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  1. 1. Whole-Genome Prokaryote Phylogeny without Sequence Alignment Bailin HAO and Ji QI T-Life Research Center, Fudan University Shanghai 200433, China Institute of Theoretical Physics, Academia Sinica Beijing 100080, China
  2. 2. Classification of Prokaryotes: A Long-Standing Problem <ul><li>Traditional taxonomy: too few features </li></ul><ul><ul><ul><li>Morphology : spheric, helices, rod-shaped…… </li></ul></ul></ul><ul><ul><ul><li>Metabolism : photosythesis, N-fixing, desulfurization…… </li></ul></ul></ul><ul><ul><ul><li>Gram staining : positive and negative </li></ul></ul></ul><ul><li>SSU rRNA Tree (Carl Woese et al., 1977): </li></ul><ul><ul><li>16S rRNA: ancient conserved sequences of about 1500kb </li></ul></ul><ul><ul><li>Discovery of the three domains of life: Archaea, Bacteria and Eucarya </li></ul></ul><ul><ul><li>Endosymbiont origin of mitochondria and chloroplasts </li></ul></ul>
  3. 3. The SSU rRNA Tree of Life: A big progress in molecular phylogeny of prokaryotes as evidenced by the history of the Bergey’s Manual
  4. 4. Bergey’s Manual Trust: Bergey’s Manual <ul><li>1st Ed. “ Determinative Bacteriology”: 1923 </li></ul><ul><li>8th Ed. “ Determinative Bacteriology”: 1974 </li></ul><ul><li>1 st Ed. “ Systematic Bacteriology”: 1984-1989, 4 volumes </li></ul><ul><li>9 th Ed. “ Determinative Bacteriology”: 1994 </li></ul><ul><li>2 nd Ed. “ Systematic Bacteriology”: 2001-200?, 5 volumes planned; On-Line “ Taxonomic Outline of Procarytes ” by Garrity et al. (October 2003) </li></ul><ul><li>(26 phyla: A1-A2, B1-B24) </li></ul>
  5. 5. Our Final Result <ul><li>132 organisms (16A + 110B + 6E) </li></ul><ul><li>Input: genome data </li></ul><ul><li>Output: phylogenetic tree </li></ul><ul><li>No selection of genes, no alignment of sequences, no fine adjustment whatsoever </li></ul><ul><li>See the tree first. Story follows. </li></ul>
  6. 7. Protein Tree for 145 Organisms From 82 Genera (K=5) 16 Archaea (11 genera, 16 species) 123 Bacteria (65 genera, 98 species) 6 Eukaryotes
  7. 9. Complete Bacterial Genomes Appeared since 1995 Early Expectations: <ul><li>More support to the SSU rRNA Tree of Life </li></ul><ul><li>Add details to the classification (branchings and groupings) </li></ul><ul><li>More hints on taxonomic revisions </li></ul>
  8. 10. <ul><li>Confusion brought by the hyperthermophiles </li></ul><ul><ul><li>Aquifex aeolicus (Aquae) 1998: 1551335 </li></ul></ul><ul><ul><li>Thermotoga maritima (Thema) 1999: 1860725 </li></ul></ul><ul><ul><li>“ Genome Data Shake tree of life ” </li></ul></ul><ul><ul><ul><ul><li>Science 280 (1 May 1998) 672 </li></ul></ul></ul></ul><ul><ul><li>“ Is it time to uproot the tree of life? ” </li></ul></ul><ul><ul><li>Science 284 (21 May 1999) 130 </li></ul></ul><ul><ul><li>“ Uprooting the tree of life ” </li></ul></ul><ul><ul><ul><li>W. Ford Doolittle, Scientific American (February 2000) 90 </li></ul></ul></ul>
  9. 11. Debate on Lateral Gene Transfer <ul><li>Extreme estimate: 17% in E. Coli </li></ul><ul><li>Limitations of the above approach </li></ul><ul><li>B. Wang, J. Mol. Evol. 53 (2001) 244 </li></ul><ul><li>“ Phase transition” and “crystalization” of species (C. Woese 1998) </li></ul><ul><li>Lateral transfer within smaller gene pools as an innovative agent </li></ul><ul><li>Composition vector may incorporate LGT within small gene pools </li></ul>
  10. 12. <ul><li>Alignment-Based Molecular Phylogeny </li></ul><ul><ul><ul><li>TCAGACGC </li></ul></ul></ul><ul><ul><ul><li>TCGGAGT </li></ul></ul></ul><ul><ul><ul><ul><li>T C A G A C G C </li></ul></ul></ul></ul><ul><ul><ul><ul><li>T C G G A - G T </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Scoring scheme </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Gap penalty </li></ul></ul></ul></ul><ul><ul><ul><ul><li>16S rRNA tree was based on sequence alignment </li></ul></ul></ul></ul>
  11. 13. <ul><ul><li>Problem: sequence alignment cannot be readily applied to complete genomes </li></ul></ul><ul><ul><li>Homology -> alignment </li></ul></ul><ul><ul><li>Different genome size, gene content and gene order </li></ul></ul>Gene A A ’ B Gene B ’ C ? 1st species 2nd species
  12. 14. Our Motivations: <ul><li>Develop a molecular phylogeny method that makes use of complete genomes – no selection of particular genes </li></ul><ul><li>Avoid sequence alignment </li></ul><ul><li>Try to reach higher resolution to provide an independent comparison with other approaches such as SSU tRNA trees </li></ul><ul><li>Make comparison with bacteriologists’ systematics as reflected in Bergey’s Manual (2001, 2002) </li></ul><ul><li>Our paper accepted by J. Molecular Evolution </li></ul>
  13. 15. Other Whole-Genome Approaches <ul><li>Gene content </li></ul><ul><li>Presence or absence of COGs </li></ul><ul><li>Conserved Gene Pairs </li></ul><ul><li>“ Information” distances </li></ul><ul><li>Domain order in proteins (Ken Nishikawa’s talk at InCoB2003) </li></ul><ul><li>… </li></ul>
  14. 16. Comparison of Complete Genomes/Proteomes <ul><li>Compositional vectors </li></ul><ul><ul><li>Nucleotides: a 、 t 、 c 、 g </li></ul></ul><ul><ul><li>aatcgcgcttaagtc </li></ul></ul><ul><ul><li>Di-nucleotide (K=2) distribution: </li></ul></ul><ul><li>{aa at ac ag ta tt tc tg ca ct cc cg ga gt gc gg} </li></ul><ul><li>{ 2 ,1 ,0 , 1 , 1 ,1, 1, 0, 0, 1, 0, 2, 0, 1 ,2 , 0} </li></ul>} }
  15. 17. <ul><li>K-strings make a composition vector </li></ul><ul><ul><ul><li>DNA sequence  vector of dimension 4 K </li></ul></ul></ul><ul><ul><ul><li>Protein sequence  vector of dimension 20 K </li></ul></ul></ul><ul><ul><ul><li>Given a genomic or protein sequence  a unique composition vector </li></ul></ul></ul><ul><ul><ul><li>The converse: a vector  one or more sequences ? </li></ul></ul></ul><ul><ul><ul><li>K big enough -> uniqueness </li></ul></ul></ul><ul><ul><ul><li>Connection with the number of Eulerian loops in a graph (a separate study available as a preprint at ArXiv:physics/0103028 and from Hao’s webpage) </li></ul></ul></ul>↑
  16. 18. A Key Improvement: Subtraction of Random Background <ul><li>Mutations took place randomly at molecular level </li></ul><ul><li>Selection shaped the direction of evolution </li></ul><ul><li>Many neutral mutations remain as random background </li></ul><ul><li>At single amino acid level protein sequences are quite close to random </li></ul><ul><li>Highlighting the role of selection by subtraction a random background </li></ul>
  17. 19. Frequency and Probability <ul><li>A sequence of length </li></ul><ul><li>A K-string </li></ul><ul><li>Frequency of appearance </li></ul><ul><li>Probability </li></ul>
  18. 20. Predicting #(K-strings) from that of lengths (K-1) and (K-2) strings <ul><li>Joint probability vs. conditional probability </li></ul><ul><li>Making the weakest Markov assumption: </li></ul><ul><li>Another joint probability: </li></ul>
  19. 21. (K-2)-th Order Markov Model <ul><li>Change to frequencies: </li></ul><ul><li>Normalization factor may be ignored when L>>K </li></ul>
  20. 22. <ul><li>Construct compositional vectors using these modified string counts: </li></ul><ul><ul><li>For the i-th string type of species A we use </li></ul></ul>
  21. 23. Composition Distance <ul><li>Define correlation between two compositional vectors by the cosine of angle </li></ul><ul><ul><li>From two complete proteomes: </li></ul></ul><ul><ul><ul><li>A : {a 1 ,a 2 ,……,a n } n=20 5 = 3 200 000 </li></ul></ul></ul><ul><ul><ul><li>B : {b 1 ,b 2 ,……,b n } </li></ul></ul></ul><ul><ul><ul><li>C(A,B) ∈[-1,1] </li></ul></ul></ul><ul><li>Distance </li></ul><ul><ul><li>D(A,B)∈[0,1] </li></ul></ul>
  22. 24. Materials: Genomes from NCBI ( ) Not the original GenBank files 6 Eucaryote genomes were included for reference Tree construction: Neighbor-Joining in Phylip
  23. 25. Protein Tree for 132 species (K=5) 16 Archaea (11 genera, 16 species) 110 Bacteria (57 genera, 88 species) 6 Eukaryotes
  24. 27. Protein Tree for 132 species K=6 16 Archaea (11 genera, 16 species) 110 Bacteria (57 genera, 88 species) 6 Eukaryotes
  25. 29. Protein Class vs. Whole Proteome <ul><li>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 </li></ul><ul><li>Trees based on collection of aminoacyl-tRNA synthetases (AARS). Trees based on single AARS were not good. Trees based on all 20 AARSs much better but not as good as that based on rProteins. </li></ul>
  26. 30. Genus Tree based on Ribosomal Proteins
  27. 31. A Genus Tree based on Aminoacyl tRNA synthetases
  28. 32. Chloroplast Tree <ul><li>Sequences of about 100 000 bp </li></ul><ul><li>Tree of the endosymbiont partners </li></ul><ul><li>Paper accepted by Molecular Biology and Evolution on 12 August 2003 </li></ul>
  29. 33. Chloroplast tree
  30. 34. Coronaviruses including Human SARS-CoV <ul><li>Sequences of tens kilo bases </li></ul><ul><li>SARS squence: about 29730 bases </li></ul><ul><li>Paper published in Chinese Science Bulletin on 26 June 2003 </li></ul>
  31. 35. Coronavirus tree
  32. 36. Understanding the Subtraction Procedure: Analysis of Extreme Cases in E. coli <ul><li>There are 1 343 887 5-strings belonging to 841832 different types. </li></ul><ul><li>Maximal count before subtraction: 58 for the </li></ul><ul><li>5-peptide GKSTL. 58 reduces to 0.646 after subtraction. </li></ul><ul><li>Maximal component after subtraction: 197 for the 5-peptide HAMSC. The number 197 came from a single count 1 before the subtraction. </li></ul>
  33. 37. GKSTL: how 58 reduces to 0.646? <ul><li>#(GKST)=113 </li></ul><ul><li>#(KSTL)=77 </li></ul><ul><li>#(KST)=247 </li></ul><ul><li>Markov prediction: 113*77/247=35.23 </li></ul><ul><li>Final result: (58-35.23)/35.23=0.646 </li></ul>
  34. 38. HAMSC: how 1 grows to 197? <ul><li>#(HAMS)=1 </li></ul><ul><li>#(AMSC)=1 </li></ul><ul><li>#(AMS)=198 </li></ul><ul><li>Markov prediction: 1*1/198=1/198 </li></ul><ul><li>Final result: (1-1/198)/(1/198)=197 </li></ul>
  35. 39. 6121 Exact Matches of GKSTL In PIR Rel.1.26 with >1.2 Mil Proteins <ul><li>These 6121 matches came from a diverse taxonomic assortment from virus to bacteria to fungi to plants and animals including human being </li></ul><ul><li>In the parlance of classic cladistics GKSTL contributes to plesiomorphic characters that should be eliminated in a strict phylogeny </li></ul><ul><li>The subtraction procedure did the job. </li></ul>
  36. 40. 15 Exact Matches of HAMSC: In PIR Rel.1.26 with >1.2 Mil Proteins <ul><li>1 match from Eukaryotic protein </li></ul><ul><li>4 matches (the same protein) from virus </li></ul><ul><li>10 matches from prokaryotes, among which </li></ul><ul><li>3 from Shegella and E. coli (HAMSCAPDKE) </li></ul><ul><li>3 from Samonella (HAMSCAPERD) </li></ul><ul><li>HAMSC is characteristic for prokaryotes </li></ul><ul><li>HAMSCA is specific for enterobacteria </li></ul>
  37. 41. Stable Topology of the Tree <ul><li>K=1: makes some sense! </li></ul><ul><li>K=2,3,4: topology gradually converges </li></ul><ul><li>K=5 and K=6: present calculation </li></ul><ul><li>K=7 and more: too high resolution; star-tree or bush expected </li></ul>
  38. 42. Statistical Test of the Tree <ul><li>Bootstrap versus Jack knife </li></ul><ul><li>Bootstrap in sequence alignments </li></ul><ul><li>“Bootstrap” by random selections </li></ul><ul><li>from the AA-sequence pool </li></ul><ul><li>A time consuming job </li></ul><ul><li>180 bootstraps for 72 species </li></ul>
  39. 43. About 70% genes for every species were selected in one bootstrap
  40. 44. “ K-string Picture” of Evolution <ul><li>K=5 ->3 200 000 points in space of </li></ul><ul><li>5-strings </li></ul><ul><li>K=6 ->64 000 000 points </li></ul><ul><li>In the primordial soup: short polypeptides of a limited assortment </li></ul><ul><li>Evolution by growth, fusion, mutation leads to diffusion in the string space </li></ul><ul><li>String space not saturated yet </li></ul>
  41. 45. The Problem of Higher Taxa <ul><li>1974: Bacteria as a separate kingdom </li></ul><ul><li>1994: Archaea and Bacetria as two domains </li></ul><ul><li>The relation of higher taxa? </li></ul>
  42. 46. <ul><li>Summary </li></ul><ul><li>As composition vectors do not depend on genome size and gene content. The use of whole genome data is straightforward </li></ul><ul><li>Data independent on that of 16S rRNA </li></ul><ul><li>Method different from that based on SSU rRNA </li></ul><ul><li>Results agree with SSU rRNA trees and the Bergey’s Manual </li></ul><ul><li>Hint on groupings of higher taxa </li></ul><ul><li>A method without “free parameters”: data in, tree out </li></ul><ul><li>Possibility of an automatic and objective classification tool for prokaryotes </li></ul>
  43. 47. Conclusion: The Tree of Life is saved! There is phylogenetic information in the prokaryotic proteomes. Time to work on molecular definition of taxa. Thank you!
  44. 50. Protein Tree for 132 species (K=5) 16 Archaea (11 genera, 16 species) 110 Bacteria (57 genera, 88 species) 6 Eukaryotes
  45. 53. A Failed Attempt Using Avoidance Sinatures
  46. 55. Comparison with the Bergey’s Manual
  47. 56. <ul><li>Tree Construction </li></ul><ul><ul><li>phylip package of J. Felsenstein (Neighbor-Joining) </li></ul></ul><ul><ul><li>The Fitch method is not </li></ul></ul><ul><ul><li>feasible here, </li></ul></ul><ul><ul><li>Nondistance-matrix method (MP, ML et al) </li></ul></ul><ul><li>Material </li></ul><ul><ul><li> </li></ul></ul>  Phyla Classes Orders Families Genera Species Strains Archaea 2 7 9 9 9 13 13 Bacteria 9 14 23 28 37 46 57 Total 11 21 32 37 46 59 70
  48. 57. Early expectation from genome data <ul><li>Was there intensive lateral gene transfer? </li></ul><ul><li>Gene tree cannot be equated to the real tree of life </li></ul><ul><li>Genome data: 10 6 to 10 7 </li></ul><ul><li>Difficult to align whole genome data </li></ul>
  49. 58. <ul><li>Prokaryote and Eukaryote </li></ul><ul><li>Three Kingdoms( Carl Woese ,16S rRNA ) </li></ul><ul><ul><li>Archaea </li></ul></ul><ul><ul><li>Eubacteria </li></ul></ul><ul><ul><li>Eukarya </li></ul></ul><ul><li>Five Kingdoms ( Lynn Margulis ) </li></ul><ul><ul><li>Bacteria ( Archaea, Eubacteria ) </li></ul></ul><ul><ul><li>Protoctista </li></ul></ul><ul><ul><li>Animalia </li></ul></ul><ul><ul><li>Fungi </li></ul></ul><ul><ul><li>Plantae </li></ul></ul>
  50. 59. <ul><li>Common features of Archaea and Eubacteria: </li></ul><ul><li>Small cells, no nucleus membrane, ring DNA, </li></ul><ul><li>no CAP at 5’end of mRNA, presence of S-D </li></ul><ul><li>segments </li></ul><ul><li>Many proteins associated with replication, transcription, and translation are common in Archaea and Eukaryote </li></ul><ul><li>Features of Archaea: lack of some enzymes, insensitive to some antibiotics </li></ul>
  51. 60. <ul><li>《 Compositional Representation of Protein Sequences and the Number of Eulerian Loops 》 </li></ul><ul><li>by Bailin Hao, Huimin Xie, Shuyu Zhang </li></ul><ul><ul><li>K=5: 76.7% proteins have unique reconstruction </li></ul></ul><ul><ul><li>K=6:  94.0% </li></ul></ul><ul><ul><li>K=10: >99% </li></ul></ul><ul><ul><ul><li>Checked 2820 AA-seqs from pdb.seq, a special selection of SWISS-PROT </li></ul></ul></ul><ul><ul><ul><li>See Los Alamos National Lab E-Archive: physics/0103028 </li></ul></ul></ul>
  52. 61. Subtraction of Random Background <ul><li>Using a (K-2)-order Markov Model </li></ul><ul><li>K=2: genomic signature by Karlin and Burge </li></ul><ul><li>May be justified by using Maximal Entropy Principle with appropriate constraints (Hu & Wang, 2001) </li></ul>
  53. 62. What to do next <ul><li>Detailed comparison with traditional taxonomy </li></ul><ul><li>Add more eukaryotes </li></ul><ul><li>Elucidation of the foundatrion and limitation of compositional approach </li></ul><ul><li>Software and web interface </li></ul><ul><li>Problem of lateral gene transfer </li></ul><ul><li>Viruses ? </li></ul>
  54. 63. <ul><li>Confusion brought by the hyperthermophiles </li></ul><ul><ul><li>Aquifex aeolicus (Aqua) 1998: 1551335 </li></ul></ul><ul><ul><li>Thermotoga maritima (Tmar) 1999: 1860725 </li></ul></ul><ul><ul><li>“ Genome Data Shake tree of life” </li></ul></ul><ul><ul><ul><ul><li>Science 280 (1 May 1998) 672 </li></ul></ul></ul></ul><ul><ul><li>“ Is it time to uproot the tree of life?” </li></ul></ul><ul><ul><li>Science 284 (21 May 1999) 130 </li></ul></ul><ul><ul><li>“ Uprooting the tree of life” </li></ul></ul><ul><ul><ul><li>Sci. Amer. (February 2000) 9 </li></ul></ul></ul><ul><ul><ul><li>Problem of Lateral Gene Transfer (LGT): tree or network </li></ul></ul></ul><ul><ul><ul><li>Problem of higher taxa </li></ul></ul></ul>