Environmental Microbial Genomics GroupEnvironmental Microbial Genomics GroupLaboratoire Ampère . Ecole Centrale de Lyon . Université de Lyon

Is there a limit to the extent of the rare (soil) biosphere?

Complete sequencing of the soil metagenome: An attainable utopia?

Pascal Simonet

La biosphère rare du sol, définition, importance, rôle mais comment l’atteindre?

Number of bacterial cells: 2.6x1029

Soil

Torsvik et al., 2002 DNA reassociation method104 different prokaryotic species of equivalent abundances (predicted).

Gans et al., 2005DNA reassociation method 107 microbial species per gram of soil (predicted).

Roesh et al., 2007 pyrosequencing <104 species (detected)

Number of species ??:

Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. Elshahed et al., 2008 AEM: 74: 5422–5428

Kessler Farm soil

Distribution of various phyla

Species distribution Rarefaction curve

Rare biosphere.

Analysis of species distribution patterns usually indicates that while a significant fraction of bacterial biomass belongs to a relatively small number of species, the majority of bacterial species within a complex microbial community are present in extremely low numbers.

•Elshahed et al. 2008. Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. AEM;74: 5422–542

•Ashby et al 2007. Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities. AEM 73:4532–4542.

•Pedros-Alio 2006. Marine microbial diversity: can it be determined. Trends Microbiol. 14:257–263.

•Sogin et al 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere.” Proc. Natl. Acad. Sci. USA 103:12115–12120

Official definition

Role of the rare biosphere ?

•Genes can be strongly expressed (numerous examples in the literature)

•Rare taxa can become dominant when environmental conditions change

•Rare taxa are a reservoir of transferable genetic information

FingerprintsDNA microarrays

Sequencing metagenome

Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. Elshahed et al., 2008 AEM: 74: 5422–5428

The rare biosphere and sensitivity of techniques

Threshold between abundant and rare bacteria ??

RARE BACTERIA

Metagenome DNA extraction :

•Soil heterogeneity•In situ lysis•Bacteria extraction (Nycodenz)•Cell lysis•DNA adsorption•DNA degradation

•Cloning bias•PCR bias•Sequencing bias

Rare, protected, lysis recalcitrant bacteria?

The right definition of the « Rare biosphere » in soil ?

Rare bacteria or/and inaccessible bacteria or DNA?

Number of colonies increased with the stringency of the lysis treatment!!

Recovery of added lambda phage DNA?

Max. recovery: 25% Most treatments and soils: less than 10%

The clay soil « A black hole »

Rare biosphere in soil ?

• Rare taxa ?

• Inaccessible bacteria, unavailable DNA ?

What is the rare biosphere ??

DNA extraction: critical bias !!!!

Not only to determine the extent of the rare biosphere but this of bacterial diversity.

What is the rare biosphere ??

What can we expect from sequencing?

«  METAGENOMICS « 

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Genomics:

“core-genome” : the genes existing in all strains

“dispensable genome” : genes present in two or more strains and genes unique to single strains

“pan-genome” : “core-genome” + “dispensable genome”

Given that the number of unique genes is vast, the pan-genome of a bacterial species might be orders of magnitude larger than any single genome.

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Core-metagenome : genes existing in all soilsCore-metapopulation : species found in all soils

Pan-metagenome : Core-metagenome + Specific metagenomePan-metapopulation :Core-metapopulation + Specific metapopulation

Specific-metagenome : genes present in two or more soils and genes unique to single soils Specific-metapopulation : species « «« and species «  « 

Fundamental questions: The actual ratio Pan/Core(the actual size of specific)

Soil metagenomics

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Specific-meta-(genome/pop.)

Soil 1S

pecific-meta-(genom

e/pop.)

Soil 2

Specific-

meta-(genome/pop.))

Soil 3S

peci

fic-m

eta-

(gen

ome/

pop.

)

So

il 5

SoilCore-metagenome

Core-metapopulation

Rare and very numerous species

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Everything is everywhere !

Only distribution differs

Pan-meta-(genome/population)Soil 1

Pan-meta-(genome/population)Soil 2

Pan-meta-(genome/population)Soil 3

Core

meta-(genome/population)

Core = Pan

«everything is everywhere, but, the environment selects» (Bas-Becking)

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Pan-meta-(genome/population)Soil 1

Pan-m

eta-(genome/population)

Soil 2

Pan-meta-(g

enome/population)

Soil 3

Pan

-met

a-(g

enom

e/po

pula

tion)

So

il 5

SoilCore-metagenome

Core-metapopulation

Pan-meta-(genome/population)Soil 1

Pan-meta-(genome/population)Soil 2

Pan-meta-(genome/population)

Soil 3

Core

meta-(genom

e/population)Core

Rare and very numerous species: Do they really matter?

The initial support for Terragenome (complete sequencing of a reference soil metagenome) :

Objective:

•Optimization of bacterial DNA recovery.

•Metagenomic DNA library construction

•Pyrosequencing of directly extracted DNA

Park Grass, Rothamsted: an internationally recognized agroecology field experiment for 150 years

Sampling strategies

•Time of the year

•Depth

Improvement of cell recovery (Nycodenz)

Improvement of DNA recovery

(sensitivity to lysis treatments)

Improvement of DNA recovery (DNA degradation)

Stringency of the lysis

Bead beating

Agarose plug

Cell ring

density

Fraction 1

Fraction 4

Fraction 3

Fraction 2

P

R

O

K

A

R

Y

O

T

E

s

E

U

K

A

R

Y

O

T

E

S

Optimization of bacterial DNA recovery

16S rDNA MICROARRAY

(8x15K) Agilent

• 3 186 targets (>20 000 probes)

• Agilent technologies

• Cover all phylogenetic bacterial groups

• Lenght: 20 nucleotides

Sampling Density gradient

Lysis stringency

DNA size

Yiel

d

Cell ring

Density

Fraction 1

Fraction 4

Fraction 3

Fraction 2

DN

A qu

ality

Stringent Lyses

Soft LysesEukaryotes (density > 1.3)

Numberof cells

Bacterial genera

Ph

ylo

chip

pro

bes

in

ten

sity

Undetected with one DNA extraction method

0

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90

100

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

Number of probes

% of

pos

itive

ph

yloge

netic

prob

es

Only one DNA extraction method (~40% of probes)

15 DNA extraction methods (about 99% of probes)

Rothamsted soil phylochip saturation curve

1. technological reproducibility 2. comparison with an ocean 3. comparison with another soil

11.67% of functions statistically different (Bootstrap)

4. Cell lysis stringency effect

72.63% 39.83%

34.69%

•Metagenomic DNA library construction: 2 000 000 clones

(16 000 equ. bacterial genomes)

•Pyrosequencing of metagenome DNA: 60 runs (depth, lysis, season etc.)

60Gbp (15 000 equ. bacterial genomes)

Park Grass: Rothamsted

Sufficient effort to reach the rare biosphere???

Rare biosphere and pyrosequencing sensitivity ??Redundancy of sequences in the DNA solution

DNA

Extraction

Culture in vitro

CloningTransformation

vector

Clone Library

PCR

Cloning and/or sequencing

RISA, T-RFLP, DGGE,

Phylochip

Functional microarrays

Molecular screening

Chemical screening Biological

screening

OMe CH3O

O

CH3

CH3

O

OHOH

OMe

OMe

Lombard et al., 2006

METAGENOME EXPLOITATION

Direct

Sequencing (454)

Cultivable bacteria: less than 1%

Direct or indirect

Domesticated bacterial host

Hybridization based gene detection Chemical structure of

produced compounds

Direct detection of enzymatic activity

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Hybridization screening of metagenomic DNA libraries

Metagenomic DNA library construction

December 2010: 2 000 000 clones (16 000 equ. bacterial genomes)

Molecular screening

Abundant/Rare taxa ? The right question ?

Extent of the Soil Bacterial Diversity

….independently of the species distribution ?

SOIL MICROFLORA

Extent of the soil bacterial diversity?

•Genes can be strongly expressed (numerous examples in the literature)

•Rare (or unavailable) taxa can become dominant (or accessible) when environmental conditions change

•Rare taxa are a reservoir of transferable genetic information

How to get it?

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INTRODUCTION

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Bacterial community extracted from soil A

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or

Soil A

Conceptual approach:

Sterilized Soil B

Diversity in soil A

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Provide new developing conditions to soil bacterial communities

Congo: Black PointKenya: EmbuMartinique New Caledonia

CSA

Brévil Talmont St-Hilaire Chinon

Montrond

Nine soils selected

CONCEPTUAL APPROACH

1. Extraction of the 9 bacterial communities

2. Inoculation of each bacterial community into the nine sterilized soils

4. Monitoring of bacterial community structure evolution (direct DNA extraction, PCR and phylochip)

Nycodenz density gradient

3. Incubation at RT for 1 day, 2 months, 6 months

Two questions:

•Are new developing community structures different from the donor ones and from these of the recipient soils?

•Are new taxa detected?

Inoculated Community

Recipient Soil

« inoculated community » stronger effect than « recipient soil »

« Recipient Soils S7 and S9 »: stronger effect

Are new developing community structures different from the original donor one and from the one of the recipient soil?

Yes: With both a recipient soil and an inoculated

community structuring effect.

A bacterial community inoculated into new (sterilized) soils reveals bacteria genera undetected in the original inoculum

Each inoculated community: Extent of the diversity increases when considering the different recipient soils.

Are new taxa detected?

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Cum

ulati

ve p

erce

ntag

e of

new

ly d

etec

ted

gene

ra (N

max

=147

5)

Number of soils

CS5

CS1

CS2

CS3

Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)

T2 = 6 months

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Number of soil communities

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Incr

ease

in d

etec

ted

gene

ra(c

umul

ative

%)

Number of soil communities

Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)

S9

S2

S4

S7

S1

T2 = 6 months T2 = 6 months

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etec

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gene

ra (N

max

=147

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Number of soils

Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)

55% (max) of the characterized genera detected (9 soils)

Rarefaction curves show a limit

Conclusion: Diversity in the rare biosphere very limited?

T2 = 6 months

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etec

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gene

ra (N

max

=147

5)

Number of soils

Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)

However:

Diversity of conditions offered by the recipient sterilized soils?

T2 = 6 months

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1 2 3 4 5

Cum

ulati

ve p

erce

ntag

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new

ly d

etec

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gene

ra (N

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Number of soil communities

Cumulative percentage of genera detected at T0 + T1 + T2

CS: Extracted (and inoculated) communityT0: 1 dayT1: 2 monthsT2: 6 months

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Number of soil communities

T2 onlyT0 + T1 +T2

Genera detected in CS and not laterGenera detected at T0, T1, T2 and not in CSGenera detected only at T1

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gene

ra

(Nm

ax=1

475)

Number of soils

Cumulative percentage of newly detected genera

Individual communities1 sampling time (6 months)

All soil communities (n=4)All sampling times (n=3)

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0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

Number of probes

% of

pos

itive

ph

yloge

netic

prob

es

One DNA extraction approach (~40% of probes)

15 DNA extraction approaches (about 99% of probes)

Rothamsted soil phylochip saturation curve

A bacterial community inoculated into new (sterilized) soils reveals bacteria genera undetected in the original inoculum

Each inoculated community: Extent of the diversity increases when considering the different recipient soils

Are new taxa detected?

the different incubation times

the different extraction techniques…

the different DNA analysis methods…

Italian forest soil /Rothamsted soil (UK)

Paolo NannipieriMaria-Teresa CeccheriniGiacomo PietramellaraDavide Francioli Tom Delmont

Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Universita` degli Studi di Firenze, Firenze, Italy

Identification of « Italy » and « Rothamsted » specific bacteria.

(Taxonomic microarrays/454/Illumina)

Extent of the bacterial (soil) diversity / extent of the soil (rare) biosphere?

Combination of conceptual and methodological approaches.

Conceptual approach:

Increase the range of conditions offered to developing communities

Methodological approach:

Phylogenetic microarrays: Limited by the number of probes and specificity /sensitivity of hybridization.

Pyrosequencing approaches required.

Conclusion

Diversity of Bacteria (rare and abundant) : Huge

•Collaboration at the international level

•Focus on one « reference » soil

Attainable ifAttainable if

Environmental Microbial

Genomics

www.GenomEnviron.org

Aurélie Faugier, Sébastien

Cécillon, Davide Francioli, Tom

Delmont,

Emmanuel Prestat, Jean-Michel

Monier, Timothy M Vogel,