Genome Paleontology:
Discoveries from complete
genomes
Steven L. Salzberg
The Institute for Genomic Research (TIGR)
and Johns Hopkins University
What is genome paleontology?
Compare genomes
to uncover:
• history of species
• genome
transformations
• recent mutations
such as SNPs
• evolution
© 2003 Steven L. Salzberg
2
Outline (time permitting)

An algorithm for rapid large-scale alignment
A.L. Delcher, S. Kasif, R.D. Fleischmann, J. Peterson, O. White, and S.L.
Salzberg. Alignment of whole genomes. Nucleic Acids Res 27:11
(1999), 2369-76. MUMmer 2: Delcher et al., NAR, 2002.

Alignments and analyses of bacterial genomes
J.A. Eisen, J.F. Heidelberg, O. White, and S.L. Salzberg. Evidence for
symmetric chromosomal inversions around the replication origin in
bacteria. Genome Biology 1:6 (2000), 1-9.

Large-scale genome duplications: plant and human
• The Arabidopsis Genome Initiative. Analysis of the genome sequence
of the flowering plant Arabidopsis thaliana. Nature 408 (2000), 796815.
• J.C. Venter et al. The sequence of the human genome. Science 291
(2001), 1304-1351.
● Lateral gene transfer between humans and bacteria
• S.L. Salzberg, O. White, J. Peterson, and J.A. Eisen. Microbial genes
in the human genome: lateral transfer or gene loss? Science 292
(2001), 1903–1906.
Organism
Arabidopsis thaliana
Archaeoglobus fulgidus
Bacillus anthracis Ames
Bacillus anthracis Florida
Borrelia burgdorferi
Brucella suis
Caulobacter crescentus
Chlamydia pneumoniae
Chlamydia muridarum
Chlamydophila caviae
Chlorobium tepidum
Coxiella burnetii RSA 493
Deinococcus radiodurans
Enterococcus faecalis
Haemophilus influenzae
Helicobacter pylori
Methanococcus jannaschii
Mycobacterium tuberculosis
Mycoplasma genitalium
Neisseria meningitidis
Oryza sativa (rice) chr 10
Plasmodium falciparum
Plasmodium yoelii
Porphyromonas gingivalis
Pseudomonas putida
Shewanella oneidensis
Streptococcus agalactiae
Streptococcus pneumoniae
Sulfolobus islandicus virus
Thermotoga maritima
Treponema pallidum
Vibrio cholerae
Reference
Lin et al., Nature 402: 761-8 (2000)
Klenk et al., Nature 390:364-370 (1997)
Read et al., Nature 423: 81-86 (2003)
Read et al., Science 296, 2028-33 (2002)
Fraser et al., Nature 390: 580-586 (1997)
Paulsen et al., PNAS 99 (2002)
Nierman et al., PNAS 98 (2001)
Read et al., Nucl. Acids Res. 28, (2000)
Read et al., Nucl. Acids Res. 28, (2000)
Read et al., Nucl. Acids Res. 31, (2003)
Eisen et al., PNAS 99: 9509-9514 (2002)
Seshadri et al., PNAS 100: 5455-60 (2003)
White et al., Science 286 (1999)
Paulsen et al., Science 299: 2071-2074 (2003)
Fleischmann et al., Science 269, (1995)
Tomb et al., Nature 388:539-547 (1997)
Bult et al., Science 273:1058-1073 (1996)
Fleischmann et al., J. Bact.184, (2002)
Fraser et al., Science 270:397-403 (1995)
Tettelin et al., Science 287 (2000)
Wing et al., Science 300: 1566-1569 (2003)
Gardner et al., Nature 419:531-534 (2002)
Carlton et al., Nature 419:512-519(2002)
Nelson et al., J. Bact., in revision.
Nelson et al., Envir. Microbiol. (2002)
Heidelberg et al., Nat. Biotech. 20 (2002)
Tettelin et al., PNAS. 99 (2002)
Tettelin et al., Science 293 (2001)
Arnold et al., Virology 15:252-66 (2000)
Nelson et al., Nature 399: 323-329 (1999)
Fraser et al., Science 281: 375-388 (1998)
Heidelberg et al., Nature 406, (2000)
Genomes
completed and
published by
TIGR and our
collaborators,
1995-present
4
Genomes in progress or recently completed
Acidithiobacillus ferrooxidans
Bacillus anthracis Kruger B
Burkholderia mallei
Clostridium perfringens ATCC13124
Dehalococcoides ethenogenes
Desulfovibrio vulgaris
Ehrlichia chaffeensis
Ehrlichia sennetsu
Geobacter sulfurreducens
Listeria monocytogenes
Methylococcus capsulatus
Mycobacterium avium 104
Mycobacterium smegmatis
Pseudomonas syringae
Staphylococcus aureus
Staphylococcus epidermidis
Treponema denticola
Wolbachia sp.
Anaplasma phagocytophila
Bacillus cereus 10987
Bacteroides forsythes
Brucella ovis
Baumannia cicadellinicola
Campylobacter jejuni
Carboxydothermus hydrogenoformans
Colwellia sp. 34H
Dichelobacter nodosus
Fibrobacter succinogenes
Prevotella intermedia
Pseudomonas fluorescens
Silicibacter pomeroyi DSS-3
Streptococcus agalactiae A909
Streptococcus gordonii
Streptococcus mitis
Streptococcus pneumoniae 670
Acidobacterium capsulatum
Bacillus anthracis A01055
Bacillus anthracis A0402
Bacillus anthracis Ames 0581
Burkholderia thailandensis
Campylobacter coli RM2228
Campylobacter upsaliensis RM3195
Clostridium perfringens SM101
Epulopiscium fishelonii
Hyphomonas neptunium
Listeria monocytogenes F6854
Listeria monocytogenes H7858
Mycoplasma arthritidis
Mycoplasma capricolum
Myxococcus xanthus
Prevotella ruminicola
Pyrococcus furiosus
Verrucomicrobium spinosum
Actinomyces naeslundii
Bacillus anthracis A0071
Bacillus anthracis Kruger B
Erwinia chrysanthemi
Gemmata obscuriglobus
Mycobacterium tuberculosis
Ruminococcus albus
Streptococcus sobrinus
Aspergillus fumigatus
Brugia malayi
Coccidioides immitis
Cryptococcus neoformans
Entamoeba histolytica
Oryza sativa Chromosome 3 & 10
Plasmodium vivax
Schistosoma mansoni
Solanum spp.
Tetrahymena thermophila
Toxoplasma gondii
Theileria parva
Trichomonas vaginalis
Trypanosoma brucei
Trypanosoma cruzi
5
Genome-Scale Sequence
Alignment
•
Efficiently compute alignments between entire
genomes and chromosomes, for example:
• Two strains of B. anthracis,
each 5.1 Mb (<30 CPU seconds)
• Two chromosomes of A. thaliana,
each 20-30 Mb (< 5 minutes)
• Two chromosomes of human,
100+ Mb each (< 30 minutes)
© 2003 Steven L. Salzberg
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MUMmer alignments

MUMs: Maximal Unique Matches
Algorithm finds ALL matches
 String them together and align gaps


Suffix trees
Very fast alignment of long DNA sequences
 Linear time and space requirements
 Software at:
http://www.tigr.org/software/mummer/

TIGR
© 2003 Steven L. Salzberg
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Suffix Trees

A trie
A tree with edges labelled by strings
 Each leaf represents a sequence—the
labels on the path to it from the root


The suffix tree for sequences A and B :
Contains |A | + |B | leaf nodes.
 Can be constructed in O (|A | + |B |) time!


Holds all suffixes of a set of sequences
© 2003 Steven L. Salzberg
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Maximal Unique Matches (MUMs)
Sequences in genomes A and B that:
Occur exactly once in A and in B
Are not contained in any larger matching sequence
A:
B:
© 2003 Steven L. Salzberg
Occurs only here
Mismatch at both ends
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MUMmer 2 streaming algorithm
Streaming String ...atgtcc...
atgtgtgtc$
$
c$
1
t
gt
9
10
i+1
c$
gt
c$
7
8
c$
Suffix Tree for String atgtgtgtc$
1 2 3 4 5 6 7 8 9 10
gt
5
gtc$
3
i
gt
c$
6
c$
4
© 2003 Steven L. Salzberg
gtc$
2
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MUMmer results: M. tuberculosis CDC1551 vs. H37Rv
a MUM
A
A
C
48
G 164
T
11
C
66
89
159
G
164
81
61
T
9
169
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Helicobacter pylori strain 26695 vs. J99
© 2003 Steven L. Salzberg
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V. cholera vs. E. coli (forward)
V. cholera vs. E. coli (reverse)
V. cholera vs. E. coli (both strands)
Duplication and Gene Loss?
© 2003 Steven L. Salzberg
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V. cholera vs. itself
© 2003 Steven L. Salzberg
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S. pyogenes vs. itself
© 2003 Steven L. Salzberg
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Symmetric Inversions Model
A3
A2
32 1 2
30 31
3
29
4
28
5
27
6
26
7
25
8
24
9
23
10
22
11
21
12
20
13
19
14
18 17 16 15
A1
30 31
A1
Inversion
around
terminus
32 1 2
30 31
3
29
4
28
5
27
6
26
7
25
8
24
9
23
10
22
11
21
12
20
13
14
19
15 16 17 18
A2
*
*
1 3231
3 2
30
4
5
27
26
25
24
23
22
21
20
*
A2
Inversion
around
origin
29
28
6
7
8
9
10
11
12
13
19
*
A3
14
15 16 17 18
32 1 2
3
29
4
28
5
27
6
26
7
Common
25
8
Ancester of 9
24
23
10
A and B
22
11
21
12
20
13
19
14
18 17 16 15
B1
A1
A3
A2
32 1 2
30 31
3
29
4
28
5
27
6
26
7
25
8
24
9
23
10
22
11
21
12
20
13
19
14
18 17 16 15
B1
32 1 2
30 31
3
Inversion
29
4
28
5
around
27
6
26
7
25*
terminus * 89
24
10
11
12
13
14
B2
15 16 17 18
Inversion
around
origin
23
22
21
20
19
1 3231
3 2
30
29
4
28
5
27
6
26
7
8
25
9
24
10
23
11
22
12
21
13
20
14
19
15 16 17 18
*
*
B3
B3
B2
B1
© 2003 Steven L. Salzberg
B3
B2
B2
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M. tuberculosis
M. leprae vs M. tuberculosis
M. leprae
© 2003 Steven L. Salzberg
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The “X-files” paper
© 2003 Steven L. Salzberg
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Arabidopsis genome paleontology
Compare all chromosomes to each other....
Diorama by B.E. Dahlgren, © The Field Museum, Chicago
© 2003 Steven L. Salzberg
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The hunt for genome-scale
duplications





S. cerevisiae?
 16% duplicated (Seoighe & Wolfe, 1999)
Maize?
 10 chromosomes vs. 5 in some related
grasses; segmental allotetraploid? (Gaut &
Dobley, 1997)
Drosophila melanogaster - no duplications
Vertebrates: much speculation but little
evidence (Skrabanek & Wolfe, 1998)
Arabidopsis thaliana: yes!
© 2003 Steven L. Salzberg
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chr.4
First discovery: large-scale
duplication between chromosomes
2 and 4 (Lin et al., 1999)
chr.2
chr.1
Tandem duplications
chr.1
• Over 60% of the
genome is covered
by duplicated
regions
• Centromeres cover
much of the rest
• Strikingly, only
about 1/3 of the
genes in each block
remain as duplicates
No triplications!
19-24 large-scale duplications
 >60% of the genome duplicated
 If duplications occurred over time,
triplications highly likely
 Duplications likely happened as one
event (on evolutionary time scale)
 Conclusion: whole genome duplication

© 2003 Steven L. Salzberg
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Start with 4 ancestral chromosomes
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Warning: data quality control

Until December 2000, Arabidopsis data in
GenBank was all BAC-based
 Errors included:




BACs on the wrong chromosome
BACs entered twice with different IDs, different
annotation (sequenced twice), slightly different
sequence
For duplications analysis, these errors would
prove disastrous
Many of these errors are still in GenBank

© 2003 Steven L. Salzberg
Old BACs are not automatically deleted
54
Human Genome analysis
 used Celera’s assembly and annotation
 26,588 genes, ordered along each of 24
chromosomes
 MUMmer 2.0 used to align whole
chromosomes
 Nothing found in DNA-level alignments
 Proteome alignments used instead
 Recently re-computed using latest
human genome annotation (Ensembl)
© 2003 Steven L. Salzberg
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Human whole-genome aligment
 Create 24 “mini-proteomes” by concatenating
all proteins on each chromosome
 Use MUMmer to align each mini-proteome to
the complete proteome (9,675,713 amino
acids)
 Search for conserved clusters of proteins
 Confirmed analysis by looking at Blast hits of
all vs. all
© 2003 Steven L. Salzberg
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What we’re looking for
 Not looking for
 tandem duplications
 domain hits (very common, often give highly
significant Blast hits)
© 2003 Steven L. Salzberg
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Summary results
 1077 duplicated blocks
 10,310 “gene pairs”
 “pair” = 2 genes that match between two blocks
 296 blocks with 3-4 gene pairs
 781 blocks with 5 or more gene pairs
 3522 distinct genes, many duplicated more than once
 Large block: 33 genes on chr 2 and chr 14
 spans 63Mbp on chr 14, over 70% of chr 14’s length
 spread over 97 genes on chr 2 and 332 genes on 14
 includes two of four known Hox clusters, an ancient duplication
 Large block: 64 genes on chr 18 and chr 20
 previously undiscovered
 Shuffled data: 370 gene pairs (3.6% false positive rate)
© 2003 Steven L. Salzberg
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© 2003 Steven L. Salzberg
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Duplications in Human Chromosome 2
© 2003 Steven L. Salzberg
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Human-mouse genome mapping
 Close evolutionary distance permits
DNA-level alignments
 Protein similarity even greater than DNA
 MUMmer quickly aligns each mouse miniproteome to its human counterparts
 Blast finds most (not all) of the same
matches (and is far slower)
 77% (566/731) of Mouse16 genes are
found in syntenic regions of human
 2.5% (18/731) of Mouse16 genes are
unique to mouse, not found in human
© 2003 Steven L. Salzberg
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Mouse chr 16 maps to human chromosomes
3, 8, 12, 16, 21, and 22
© 2003 Steven L. Salzberg
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Have bacteria transferred their
genes directly into the human
genome?
“Startling” discovery, Feb. 2001: 223
bacterial genes were laterally transferred
into a vertebrate ancestor of humans
(from the Nature human genome paper)
© 2003 Steven L. Salzberg
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Horizontal (Lateral) Gene
Transfer
© 2003 Steven L. Salzberg
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Vertical Inheritance
© 2003 Steven L. Salzberg
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Horizontal Gene Transfer ???
© 2003 Steven L. Salzberg
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Horizontal gene transfer in
Arabidopsis thaliana chr 2
(Lin et al., Nature, 1999)
135 genes most closely related to
cyanobacterial genes and thus likely
were transferred from chloroplast to the
nucleus
 Very recent transfer of > 250 kb section
of mitochondrial genome
 Many additional older mitochondrial →
nuclear gene transfers

© 2003 Steven L. Salzberg
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Examples of Horizontal Transfers
Antibiotic resistance genes on plasmids
 Pathogenicity islands
 Toxin resistance genes on plasmids
 Agrobacterium Ti plasmid
 Viruses and viroids
 Organelle to nucleus transfers

© 2003 Steven L. Salzberg
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Mechanisms of Horizontal Transfer
Plasmid exchange (prokaryotes)
 Mating/conjugation (prokaryotes)
 Viruses and viroids
 Organelle to nucleus exchange
(eukaryotes)
 Scavenging from environment
 Passive absorption
 Fusion of cells

© 2003 Steven L. Salzberg
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Nature human genome paper (2001):
Evidence for transfer?
Evidence: Genes match bacteria, but do
not match non-vertebrate eukaryotes
 Or, genes really are in non-vertebrates,
but have stronger match to bacteria



Measured by BLAST E-value
113 of the 223 genes found in a broad
spectrum of prokaryotic species
© 2003 Steven L. Salzberg
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Alternative explanations

Gene loss from a small sample of non-vertebrate
eukaryotes






Only 4 non-vertebrates used for analysis: fruit fly, nematode,
yeast, and mustard weed (Arabidopsis)
Large and diverse set of prokaryotes (over 30 organisms,
including extremophiles) used as well
Rapid divergence in non-vertebrate eukaryotes
(evolutionary rate variation)
Still-incomplete genomes (e.g., D. melanogaster)
Erroneous annotation/gene finding
Contamination
© 2003 Steven L. Salzberg
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Re-analysis: number of “transfers”
decreases with # of genomes analyzed
2000
1800
Number of genes in lateral transfer candidate set
1600
1400
Fruit fly
C. elegans
Arabidopsis
Yeast
Parasites
1200
1000
800
600
400
200
0
1
2
3
4
Number of protein sets removed
© 2003 Steven L. Salzberg
5
Other
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Evolutionary Rate Variation
© 2003 Steven L. Salzberg
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Trees Don’t Support Transfer
© 2003 Steven L. Salzberg
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Birney et al., Nature special issue
on human genome
“The unfinished human genomic DNA may
contain contamination, particularly from bacteria
but also from other sources. .... If the predicted
gene matches a bacterial gene more closely
than any vertebrate gene then it will almost
always be a contaminant.”
© 2003 Steven L. Salzberg
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Were genes really transferred?


NO
Our re-analysis finds just 41 genes (Ensembl) or 46
(Celera) with best hits to bacteria – not 223




Great care is needed in order to make assertions of
transfer from bacteria to humans



All of these could be explained by alternative mechanisms
More genomes will likely eliminate these remaining
candidates
At least 3 have already been found in Drosophila, 10 more in
other species
Implications would be significant; e.g., GMOs
Even more care is needed when working with
unfinished data
Nature erratum to human genome paper, August
2001: “We agree.”
© 2003 Steven L. Salzberg
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Acknowledgements
MUMmer: Arthur Delcher, Jeremy Peterson, Rob Fleischmann, Owen
White, Simon Kasif, Jonathan Allen, Sam Angiuoli, Adam Phillippy
X alignments: Jonathan Eisen, Owen White, John Heidelberg
Arabidopsis duplications:
TIGR: Maria Ermolaeva, Owen White, Jonathan Eisen, Xiaoying
Lin, Samir Kaul
AGI collaborators: Klaus Meyer and all his MIPS colleagues, Mike
Bevan
Human duplications: Mark Yandell, Mark Adams, Mani
Subramanian, Craig Venter (all formerly Celera), Ron Wides (BarIlan University), Art Delcher
Lateral transfer: Jonathan Eisen, Owen White, Jeremy Peterson
Funding support:
National Institutes of Health (NHGRI, NLM)
National Science Foundation (CISE, BIO)
© 2003 Steven L. Salzberg
77