Whole-Genome Prokaryote Phylogeny without Sequence Alignment

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Whole-Genome Prokaryote Phylogeny
withoutSequence Alignment
Bailin HAO and Ji QI T-Life Research
Center, Fudan University Shanghai 200433,
China Institute of Theoretical Physics, Academia
Sinica Beijing 100080, China http//www.itp.ac.cn/
hao/
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Classification of ProkaryotesA Long-Standing
Problem

• Traditional taxonomy too few features
• Morphologyspheric, helices, rod-shaped
• Metabolismphotosythesis, N-fixing,
  desulfurization
• Gram stainingpositive and negative
• SSU rRNA Tree (Carl Woese et al., 1977)
• 16S rRNA ancient conserved sequences of about
  1500kb
• Discovery of the three domains of life Archaea,
  Bacteria and Eucarya
• Support to endosymbiont origin of mitochondria
  and chloroplasts

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The SSU rRNA Tree of LifeA big progress in
molecular phylogeny of prokaryotes as evidenced
by thehistory of theBergeys Manual
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Bergeys Manual TrustBergeys Manual

• 1st Ed. Determinative Bacteriology 1923
• 8th Ed. Determinative Bacteriology 1974
• 1st Ed. Systematic Bacteriology 1984-1989, 4
  volumes
• 9th Ed. Determinative Bacteriology 1994
• 2nd Ed. Systematic Bacteriology 2001-200?, 5
  volumes planned On-Line Taxonomic Outline of
  Procarytes by Garrity et al. Rel.4.0 (October
  2003) 26 phyla A1-A2, B1-B24

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Phylogeny versus Taxonomy

• Phylogeny and taxonomy are not synonyms
• Taxonomy classification, systematics of extant
  species
• Phylogeny the history of evolution since the
  origin of species
• One should not contradict the two with each other
• From the Preface to Outline of Procaryotes
  (Rel.4.0, October 2003) The primary objective
  was to devise a classification that would reflect
  the phylogeny of procaryotes,

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Our Latest Result

• NCBI Genome data as of 31 December 2004
• 222 organisms (21A 193B 8E)
• Input genome data (the .faa files)
• Output a phylogenetic tree
• No selection of genes, no alignment of sequences,
  no fine adjustment whatsoever
• See the tree first. Story follows.

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??222??????????(K5)

• 21 ????
• 193 ????
• 8 ?????

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Complete Bacterial Genomes Appeared since
1995Early Expectations

• More support to the SSU rRNA Tree of Life
• Add details to the classification (branchings and
  groupings)
• More hints on taxonomic revisions

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• Confusion brought by the hyperthermophiles
• Aquifex aeolicus (Aquae) 1998 1551335
• Thermotoga maritima (Thema) 1999 1860725
• 
  
  
• Genome Data Shake tree of life
• Science 280 (1 May 1998) 672
• Is it time to uproot the tree of life?
• Science 284 (21 May 1999) 130
• Uprooting the tree of life
• W. Ford Doolittle, Scientific American
  (February 2000) 90

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Debate on Lateral Gene Transfer

• Extreme estimate 17 in E. Coli
• Limitations of the above approach
• B. Wang, J. Mol. Evol. 53 (2001) 244
• Phase transition and crystalization of
  species (C. Woese 1998)
• Lateral transfer within smaller gene pools as an
  innovative agent
• Composition vector may incorporate LGT within
  small gene pools

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Our Motivations

• Develop a molecular phylogeny method that makes
  use of complete genomes no selection of
  particular genes
• Avoid sequence alignment
• Try to reach higher resolution to provide an
  independent comparison with other approaches such
  as SSU rRNA trees
• Make comparison with bacteriologists systematics
  as reflected in Bergeys Manual (2001 - 2003)
• Qi, Wang, Hao, J. Molecular Evolution, 58 (1)
  (Jaunary 2004) 1 11. (10916A87B6E)

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Comparison of Complete Genomes/Proteomes

• Compositional vectors
• Nucleotides a?t?c?g
• aatcgcgcttaagtc
• Di-nucleotide (K2) distribution
• aa at ac ag ta tt tc tg ca ct cc cg ga gt
  gc gg
• 2 ,1 ,0 , 1 , 1 ,1, 1, 0, 0, 1, 0,
  2, 0, 1 ,2 , 0

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• K-strings make a composition vector
• DNA sequence ? vector of dimension 4K
• Protein sequence ? vector of dimension 20K
• Given a genomic or protein sequence ? a unique
  composition vector
• The converse a vector ? one or more sequences?
• K big enough -gt uniqueness
• Connection with the number of Eulerian loops in a
  graph (a separate study available as a preprint
  at ArXivphysics/0103028 and from Haos webpage)

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A Key ImprovementSubtraction of Random
Background

• Mutations took place randomly at molecular level
• Selection shaped the direction of evolution
• Many neutral mutations remain as random
  background
• At single amino acid level protein sequences are
  quite close to random
• Highlighting the role of selection by subtraction
  a random background

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Frequency and Probability

• A sequence of length
• A K-string
• Frequency of appearance
• Probability

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Predicting (K-strings) from that of lengths
(K-1) and (K-2) strings

• Joint probability vs. conditional probability
• Making the weakest Markov assumption
• Another joint probability

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(K-2)-th Order Markov Model

• Change to frequencies
• Normalization factor may be ignored when LgtgtK

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• Construct
• composition vectors
• using these modified string counts
• For the i-th string type of species A we use

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Composition Distance

• Define correlation between two composition
  vectors by the cosine of angle
• From two complete proteomes
• Aa1,a2,,an n205 3 200 000
• Bb1,b2,,bn
• 
  
• C(A,B) ?-1,1
• Distance
• 
  D(A,B)?0,1

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Protein Class vs. Whole Proteome

• Trees based on collection of ribosomal proteins
  (SSU LSU) ribosomal proteins are interwoven
  with rRNA to form functioning complex results
  consistent with SSU rRNA trees
• Trees based on collection of aminoacyl-tRNA
  synthetases (AARS). Trees based on single AARS
  were not good. Trees based on all 20 AARSs taken
  together much better but not as good as that
  based on rProteins.

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Genus Tree based on Ribosomal Proteins
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A Genus Tree based on Aminoacyl tRNA synthetases
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Chloroplast Tree

• Sequences of about 100 000 bp
• Tree of the endosymbiont partners
• Paper appeared in Molecular Biology and
  Evolution, 21 (2004), 200-206.

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Chloroplast tree
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Coronaviruses includingHuman SARS-CoV

• Sequences of tens kilo bases
• SARS squence about 29730 bases
• Paper published in Chinese Science Bulletin,
  48(12), 1170-1174 (26 June 2003)

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Coronavirus tree
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Understanding the Subtraction ProcedureAnalysis
of Extreme Cases in E. coli K12

• There are 1 343 887 5-strings belonging to
  841832 different types.
• Maximal count before subtraction 58 for the
• 5-peptide GKSTL. 58 reduces to 0.646 after
  subtraction.
• Maximal component after subtraction 197 for the
  5-peptide HAMSC. The number 197 came from a
  single count 1 before the subtraction.

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GKSTL how 58 reduces to 0.646?

• (GKST)113
• (KSTL)77
• (KST)247
  
• Markov prediction 11377/24735.23
• Final result (58-35.23)/35.230.646

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HAMSC how 1 grows to 197?

• (HAMS)1
• (AMSC)1
• (AMS)198
• Markov prediction 11/1981/198
• Final result (1-1/198)/(1/198)197

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6121 Exact Matches of GKSTLIn PIR Rel.1.26 with
gt1.2 Mil Proteins

• These 6121 matches came from a diverse taxonomic
  assortment from virus to bacteria to fungi to
  plants and animals including human being
• In the parlance of classic cladistics GKSTL
  contributes to plesiomorphic characters that
  should be eliminated in a strict phylogeny
• The subtraction procedure did the job.

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15 Exact Matches of HAMSCIn PIR Rel.1.26 with
gt1.2 Mil Proteins

• 1 match from Eukaryotic protein
• 4 matches (the same protein) from virus
• 10 matches from prokaryotes, among which
• 3 from Shegella and E. coli (HAMSCAPDKE)
• 3 from Samonella
  (HAMSCAPERD)
• HAMSC is characteristic for prokaryotes
• HAMSCA is specific for enterobacteria

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Stable Topology of the Tree

• K1 makes some sense!
• K2,3,4 topology gradually converges
• K5 and K6 present calculation
• K7 and more beyond our computing capability at
  present too high resolution star-tree or bush
  expected

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Statistical Test of the Tree

• Bootstrap versus Jack knife
• Bootstrap in sequence alignments
• Bootstrap by random selections
• from the AA-sequence pool
• A time consuming job
• 180 bootstraps for 72 species

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About 70 genes for every species were selected
in one bootstrap
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K-string Picture of Evolution

• K5 -gt3 200 000 points in space of
• 5-strings
• K6 -gt64 000 000 points
• In the primordial soup short polypeptides of a
  limited assortment
• Evolution by growth, fusion, mutation leads to
  diffusion in the string space
• String space not saturated yet

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The Problem of Higher Taxa

• 1974 Bacteria as a separate kingdom
• 1994 Archaea and Bacetria as two domains
• The relation of higher taxa? Much debate among
  bacteriologists but some hints from our trees
  and other whole-genome trees
• No wonder taxonomists of all walks disagree on
  grouping and palcing higher taxa

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References

• J Qi, B Wang, BL Hao, J. Mol. Evol. 58 (2004)
  1-11. (10916A 87B 6E)
• KH Chu, J Qi, ZG Yu, V Ahn, Mol. Biol. Evol.
  21(2004) 200-206. (Chloroplasts)
• L Gao, HB Wei, J Qi, YG Sun, BL Hao, Chinese Sci.
  Bull. 48(2003) 1170-1174. (Coronavirus, SARSCoV)
• HB Wei, J Qi, BL Hao, Science in China, 34(2)
  (2004) 186-199. (Using ribosomal and aminoacyl
  tRNA synthetases)
• BL Hao, J Qi, J. Bioinf. Comput. Biol. 2 (2004)
  1-19. (A review with 13216A 110B 6E)

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• Summary
• As composition vectors do not depend on genome
  size and gene content. The use of whole genome
  data is straightforward
• Data independent on that of 16S rRNA
• Method different from that based on SSU rRNA
• Results agree with SSU rRNA trees and the
  Bergeys Manual
• Hint on groupings of higher taxa
• A method without free parameters data in, tree
  out
• Possibility of an automatic and objective
  classification tool for prokaryotes

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ConclusionThe phylogeny has met taxonomy. The
Tree of Life is saved!There is phylogenetic
information in the prokaryotic proteomes.Time to
work on molecular definition of taxa.Thank you!
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A Protein Tree for 154 OrganismsFrom 88
Genera(K5)

• 17 Archaea (12 genera, 17 species)
• 131 Bacteria (70 genera, 105 species)
• 6 Eukaryotes

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