eisen.csb2009
DESCRIPTION
Talk by Jonathan Eisen on JGI-GEBA "Genomic encyclopedia of bacteria and archaea" projectTRANSCRIPT
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Seeking out the Dark Matter of the Biological Universe
& The Need for a Phylogeny Driven
Genomic Encyclopedia
Jonathan A. EisenAugust 11, 2009
CSB 2009
Tuesday, August 11, 2009
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Tuesday, August 11, 2009
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“Nothing in biology makes senseexcept in the light of evolution.”
T. Dobzhansky (1973)
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Fleischmann et al. 1995
Image removed because it comes from a non open access journal
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From http://genomesonline.orgTuesday, August 11, 2009
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rRNA Tree of Life
Tuesday, August 11, 2009
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The Tree is not Happy
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
As of 2002
Based on Hugenholtz, 2002
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
As of 2002
Based on Hugenholtz, 2002
Tuesday, August 11, 2009
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
As of 2002
Based on Hugenholtz, 2002
Tuesday, August 11, 2009
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Same trend in Archaea
As of 2002
Based on Hugenholtz, 2002
Tuesday, August 11, 2009
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Need for Tree Guidance Well Established
• Common approach within some eukaryotic groups
• Many small projects funded to fill in some bacterial or archaeal gaps
• Phylogenetic gaps in bacterial and archaeal projects commonly lamented in literature
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Solution I: sequence more phyla
• NSF-funded Tree of Life Project
• A genome from each of eight phyla
Eisen, Ward, Badger, Wu, Wu, et al.
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Phylum
Species selected
Chrysiogenes
Chrysiogenes arsenatis (GCA)
Coprothermobacter
Coprothermobacter proteolyticus (GCBP)
Dictyoglomi
Dictyoglomus thermophilum (GD T )
Thermodesulfobacteria
Thermodesulfobacterium commune (GTC)
Nitrospirae
Thermodesulfovibrio yellowstonii (GTY)
Thermomicrobia
Thermomicrobium roseum (GTR )
Deferribacteres
Geovibrio thiophilus (GGT)
Synergistes
Synergistes jonesii (GSJ)
Organisms Selected
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Bacterial aTOL Project AIMS
• Improve resolution of deep branches in the bacterial tree
• Launch biological studies of these phyla
• Leverage data for interpreting environmental surveys
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T. roseum genome
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The Tree of Life is Still Angry
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Major Lineages of Actinobacteria2.5.1 Acidimicrobidae2.5.1.1 Unclassified2.5.1.2 "Microthrixineae2.5.1.3 Acidimicrobineae2.5.1.4 BD2-102.5.1.5 EB10172.5.2 Actinobacteridae2.5.2.1 Unclassified2.5.2.10 Ellin306/WR1602.5.2.11 Ellin50122.5.2.12 Ellin50342.5.2.13 Frankineae2.5.2.14 Glycomyces2.5.2.15 Intrasporangiaceae2.5.2.16 Kineosporiaceae2.5.2.17 Microbacteriaceae2.5.2.18 Micrococcaceae2.5.2.19 Micromonosporaceae2.5.2.2 Actinomyces2.5.2.20 Propionibacterineae2.5.2.21 Pseudonocardiaceae2.5.2.22 Streptomycineae2.5.2.23 Streptosporangineae2.5.2.3 Actinomycineae2.5.2.4 Actinosynnemataceae2.5.2.5 Bifidobacteriaceae2.5.2.6 Brevibacteriaceae2.5.2.7 Cellulomonadaceae2.5.2.8 Corynebacterineae2.5.2.9 Dermabacteraceae2.5.3 Coriobacteridae2.5.3.1 Unclassified2.5.3.2 Atopobiales2.5.3.3 Coriobacteriales2.5.3.4 Eggerthellales2.5.4 OPB412.5.5 PK12.5.6 Rubrobacteridae2.5.6.1 Unclassified2.5.6.2 "Thermoleiphilaceae2.5.6.3 MC472.5.6.4 Rubrobacteraceae
2.5 Actinobacteria2.5.1 Acidimicrobidae2.5.1.1 Unclassified2.5.1.2 "Microthrixineae2.5.1.3 Acidimicrobineae2.5.1.3.1 Unclassified2.5.1.3.2 Acidimicrobiaceae2.5.1.4 BD2-102.5.1.5 EB10172.5.2 Actinobacteridae2.5.2.1 Unclassified2.5.2.10 Ellin306/WR1602.5.2.11 Ellin50122.5.2.12 Ellin50342.5.2.13 Frankineae2.5.2.13.1 Unclassified2.5.2.13.2 Acidothermaceae2.5.2.13.3 Ellin60902.5.2.13.4 Frankiaceae2.5.2.13.5 Geodermatophilaceae2.5.2.13.6 Microsphaeraceae2.5.2.13.7 Sporichthyaceae2.5.2.14 Glycomyces2.5.2.15 Intrasporangiaceae2.5.2.15.1 Unclassified2.5.2.15.2 Dermacoccus2.5.2.15.3 Intrasporangiaceae2.5.2.16 Kineosporiaceae2.5.2.17 Microbacteriaceae2.5.2.17.1 Unclassified2.5.2.17.2 Agrococcus2.5.2.17.3 Agromyces2.5.2.18 Micrococcaceae2.5.2.19 Micromonosporaceae2.5.2.2 Actinomyces2.5.2.20 Propionibacterineae2.5.2.20.1 Unclassified2.5.2.20.2 Kribbella2.5.2.20.3 Nocardioidaceae2.5.2.20.4 Propionibacteriaceae2.5.2.21 Pseudonocardiaceae2.5.2.22 Streptomycineae2.5.2.22.1 Unclassified2.5.2.22.2 Kitasatospora2.5.2.22.3 Streptacidiphilus2.5.2.23 Streptosporangineae2.5.2.23.1 Unclassified2.5.2.23.2 Ellin51292.5.2.23.3 Nocardiopsaceae2.5.2.23.4 Streptosporangiaceae2.5.2.23.5 Thermomonosporaceae2.5.2.3 Actinomycineae2.5.2.4 Actinosynnemataceae2.5.2.5 Bifidobacteriaceae2.5.2.6 Brevibacteriaceae2.5.2.7 Cellulomonadaceae2.5.2.8 Corynebacterineae2.5.2.8.1 Unclassified2.5.2.8.2 Corynebacteriaceae2.5.2.8.3 Dietziaceae2.5.2.8.4 Gordoniaceae2.5.2.8.5 Mycobacteriaceae2.5.2.8.6 Rhodococcus2.5.2.8.7 Rhodococcus2.5.2.8.8 Rhodococcus2.5.2.9 Dermabacteraceae2.5.2.9.1 Unclassified2.5.2.9.2 Brachybacterium2.5.2.9.3 Dermabacter2.5.3 Coriobacteridae2.5.3.1 Unclassified2.5.3.2 Atopobiales2.5.3.3 Coriobacteriales2.5.3.4 Eggerthellales2.5.4 OPB412.5.5 PK12.5.6 Rubrobacteridae2.5.6.1 Unclassified2.5.6.2 "Thermoleiphilaceae2.5.6.2.1 Unclassified2.5.6.2.2 Conexibacter2.5.6.2.3 XGE5142.5.6.3 MC472.5.6.4 Rubrobacteraceae
Tuesday, August 11, 2009
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 100 phyla of bacteria
• Genome sequences are mostly from three phyla
• Most phyla with cultured species are sparsely sampled
• Lineages with no cultured taxa even more poorly sampled
• Solution - use tree to really fill gaps
Well sampled phyla
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http://www.jgi.doe.gov/programs/GEBA/pilot.htmlTuesday, August 11, 2009
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GEBA Pilot Project Overview
• Identify major branches in rRNA tree for which no genomes are available
• Identify which lineages have type species available in DSMZ
• Grow > 200 of these and prep. DNA• Sequence and finish 100 • Annotate, analyze, release data• Assess benefits of tree guided sequencing
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GEBA Pilot Project: Components• Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan Eisen)• Project management (David Bruce, Lynne Goodwin et al)• Culture collection and DNA prep (DSMZ, Hans-Peter Klenk)• Libraries and DNA (Eileen Dalin et al)• Sequencing and closure (Susan Lucas, Alla Lapidus et al.)• Annotation and data release (Nikos Kyrpides)• Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Jenna
Morgan, Victor Kunin, Marcel Huntemann, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson)
• Adopt a microbe education project (Cheryl Kerfeld)• Outreach (David Gilbert)• $$$ (DOE, Eddy Rubin, Jim Bristow)
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GEBA Pilot: Selecting Targets
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GEBA Pilot Target List
0
5
10
15
20
25
30
35
B: A
ctinob
acteria
(High GC)
B: A
minan
aero
bia
B: A
quifica
e
B: B
actero
idetes
B: C
hlor
oflexi
B: D
efer
ribac
tere
s
B: D
efer
ribac
tere
s
B: D
eino
cocc
i
B: D
elta Pro
teob
acteria
B: Eps
ilon Pr
oteo
bacter
ia
B: Firm
icutes
B: Fus
obac
teria
B: G
amma Pr
oteo
bacter
ia
B: G
emmatim
onad
etes
B: H
aloa
naer
obiales
B: Planc
tomyc
etes
B: S
piro
chae
tes
B: The
rmod
esulfoba
cter
ia
B: The
rmod
esulfobia
B: The
rmov
enab
ulae
A: H
alob
acteria
A: A
rcha
eoglob
i
A: M
etha
noba
cter
ia
A: M
etha
nomicr
obia
A: The
rmoc
occi
A: The
rmop
rotei
Phyla
# o
f G
en
om
es
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GEBA Current Status
• >100 in progress
• GEBA 56 (focus of first paper)– 34 finished genomes– Released to IMG-GEBA page, JGI-FTP site,
and Genbank
• All data is completely Open for anyone to use
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Assess Benefits of GEBA
• All genomes have some value
• But what, if any, is the benefit of tree-guided sequencing over other selection methods
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Why Increase Taxonomic Coverage?
• Gene discovery• Annotation, functional prediction• Metagenomic analysis• Mechanisms of diversification• Species phylogeny and classification
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GEBA Lesson 1
rRNA Tree of Life is a Useful Guide for Genome Core Phylogenetic
Diversity
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rRNA Tree of Life
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16s Says Hyphomonas is in Rhodobacteriales
Badger et al. 2005
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WGT Says Its Related to Caulobacterales
Badger et al. 2005
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GEBA Lesson 2
Phylogenetically Guided Selection Can Help Many Aspects of Genome
Analysis
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Annotation Improves
• Conversion of hypothetical into conserved hypotheticals
• Linking distantly related members of protein families
• Non-homology functional prediction methods
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Improved Rosetta Stone Predictions
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Phylogenetic Metagenomics
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Alph
apro
teob
acteria
Betapr
oteo
bacter
ia
Gammap
roteob
acteria
Deltapr
oteo
bacter
ia
Epsil
onpr
oteo
bacter
ia
Uncla
ssified
Pro
teob
acteria
Cyan
obac
teria
Chlamyd
iae
Acidob
acteria
Bacter
oide
tes
Actin
obac
teria
Aquific
ae
Plan
ctom
ycetes
Spiro
chae
tes
Firmicu
tes
Chloro
flexi
Chloro
bi
Uncla
ssified
Bac
teria
dnaGfrrinfCnusApgkpyrGrplArplBrplCrplDrplErplFrplKrplLrplMrplNrplPrplSrplTrpmArpoBrpsBrpsCrpsErpsIrpsJrpsKrpsMrpsSsmpBtsf
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16s Says Hyphomonas is in Rhodobacteriales
Badger et al. 2005
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WGT Says Its Related to Caulobacterales
Badger et al. 2005
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GEBA Lesson 3
We have still only scratched the surface of microbial diversity
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Protein Family Rarefaction Curves
• Take data set of multiple complete genomes• Identify all protein families using MCL• Plot # of genomes vs. # of protein families
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0
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EnterobacteriaceaeActinobacteria
Bacteria from GEBA project
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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)
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Within Family Novelty Example: Transporter Profiles
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inorganic ions amino acids, nitro compounds and peptides drugs/ toxins sugars carboxylates nucleosides/ tides, bases siderophores other
Sebaldella termitidis ATCC 33386 has 2x number of sugar PTS transporters of any genome
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Phylogenetic Distribution Novelty: 1st Bacterial Actin Related Protein
Haliangium ochraceum DSM 14365Tuesday, August 11, 2009
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Phylogenetic Diversity: Sequenced Bacteria & Archaea
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Phylogenetic Diversity with GEBA
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Phylogenetic Diversity: Isolates
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Phylogenetic Diversity: All
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Most phyla with cultured species are sparsely sampled
• Lineages with no cultured taxa even more poorly sampled
Well sampled phylaPoorly sampled
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Uncultured Lineages:Technical Approaches
• Get into culture• Enrichment cultures• If abundant in low diversity ecosystems• Flow sorting• Microbeads• Microfluidic sorting• Single cell amplification
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GEBA Lesson 4
Need Experiments from Across the Tree of Life too
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
As of 2002
Based on Hugenholtz, 2002
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Experimental studies are mostly from three phyla
As of 2002
Based on Hugenholtz, 2002
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Experimental studies are mostly from three phyla
• Some studies in other phyla
As of 2002
Based on Hugenholtz, 2002
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Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
Need experimental studies from across the tree too
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Adopt a Microbe
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GEBA Lesson 5
The Importance of Culture(Collections that is)
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GEBA Biggest Challenge:Getting DNA
• Getting quality DNA is biggest bottleneck• Sharing strains is also a bottleneck• Solution: Beg Borrow and Steal
• DSMZ offered to do for free• ATCC is doing a small number for a fee• In discussions with other PCC and other
collections
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MicroorganismsQuantification gel of the genomic DNA isolated from Conexibacter woesei (DSM 14684T)
Conexibacter woesei (DSM 14684T) was taken from the German Collection of Microorganisms and Cell Cultures (DSMZ). The genomic DNA was isolated using the Qiagen Genomic 500 DNA Kit (Qiagen 10262). The genomic DNA was 10-250 kb in size as determined by Pulsed Field Gel Electrophoresis (PFGE). The bulk of DNA had a size of 50-250 kb (see attached PFGE image). The DNA concentration is 500 ng/µl as estimated from the gel. Spectrophotometric measurements yielded a DNA concentration of 450 µg/ml; 300 µl of genomic DNA are shipped (150 µg).
1 2 3 4 5 6 7 8
Lane 1: c(λ-Marker)= 15 ngLane 2: c(λ-Marker)= 30 ngLane 3: c(λ-Marker)= 50 ngLane 4: DNA Molecular Weight Marker II (Roche
236250)Lane 5: DSM 13279, Collinsella stercorisLane 6: DSM 43043, Intrasporangium calvumLane 7: DSM 18053, Dyadobacter fermentansLane 8: DSM 20476, Slackia heliotrinireducens
Lane 9: DSM 18081, Patulibacter minatonensisLane 10: DSM 14684, Conexibacter woeseiLane 11: DSM 11002, Dethiosulfovibrio peptidovoransLane 12: DSM 11551, Halogeometricum borinquenseLane 13: DNA Molecular Weight Marker II (Roche
236250)Lane 14: c(λ-Marker)= 125 ngLane 15: c(λ-Marker)= 250 ng Lane 16: c(λ-Marker)= 500 ng
9 10 11 12 13 14 15 16
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Related Lesson 1
METADATA ROCKS
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SIGS
• The Genomic Standards Consortium • The GSC is an open-membership working body which
formed in September 2005. • The goal of this international community is to promote
mechanisms that standardize the description of genomes and the exchange and integration of genomic data.
• See http://gensc.org/gc_wiki/index.php/Main_Page
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Additional Lessons
• Completeness matters• Computational methods need to be more
automated• Need to limit analyses to subsets of all
available data• Need for people to help interpret and study
data is increasing not decreasing• Sequence is just the beginning• Need to train more students
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MICROBES
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A Happy Tree of Life
Tuesday, August 11, 2009