufferfish, Tetraodon, that was sequenced
the following year..
-physical mapping to chromosomes was complete
Tetraodon-Human Synteny
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Comparative Genomics – Synetny
Human Chrom.1 vs. Chimp
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Comparative Genomics – Synetny
Human Chrom.1 vs. Mouse
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Comparative Genomics – Synetny
Human Chrom.1 vs. Cow
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Comparative Genomics – Synetny
Human Chrom.1 vs. Opossum
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Comparative Genomics – Synetny
Human Chrom.1 vs. Platypus
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Comparative Genomics – Synetny
Human Chrom.1 vs. Chicken
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Synteny
• Large blocks of synteny exist even at
great phylogenetic distance
• Also substantial scrambling, even at
short distance…
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Whole Genome Alignments
• Functional sequences often evolve more slowly than
non-functional sequences, therefore sequences that
remain conserved may perform a biological function.
• Comparing genomic sequences from species at
different evolutionary distances allows us to identify:
– Coding genes
– Non-coding genes
– Non-coding regulatory sequences
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The Rate of Evolution Depends on
Constraints
Human vs. Rodent Comparison
Highest substitution rates:
pseudogenes
introns
3’ flanking (not transcribed
to mature mRNA)
4-fold degenerate sites
Intermediate substitution rates:
5’ flanking (contains promoter)
3’, 5’ untranslated (transcribed
to mRNA)
2-fold degenerate sites
Lowest substitution rates:
Nondegenerate sites
Selection of Species for DNA comparisons
Human vs..
Chimpanzee
Mouse
Opossum
Pufferfish
Size (Gbp)
3.0
2.5
4.2
0.4
Time since
divergence
~5 MYA
~ 65 MYA
~150 MYA
~450 MYA
Sequence
conservation (in
coding regions)
>99%
~80%
~70-75%
~65%
Aids identification
of…
Recently
changed
sequences and
genomic
rearrangements
Both
Both
Primarily
coding and coding and
coding
non-coding non-coding sequences
sequences sequences
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Comparative Analyses of Sequence Conservation
Hypothesis: areas with high sequence similarity are likely to contain
functionally important elements:
protein-coding exons
transcription factor binding sites
These two are conceptually the same…
Phylogenetic Shadowing (fine scale)
Identifying regions that do not accumulate change
Phylogenetic Footprinting (large scale)
Identifying which regions stay somewhat conserved (identifiable)
across larger evolutionary distances
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UCSC Genome
Browser
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In these comparative genomic charts, it is easy to see why meaningful comparisons between humans and
other primates have been difficult.
The pink areas represent regions of high conservation between the two species being compared,
(meaning the sequences are the same in both), the blue areas represent the positions of protein-coding
regions and the purple areas represent the non-protein coding parts of a gene.
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Phylogenetic shadowing analyses
sequence variation in a multiple alignment
to identify regions that accumulate
variation at a slower rate.
Each position of an alignment is fitted to a
phylogenetic model to calculate the
likelihood that the position is evolving at a
fast or a slow rate (a).
Generally, positions with several sequence
differences across species are more likely
to be evolving at a fast rate, and in turn
identify the least variable regions (b).
The slowly evolving regions often
correspond to functional sequences.
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Phylogenetic Footprinting (VISTA)
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Identification of Conserved
Regulatory Elements
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Comparative analysis of multi-species sequences
from targeted genomic regions
Nature, 2003
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CFTR Locus
Encodes the protein:
Cystic Fibrosis Transmembrane Conductance Regulator
– An ion channel across the cell membrane
– The transport of chloride through CFTR helps control
the movement of water in tissues and maintain the
fluidity of mucus and other secretions
– Normal functioning ensures that organs such as the
lungs and pancreas function properly
– Most CF patients show a deletion that either leads to
an amino acid substitution, or a deletion of part of an
exon of CFTR
Comparative Genomics of the CFTR Locus
• CFTR = 1.8 Mb of human Ch7, Sequenced for 12 ssp.
• How does a single locus change over evolutionary time?
• How much does it change?
• What types of changes are more/less common?
• Do some lineages have more of certain changes than
others?
• How much comparative genomic data do we need???
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Sequence Conservation
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Looking backward from the human genome
How much is still there after 450my (Fugu)
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Differences in exon length
Data like this sure makes you wonder about
mouse models of human disease, eh?
Differences in exon lengths:
+ = insertion
-= deletion
e = extension due to
alteration of splice site or
stop codon
s = early stop codon
Transposable Elements
Gone Wild!
High Turnover in TEs
despite gene
conservation
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Nucleotide Changes
Big insertions/deletions
More common
Than nucleotide changes!
In primates, large indels are the
principal mechanism
accounting for
the observed sequence
differences
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Using evolutionary conservation to ID functionally important
conserved human genome segments
How many comparative genomes do we need – can’t we just use
the mouse? (Lots, and NO)…
Using all 12 species, they
found 561 Multi-Species
Conserved
Sequences (MCSs)
So, how many could we find
using just the Mouse genome
(rather than all 12)
False Pos.
True Pos.
False Neg.
Less than half even with high
false positives…!!!
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Multi-Species Conserved Sequences
950 of the 1,194 MCSs
are neither exonic nor lie
less than 1-kb upstream
of transcribed sequence.
Meaning they are
otherwise hard to predict
(= Evolutionary Distance)
Strong argument for comparative genomics:
Need many species, and distant species – like cat, dog, fish - to
ID conserved possibly-functional regions in humans!
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Take Home Messages…
• Identification of conserved non-coding segments beyond those previously
identified experimentally, and evidence we can find more with even more
genomes!!!
• These were not detectable by pair-wise sequence comparisons alone
– Underscores importance of comparative genomics
• Need many diverse species to figure out these questions!
• Analysis of TE insertions highlights variation in genome dynamics among
species
– The rate of TE evolutionary dynamics in vertebrates is amazing, and hugely
important for the structure and evolution of the genome
• Importance of large insertion-deletion (not necessarily nucleotide
changes) between closely related species, including humans and primates
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ENCODE Project
• Cross-reference existing with new data on human
genome function
• Identify the functional relevance of as many bases of
human genome as possible.
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ENCODE Project Findings (2007)
• A total of 5% of the bases in the genome can be confidently identified as
being under evolutionary constraint in mammals
• For ~60% of these conserved bases, evidence of function based on
experimental assays
• However, not all bases within known functional regions are evolutionarily
conserved
• Much of the variation, while functional, appears to be evolving under little
selective constraint!
– While functional, must not be important enough for “fitness” to be
highly conserved….
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Evolutionarily Conserved
Regions
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Comparative Genomics
Where do babies come from? (ask your parents)
Where do genes come from?
Evolution of Gene Families in Vertebrates
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Gene Duplication
Orthologous genes: in different
organisms, diverged from common
ancestral gene by speciation
A1 – A2 or B1 – B2
Paralogous genes: originated from
common ancestral gene via gene
duplication
A1 – B1 or A1 – B2, etc…
Homologs: genes that have the same ancestor
Orthologues and Paralogues
The Fate of Gene Duplicates
Functional Conservation – both copies can retain original function
Gene Loss – one (or both) copies can be lost either by complete
deletion or by mutation leading to a pseudogene (non-functional
copy)
Neofunctionalization – e.g., one copy may take on a new function
while the other copy retains the original function
Subfunctionalization - each copy becomes specialized for a subset
of ancestral gene’s roles (Hox genes seem to be an example)
Humans
genome
duplication
Van de Peer et al. Nature Reviews Genetics (2009)
Gene Duplication
Most gene families are small; exceptions often have an adaptive basis: immunoglobulin genes
(1000 copies in humans), olfactory receptor genes (100’s of copies in mammals)
Rho GTPases – Molecular Switches
Control cytoskeletal architecture, survival, adhesion, proliferation, motility, etc.
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Gene Gain and Loss…. In 550MY
Sea urchin is estimated to have 23,300 genes
with representatives of nearly all vertebrate gene families
•Gene families are not as large as in vertebrates
•Some genes thought to be vertebrate-specific were found in the sea
urchin
•Others were identified in sea urchin but not the chordate lineage, which
suggests loss in the vertebrates.
•The sea urchin has orthologs of genes associated with
•Vision
•Hearing
•Balance
•chemosensation in vertebrates
•raw material for current vertebrate complex sensory gene
programs)..
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Expansion of urchin-specific Rho GTPases
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Gain and loss of genes in gene families
GAIN
LOSS
Human genome has 689 genes not present in the chimp and
the chimp has 729 genes not present in humans.
Demuth et al., 2006, PLoS 1
Despite expansion-contraction of gene families, there
is little novel gain or complete loss
Opossum genome… 180MY of change
• The opossum genome contains ~18,000–20,000 protein-coding genes, the
vast majority have eutherian orthologues.
• Lineage-specific genes largely originate from expansion and rapid turnover
in gene families involved in immunity, sensory perception and
detoxification.
• Only eight currently have strong evidence of representing functional genes
without homologues in humans!
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Conclusions
• Studying biology and medicine means
studying recycled genomic material
• Studying evolution informs genomics
– Studying genomics informs evolution
• Knowing how genomes evolve can directly
inform on how they function
• More genomes = more data points for
studying how they change through
evolution, thus how they function
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