Transcript Whole-Genome Prokaryote Phylogeny without Sequence Alignment

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
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
1500kb
– Discovery of the three domains of life: Archaea,
Bacteria and Eucarya
– Support to endosymbiont origin of mitochondria and
chloroplasts
The SSU rRNA Tree of Life:
A big progress in molecular phylogeny
of prokaryotes as evidenced by the
history 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
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, …”
Our Latest Result
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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
innovative agent
• Composition vector may incorporate LGT within
small gene pools
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 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 vectors
}}
Nucleotides: 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
graph (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
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A sequence of length L
A K-string 1 2  K
Frequency of appearance
Probability
f (1 2  K )
f (1 2  K )
P (1 2  K ) 
L  K 1
Predicting #(K-strings) from that of
lengths (K-1) and (K-2) strings
Joint probability vs. conditional probability
p(1 2  K )  p( K 1 2  K 1 ) p(1 2  K 1 )
Making the weakest Markov assumption:
p(1 2  K )  p( K  2  K 1 ) p(1 2  K 1 )
Another joint probability:
p( 2  K 1 K )  p( K  2  K 1 ) p( 2  K 1 )
(K-2)-th Order Markov Model
p(1 2  K 1 ) p( 2  K 1 K )
p (1 2  K 1 K ) 
p( 2  K 1 )
0
Change to frequencies:
f (1  K 1 ) f ( 2  K ) ( L  K  1)( L  K  3)
f (1  K ) 
f ( 2  K 1 )
( L  K  2) 2
0
Normalization factor may be ignored when L>>K
Construct
composition vectors
using these modified string
counts:
For the i-th string type of species A we use
ai  a
 ai
0
ai
0
i
Composition Distance
• Define correlation between two composition
vectors by the cosine of angle
– From two complete proteomes:
A：{a1,a2,……,an}
B：{b1,b2,……,bn}
C ( A, B ) 
n=205 ＝ 3 200 000
a
i
 bi
i
( a 2j  b 2j )
j
1
2
j
C(A,B) ∈[-1,1]
• Distance
–
1 C
D( A, B) 
2
D(A,B)∈[0,1]
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 including
Human 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 prokaryotes
HAMSCA is specific for enterobacteria
Stable Topology of the Tree
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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
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 Bergey’s
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
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 Organisms
From 88 Genera
(K=5)
17 Archaea (12 genera, 17 species)
131 Bacteria (70 genera, 105 species)
6 Eukaryotes