Whole-Genome Prokaryote Phylogeny without

Sequence Alignment

Bailin HAO and Ji QI

T-Life Research Center, Fudan UniversityShanghai 200433, China

Institute of Theoretical Physics, Academia SinicaBeijing 100080, China

http://www.itp.ac.cn/~hao/

Classification of Prokaryotes:A Long-Standing Problem

• Traditional taxonomy: too few features• Morphology ： spheric, helices, rod-shaped……• Metabolism ： photosythesis, N-fixing, desulfurization…

…• Gram staining ： positive and negative

• SSU rRNA Tree (Carl Woese et al., 1977):– 16S rRNA: ancient conserved sequences of about 15

00kb– Discovery of the three domains of life: Archaea, Bact

eria and Eucarya– Support to endosymbiont origin of mitochondria and

chloroplasts

The SSU rRNA Tree of Life:A big progress in molecular phylogeny o

f prokaryotes as evidenced by thehistory of the

Bergey’s Manual

Bergey’s Manual Trust:

Bergey’s 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

Phylogeny versus Taxonomy• Phylogeny and taxonomy are not synonyms• Taxonomy – classification, systematics of extant sp

ecies• Phylogeny – the history of evolution since the origi

n 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, …”

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.

基于 222 个完全基因组的亲缘树(K=5)

21 个古细菌193 个真细菌8 个真核生物

Complete Bacterial Genomes Appeared since 1995

Early Expectations:

• More support to the SSU rRNA Tree of Life

• Add details to the classification (branchings and groupings)

• More hints on taxonomic revisions

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

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 in

novative agent• Composition vector may incorporate LGT within s

mall gene pools

Our Motivations:• Develop a molecular phylogeny method that make

s use of complete genomes – no selection of particular genes

• Avoid sequence alignment • Try to reach higher resolution to provide an indepe

ndent comparison with other approaches such as SSU rRNA trees

• Make comparison with bacteriologists’ systematics as reflected in Bergey’s Manual (2001 - 2003)

• Qi, Wang, Hao, J. Molecular Evolution, 58 (1) (Jaunary 2004) 1 – 11. (109=16A+87B+6E)

Comparison of Complete Genomes/Proteomes

• Compositional vectorsNucleotides: a 、 t 、 c 、 g

aatcgcgcttaagtc

Di-nucleotide (K=2) 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}

} }

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 -> uniqueness• Connection with the number of Eulerian loops in a gra

ph (a separate study available as a preprint at ArXiv:physics/0103028 and from Hao’s webpage)

↑

A Key Improvement:Subtraction 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

Frequency and Probability

• A sequence of length

• A K-string

• Frequency of appearance

• Probability

L

K 21

)( 21 Kf

1

)()( 21

21

KL

fP K

K

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:

)()()( 12112121 KKKK ppp ) ( ) ( ) (1 2 1 1 2 2 1 K K K Kp p p

)()()( 121212 KKKKK ppp

(K-2)-th Order Markov Model

Change to frequencies:

Normalization factor may be ignored when L>>K

)(

)()()(

12

12121121

0

K

KKKKK p

ppp

212

2111

0

)2(

)3)(1(

)(

)()()(

KL

KLKL

f

fff

K

KKK

Construct

composition vectors

using these modified string counts:

For the i-th string type of species A we use

ii

ii aa

aa

0

0

Composition Distance

• Define correlation between two composition vectors by the cosine of angle – From two complete proteomes:

A ： {a1,a2,……,an} n=205 ＝ 3 200 000

B ： {b1,b2,……,bn}

C(A,B) [-1,1]∈• Distance

– D(A,B) [0,1]∈

jj

jj

iii

ba

baBAC

2

122 )(

),(

2

1),(

CBAD

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.

Genus Tree based on Ribosomal

Proteins

A Genus Tree based on Aminoacyl tRNA synthetases

Chloroplast Tree

• Sequences of about 100 000 bp

• Tree of the endosymbiont partners

• Paper appeared in Molecular Biology and Evolution, 21 (2004), 200-206.

Chloroplast tree

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)

Coronavirus tree

Understanding the Subtraction Procedure:Analysis 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.

GKSTL: how 58 reduces to 0.646?

• #(GKST)=113

• #(KSTL)=77

• #(KST)=247

• Markov prediction: 113*77/247=35.23

• Final result: (58-35.23)/35.23=0.646

HAMSC: how 1 grows to 197?

• #(HAMS)=1

• #(AMSC)=1

• #(AMS)=198

• Markov prediction: 1*1/198=1/198

• Final result: (1-1/198)/(1/198)=197

6121 Exact Matches of GKSTL

In PIR Rel.1.26 with >1.2 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.

15 Exact Matches of HAMSC:

In PIR Rel.1.26 with >1.2 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 prokaryotesHAMSCA is specific for enterobacteria

Stable Topology of the Tree• K=1: makes some sense!

• K=2,3,4: topology gradually converges

• K=5 and K=6: present calculation

• K=7 and more: beyond our computing capability at present; too high resolution; star-tree or bush expected

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

About 70% genes for

every species were selected

in one bootstrap

“K-string Picture” of Evolution

• K=5 ->3 200 000 points in space of

5-strings

• K=6 ->64 000 000 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

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

References

• J Qi, B Wang, BL Hao, J. Mol. Evol. 58 (2004) 1-11. (109=16A + 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 132=16A + 110B + 6E)

Summary As composition vectors do not depend on genome size a

nd 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 Bergey’s Ma

nual Hint on groupings of higher taxa A method without “free parameters”: data in, tree out Possibility of an automatic and objective classification to

ol for prokaryotes

Conclusion:

The 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!

A Protein Tree for 154 OrganismsFrom 88 Genera

(K=5)

17 Archaea (12 genera, 17 species)

131 Bacteria (70 genera, 105 species)

6 Eukaryotes