Jonathan Eisen talk "Phylogneomic approaches to functional prediction"a #AFP2012 #ISMB

1. Phylogenomic Approaches to Functional Prediction
Automated Function Prediction SIG
ISMB 2012
July 13, 2012
Jonathan A. Eisen
University of California, Davis
@phylogenomics
Saturday, July 14, 12

2. PAFP
Automated Function Prediction SIG
ISMB 2012
July 13, 2012
Jonathan A. Eisen
University of California, Davis
@phylogenomics
Saturday, July 14, 12

3. PAFP
AFP SIG
ISMB 2012
July 13, 2012
Jonathan A. Eisen
University of California, Davis
@phylogenomics
Saturday, July 14, 12

4. PAFP AFP SIG ISMB 2012
July 13, 2012
Jonathan A. Eisen
University of California, Davis
@phylogenomics
Saturday, July 14, 12

5. Acknowledgements
• $$$
• DOE
• NSF
• GBMF
• Sloan
• DARPA
• DSMZ
• DHS
• People, places
• DOE JGI: Eddy Rubin, Phil Hugenholtz, Nikos Kyrpides
• UC Davis: Aaron Darling, Dongying Wu, Holly Bik, Russell
Neches, Jenna Morgan-Lang
• Other: Jessica Green, Katie Pollard, Martin Wu, Tom Slezak,
Jack Gilbert, Steven Kembel, J. Craig Venter, Naomi Ward,
Hans-Peter Klenk, Phil Hanawalt
Saturday, July 14, 12

6. Phylogenomics of Novelty
Variation in
Mechanisms of
Mechanisms:
Origin of New
Patterns, Causes
Functions
and Effects
Species Evolution
Saturday, July 14, 12

7. Origin of Novelty
• How does novelty originate?
• What are the constraints on evolvability?
• What leads to variation within the genome
and within and between species in
evolvability
• This information helps interpret the past,
understand the present and (maybe)
predict the future
Saturday, July 14, 12

8. History
Saturday, July 14, 12

9. Whatever the History:
Trying to Incorporate it is Critical
from Lake et al. doi: 10.1098/rstb.2009.0035
Saturday, July 14, 12

10. PAFP AFP SIG ISMB 2012 I:
Predicting Functions with Evolutionary Trees
Saturday, July 14, 12

11. SNF2 Family of Proteins (1995)
• SNF2 family defined by presence of conserved
DNA-dependent ATPase domain Bork and Koonin 1993
• 100s of proteins
• Diversity of functions:
• transcriptional activation (SNF2)
• transcriptional repression (MOT1)
• Recombination (RAD54)
• transcription-coupled repair (CSB)
• post-replication repair (RAD5)
• chromosome segregation (lodestar)
• Many with unknown functions
• Some species have 15+ representatives
Saturday, July 14, 12

12. Sub-
Family Protein Helicase Motifs -- I Ia Ib II III IV V VI
BRM
hBRM
hBRG1
SNF2 Alignment Br
Br
Br
SNF2
mBRG1 Br
STH1 Br
SNF2 Br
YB95
F37A4
SNF2L
ISWI
SNF2L
CHD1 C
CHD1
SYGP C
ETL1
ETL1
FUN30
MOT1 MOT1
ERCC6
ERCC6
RAD26
YB53
RAD54
RAD54 DNRPPX
hNUCP
mNUCP
RAD5 R
spRAD8 R
RAD16
HIP116 R
RAD16 R
LODE
NPH42
HepA
B.cereus ORF SCALE (aa)
0 500
Saturday, July 14, 12

13. Saturday, July 14, 12

14. SNF2 Subfamilies
Subfamily Function
SNF2 Transcription activation (Swi/Snf complex)
SNF2L Transcription activation (NURF complex)
CHD1 Chromatin remodelling
ETL1 Unknown
MOT1 Transcription repression
CSB Transcription-coupled repair
Rad54 Recombinational repair
Rad16 Chromatin access for DNA repair
HepA Bacterial RNA polymerase subunit
Saturday, July 14, 12

15. SNF2 Tree and F(x) Prediction
• Function conserved within but not
between subfamilies/orthology groups
• Therefore, assignment of genes to
subfamilies can be used to predict
functions of unknowns
• Grouping into subfamilies helps identify
motifs conserved within groups
• Phylogeny recovers subfamilies better
than similarity searches
Saturday, July 14, 12

16. From Eisen et al.
1997 Nature
Medicine 3:
1076-1078.
Saturday, July 14, 12

17. Blast Search of H. pylori “MutS”
• Blast search pulls up Syn. sp MutS#2 with much higher p
value than other MutS homologs
• Based on this TIGR predicted this species had mismatch
repair
Based on Eisen
• Assumes functional constancy et al. 1997 Nature
Medicine 3:
1076-1078.
Saturday, July 14, 12

18. MutL??
From http://asajj.roswellpark.org/huberman/dna_repair/mmr.html
Saturday, July 14, 12

19. Phylogenetic Tree of MutS Family
Aquae
Strpy
BacsuSynsp
Deira Helpy
Yeast
Human Borbu Metth
Celeg
mSaco
Yeast
Human Yeast
Mouse
Arath Celeg
Human
Arath
Human
Mouse
Spombe Fly
Yeast Xenla
Rat
Mouse
Yeast Human
Spombe Yeast
Neucr
Arath
Aquae Trepa
Chltr
DeiraTheaq
Thema Bacsu Borbu Based on Eisen,
Synsp Strpy 1998 Nucl Acids Res
Ecoli
Neigo 26: 4291-4300.
Saturday, July 14, 12

20. MutS Subfamilies
MSH5 MutS2
Aquae
StrpyBacsuSynsp
Deira Helpy
Yeast
Human Borbu
Celeg Metth
mSaco
MSH6 Yeast
Human
Mouse
Arath
Yeast MSH4
Celeg
Human
Arath
Human
MSH3 Mouse
Fly
Spombe
Yeast Xenla
Rat
Mouse
Yeast
MSH1 Spombe
Human
Yeast
MSH2
Neucr
Arath
Aquae Trepa
Chltr
DeiraTheaq
Bacsu Borbu
Thema
Synsp Strpy
Ecoli
Neigo Based on Eisen,
1998 Nucl Acids Res
MutS1
26: 4291-4300.
Saturday, July 14, 12

21. Overlaying Functions onto Tree
MutS2
Aquae
MSH5 StrpyBacsuSynsp
Deira Helpy
Yeast
Human Borbu
Celeg Metth
MSH6 mSaco
Yeast
Human
Mouse
Arath
Yeast MSH4
Celeg
Human
Arath
Human
MSH3 Mouse
Spombe Fly
Yeast Xenla
Rat
Mouse
Yeast Human
MSH1 Spombe Yeast MSH2
Neucr
Arath
Aquae Trepa
Chltr
DeiraTheaq
Bacsu Borbu
Thema
Synsp Strpy
Ecoli Based on Eisen,
Neigo
1998 Nucl Acids Res
MutS1 26: 4291-4300.
Saturday, July 14, 12

22. MutS Subfamilies
• MutS1
Bacterial MMR
• MSH1
Euk - mitochondrial MMR
• MSH2
Euk - all MMR in nucleus
• MSH3
Euk - loop MMR in nucleus
• MSH6
Euk - base:base MMR in nucleus
• MutS2
Bacterial - function unknown
• MSH4
Euk - meiotic crossing-over
• MSH5
Euk - meiotic crossing-over
Saturday, July 14, 12

23. Functional Prediction Using Tree
MSH5 - Meiotic Crossing Over MutS2 - Unknown Functions
Aquae
StrpyBacsuSynsp
Deira Helpy
Yeast
Human Borbu
Celeg Metth
MSH6 - Nuclear mSaco
Repair
Of Mismatches Yeast
Human MSH4 - Meiotic Crossing
Mouse Yeast Over
Arath
Celeg
Human
Arath
MSH3 - Nuclear Human
Mouse
RepairOf Loops Spombe Fly
Yeast Xenla
Rat
Mouse MSH2 - Eukaryotic Nuclear
Yeast Human Mismatch and Loop Repair
MSH1 Spombe Yeast
Mitochondrial Neucr
Arath
Repair
Aquae Trepa
Chltr
DeiraTheaq
Bacsu Borbu
Thema
Synsp Strpy
Ecoli Based on Eisen,
Neigo
1998 Nucl Acids Res
MutS1 - Bacterial Mismatch and Loop Repair 26: 4291-4300.
Saturday, July 14, 12

24. Ancient MutS Duplication
Saturday, July 14, 12

25. MutS1,2 vs MutL
Table 3. Presence of MutS Homologs in Complete Genomes Sequences
Species # of MutS Which MutL
Homologs Subfamilies? Homologs
Bacteria
Escherichia coli K12 1 MutS1 1
Haemophilus influenzae Rd KW20 1 MutS1 1
Neisseria gonorrhoeae 1 MutS1 1
Helicobacter pylori 26695 1 MutS2 -
Mycoplasma genitalium G-37 - - -
Mycoplasma pneumoniae M129 - - -
Bacillus subtilis 169 2 MutS1,MutS2 1
Streptococcus pyogenes 2 MutS1,MutS2 1
Mycobacterium tuberculosis - - -
Synechocystis sp. PCC6803 2 MutS1,MutS2 1
Treponema pallidum Nichols 1 MutS1 1
Borrelia burgdorferi B31 2 MutS1,MutS2 1
Aquifex aeolicus 2 MutS1,MutS2 1
Deinococcus radiodurans R1 2 MutS1,MutS2 1
Archaea
Archaeoglobus fulgidus VC-16, DSM4304 - - -
Methanococcus janasscii DSM 2661 - - -
Methanobacterium thermoautotrophicum ΔH 1 MutS2 -
Eukaryotes
Saccharomyces cerevisiae 6 MSH1-6 3+
Homo sapiens 5 MSH2-6 3+
Saturday, July 14, 12

26. Saturday, July 14, 12

27. PHYLOGENENETIC PREDICTION OF GENE FUNCTION
EXAMPLE A METHOD EXAMPLE B
2A CHOOSE GENE(S) OF INTEREST 5
3A 1 3 4
2B 2
IDENTIFY HOMOLOGS 5
1A 2A 1B 3B 6
ALIGN SEQUENCES
1A 2A 3A 1B 2B 3B 1 2 3 4 5 6
CALCULATE GENE TREE
Duplication?
1A 2A 3A 1B 2B 3B 1 2 3 4 5 6
OVERLAY KNOWN
FUNCTIONS ONTO TREE
Duplication?
2B 3B 1 2 3 4 5 6
1A 2A 3A 1B
INFER LIKELY FUNCTION
OF GENE(S) OF INTEREST
Ambiguous
Duplication?
Species 1 Species 2 Species 3
1A 1B 1 2 3 4 5 6
2A 2B 3A 3B
ACTUAL EVOLUTION
(ASSUMED TO BE UNKNOWN) Based on Eisen,
1998 Genome
Duplication
Res 8: 163-167.
Saturday, July 14, 12

28. Evolutionary Rate Variation
1 2
4 6
3
5
Saturday, July 14, 12

29. Functional Diversity of Proteorhodopsins?
Venter et al., Science
304: 66. 2004
Saturday, July 14, 12

30. Phylogenetic Challenge
A single tree with everything?
Saturday, July 14, 12

31. Phylosift/ pplacer
Saturday, July 14, 12

32. rRNA Phylotyping
DNA
extraction PCR
Makes lots of Sequence
PCR copies of the rRNA genes
rRNA genes
in sample
rRNA1
5’...ACACACATAGGTGGAGCTA
GCGATCGATCGA... 3’
Phylogenetic tree Sequence alignment = Data matrix
rRNA2
rRNA1 rRNA2
rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCTAG
CGACGATCGA... 3’
rRNA4
rRNA3 rRNA2 T A C A G T
rRNA3
rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGATT
E. coli Humans rRNA4 C A C A G T CTAGCGATATAGA... 3’
Yeast E. coli A G A C A G rRNA4
5’...ACGGCCCGATAGGTGGATT
Humans T A T A G T CTAGCGCCATAGA... 3’
Yeast T A C A G T
Saturday, July 14, 12

33. Eisen et al. 2002
Eisen et
al. 1992
Saturday, July 14, 12

34. PAFP AFP SIG ISMB 2012 II:
Every gene family is unique ...
Saturday, July 14, 12

35. Saturday, July 14, 12

36. Steps in Phylogenomics
• Create database of genes of interest
• Presence/absence of homologs in complete genomes
• Phylogenetic trees of each gene family
• Infer evolutionary events (gene origin, duplication, loss and
transfer)
• Refine presence/absence (orthologs, paralogs, subfamilies)
• Functional predictions and functional evolution
• Analysis of pathways
Saturday, July 14, 12

37. Photoreactivation/Photolyases
• All photoreactivation is carried out by enzymes in the photolyase
family
• Two main classes of photolyases – class I and class II – are distantly
related to each other and likely the result of an ancient duplication
• PhrI and PhrII missing from most species for which complete genomes
are available.
• Many cases of functional change (e.g., CPD -> 6-4) and some are not
even involved in DNA repair
• Many of the eukaryotic proteins appear to be of an organellar
ancestry
Saturday, July 14, 12

38. Photoreactivation
• All known enzymes that perform photoreactivation are part of
a single large photolyase gene family
• Some members of the family do not function as photolyases, but
instead work as blue-light receptors
• If a species does not encode a member of the photolyase gene
family, it likely does not have photoreactivation capability
• If a species encodes a photolyase, one cannot conclude it has
photolyase activity
• Position of photolyase homologs within photolyase tree helps
predict what activities they have
Saturday, July 14, 12

39. Alkyltransferases
• All known alkyltransferases are members of a single gene
family
• Found in most but not all species
• Likely present in LUCA
• Ada protein in E. coli originated by fusion between an
alkyltransferase and a transcription-regulatory domain
• Gram-positive bacteria have the Ada domain fused to an
alkylation glycosylase instead of alkyltransferase
Saturday, July 14, 12

40. BER Glycosylases
• Distribution patterns highly uneven but some glycosylases
have been found in all species
• Some are ancient enzymes, probably presence in LUCA (e.g.,
MutY-Nth), others more recent (e.g., TagI).
• Many families are distantly related to each other (e.g., Ogg,
AlkA, MutY-Nth)
• Many cases of gene duplication, loss and possibly transfer,
especially from organellar genomes to nucleus
• Orthologs frequently have different specificity
Saturday, July 14, 12

41. AP Endonucleases
• All species encode either Nfo or Xth homologs. Some encode
both.
• Only Nfo: mycoplasmas, Aquifex, M. jannascii, yeast
• Only Xth: many bacteria, A. fulgidus, humans (so far)
• Both: E. coli, B. subtilis, M. tuberculosis, M.
thermoautotrophicum
• Both Nfo and Xth are likely ancient.
• Many cases of gene loss of one or the other, but never both
Saturday, July 14, 12

42. Uracil Glycosylase
• Many non-homologous proteins have uracil-
DNA glycosylase activity (Ung, GPADH, MUG,
cyclin)
• Therefore, absence of homologs of these
genes should not be used to infer likely
absence of activity
• However, presence of homologs of Ung and
MUG genes can be used to indicate presence
of activity because all homologs of these
genes have this activity
Saturday, July 14, 12

43. Not Open Access
Saturday, July 14, 12

44. Saturday, July 14, 12

45. PAFP AFP SIG ISMB 2012 III:
When phylogeny is not enough ...
Saturday, July 14, 12

46. But ...
• Many powerful and automated similarity based
methods for assigning genes to protein families
• COGs
• PFAM HMM searches
• Some limitations of similarity based methods can
be overcome by phylogenetic approaches
• Automated methods now available
• Sean Eddy
• Steven Brenner
• Kimmen Sjölander
• But …
Saturday, July 14, 12

47. Example: Recent Changes
• Phylogenomic functional prediction may NJ
* **
V.cholerae0512
VC
V.cholerae
VCA1034
V.cholerae
VC
V.cholerae
VC
A0974
A0068
V.cholerae
VC
0825
not work well for very newly evolved
V.cholerae
VC0282
V.cholerae
VCA0906
V.cholerae
VCA0979
V.cholerae
VCA1056
V.cholerae
VC1643
V.cholerae
VC2161
** V.cholerae
VC0923
A
** V.cholerae
VC0514
V.cholerae
VC
1868
V.cholerae
VC
A0773
functions
V.cholerae
VC1313
V.cholerae
VC
1859
V.cholerae
VC1413
V.cholerae
VCA0268
** V.cholerae
VC
A0658
V.cholerae
VC
1405
* V.cholerae
VC1298
V.cholerae
VC1248
V.cholerae
VCA0864
V.cholerae
VCA0176
** V.cholerae
VCA0220
V.cholerae
VC
1289
V.cholerae
VC1069
A
• Can use understanding of origin of novelty
** V.cholerae
VC2439
V.cholerae
VC967
1
V.cholerae
VC
A0031
V.cholerae
VC1898
V.cholerae
VCA0663
V.cholerae
VC0988
A
V.cholerae
VC0216
* V.cholerae
VC0449
V.cholerae
VCA0008
V.cholerae
VC1406
to better interpret these cases?
V.cholerae
VC1535
V.cholerae
VC0840
B.subtilis
gi2633766
Synechocystis
sp.
gi1001299
* Synechocystis
sp.gi1001300
* Synechocystis
sp.
gi1652276
* Synechocystis
sp.
gi1652103
H.pylori
gi2313716
H.pylori
99 gi4155097
** C.jejuni
** Cj1190c
C.jejuni
Cj1110c
A.fulgidus
gi2649560
A.fulgidus
gi2649548
• Screen genomes for genes that have
** B.subtilis
gi2634254
B.subtilis
gi2632630
B.subtilis
gi2635607
B.subtilis
gi2635608
** B.subtilis
gi2635609
** ** B.subtilis gi2635610
B.subtilis
gi2635882
E.coli
gi1788195
E.coli
gi2367378
* ** E.coli
gi1788194
changed recently
E.coli A1092
gi1787690
V.cholerae
VC
V.cholerae
VC
0098
E.coli
gi1789453
H.pylori
gi2313186
H.pylori
99 gi4154603
** C.jejuni
Cj0144
C.jejuni
Cj1564
**C.jejuni
C.jejuni
Cj0262c
Cj1506c
** H.pylori
gi2313163
* ** H.pylori
99 gi4154575
– Pseudogenes and gene loss ** H.pylori
gi2313179
** H.pylori
99 gi4154599
C.jejuni
Cj0019c
C.jejuni
Cj0951c
C.jejuni
Cj0246c
B.subtilis
gi2633374
T.maritima
TM0014
V.cholerae
VC1403
V.cholerae
VC
A1088
T.pallidum
gi3322777
T.pallidum
gi3322939
– Contingency Loci **** T.pallidum
gi3322938
B.burgdorferi
gi2688522
T.pallidum
gi3322296
B.burgdorferi
gi2688521
* T.maritima
TM0429
**T.maritima
TM0918
* ** T.maritima
T.maritima
TM0023
TM1428
T.maritima
TM1143
T.maritima
TM1146
P.abyssi
PAB1308
– Acquisition (e.g., LGT)
P.horikoshii
** P.abyssi gi3256846
** PAB1336
P.horikoshii
**P.abyssi gi3256896
** PAB2066
** * ** P.horikoshii
P.abyssi
gi3258290
PAB1026
** P.horikoshii DRA00354
gi3256884
D.radiodurans
D.radiodurans
** D.radioduransDRA0353
** DRA0352
** V.cholerae
VC
1394
P.abyssi
PAB1189
– Unusual dS/dN ratios P.horikoshii
gi3258414
** B.burgdorferi
gi2688621
M.tuberculosis
gi1666149
V.cholerae
VC
0622
– Rapid evolutionary rates
– Recent duplications
Saturday, July 14, 12

48. Non-Homology Predictions:
Phylogenetic Profiling
• Step 1: Search all genes in
organisms of interest against all
other genomes
• Ask: Yes or No, is each gene found
in each other species
• Cluster genes by distribution
patterns (profiles)
Pelligrini et al. 1999. PNAS 96: 4285.
Saturday, July 14, 12

49. Correlated gain/loss of genes
• Microbial genes are lost rapidly when not
maintained by selection
• Genes can be acquired by lateral transfer
• Frequently gain and loss occurs for entire
pathways/processes
• Thus might be able to use correlated presence/
absence information to identify genes with
similar functions
Saturday, July 14, 12

50. Carboxydothermus hydrogenoformans
• Isolated from a Russian hotspring
• Thermophile (grows at 80°C)
• Anaerobic
• Grows very efficiently on CO
• Produces hydrogen gas
• Low GC Gram + (Firmicute)
• Genome Determined
Wu et al. 2005
PLoS Genetics 1: e65.
Saturday, July 14, 12

51. Homologs of Sporulation Genes
Wu et al. 2005
PLoS Genetics 1: e65.
Saturday, July 14, 12

52. Carboxydothermus sporulates
Wu et al. 2005
PLoS Genetics 1: e65.
Saturday, July 14, 12

53. Wu et al. 2005 PLoS Genetics 1: e65.
Saturday, July 14, 12

54. PG Profiling Works Better with Families
Saturday, July 14, 12

55. PAFP AFP SIG ISMB 2012 IV:
Knowing What You Don’t Know
Saturday, July 14, 12

56. As of 2002 Proteobacteria
TM6
OS-K • At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
WS3
Gemmimonas
Firmicutes
Fusobacteria
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

57. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Most genomes
WS3
Gemmimonas from three
Firmicutes
Fusobacteria phyla
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

58. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Most genomes
WS3
Gemmimonas from three
Firmicutes
Fusobacteria phyla
Actinobacteria
OP9
Cyanobacteria
Synergistes
• Some studies
Deferribacteres
Chrysiogenetes in other phyla
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

59. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Most genomes
WS3
Gemmimonas from three
Firmicutes
Fusobacteria phyla
Actinobacteria
OP9
Cyanobacteria
Synergistes
• Some other
Deferribacteres
Chrysiogenetes phyla are only
NKB19
Verrucomicrobia
Chlamydia
sparsely
OP3
Planctomycetes
Spriochaetes
sampled
Coprothmermobacter
OP10 • Same trend in
Thermomicrobia
Chloroflexi
TM7
Eukaryotes
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

60. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Most genomes
WS3
Gemmimonas from three
Firmicutes
Fusobacteria phyla
Actinobacteria
OP9
Cyanobacteria
Synergistes
• Some other
Deferribacteres
Chrysiogenetes phyla are only
NKB19
Verrucomicrobia
Chlamydia
sparsely
OP3
Planctomycetes
Spriochaetes
sampled
Coprothmermobacter
OP10 • Same trend in
Thermomicrobia
Chloroflexi
TM7
Viruses
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

61. TIGR TOL 2002
Saturday, July 14, 12

62. GEBA
Saturday, July 14, 12

63. GEBA Lesson 1:
Improves genome annotation
• Took 56 GEBA genomes and compared results vs. 56
randomly sampled new genomes
• Better definition of protein family sequence “patterns”
• Greatly improves “comparative” and “evolutionary”
based predictions
• Conversion of hypothetical into conserved hypotheticals
• Linking distantly related members of protein families
• Improved non-homology prediction
Saturday, July 14, 12

64. GEBA Lesson 2:
Metadata Important
Saturday, July 14, 12

65. GEBA Lesson 3:
Improves discovering new genetic diversity
Saturday, July 14, 12

66. Protein Family Rarefaction
• Take data set of multiple complete
genomes
• Identify all protein families using MCL
• Plot # of genomes vs. # of protein families
Saturday, July 14, 12

67. Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

68. Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

69. Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

70. Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

71. Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

72. Synapomorphies exist
Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

73. Families/PD not uniform
31
6
Saturday, July 14, 12

74. Structural Novelty
• Of the 17000 protein families in the GEBA56, 1800 are
novel in sequence (Wu)
• Structural modeling suggests many are structurally novel
too (D'haeseleer)
• 372 being crystallized by the PSI (Kerfeld)
Saturday, July 14, 12

75. Needed Reference Tree
Saturday, July 14, 12

76. GEBA Lesson 4:
Much diversity untouched
Saturday, July 14, 12

77. rRNA Tree of Life
FIgure from Barton, Eisen et al.
“Evolution”, CSHL Press.
Based on tree from Pace NR, 2003.
Saturday, July 14, 12

78. Phylogenetic Diversity:
From Wu et
al. 2009
Nature 462,
1056-1060
Saturday, July 14, 12

79. Phylogenetic Diversity with
From Wu et
al. 2009
Nature 462,
1056-1060
Saturday, July 14, 12

80. Phylogenetic Diversity: Isolates
From Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

81. Haloarchaeal GEBA-like
Saturday, July 14, 12

82. Phylogenetic Diversity: All
From Wu et al. 2009 Nature 462, 1056-1060
Saturday, July 14, 12

83. Uncultured Lineages:
• Get into culture
• Enrichment cultures
• If abundant in low diversity ecosystems
• Flow sorting
• Microbeads
• Microfluidic sorting
• Single cell amplification
Saturday, July 14, 12

84. GEBA uncultured
Number of SAGs from Candidate Phyla
406
1
OD1
OP1
OP3
SAR
Site A: Hydrothermal vent 4 1 - -
Site B: Gold Mine 6 13 2 -
Site C: Tropical gyres (Mesopelagic) - - - 2
Site D: Tropical gyres (Photic zone) 1 - - -
Sample collections at 4 additional sites are underway.
Phil Hugenholtz
80
Saturday, July 14, 12

85. RecA, RpoB in GOS
GOS 1
GOS 2
GOS 3
GOS 4
GOS 5
Wu et al PLoS One 2011
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86. GEBA Lesson 6:
Experimental diversity
Saturday, July 14, 12

87. As of 2002 Proteobacteria
TM6
OS-K • At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
WS3
Gemmimonas
Firmicutes
Fusobacteria
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

88. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Experimental
WS3
Gemmimonas studies are
Firmicutes
Fusobacteria mostly from
Actinobacteria
OP9
Cyanobacteria
three phyla
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

89. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Experimental
WS3
Gemmimonas studies are
Firmicutes
Fusobacteria mostly from
Actinobacteria
OP9
Cyanobacteria
three phyla
Synergistes
Deferribacteres
Chrysiogenetes • Some studies
NKB19
Verrucomicrobia
Chlamydia
in other phyla
OP3
Planctomycetes
Spriochaetes
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

90. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Genome
WS3
Gemmimonas sequences are
Firmicutes
Fusobacteria mostly from
Actinobacteria
OP9
Cyanobacteria
three phyla
Synergistes
Deferribacteres
Chrysiogenetes • Some other
NKB19
Verrucomicrobia
Chlamydia
phyla are only
OP3
Planctomycetes
Spriochaetes
sparsely
Coprothmermobacter
OP10
sampled
Thermomicrobia
Chloroflexi
TM7
• Same trend in
Deinococcus-Thermus
Dictyoglomus
Aquificae
Eukaryotes
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

91. As of 2002 Proteobacteria
TM6
OS-K
• At least 40
Acidobacteria
Termite Group
OP8
phyla of
Nitrospira
Bacteroides
bacteria
Chlorobi
Fibrobacteres
Marine GroupA
• Genome
WS3
Gemmimonas sequences are
Firmicutes
Fusobacteria mostly from
Actinobacteria
OP9
Cyanobacteria
three phyla
Synergistes
Deferribacteres
Chrysiogenetes • Some other
NKB19
Verrucomicrobia
Chlamydia
phyla are only
OP3
Planctomycetes
Spriochaetes
sparsely
Coprothmermobacter
OP10
sampled
Thermomicrobia
Chloroflexi
TM7
• Same trend in
Deinococcus-Thermus
Dictyoglomus
Aquificae
Viruses
Thermudesulfobacteria
Thermotogae
OP1 Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

92. Proteobacteria
TM6
OS-K
Need
Acidobacteria
Termite Group
OP8
experimental
Nitrospira
Bacteroides
Chlorobi
studies from
Fibrobacteres
Marine GroupA
WS3
across the tree
Gemmimonas
Firmicutes
too
Fusobacteria
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes 0.1
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1
Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

93. Proteobacteria
TM6
OS-K
Adopt a
Acidobacteria
Termite Group
OP8
Microbe
Nitrospira
Bacteroides
Chlorobi
Fibrobacteres
Marine GroupA
WS3
Gemmimonas
Firmicutes
Fusobacteria
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes 0.1
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae
Thermudesulfobacteria
Thermotogae
OP1
Based on Hugenholtz, 2002
OP11
Saturday, July 14, 12

94. Acknowledgements
• $$$
• DOE
• NSF
• GBMF
• Sloan
• DARPA
• DSMZ
• DHS
• People, places
• DOE JGI: Eddy Rubin, Phil Hugenholtz, Nikos Kyrpides
• UC Davis: Aaron Darling, Dongying Wu, Holly Bik, Russell
Neches, Jenna Morgan-Lang
• Other: Jessica Green, Katie Pollard, Martin Wu, Tom Slezak,
Jack Gilbert, Steven Kembel, J. Craig Venter, Naomi Ward,
Hans-Peter Klenk
Saturday, July 14, 12