This document discusses comparative genomics and gene family analysis. It describes clustering proteins into families to study evolution, orthology, paralogy, gene duplication and loss. Gene family analysis allows detection of errors in gene structure annotation by comparing sequences. Phylogenetic trees classify gene families and reveal functional divergence. Resources for orthology analysis include Ensembl, OrthoMCLDB and YGOB. The goal of hands-on analysis is to characterize the talin 2 gene family.

1. Comparative genomics
in eukaryotes
Gene family analysis
Klaas Vandepoele, PhD
Professor Ghent University
Comparative & Integrative Genomics
VIB – Ghent University, Belgium
http://www.bits.vib.be

2. Workflow
2

3. Applications of clustering the
proteome(s)
 Gene families form the basis for the evolutionary
(or phylogenetic) analysis of
 Detection of orthologs and paralogs
 Gene duplication, family expansions,
pseudogene formation and gene loss
 Species taxonomies
 Horizontal Gene Transfer (HGT)
 Evolution of gene structure
• Introns
• Protein domain organisation &
(re)arrangements
 Base composition and codon usage
3

4. I. Structural annotation: genome-
wide versus family-wise
 Rationale family-wise annotation
 Since every gene has different (sequence)
characteristics and different genes evolve at
different rates, using these characteristics to
determine homologous gene models will
improve the overall structural annotation
quality
 Properties:
 Slow & nearly-manual procedure
 High-quality gene models revealing biological
novel findings
4

5. Workflow family-wise annotation
procedure
Collecting experi- MSA experimental Family
HMMbuild
mental representatives representatives HMM profile
EST/cDNA
BLAST Species X
proteome
Protein motifs Ab initio gene prediction
Correction gene model Putative
HMMsearch
Homologs
Classification using
Phylogenetic trees
5 Detailed characterization http://hmmer.janelia.org/

6. Experimental representatives
InterProScan
PFAM HMM logo
Clustalw + JalView
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7. BLAST / HMMsearch
1. Use multiple sequence
alignment to create HMM profile
2. Use HMM profile to search for
similar proteins
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8. Representatives + putative homologs
BioEdit Sequence Editor
Suffix finalcds indicates corrected gene model compared to the original gene model
generate by the ab-initio gene prediction
 Multiple sequence alignments assist in the detection and
correction of errors in the structural annotation (missed exon)
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9. Representatives + putative homologs
Suffix finalcds indicates corrected gene model compared to the original gene model
generate by the ab-initio gene prediction
 Multiple sequence alignments assist in the detection of errors
in the structural annotation (false first exon)
9

10. Examples of family-specific protein
motifs
 B-type cyclins have HxKF signature
 Cyclin destruction boxes (B1-type cyclin R-[AV]LGDIGN)
10

11. Examples of family-specific protein
Arabidopsis
Rice
motifs
 D-type cyclins contain LxCxE Rb-binding motif
 Low conservation of phylogenetic signal at primary sequence level
 General rules are rarely general: exceptions (i.e. missing protein
motifs) are frequent and might indicate functional divergence
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12. Classification using phylogenetic
tree construction
A- and B-type cyclins
are mitotic cyclins
D-type cyclins are
G1-specific
H-type cyclins regulate activity
of CDK-activating kinases
• The complexity of the cyclin gene family appears to be higher in plants than in
mammals
• Whether there is functional redundancy within A- and B-type cyclins or different
regulation (and expression) of some cyclin subclasses remains to be analyzed
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13. Unraveling functional divergence using
Genes large-scale expression compendia
13
Plant tissues

14. Unraveling functional divergence using
large-scale expression compendia
A-type cyclin
B-type cyclin
Genes
D-type cyclin
14
Plant tissues Genevestigator

15. II. Orthology & paralogy
 A major goal of sequence analysis is evolutionary
reconstruction. It is critical to distinguish between two
principal types of homologous relationships, which differ
in their evolutionary history and functional implications.
 Orthologs, defined as homologous genes evolved
through speciation (~evolutionary counterparts derived
from a single ancestral gene in the last common ancestor
of the given two species)
 Paralogs, which are homologous genes evolved through
duplication within the same (perhaps ancestral) genome.
 These definitions were first introduced by Fitch (1970)
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16. Orthology & paralogy inference
Organism phylogeny Gene phylogenies
(species tree) gene duplication
a1
A
b1
B c1
a1
b) a2
a2
C b2
b1
c2
a) b2
speciation Outparalogs
16 Inparalogs c1

17. In- and outparalogy
17 Sonnhammer & Koonin: Orthology, paralogy and proposed classification for paralog subtypes

18. Tree reconciliation
 The automatic detection of speciation and duplication
events using a species tree and gene family tree
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19. III. Types of proteome analysis
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20. The evolution of multi-domain
proteins
20

21. Interpreting the output of an all-
against-all similarity search
Metrics for sequence similarity:
• E-value, Bit score or percent identity
21 • alignment coverage

22. Clustering of similar sequences
Proteins = vertices ~ nodes
Sequence similarity relationship = edges
22

23. Clustering of similar sequences
23

24. Advanced methods for protein
(orthology) clustering
 Sequence similarity-based
 COG (RBH) [Tatusov 1997]
 InParanoid [Remm et al., 2001]
 Tribe-MCL [Van Dongen 2000]
 OrthoMCL [Li et al., 2003]
 Phylogenetic tree-based
 PhylomeDB [Huerta-Cepas et al., 2007]
 Ensembl Compara [Vilella et al., 2008]
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25. Overview methodologies
BBH
Inparanoid
COG
species overlap
25 Gabaldon, 2008
reconciliation

26. IV. Resources
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27. Resources (bis)
 Ensembl (Vertebrates)
 EnsembGenomes (Metazoa, Protists,
Fungi, Plants & Bacteria)
 OrthoMCLDB 5 (150 genomes)
 YGOB (>15 Fungi)
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28. Hands-on
 Goal: identify and characterize gene family
members encoding for talin 2 (TLN2)
1. Select Query gene
2. Retrieve homo/orthologs
3. Create multiple sequence alignment
4. Identify conserved positions
5. Create phylogenetic tree and identify
ortho/paralogous genes
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