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Phylogenomics:
A Genome Level Approach to
Assembling the Bacterial Branches
of the Tree of Life
TIGR:
Jonathan A. Eisen
Naomi Ward
Karen E. Nelson
Jonathan H. Badger
James Sakwa
Martin Wu
Dongying Wu
Kevin Penn
COMB:
Frank T. Robb
Elizabeth M. O’Connor
Julie Enticknap
Tim Steppe

http://www.tigr.org/tol

TIGR

QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Background I: rRNA Tree

• Phenotype not very useful for
bacterial phylogeny
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.

• Most molecular studies based on
16s rRNA sequence analysis
• Studies of other genes do not
always agree with rRNA,
especially for deep branches

from Woese 1987

TIGR
Background II:
Most Bacteria Have Never Been Cultured
• Microscopic and molecular studies show
that <1% of the microbes in most
environments have been grown in pure
culture
• True in terms of #s and phylogenetic
diversity
• This means we know little about their
biology
TIGR
Background III:
Genomics Has Revolutionized Bacteriology
•
•
•
•

Predictions of biology
Drug design, vaccine development
Functional genomic studies
Evolutionary reconstructions
– Whole genome phylogeny
– Lateral gene transfer
– Population genomics

TIGR
Biased Sampling of Bacterial Genomes

TIGR

Hugenholtz
2002
TIGR

Automated
Whole
Genome
Phylogeny
TIGR Tree of Life Project
• Major goal
– Increase phylogenetic diversity of genome sequences

• Three sub-goals
– Resolve relationships among the phyla
– Launch experimental studies of these phyla
– Inform environmental studies of uncultured microbes

• The Players
– TIGR (Jonathan Eisen, Naomi Ward, Karen Nelson et
al.)
– COMB (Frank Robb et al.)

TIGR
TIGR

Proteobacteria
TM6
OS-K
Acidobacteria
Termite Group
OP8
Nitrospira
CFB
Chlorobi
Fibrobacteres
Marine GroupA
WS3
Gemmimonas
Firmicutes
Fusobacteria
This project
Actinobacteria
OP9
Published
Cyanobacteria
Synergistes
In progress
Deferribacteres
Uncultured lineage
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes
0.1
Coprothermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificacae
Tree based on
Thermodesulfobacteria
Hugenholtz (2002)
Thermotogae
with some
OP1
modifications.
OP11
Genome Sequencing Progress
Phylum

Sp
eciesse
lected

Libr
aries

Chry
siogenesars tis
ena

Grow
th,
DNA
iso
lation
+

+

Sho
tgun Estim
ated # of
Aut
oCover
age Gen e Contigs Ann
om
otated
Size (Mb)
4x
2.5
155
+

Chry
siogenes
Cop hermobacter
rot

Copr
otherm cter pro
oba
teolyticus(CP)

+

+

8x

1.38

3

+

Dicty
oglomi

Dictyoglomus m hilum(DT)
ther op

+

+

8x

2.0

9

+

Thermo ulfobacteria
des

Therm sulfobacte
ode
riumcomm (TC)
une

+

+

8x

1.78

26

+

Nitrosp
irae

Therm sulfovib ye
ode
rio llows ii (TY)
ton

+

+

8x

1.98

27

+

Thermomicrobia

Therm icro
om biumroseum

+

+

8x

3.4

82

+

De ibacteres
ferr

Se
lectingfrom
Deferr cter thermohilus,
iba
p
Ge
ovib thiop
rio
hilus,
Flexistipessinusara
bici
Se
lectingfrom
Sy
nergistesjones
ii,
Am bacter co biense,
ino
lom
Therm
anaero
vibrio acida inovor
m
ans,
Am
inom
onas p
aucivorans,
Dethio
sulfovib peptido
rio
vorans

+

In
prog
ress

+

In
prog
ress

Syn stes
ergi

TIGR

Type Strain or Species Selected When Possible
Goal I:
Relationships Among Phyla

TIGR
Concatenated Alignment ML Tree

TIGR
Proteobacteria

TIGR
Green Non Sulfur Bacteria

TIGR
Goal II:
Biology of These Phyla

TIGR
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.

TIGR
Goal III:
Uncultured Microbes

TIGR
Key Issues in Uncultured Microbes
• Questions
– 1. Who is out there?
– 2. What are they doing?
– 3. Need to connect 1 and 2.

• Answers
– 1. Phylogenetic anchors
– 2. Genomics
– 3. Linking anchors to genomics contigs

TIGR
Phylogenetic Anchors and the Sargasso
Sea Shotgun Sequencing

Warner Brothers, Inc.

sequence

TIGR

shotgun
rRNA as a Phylogenetic Anchor

TIGR

Venter et al., 2004
Shotgun Sequencing Allows Use of
Alternative Anchors (e.g., RecA)

TIGR

Venter et al., 2004
Biased Sampling of Bacterial Genomes

Hugenholtz
2002

TIGR
Our Tree of Life
Genomes Allow
Anchoring of 100s
of clones from
Yellowstone Mats
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.

TIGR
Tree of GYOAP59TF

TIGR
Blast of GYOAP59TF
>ORF02612-TG_gtr_857 rho transcription termination factor Rho
{Thermomicrobium_roseum_DSM5159}
Length = 426
Score = 1177 (419.4 bits), Expect = 2.9e-119, P = 2.9e-119
Identities = 232/232 (100%), Positives = 232/232 (100%)
Query:
Sbjct:
Query:
Sbjct:
Query:
Sbjct:
Query:
Sbjct:

TIGR

1 VAPIGRGQRGLIVSPPKAGKTVLLKHIANGITTNYKDIHLIVLLIGERPEEVTDMRRSVD 60
VAPIGRGQRGLIVSPPKAGKTVLLKHIANGITTNYKDIHLIVLLIGERPEEVTDMRRSVD
191 VAPIGRGQRGLIVSPPKAGKTVLLKHIANGITTNYKDIHLIVLLIGERPEEVTDMRRSVD 250
61 GEVISSTFDEPVEDHIRVAEMTLERAKRLVECGMDVVILLDSITRLARAYNLSVPPSGRT 120
GEVISSTFDEPVEDHIRVAEMTLERAKRLVECGMDVVILLDSITRLARAYNLSVPPSGRT
251 GEVISSTFDEPVEDHIRVAEMTLERAKRLVECGMDVVILLDSITRLARAYNLSVPPSGRT 310
121 LSGGIDPVALYPPKRFFGAARNIEGGGSLTIIATCLVDTGSRMDDVIYEEFKGTGNMELH 180
LSGGIDPVALYPPKRFFGAARNIEGGGSLTIIATCLVDTGSRMDDVIYEEFKGTGNMELH
311 LSGGIDPVALYPPKRFFGAARNIEGGGSLTIIATCLVDTGSRMDDVIYEEFKGTGNMELH 370
181 LDRKLAERRIFPAIDIQRSGTRREELLLDEQTLRQVWTMRRMVSMLGGTDGT 232
LDRKLAERRIFPAIDIQRSGTRREELLLDEQTLRQVWTMRRMVSMLGGTDGT
371 LDRKLAERRIFPAIDIQRSGTRREELLLDEQTLRQVWTMRRMVSMLGGTDGT 422
Broader Impact
• Genome sequence data released to TIGR web site
• Genome Users Workshop to be held at TIGR in
2005
• Project used as model for evolution teaching in
talks to HHMI, MD Science Teachers Assocation,
and Montgomery County high school science
teachers
• Project promoted to public through radio and print
media

TIGR
Training
• Student involvement
– Kevin Penn, technician at TIGR
– Temylope Adeyefa-Olasupo, U. Md Undergrad working
at COMB
– Rebecca Brocato, high school intern, COMB
– Joseph Wister, TIGR Summer Fellow with Karen Nelson
– Ryan Corces-Zimmerman, high school intern working at
TIGR

• Teaching using this project
– MBL Molecular Evolution Workshop
– MBL Genomics Workshop
– Jackson Lab Genomics Workshop

TIGR
Future Issues
• New estimates suggests > 100 bacterial
phyla
• Someone needs to cover Archaeal diversity
• Need DNA repository

TIGR

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TIGR "Tree of Life" Project Slides (from 2004)

  • 1. Phylogenomics: A Genome Level Approach to Assembling the Bacterial Branches of the Tree of Life TIGR: Jonathan A. Eisen Naomi Ward Karen E. Nelson Jonathan H. Badger James Sakwa Martin Wu Dongying Wu Kevin Penn COMB: Frank T. Robb Elizabeth M. O’Connor Julie Enticknap Tim Steppe http://www.tigr.org/tol TIGR QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture.
  • 2. Background I: rRNA Tree • Phenotype not very useful for bacterial phylogeny QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. • Most molecular studies based on 16s rRNA sequence analysis • Studies of other genes do not always agree with rRNA, especially for deep branches from Woese 1987 TIGR
  • 3. Background II: Most Bacteria Have Never Been Cultured • Microscopic and molecular studies show that <1% of the microbes in most environments have been grown in pure culture • True in terms of #s and phylogenetic diversity • This means we know little about their biology TIGR
  • 4. Background III: Genomics Has Revolutionized Bacteriology • • • • Predictions of biology Drug design, vaccine development Functional genomic studies Evolutionary reconstructions – Whole genome phylogeny – Lateral gene transfer – Population genomics TIGR
  • 5. Biased Sampling of Bacterial Genomes TIGR Hugenholtz 2002
  • 7. TIGR Tree of Life Project • Major goal – Increase phylogenetic diversity of genome sequences • Three sub-goals – Resolve relationships among the phyla – Launch experimental studies of these phyla – Inform environmental studies of uncultured microbes • The Players – TIGR (Jonathan Eisen, Naomi Ward, Karen Nelson et al.) – COMB (Frank Robb et al.) TIGR
  • 8. TIGR Proteobacteria TM6 OS-K Acidobacteria Termite Group OP8 Nitrospira CFB Chlorobi Fibrobacteres Marine GroupA WS3 Gemmimonas Firmicutes Fusobacteria This project Actinobacteria OP9 Published Cyanobacteria Synergistes In progress Deferribacteres Uncultured lineage Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes 0.1 Coprothermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificacae Tree based on Thermodesulfobacteria Hugenholtz (2002) Thermotogae with some OP1 modifications. OP11
  • 9. Genome Sequencing Progress Phylum Sp eciesse lected Libr aries Chry siogenesars tis ena Grow th, DNA iso lation + + Sho tgun Estim ated # of Aut oCover age Gen e Contigs Ann om otated Size (Mb) 4x 2.5 155 + Chry siogenes Cop hermobacter rot Copr otherm cter pro oba teolyticus(CP) + + 8x 1.38 3 + Dicty oglomi Dictyoglomus m hilum(DT) ther op + + 8x 2.0 9 + Thermo ulfobacteria des Therm sulfobacte ode riumcomm (TC) une + + 8x 1.78 26 + Nitrosp irae Therm sulfovib ye ode rio llows ii (TY) ton + + 8x 1.98 27 + Thermomicrobia Therm icro om biumroseum + + 8x 3.4 82 + De ibacteres ferr Se lectingfrom Deferr cter thermohilus, iba p Ge ovib thiop rio hilus, Flexistipessinusara bici Se lectingfrom Sy nergistesjones ii, Am bacter co biense, ino lom Therm anaero vibrio acida inovor m ans, Am inom onas p aucivorans, Dethio sulfovib peptido rio vorans + In prog ress + In prog ress Syn stes ergi TIGR Type Strain or Species Selected When Possible
  • 13. Green Non Sulfur Bacteria TIGR
  • 14. Goal II: Biology of These Phyla TIGR
  • 15. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. TIGR
  • 17. Key Issues in Uncultured Microbes • Questions – 1. Who is out there? – 2. What are they doing? – 3. Need to connect 1 and 2. • Answers – 1. Phylogenetic anchors – 2. Genomics – 3. Linking anchors to genomics contigs TIGR
  • 18. Phylogenetic Anchors and the Sargasso Sea Shotgun Sequencing Warner Brothers, Inc. sequence TIGR shotgun
  • 19. rRNA as a Phylogenetic Anchor TIGR Venter et al., 2004
  • 20. Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA) TIGR Venter et al., 2004
  • 21. Biased Sampling of Bacterial Genomes Hugenholtz 2002 TIGR
  • 22. Our Tree of Life Genomes Allow Anchoring of 100s of clones from Yellowstone Mats QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. TIGR
  • 24. Blast of GYOAP59TF >ORF02612-TG_gtr_857 rho transcription termination factor Rho {Thermomicrobium_roseum_DSM5159} Length = 426 Score = 1177 (419.4 bits), Expect = 2.9e-119, P = 2.9e-119 Identities = 232/232 (100%), Positives = 232/232 (100%) Query: Sbjct: Query: Sbjct: Query: Sbjct: Query: Sbjct: TIGR 1 VAPIGRGQRGLIVSPPKAGKTVLLKHIANGITTNYKDIHLIVLLIGERPEEVTDMRRSVD 60 VAPIGRGQRGLIVSPPKAGKTVLLKHIANGITTNYKDIHLIVLLIGERPEEVTDMRRSVD 191 VAPIGRGQRGLIVSPPKAGKTVLLKHIANGITTNYKDIHLIVLLIGERPEEVTDMRRSVD 250 61 GEVISSTFDEPVEDHIRVAEMTLERAKRLVECGMDVVILLDSITRLARAYNLSVPPSGRT 120 GEVISSTFDEPVEDHIRVAEMTLERAKRLVECGMDVVILLDSITRLARAYNLSVPPSGRT 251 GEVISSTFDEPVEDHIRVAEMTLERAKRLVECGMDVVILLDSITRLARAYNLSVPPSGRT 310 121 LSGGIDPVALYPPKRFFGAARNIEGGGSLTIIATCLVDTGSRMDDVIYEEFKGTGNMELH 180 LSGGIDPVALYPPKRFFGAARNIEGGGSLTIIATCLVDTGSRMDDVIYEEFKGTGNMELH 311 LSGGIDPVALYPPKRFFGAARNIEGGGSLTIIATCLVDTGSRMDDVIYEEFKGTGNMELH 370 181 LDRKLAERRIFPAIDIQRSGTRREELLLDEQTLRQVWTMRRMVSMLGGTDGT 232 LDRKLAERRIFPAIDIQRSGTRREELLLDEQTLRQVWTMRRMVSMLGGTDGT 371 LDRKLAERRIFPAIDIQRSGTRREELLLDEQTLRQVWTMRRMVSMLGGTDGT 422
  • 25. Broader Impact • Genome sequence data released to TIGR web site • Genome Users Workshop to be held at TIGR in 2005 • Project used as model for evolution teaching in talks to HHMI, MD Science Teachers Assocation, and Montgomery County high school science teachers • Project promoted to public through radio and print media TIGR
  • 26. Training • Student involvement – Kevin Penn, technician at TIGR – Temylope Adeyefa-Olasupo, U. Md Undergrad working at COMB – Rebecca Brocato, high school intern, COMB – Joseph Wister, TIGR Summer Fellow with Karen Nelson – Ryan Corces-Zimmerman, high school intern working at TIGR • Teaching using this project – MBL Molecular Evolution Workshop – MBL Genomics Workshop – Jackson Lab Genomics Workshop TIGR
  • 27. Future Issues • New estimates suggests > 100 bacterial phyla • Someone needs to cover Archaeal diversity • Need DNA repository TIGR

Notas do Editor

  1. Phil Hugenholtz wrote an excellent review paper in Genome Biology tracing which Phyla had genome sequences available. Proteobacteria have the most with Firmicutes next (these are the low GC gram positives like B. subtilis) and Actinobacteria (the high GC gram positives like M. tuberculosis) third.
  2. In Red are the Phyla of bacteria with cultured species but no genome sequences that we are sequencing as part of a NSF Tree of Life project. NOTE **** In White are the Phyla with no cultured representatives. ALSO NOTE **** This project is contingent upon having a good tree of bacteria.
  3. Shotgun genome sequencing works by breaking a genome apart into millions of little pieces and then sequencing those pieces randomly
  4. This is a tree of a rRNA gene that was found on a large DNA fragment isolated from the Monterey Bay. This rRNA gene groups in a tree with genes from members of the gamma Proteobacteria a group that includes E. coli as well as many environmental bacteria. This rRNA phylotype has been found to be a dominant species in many ocean ecosystems. clone from the Sargasso Sea. This shows that this
  5. Phil Hugenholtz wrote an excellent review paper in Genome Biology tracing which Phyla had genome sequences available. Proteobacteria have the most with Firmicutes next (these are the low GC gram positives like B. subtilis) and Actinobacteria (the high GC gram positives like M. tuberculosis) third.