Microbial Community Genomics at the JGI

Susannah Green Tringe, PhDJGI Metagenome Program Lead

Advancing Science with DNA Sequence

Talk outline

• Metagenomics background and history• JGI Metagenome Program

• Organization• Project portfolio

• JGI Science: Wetlands metagenomics

Talk outline

• Metagenomics background and history• JGI Metagenome Program

• Organization• Project portfolio

• JGI Science: Wetlands metagenomics

Isolate (pure culture)

Genomics

Microbial community

Metagenomics

What is metagenomics?

Why metagenomics?

• Vast uncultivated phylogenetic diversity hints at a potential reservoir of untapped functional diversity• Ecologically important processes

• nutrient cycling

• Pharmacologically valuable compounds• antibiotics

• Industrially useful enzymes • cellulases

Metagenomics

1 10 100 1000 10000

Acid mine Sargasso Sea Soil

Species complexity

?

Adaptive gene for habitat AAdaptive gene for habitat BEssential gene

A

B

Environmental Gene Tags(EGTs)

Comparative metagenomics

Tringe et al 2005

COG3459:Cellobiose phosphorylase

COG5524: Bacteriorhodopsin

COG1292: Choline-glycine betaine transporter

Tringe et al 2005

Talk outline

• Metagenomics background and history• JGI Metagenome Program

• Organization• Project portfolio

• JGI Science: Wetlands metagenomics

User Programs

Plants Fungi Microbes Metagenomes

Program organization

Scientific Advisory Board

1. Cameron Currie, University of Wisconsin

2. Ed DeLong, MIT

3. Jed Fuhrman, University of Southern California

4. George Garrity, MSU

5. Steve Hallam, University of British Columbia

6. Bob Landick, Great Lakes BRC

7. Folker Meyer, Argonne National Laboratory

8. Nancy Moran, Yale University

9. Mary Ann Moran, University of Georgia

10. Karen Nelson, JCVI

11. Rich Roberts, NEB

12. Doug Rusch, J. Craig Venter Institute

13. Ramunas Stepanauskas, Bigelow Laboratory

14. Niels van der Lelie, RTI

15. Phil Hugenholtz, University of Queensland

2011- one super program

Prokaryote Super Program

N. Kyrpides

Single Cells GroupT. Woyke

Omics GroupK. Mavrommatis

Functional Annotation Group

N. Ivanova

Microbial Systems Group

S. Tringe

Metadata GroupD. Liolios

Comparative Analysis Systems

A. Chen

LANLP. Chain

Metagenome ProgramS. Tringe

Microbial ProgramT. Woyke

Major Collaborators

• DSMZ

• BIGELOW

Science ProgramsJ. Bristow

2004 2005 2006 2007 2008 2009 2010 2011 201210

100

1000

10000

100000

JGI sequence outputSe

quen

ce o

utpu

t (G

b)

Fiscal Year

40 Gb

30 Tb

2012 projected:47.1 Tb

JGI Project Portfolio

2008 2009 2010 2011 2012 (target)

2013 (target)

0

50

100

150

200

250

300

350

400Standard draft

Minimal draft

Metatranscriptome

Metatranscriptome counting

FY13 Program Targets

YTD

Genomics for Bioenergy

SugarCellulose

MicrobesEnzymesPlants

CO2

Pre-treatment

Biomass

Poplar (Science)Sorghum bicolor (Nature)SwitchgrassMiscanthusPinus taedaFoxtail milletBrachypodium distachyon

Feedstock improvement

Trichoderma reesei (PNAS)Postia placenta (PNAS)Termite gut (Nature)Cow rumen (Science)Leaf cutter ant gardenShipworm mollusk

Biomass degradation

T. ethanolicusPichia stipitus Biogas bioreactorMixed alcohols bioreactorButanol producing E coli

Fuels synthesis

Termite metagenome

Warnecke et al Nature 2007

Biogeochemistry and bioremediation

Enhanced BiologicalPhosphate RemovalSludge (Nat Biotech)

Anammox bioreactors(Env Microbiol)

Terephthalate degradingcommunity (ISME)

Gulf oil spill (ISME)

Terephthalate-degrading community

Lykidis et al, ISME 2011

Carbon Cycling & Environment

Picoprymnesiophytes

Lake Washington Methylotrophs

Prairie soil metagenome

Deep subsurface ecosystem

(Science)(PNAS) (Nat Biotech)

Permafrost metagenome(Nature)Wetlands metagenome

Talk outline

• Metagenomics background and history• JGI Metagenome Program

• Organization• Project portfolio

• JGI Science: Wetlands metagenomics

Why study wetlands?

Wetlands store a lot of carbon (IPCC, 2000)

…but their sequestration potential is uncertain (USGS, 2010)

Peat island subsidence

Wetland “carbon farming”

CO2

O2

CH4

LisamarieWindham-Myers

Major microbial processes in wetland sediments

• Plant biomass decomposition• Denitrification • Mn(IV) reduction• Fe(III) reduction• Sulfate reduction • Methanogenesis• Methane oxidation (aerobic or anaerobic)

Laanbroek, Annals of Botany

How do these processes impact “carbon farming”?

Sampling site gradients

PeataccretionOxygen,Nitrate,Sulfate

Methaneflux

Waterinlet

Wateroutflow

Site A B C/L

Does microbial community composition change with nutrient gradients, primary production and methane release?

Sample Collection

Caffrey & Kemp 1991

Illumina shotgun sequencing

Shotgun Metagenome

Community composition

454 Titanium Pyrotag sequencing

Functional analysis

Sequencing strategy

Wetland microbial communities

-Sampling site is major driver ofcommunity composition

-Sample type is next largest factor-Depth effect is subtle

Site LHigh biomassaccumulationSite A

Low biomassaccumulation

Site BMedium biomass

accumulation

February 2011

Shaomei He

-Similar resultsin August

Indicator OTUsDechloromonas OTUMethanoregula OTU

Archaeal methanogen Correlates with CH4 production

170X coverage in one metagenome dataset

Denitrifying BetaproteobacteriumCorrelates with nitrate abundance

A Rhizobiales OTU

AlphaproteobacteriumKnown plant-associated

bacteria

A Crenarchaeota subphylum 2 OTU

Uncharacterized crenarchaeote

A, B, L

RhizomeBulk

Shaomei He

Metagenome Sequencing and Assembly

More complex community, less

assembly

5.2 5.6 6 6.4 6.80%

5%

10%

15%

20%

25%

30%

Shannon diversity index from 16S pyrotag

Perc

ent r

eads

map

ped

to c

ontig

s

Shotgundata

Assembly

Contigs

Singlets

Shaomei He

Relative gene family abundances

Samples with more methanogenesis genes have less dissimilatory sulfate/nitrate reduction genes

Methane oxidation genes were more abundant in rhizomes

Shaomei He

Metagenome assemblyG

+C c

onte

nt

100 500 1000

Average read depth5 10 50

Alphaproteobacteria~850X

Methanoregula~170X

Clostridia

Draft methanogengenome

Single-copy phylogenetic marker COGs in finishedMethanomicrobial genomes

M. boonei

Wetland OTU

High coverage and low redundancy of the draft genome

Methanogenesis Pathways

HydrogenotrophicAcetoclastic Methylotrophic

Red: present in wetland MethanoregulaGrey: absent in wetland Methanoregula

Conclusions

• Metagenomics provides a means to study uncultivated communities of microbes

• Recent advances in sequencing technologies allow us to explore these communities at unprecedented depth

• Complex communities found in soils and sediments still present significant challenges to genome reconstruction

• Appropriate library construction, sequencing and analysis methods enable greater functional insight into complex communities

Questions?