Eisen.Csb2009

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

“Nothing in biology makes senseexcept in the light of evolution.”

T. Dobzhansky (1973)

Fleischmann et al. 1995

Image removed because it comes from a non open access journal

From http://genomesonline.orgTuesday, August 11, 2009

rRNA Tree of Life

The Tree is not Happy

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

FirmicutesFusobacteria Actinobacteria

Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

SynergistesDeferribacteres

Thermudesulfobacteria

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• At least 40 phyla of bacteria

As of 2002

Based on Hugenholtz, 2002

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• Genome sequences are mostly from three phyla

As of 2002

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• Some other phyla are only sparsely sampled

As of 2002

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• Same trend in Archaea

As of 2002

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

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• 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.

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

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

T. roseum genome

The Tree of Life is Still Angry

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

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• 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

http://www.jgi.doe.gov/programs/GEBA/pilot.htmlTuesday, August 11, 2009

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

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)

GEBA Pilot: Selecting Targets

GEBA Pilot Target List

ctinob

acteria

(High GC)

quifica

actero

idetes

oflexi

elta Pro

acteria

B: Eps

ilon Pr

bacter

B: Firm

icutes

B: Fus

amma Pr

bacter

emmatim

obiales

B: Planc

B: The

esulfoba

B: The

esulfobia

B: The

acteria

eoglob

nomicr

A: The

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

Assess Benefits of GEBA

• All genomes have some value

• But what, if any, is the benefit of tree-guided sequencing over other selection methods

Why Increase Taxonomic Coverage?

• Gene discovery• Annotation, functional prediction• Metagenomic analysis• Mechanisms of diversification• Species phylogeny and classification

GEBA Lesson 1

rRNA Tree of Life is a Useful Guide for Genome Core Phylogenetic

Diversity

rRNA Tree of Life

16s Says Hyphomonas is in Rhodobacteriales

Badger et al. 2005

WGT Says Its Related to Caulobacterales

Badger et al. 2005

GEBA Lesson 2

Phylogenetically Guided Selection Can Help Many Aspects of Genome

Analysis

Annotation Improves

• Conversion of hypothetical into conserved hypotheticals

• Linking distantly related members of protein families

• Non-homology functional prediction methods

Improved Rosetta Stone Predictions

Phylogenetic Metagenomics

acteria

Betapr

bacter

Gammap

roteob

acteria

Deltapr

bacter

ssified

acteria

Chlamyd

Acidob

acteria

Bacter

Aquific

ycetes

Firmicu

Chloro

ssified

dnaGfrrinfCnusApgkpyrGrplArplBrplCrplDrplErplFrplKrplLrplMrplNrplPrplSrplTrpmArpoBrpsBrpsCrpsErpsIrpsJrpsKrpsMrpsSsmpBtsf

16s Says Hyphomonas is in Rhodobacteriales

Badger et al. 2005

WGT Says Its Related to Caulobacterales

Badger et al. 2005

GEBA Lesson 3

We have still only scratched the surface of microbial diversity

Protein Family Rarefaction Curves

• Take data set of multiple complete genomes• Identify all protein families using MCL• Plot # of genomes vs. # of protein families

100000

150000

200000

250000

300000

350000

0 10 20 30 40 50 60 70 80

S. agalactiae

EnterobacteriaceaeActinobacteria

Bacteria from GEBA project

Genome Number

r of p

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)

Within Family Novelty Example: Transporter Profiles

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

Phylogenetic Distribution Novelty: 1st Bacterial Actin Related Protein

Haliangium ochraceum DSM 14365Tuesday, August 11, 2009

Phylogenetic Diversity: Sequenced Bacteria & Archaea

Phylogenetic Diversity with GEBA

Phylogenetic Diversity: Isolates

Phylogenetic Diversity: All

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• Most phyla with cultured species are sparsely sampled

• Lineages with no cultured taxa even more poorly sampled

Well sampled phylaPoorly sampled

No cultured taxaTuesday, August 11, 2009

Uncultured Lineages:Technical Approaches

• Get into culture• Enrichment cultures• If abundant in low diversity ecosystems• Flow sorting• Microbeads• Microfluidic sorting• Single cell amplification

GEBA Lesson 4

Need Experiments from Across the Tree of Life too

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

As of 2002

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• Experimental studies are mostly from three phyla

As of 2002

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• Experimental studies are mostly from three phyla

• Some studies in other phyla

As of 2002

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

Cyanobacteria

Chlamydia

Spriochaetes

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP1OP11

Nitrospira

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

Need experimental studies from across the tree too

Adopt a Microbe

GEBA Lesson 5

The Importance of Culture(Collections that is)

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

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

Related Lesson 1

METADATA ROCKS

• 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

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

MICROBES

A Happy Tree of Life

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