1. Genome Biology and Biotechnology
Genoom Biologie
Prof. M. Zabeau
Genome Biology and Biotechnology
3. The genome structures of vertebrates
Prof. M. Zabeau
Department of Plant Systems Biology
Flanders Interuniversity Institute for Biotechnology (VIB)
University of Gent
International course 2005
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2. The Genome Sequences of vertebrates
Fish genomes: “compact” vertebrate genomes
Fugu rubripes (2002)
Tetraodon nigroviridis (2004)
Bird genome: Interesting evolutionary intermediate
Chicken - Gallus gallus (2004)
Rodent genomes: the model organism for the human
Mouse - mus musculus (2002)
Rat – Rattus norvegicus (2004)
Primate genomes: our closest relatives
Chimpanzee
Human genome
Draft genome sequence (2001)
Finished genome sequence (2004)

3. vertebrate evolution 310 MY 450 MY
Genoom Biologie
Prof. M. Zabeau
310 MY
450 MY
Figure 1 Basal vertebrate evolution showing extant species whose genomes have been sequenced. The horizontal axis represents estimated relative species diversity. The Archosauria include the Aves, their Mesozoic dinosaur predecessors, and Crocodilia; the Lepidosauria (lizards, snakes and tuataras) are not indicated. Archaeopteryx (indicated by an asterisk) is considered to be the first known bird and lived approximately 150 Myr ago.
Reprinted from: ICGSC, Nature 432, (2004)
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4. Aparicio et al., Science, 297, 1301-1310 (2002)
Whole-Genome Shotgun Assembly and Analysis of the Genome of Fugu rubripes
Aparicio et al., Science, 297, (2002)
Paper presents
Low quality draft genome sequence of Fugu rubripes
the sequence provided a valuable reference for annotating the human and mouse genomes
Small genome (350 Mb versus 3000 Mb)
low repetitive DNA content

5. The Fugu Genome Sequence
The draft sequence covers a total of 332.5 Mb
Highly fragmented sequence (~30 Mb unassembled sequences)
The total genome size is estimated at ~365 Mb
The number of predicted genes: 31,059
similar to the number of human genes predicted from the draft sequence
Repetitive sequences
Density of <15% far below the 35 to 45% observed in mammals
Transposable elements are still very active
Reprinted from: Aparicio et al., Science, 297, (2002)

6. Protein-coding genes The gene-containing fraction is a ~ 108 Mb (30%)
The average gene density: one gene per 10.9 kb
The Fugu genome is compact because introns are shorter than in the human genome
Genome contains ~500 large introns (> 10 kb) compared > 12,000 large introns in human
Genes are scaled in proportion to the compact genome size
The number of introns is roughly the same as in human
Both gain and loss of introns in the Fugu lineage are observed
The compactness of the Fugu is accounted for by
Low abundance of repeated sequences
The small size of introns and intergenic regions
Reprinted from: Aparicio et al., Science, 297, (2002)

7. Comparison of Fugu and Human Proteomes
75% of predicted human proteins have a strong match to Fugu
Reprinted from: Aparicio et al., Science, 297, (2002)

8. Jaillon et. al., Nature 431, 946 - 957 (2004)
Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype
Jaillon et. al., Nature 431, (2004)
Paper presents
High quality draft genome sequence with long-range linkage and chromosome anchoring of Tetraodon nigroviridis
freshwater puffer fish with the smallest known vertebrate genome

9. The Tetraodon genome sequence
The draft genome sequence (8,3 x) spans 342 Mb
Largest scaffolds were mapped onto the chromosomes
Draft is much less fragmented than that of Fugu
Genome landscape
Transposable elements are very rare (<4000 copies)
Fewer than Fugu (15% of the genome)
Estimated 20,000–25,000 protein coding genes
Very similar to the recent (2004) human gene count
Much lower than reported for Fugu (current Fugu is also lower)
Gene ontology (GO) classifications shows only subtle differences between fish and mammals
Improved fish gene catalogue aids human gene predictions
Reprinted from: Jaillon et. al., Nature 431, (2004)

10. Evidence For Whole-genome Duplication
Duplicated genes cluster on paralogous chromosomes
paralogous chromosomes arising from whole-genome duplication each contain one member of duplicated gene pairs in the same order
Reprinted from: Jaillon et. al., Nature 431, (2004)

11. Evidence For Whole-genome Duplication
Blocks of doubly conserved synteny
The synteny map typically associates two regions in Tetraodon with one region in human
Tni: Tetraodon
Hsa: human
Reprinted from: Jaillon et. al., Nature 431, (2004)

12. Ancestral genome of bony vertebrates
The patterns of doubly conserved synteny are consistent with
12 ancestral chromosomes which have rearranged to form
the present day chromosomes of human and fish
Human
Fish
Reprinted from: Jaillon et. al., Nature 431, (2004)

13. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution
International Chicken Genome Sequencing Consortium, Nature 432, (2004)
Paper presents
a draft genome sequence of the red jungle fowl Gallus gallus
The first genome of non-mammalian amniote
provides a new perspective on vertebrate genome evolution
The evolutionary distance between chicken and human provides an excellent signal-to-noise ratio to detect functional elements
310 MY since the divergence of birds and mammals

14. The chicken genome sequence
Genoom Biologie
Prof. M. Zabeau
The chicken genome sequence
The draft genome sequence (6,6 x) spans Mb
Draft represents ~96% of the euchromatic part of the genome
23,212 chicken mRNAs and 485,000 ESTs
Chicken genome is 3x smaller than mammalian genomes reflecting substantially fewer
interspersed repeats
transposable elements make up <9% of the genome, markedly lower than the 40–50% observed in mammalian genomes
Pseudogenes
51 retrotransposed genes vs. > 15,000 in mammalian genomes
segmental duplications
Limited to very small (<10kb) intrachromosomal duplications
Reprinted from: ICGSC, Nature 432, (2004)
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15. Gene content of the chicken genome
Genoom Biologie
Prof. M. Zabeau
Gene content of the chicken genome
Protein-coding genes
Predict 20,000 to 23,000 protein-coding genes
Matches the current (2004) estimate for mammalian genomes
Non-coding RNA genes
571 ncRNA genes from >20 gene families
Fewer than in human: many ncRNA genes are pseudogenes
Syntenic relationships for non-coding RNA genes differ from those of protein-coding genes
implies a novel mode of evolution for some ncRNA genes
Only certain ncRNA genes are in regions of conserved synteny
microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) found in introns of protein-coding genes
Reprinted from: ICGSC, Nature 432, (2004)
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16. Evolutionary conservation of gene components
Genoom Biologie
Prof. M. Zabeau
Evolutionary conservation of gene components
Sequence conservation of chicken and human orthologs
highest in protein-coding exons
minimal in introns
Significant in the 5' and 3' flanking and untranslated regions
Figure 2 An idealized protein-coding gene structure showing average percentage alignment and average percentage identity (including gaps and unaligned regions) over 10,000 orthologous gene structures in either human–chicken, human–mouse or mouse–rat alignments (as aligned by BLASTZ156). The reference structure was taken from human or mouse, and only those with cDNA-based definitions of the structure were used. The central figure shows an idealized gene structure, with the grey exons representing coding sequence and white boxes representing 3' and 5' untranslated regions.
5’UTR
exon
3’UTR
Reprinted from: ICGSC, Nature 432, (2004)
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17. Conservation of vertebrate protein content
Genoom Biologie
Prof. M. Zabeau
Conservation of vertebrate protein content
60% of chicken genes have a single human orthologue
also have a single orthologue in the Fugu genome
Represent a conserved core present in most vertebrates
Figure 5 Chicken genes classified according to their predicted evolutionary relationships with genes of two other model vertebrates (Fugu and human). Forty-three per cent of the chicken genes are present in 1:1:1 orthology relationships for the three species. Also present in three species are n:n:n (many:many:many) orthologues; putative gene duplication events have resulted in multiple genes in at least one of the species. Pairwise orthologues are assigned when orthology is not detectable in the third species. Between Fugu and chicken, pairwise orthologues are rare (as expected), and might be indicative of gene loss in the lineage leading to humans. For a substantial number of genes, clear orthology relations cannot be described at all, but some similarity to genes in the other species remains detectable ('Homology', E-value cutoff is 10-6 in Smith–Waterman searches at the protein level). See Methods for details of orthology assignment.
Reprinted from: ICGSC, Nature 432, (2004)
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18. Conserved core orthologues in vertebrates
Core orthologues conserved in vertebrates have
Highly conserved protein sequences indicating that
They have been subject to purifying selection
Reprinted from: ICGSC, Nature 432, (2004)

19. Expansion of multigene families
Genoom Biologie
Prof. M. Zabeau
Expansion of multigene families
Expansion and contraction of multigene families were
major factors in the independent evolution of mammals and birds
Figure 7 Loss, innovation, expansions and contractions of protein families: domain counts and orthologous relations. All at-least-twofold over- and under-representations (separated by a solidus) are shown for both members of domain families (a) and 'many to many' orthology relations (b). Ranking of families and groups has been done with respect to the human genome; Fugu data are also shown for comparison. An asterisk indicates that manual analyses refined what were otherwise automatic counts. Families not subjected to twofold variations are not shown.
Reprinted from: ICGSC, Nature 432, (2004)
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20. Chromosomal dynamics in the vertebrates
Genoom Biologie
Prof. M. Zabeau
Chromosomal dynamics in the vertebrates
Maps of conserved synteny: orthologous chromosomal segments with conserved gene order show
slow rate of rearrangement in the human lineage
3-fold higher rate in the rodent lineage
The human genome is closer to the chicken in terms of synteny
Figure 14 Rates of genome structure divergence. a, Phylogenetic tree of chicken, human and mouse showing rates of genome structure divergence, where the branch length is proportional to the number of estimated chromosomal rearrangements. Details are given in the supporting table (b). AA, amniote ancestor; MA, mammalian ancestor. The pie charts show the fraction of orthologous genes that have retained their genomic neighbourhood; for example, about 85% of chicken and human orthologous genes reside in orthologous chromosomal segments, and their sizes are proportional to the number of recognizable orthologous genes. b, The table highlights a very low rate of interchromosomal shuffling in early mammalian and bird evolution, and an elevated rate of interchromosomal shuffling in the rodent lineage. It provides a summary of the number of chromosomal rearrangements estimated by counting synteny breaks where ancestral state is supported by synteny to an outgroup species, and by reconstruction of ancestral genomes through a combinatorial search for the most parsimonious rearrangement pattern. The two methods agree reasonably and have been used to estimate the relative branch lengths of the genome structure divergence tree in a. Normalization to the length of the MA–human branch and to the time of independent evolution is presented for easy comparison.
Reprinted from: ICGSC, Nature 432, (2004)
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21. Ancestral mammalian genome
Genoom Biologie
Prof. M. Zabeau
Long blocks of conserved chicken–human synteny
Entire chromosomes
Genome rearrangements
Many intrachromosomal rearrangements
Few translocations between chromosomes
Chicken has a number of micro-chromosomes
Figure 12 Putative mammalian ancestor recovered by GRIMM and MGR using the human, mouse, rat (not shown) and chicken genomes. a, Each genome is represented as an arrangement of 586 synteny blocks each drawn as one unit, regardless of its length in nucleotides. Each human chromosome is assigned a unique colour, and a diagonal line is drawn through the whole chromosome. In other genomes, this diagonal line indicates the relative order and orientation of the rearranged blocks. b, The recovered ancestral X chromosome is optimal and unique. Gene order of the ancestral X chromosome is identical to human. Numbers associated with the lines indicate the minimum number of rearrangements required to convert between two nodes.
Reprinted from: ICGSC, Nature 432, (2004)
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22. Conserved sequences in chicken and human
Genoom Biologie
Prof. M. Zabeau
Conserved sequences in chicken and human
High substitution rates between human and chicken
Can be used to detect functionally conserved sequences
70 Mb (2.5%) of human sequence aligns with chicken
44% are in protein-coding regions - exons
66% is non-coding: intronic (25%) and intergenic (31%)
Conserved non-coding segments occur clustered and far from genes
Identified 57 segments with average length of 1,1 MB
gene poor, G+C poor and have no interspersed repeats
the functional significance of these sequences is completely unknown
Reprinted from: ICGSC, Nature 432, (2004)
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23. Conclusion The chicken genome sequence
Genoom Biologie
Prof. M. Zabeau
Conclusion
The chicken genome sequence
is a key resource for comparative genomics
to distinguish derived or ancient features of mammalian biology
mammalian innovation and adaptation
conserved non-coding sequences in particular
Provides a framework for discovering the functional polymorphisms underlying
interesting quantitative traits to further exploit the genetic potential of the chicken
Figure 1 This shows the relative number of ORFs assigned to individual categories in the Gazetteer. There are eleven functional categories. Only proteins with a known function, or similarity or strong similarity to known proteins were assigned to one of the categories (similarities were measured by FASTA scores). In total, 3167 ORFs were assigned to at least one category. A single ORF can be assigned to more than one category
Reprinted from: ICGSC, Nature 432, (2004)
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24. Initial sequencing and comparative analysis of the mouse genome
Mouse Genome Sequencing Consortium, Nature 420, (2002)
Paper presents
Draft genome sequence of the mouse
comparative analysis of the mouse and human genomes
75 MY since the divergence of rodents and primates
The two genome sequences diverge by nearly one substitution for every two nucleotides
the insights that can be gleaned from the two sequences

25. The Mouse Genome Project
The laboratory mouse is an experimental model system
for studying human disease and mammalian biology
The Mouse Genome Project
International collaboration of centres in the US and the UK
Adopted mixed strategy for the draft genome sequencing
a BAC-based physical map of the mouse genome
The initial draft genome sequence was generated by
WGS sequencing to ~7-fold coverage
Hierarchical shotgun sequencing of BAC clones
The finished sequence should be completed in 2005
using the BAC clones for directed finishing
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

26. The draft mouse genome sequence
The euchromatic mouse genome is estimated ~2.5 Gb
The draft genome sequence covers ~96% of the genome
Generation of the draft genome sequence
Sequencing
41.4 Mi paired-end sequence reads derived from various clone types
Assembly
represents ~7.7-fold sequence coverage
224,713 sequence contigs
total of 7,418 supercontigs
The 200 largest supercontigs span more than 98% of the assembled sequence
Anchoring to chromosomes
Anchored all supercontigs >500 kb with the mouse genetic map
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

27. The draft mouse genome sequence
The euchromatic mouse genome is estimated ~2.5 Gb
The draft genome sequence covers ~96% of the genome
Comparative analysis of human and mouse genomes
The mouse genome is about 14% smaller than the human genome
High degree of synteny
>90% of the two genomes can be partitioned into corresponding regions of conserved synteny
At the nucleotide level, approximately 40% of the human genome can be aligned to the mouse genome.
represent orthologous sequences conserved from the common ancestor
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

28. Synteny between mouse and human
Genoom Biologie
Prof. M. Zabeau
Synteny between mouse and human
Regions containing orthologous sequence pairs define
Syntenic segments as regions in which
Orthologous sequence pairs are in the same order on a chromosome in both species
Figure 2 Conservation of synteny between human and mouse. We detected 558,000 highly conserved, reciprocally unique landmarks within the mouse and human genomes, which can be joined into conserved syntenic segments and blocks (defined in text). A typical 510-kb segment of mouse chromosome 12 that shares common ancestry with a 600-kb section of human chromosome 14 is shown. Blue lines connect the reciprocal unique matches in the two genomes. The cyan bars represent sequence coverage in each of the two genomes for the regions. In general, the landmarks in the mouse genome are more closely spaced, reflecting the 14% smaller overall genome size.
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)
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29. Synteny between mouse and human
Conservation of orthologous sequence pairs shows
Each genome can be parsed into a total of 342 conserved syntenic segments.
The segments vary greatly in length, from 303 kb to 64.9 Mb
In total, about 90.2% of the human genome and 93.3% of the mouse genome reside within conserved syntenic segments
The segments can be aggregated into a total of 217 conserved syntenic blocks
The syntenic block and segment sizes are
consistent with the random breakage model of genome evolution
the minimal number of rearrangements needed to 'transform' one genome into the other is 295 rearrangements
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

30. Blocks of conserved synteny in the human and mouse genomes
Genoom Biologie
Blocks of conserved synteny in the human and mouse genomes
Prof. M. Zabeau
Figure 3 Segments and blocks >300 kb in size with conserved synteny in human are superimposed on the mouse genome. Each colour corresponds to a particular human chromosome. The 342 segments are separated from each other by thin, white lines within the 217 blocks of consistent colour
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)
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31. Repetitive sequences in human and mouse
The most prevalent feature of mammalian genomes is their high content of repetitive sequences
Most of which are interspersed repeats representing 'fossils' of transposable elements
The repetitive sequences in mouse and human differ
Only 37.5% of the mouse genome
~46% of the human genome is transposon-derived
Insertions of transposable elements occured in the last 150–200 million years
The most notable difference is the rate of transposition over time
in mouse the rate has remained fairly constant
in human the rate increased to a peak at ~40 Myr, and then plummeted
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

32. Age distribution of interspersed repeats in the mouse and human genomes
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

33. Protein-coding genes in mouse and human
Human and mouse gene catalogues
The current human gene catalogue (Ensembl build 29) contains 22,808 predicted genes
The current mouse gene catalogue contains 22,011 predicted genes
Comparative analysis of protein coding genes shows
80% of the mouse genes have orthologues in the human genome
The proportion of mouse/human genes without any homologue is < 1%.
Many local gene family expansions have occurred in the mouse lineage
Most seem to involve genes for reproduction, immunity and olfaction
The rate of protein evolution
Most proteins evolve at fairly constant rate
Certain proteins evolve much more rapidly: positive selection
Proteins implicated in reproduction, host defence and immune response seem to be under, which drives
Reprinted from: Mouse Genome Sequencing Consortium, Nature 420, (2002)

34. Conclusions
The mouse genome provides a powerful resource to unravel the secrets of the human genome
Demonstrates the power of comparative genomics in identifying relevant genetic elements
These findings inspired additional animal genome sequencing projects to fully exploit the power of comparative genomics
As illustrated in: Thomas et. al., Nature 423, (2003)
The sequence provides a comprehensive framework for functional genomics approaches to unravel gene functions in both human and mouse

35. Genome sequence of the Brown Norway rat yields insights into mammalian evolution
RGSPC, Nature 424: (2004)
Paper presents
a high-quality 'draft' sequence covering > 90% of the genome
a three-way comparison with the human and mouse genomes to study the mammalian genome evolution
Rat - mouse common ancestor: 12–24 Myr
Rodent - human common ancestor: 75 Myr

36. The rat genome sequence
Genoom Biologie
Prof. M. Zabeau
The rat genome sequence
The draft genome sequence covers 2,75 Gb
A 'combined' sequencing strategy using
WGS sequencing and light sequence coverage of BACs
Sequential assembly of 'enriched BACs' (eBACs) joined into bactigs, superbactigs and ultrabactigs
Figure 1 The new 'combined' sequence strategy and Atlas software. a, Formation of 'eBACs'. The RGSP strategy combined the advantages of both BAC and WGS sequence data54. Modest sequence coverage ( 1.8-fold) from a BAC is used as 'bait' to 'catch' WGS reads from the same region of the genome. These reads, and their mate pairs, are assembled using Phrap to form an eBAC. This stringent local assembly retains 95% of the 'catch'. b, Creation of higher-order structures. Multiple eBACs are assembled into bactigs based on sequence overlaps. The bactigs are joined into superbactigs by large clone mate-pair information (at least two links), extended into ultrabactigs using additional information (single links, FPC contigs, synteny, markers), and ultimately aligned to genome mapping data (radiation hybrid and physical maps) to form the complete assembly.
eBAC
Reprinted from: RGSPC, Nature 424: (2004)
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37. Rat – mouse – human genome sequences
Genoom Biologie
Prof. M. Zabeau
Rat – mouse – human genome sequences
Sequence elements in human, mouse and rat genomes
40% align in all 3 species
'ancestral core' of 1 Gb
95% of the exons and regulatory regions
28% aligns only with mouse
rodent-specific repeats
29% does not align
rat-specific repeats
Figure 7 Aligning portions and origins of sequences in rat, mouse and human genomes. Each outlined ellipse is a genome, and the overlapping areas indicate the amount of sequence that aligns in all three species (rat, mouse and human) or in only two species. Non-overlapping regions represent sequence that does not align. Types of repeats classified by ancestry: those that predate the human–rodent divergence (grey), those that arose on the rodent lineage before the rat–mouse divergence (lavender), species-specific (orange for rat, green for mouse, blue for human) and simple (yellow), placed to illustrate the approximate amount of each type in each alignment category. Uncoloured areas are non-repetitive DNA—the bulk is assumed to be ancestral to the human–rodent divergence. Numbers of nucleotides (in Mb) are given for each sector (type of sequence and alignment category). Detailed results are tabulated (Supplementary Table SI-1).
Reprinted from: RGSPC, Nature 424: (2004)
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38. Evolution of genes Estimate that 90% of rat genes possess
Genoom Biologie
Prof. M. Zabeau
Evolution of genes
Estimate that 90% of rat genes possess
strict orthologues in both mouse and human
Intronic structures are well conserved
Most of the non-orthologous genes
Arose by expansions of gene families in the different lineages
Rapidly evolving genes
Rat-specific genes comprise novel genes for “life style”
pheromones, immunity, chemosensation, detoxification, proteolysis
Reprinted from: RGSPC, Nature 424: (2004)
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39. Rat – mouse – human synteny
Genoom Biologie
Prof. M. Zabeau
Rat – mouse – human synteny
orthologous chromosome segments
105 mouse–rat segments
278 human-rat segments
280 human-mouse segments
Figure 4 Map of conserved synteny between the human, mouse and rat genomes. For each species, each chromosome (x axis) is a two-column boxed pane (p arm at the bottom) coloured according to conserved synteny to chromosomes of the other two species. The same chromosome colour code is used for all species (indicated below). For example, the first 30 Mb of mouse chromosome 15 is shown to be similar to part of human chromosome 5 (by the red in left column) and part of rat chromosome 2 (by the olive in right column). An interactive version is accessible (
Reprinted from: RGSPC, Nature 424: (2004)
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40. Rat – mouse – human genome rearrangements
Genoom Biologie
Prof. M. Zabeau
Rat – mouse – human genome rearrangements
Reconstruction of the ancestral mammalian genome
Identified a total of 353 rearrangements
247 between the murid ancestor and human
50 from the murid ancestor to mouse
56 from the murid ancestor to rat
much higher (3x) rearrangement rate in the rodent than in the human lineage
247
Figure 5 Substitutions and microindels (1–10 bp) in the evolution of the human, mouse and rat genomes. a, The lengths of the labelled branches in the tree are proportional to the number of substitutions per site inferred using the REV model222 from all sites with aligned bases in all three genomes. b, The table shows the midpoint and variation in these branch-length estimates when estimated from different sequence alignment programs and different neutral sites, including sites from ancestral repeats3, fourfold degenerate sites in codons, and rodent-specific sites ('in neutral sites only' row; Supplementary Information). Other rows give midpoints and variation for micro-indels on each branch of the tree in a 50 56
Reprinted from: RGSPC, Nature 424: (2004)
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41. The Human Genome The human genome project was launched in 1990
Phase I: generation of genetic and physical maps ( )
Demonstration that large scale sequencing is feasible: yeast, worm
Phase II: large scale sequencing ( )
Pilot phase: finished sequence with 99.99% accuracy and no gaps of the human chromosomes 21 and 22 (published in ’98 and ‘99)
Draft phase : draft sequence covering >90% of the genome completed in June 2000 (published in ) – took ~1 year
Finishing phase: “finished” covering 99% of the genome sequence, completed in spring 2004 – took ~3 years
Aftermath: no end point projected
closing the last couple of hundred gaps
Sequencing the centromeres
Reprinted from: International Human Genome Sequencing Consortium Nature 409, 860 (2001)

42. The Human Genome Sequences
Draft genome sequences (2001)
International Human Genome Sequencing Consortium (Collaboration of 20 public sequencing centers in 6 countries)
Used a hierarchical shotgun sequencing strategy
Sequence published in: Nature 409, 860 (2001)
Celera Genomics - private initiative
Used a whole-genome shotgun approach
Assembly of the sequence combined their whole-genome shotgun data and the public genome sequence data
Sequence published in: Venter et. al., Science, 291, 1304 (2001)
Finished genome sequence (2004)
International Human Genome Sequencing Consortium
Sequence published in: Nature 431, (2004)

43. Finished Human Genome Sequence
Finishing process: complex iterative process
Resolving problematic sequences
From single nucleotide errors and gaps to the integrity of whole chromosomes
The finishing process involved two distinct components
producing finished maps consisting of continuous and accurate paths of overlapping large-insert clones
producing finished clone sequences, consisting of continuous and accurate nucleotide sequences for each clone
generated shotgun sequence of ~ BACs comprising a total sequence (redundant) length of 5,8 Gb
Assembled sequences of ~ BACs
Reprinted from: International Human Genome Sequencing Consortium Nature 431, (2004)

44. Finished Human Genome Sequence
Finished genome sequence
Build 35 comprises Mbp
Interrupted by “only” 341 gaps
308 gaps in the euchromatic sequence: totalling ~28 Mb
33 heterochromatic gaps (including 24 centromeres) : total ~198 Mb
The total human genome size is estimated at ~3,080 Mb
Comparison with draft sequence
Substantially fewer gaps (341 versus 147,821)
More accurate and complete sequence: error rate ~1 per 105
Confirmed local order and orientation of the sequences
Corrected artefactual duplications resulting from mixups
Verified most of the sequence with
BAC cloned overlap sequence, paired end sequence reads from fosmids, draft chimpanzee genome sequence
Reprinted from: International Human Genome Sequencing Consortium Nature 431, (2004)

45. Finished Human Genome Sequence
Importance of a completely finished genome sequence
Accurate reference for identifying genetic variation in the human population
Error rate of 10-5 << frequency of SNP of 10-3
Identification of segmental duplications
Estimated to cover >5% of the genome sequence
Located primarily in the pericentromeric and subtelomeric regions
much higher than in mouse and rat
Great medical interest: predisposes to deletion or rearrangement
Williams syndrome region (7q)
Charcot–Marie–Tooth region (17p)
DiGeorge syndrome region (22q)
Many remaining gaps involve unresolved segmental duplications
Correct identification of all protein- coding genes structures
~60% of the gene models were corrected compared to the draft
Reprinted from: International Human Genome Sequencing Consortium Nature 431, (2004)

46. Estimates of the Number of Human Genes
Reassociation kinetics (60s and 70s)
Early estimates based estimated the mRNA complexity of typical vertebrate tissues to be 10,000–20,000, and were extrapolated to suggest around 40,000 genes for the entire genome
Estimates from approximate gene and genome sizes
Calculation based on the size of a typical gene ( 3*104 bp) and the size of the genome (3*109 bp) yielded 100,000 genes (W. Gilbert, pers. Com.)
Number of CpG islands associated with known genes
An estimate of 70,000–80,000 genes was made
Estimates based on ESTs
Estimates based on ESTs varied widely, from 35,000 to 120,000 genes
Discrepancy results from contaminating genomic sequences and multiple ESTs from single genes
Whole-genome shotgun sequence from the pufferfish
Suggested around 30,000 human genes
Reprinted from: International Human Genome Sequencing Consortium Nature 409, 860 (2001)

47. Identification of Protein Coding Genes
Draft genome sequence
Initial estimate: – genes
Finished genome sequence
The human gene catalogue contains 22,287 gene loci consisting of 19,438 known genes and 2,188 predicted genes
Current estimate: – genes
is an upper limit, the actual number may be ~23.000
Consistent with gene counts in other vertebrates: fish and chicken
Reprinted from: International Human Genome Sequencing Consortium Nature 431, (2004)

48. Basic Characteristics of Gene Structures
Mean and median values of gene structures
Based on the draft sequence
In particular, the UTRs in the RefSeq database are incomplete
Reprinted from: International Human Genome Sequencing Consortium Nature 409, 860 (2001)

49. Protein Coding Genes General features of human genes
average coding length of about 1,400 bp
Similar to al eukaryotic organisms
average genomic extent of about 30 kb
Much larger than in lower eukaryotes
The variation in gene and intron size
GC-rich regions: gene-dense with many compact genes
AT-rich regions: gene-poor with genes containing large introns
Known and predicted exons: ~
1,2% of the human genome
Average of 10.4 exons per gene
Pseudogenes
Current estimates: processed and unprocessed pseudogenes
The total number of pseudogenes is thus likely to exceed the total number of functional genes
Only those of recent origin can be identified with confidence
Reprinted from: International Human Genome Sequencing Consortium Nature 431, (2004)

50. Basic Characteristics of Gene Structures
High variation in overall intron size
distribution has very long tails
Many genes are over 100 kb long
Largest gene: dystrophin gene (DMD) 2.4 Mb
longest known coding sequence: titin gene 80,780 bp, 178 exons
Reprinted from: International Human Genome Sequencing Consortium Nature 409, 860 (2001)

51. Comparison with fly, worm and yeast
Apparent homologues of human proteins
40% to 60% of the yeast, worm and fly proteomes
Human genes differ from those in worm and fly
Spread out over much larger regions of genomic DNA
Have a substantially larger number of exons
4,5 to 5 in fly and worm compared to 10,4 in human
Are used to construct more alternative transcripts
Larger number of proteins in human than in the worm or fly
Increased complexity of the proteome
Complexity of the human proteome is a consequence of large-scale protein innovation
Multi-domain proteins with multiple functions, and domain architectures
Reprinted from: International Human Genome Sequencing Consortium Nature 409, 860 (2001)

52. Protein Coding Gene Evolution in Human
Gene birth in the human lineage
gene duplications that arose after divergence from the mouse
Identified 1,183 gene clusters containing 3,300 recently duplicated genes ( with a peak 3–4 million years ago ) enriched in genes with
immune function
olfactory function
reproductive functions
Duplicated genes are the raw material for adaptive evolution:
extra copies are able to undergo functional divergence in response to positive selection
Gene death in the human lineage
Recently inactivated genes include genes in olfactory function
Reprinted from: International Human Genome Sequencing Consortium Nature 431, (2004)

53. Genoom Biologie
Prof. M. Zabeau
Future Perspectives
Vertebrate genome sequencing projects ongoing or planned (currently totaling 25)
Fish: zebrafish, salmon, tilapia, stickleback and Japanese medaka
Amphibians: Xenopus laevis and X. tropicalis
Birds: turkey
Mammals: ~15 additional species
cow, pig, cat, dog, horse, rabbit, guinea pig, elephant, kangaroo, shrew,….
Primates: chimp, orangutan, baboon and rhesus monkey
Source: GOLDTM Genomes OnLine Database
Xenopus laevis and X. tropicalis
Academiejaar

54. Recommended reading Genome sequences
The sequencing of the human genome
International Human Genome Sequencing Consortium Nature 409, 860 (2001)
International Human Genome Sequencing Consortium Nature 431, (2004)
The sequencing of the mouse genome
Mouse Genome Sequencing Consortium, Nature 420, (2002)
Chicken genome sequence
International Chicken Genome Sequencing Consortium, Nature 432, (2004)

55. Further reading Vertebrate genome sequences Fish genome sequences
Aparicio et al., Science, 297, (2002)
Jaillon et. al., Nature 431, (2004)
Rat genome sequence
RGSPC, Nature 424: (2004)
Human genome sequence - Celera Genomics - private initiative
Venter et. al., Science, 291, 1304 (2001)