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

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


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes


Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

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


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

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


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


0

50000

100000

150000

200000

250000

300000

350000

0 10 20 30 40 50 60 70 80

S. agalactiae

EnterobacteriaceaeActinobacteria

Bacteria from GEBA project

Genome Number

Tota

l Gen

e N

umbe

rN

umbe

r of p

rote

ins


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

0

100

200

300

400

500

600

700

actm

i

be

uca

braf

a

cata

c

celf

l

conw

o

dya

fe

halm

u

ha

lut

krif

l

nakm

u

pe

dh

e

sa

cv

i

sph

th

spil

i

sta

na

strr

o

suld

e

the

ac

thet

e

tsu

pa

xy

lce

de

na

c

de

tpe

ha

loc

ha

lbo

kan

ko

pla

li

aci

fe

mei

ru

mei

si

rhom

a

ali

ac

chip

i

de

sr5

de

sba

ge

oo

b

the

bi

the

cu

anap

r

ato

pa

bram

u

de

sa7

jon

de

san

ke

seb

te

sla

he

cap

oc

cryc

u

eg

gle

gorb

r

kyts

e

lep

bu

no

cda

strm

o

veip

a

Num

ber o

f tra

nspo

rters

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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

OP9

NKB19

OP3

OP10

TM7

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


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


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


Eisen.Csb2009

Technology

Transcript of Eisen.Csb2009