'6810 JLGGC

Functional Metagenomic Profiling of Nine Biomes

Elizabeth A. Dinsdale1,2*, Robert A. Edwards1,3,4,5, Dana Hall1, Florent Angly1,6, Mya

Breitbart7, Jennifer M. Brulc 8,, Mike Furlan1, Christelle Desnues1,9, Matthew Haynes1,

Linlin Li1, Lauren McDaniel7, Mary Ann Moran10, Karen E. Nelson11, Christina

Nilsson12, Robert Olson5, John Paul7, Beltran Rodriguez Brito1,6, Yijun Ruan12, Brandon

K. Swan13, Rick Stevens5, David L. Valentine13, Rebecca Vega Thurber1, Linda

Wegley1, Bryan A. White8,14, and Forest Rohwer1,3

1Department of Biology, San Diego State University, San Diego, CA 92182 USA

2School of Biological Sciences, Flinders University, Adelaide, SA 5042, Australia

3Center for Microbial Sciences, San Diego State University, San Diego, CA 92182 USA

4Department of Computer Sciences, San Diego State University, San Diego, CA 92182

USA

5Mathematics and Computer Science Division, Argonne National Laboratory, Argonne,

IL 60439 USA

6Computational Science Research Centre, San Diego State University, San Diego, CA

92182-1245 USA

7University of South Florida, College of Marine Science 140 7th Avenue S., St.

Petersburg, FL 33701 USA 8 Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA

SUPPLEMENTARY INFORMATION

doi: 10.1038/nature06810

www.nature.com/nature 1

9Current address: Unité des Rickettsies, CNRS-UMR 6020, Faculté de médecine,

13385 Marseille, France

10 Department of Marine Sciences, University of Georgia, Athens, GA, USA

30602.

11 The J. Craig Venter Institute, 9712 Medical Center Drive, Rockville, MD, 20850, USA

12 Genome Institute of Singapore, 60 Biopolis Street, #02-01, Genome, Singapore

138672

13 Department of Earth Science, University of California Santa Barbara, Santa

Barbara, CA 93106, USA 14 The Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA

doi: 10.1038/nature06810 SUPPLEMENTARY INFORMATION


Supplementary information includes four tables presenting accession numbers and

descriptions of metagenomes, phage and prophage content of each metagenome, the

motility proteins present in the microbial and viral metagenomes and statistical

comparisons of the proportions of metabolic processes across the nine biomes. Six

figures provide information about the geographic separation of samples, diversity versus

sequences number, comparison of diversity between metagenomes and sequenced

whole bacterial genomes, the fine-scale details about the sulfur metabolic processes, and

two experiments that show the strength of the CDA across multiple groupings.



Table S1. Metagenomes used in this manuscript listed using collector’s description and biome assignment. All metagenomes were sequenced using 454 Life Science GS20 pyrosequencing. Simple statistics of the individual libraries, including number of sequences, blast hits and 16Sr RNA genes are provided. M = microbial library and V = Viral library. The metagenomes used in this paper are freely available from the SEED platform and are being made accessible from CAMERA and the NCBI Short Read Archive when available. The accession numbers are shown and further material and direct links to the data is available at http://www.theseed.org/DinsdaleSupplementalMaterial/.

ID Name SEED

accession #

NCBI

Genome

project #

Type Biome # of

Sequences

# of Blast hits # of 16S

1 Soudan Red 4440281.3 17633 M Subterranean 334,386 55,069 321

2 Soudan Black 4440282.3 17635 M Subterranean 388,627 43,079 24

3 Solar Salterns low

Salinity San Diego 4440437.3

28359

M Hyper-saline 268,206 52,745 243

4 Solar Salterns medium

Salinity San Diego

4440435.3

28377

M Hyper-saline 38,929 10,151 41




Salinity San Diego

4440434.3

28379

M Hyper-saline 23,261 5,630 26

6 Solar Salterns Plasmid

component

4440090.3

28443

M Hyper-saline 111,431 19,365 129


salinity west California

4440416.3

28449

M Hyper-saline 8,062 770 3

8 Solar Salterns high

salinity west California

4440419.3

28453


9 Salton Sea 4440329.3 28613 M Hyper-saline 178,407 17,531 43


salinity west California 4440425.3

28459

M Hyper-saline 120,987 32,871 110

11 Solar Salterns low salinity

west California 4440426.3

28461




28463

V Hyper-saline 39,943 414



28465

V Hyper-saline 58,735 1,822




salinity West California 4440421.3

28457



San Diego 4440436.3

28353



San Diego 4440432.3

28373




28375



salinity West California 4440417.3

28445




28447




28451



west California 4440420.3

28455


22 Salton Sea 4440327.3 28613 V Hyper-saline 55,787 926



23 Salton Sea 4440328.3 28613 V Hyper-saline 29,970 454

24 Line Is Kingman 4440037.3 28343 M Marine 188,445 11,309 6

25 Line Is Christmas 4440041.3 28347 M Marine 227,542 11,574 18

26 Line Is Palmyra 4440039.3 28363 M Marine 289,723 26,173 97

27 Line Is Tabuaeran 4440279.3 28367 M Marine 290,844 12,631 100

28 DMSP Treated 4440364.3 19145 M Marine 54,848 11,725 24

29 DMSP Treated 4440360.3 19145 M Marine 50,313 7,198 52

30 Vanillate Treated 4440365.3 19145 M Marine 12,446 1,720 48

31 Vanillate Treated 4440363.3 19145 M Marine 33,773 6,610 7

32 Marine GOM 4440304.3 17765 V Marine 263,908 28,878

33 Marine BBC 4440305.3 17767 V Marine 416,456 20,770

34 Marine Arctic 4440306.3 17769 V Marine 688,590 197,018

35 Marine SAR 4440322.3 17771 V Marine 399,343 17,813

36 Line Is Kingman 4440036.3 28345 V Marine 94,915 6,597



37 Line Is Christmas 4440038.3 28349 V Marine 283,390 69,501

38 Line Is Palmyra 4440040.3 28365 V Marine 320,397 9,608

39 Line Is Tabuaeran 4440280.3 28369 V Marine 380,355 10,716

40 Tampa Bay Mitomycin C

induced 4440102.3

28619

V Marine 280,019 8,767

41 Skan Bay 4440330.3 28619 V Marine 31,375 417

42 Tilapia pond 4440440.3 28387 M Freshwater 381,076 58,596 177

43 Healthy fish pond 4440413.3 28405 M Freshwater 63,978 8,911 48

44 Healthy fish Prebead 4440411.3 28407 M Freshwater 44,094 6,937 32

45 Tilapia pond 3 4440422.3 28603 M Freshwater 67,612 10,549 71

46 Tilapia pond 3 4440424.3 28601 V Freshwater 267,640 9,055

47 Healthy fish pond 4440412.3 28409 V Freshwater 60,319 1,152

48 Healthy fish Prebead 4440414.3 28411 V Freshwater 67,988 1,739

49 Tilapia pond 4440439.3 28361 V Freshwater 57,134 1,226



50 Porites compressa time

zero 4440380.3

28427

M Coral 53,473 2,560 0

51 Porites compressa

control 4440378.3

28429

M Coral 65,191 2,030 2


temperature 4440373.3

28431

M Coral 61,356 1,359 13

53 Porites compressa DOC 4440372.3 28433 M Coral 62,959 1,566 7

54 Porites compressa pH 4440379.3 28435 M Coral 67,994 1,913 5


Nutrient 4440381.3

28437

M Coral 65,008 3,258 11

56 Porites asteriodes 4440319.3 28371 M Coral 316,279 39,004 393

57 Porites compressa time

zero 4440376.3

28415

V Coral 39,270 2,772


control 4440374.3

28417

V Coral 39,340 5,276

59 Porites compressa DOC 4440370.3 28421 V Coral 35,680 2,410



60 Porites compressa pH 4440371.3 28423 V Coral 50,364 2,710


nutrients 4440377.3

28425

V Coral 34,433 2,338


Temperature 4440375.3

28419

V Coral 39,036 2,141

63 Rio Mesquites 4440060.3 28351 M Microbialites 124,694 21,374 10

64 Highborne Cay 4440061.3 28383 M Microbialites 257,573 5,286 12

65 Pozas Azule II 4440067.3 28385 M Microbialites 326,146 36,468 61

66 Pozas Azules II 4440320.3 28355 V Microbialites 302,987 3,947

67 Rios Mesquites 4440321.3 28357 V Microbialites 328,656 14,561

68 Highborne Cay 4440323.3 28381 V Microbialites 150,223 3,063

69 Healthy fish slime 4440059.3 28393 M Fish 66,066 15,686 68

70 Morbid fish slime 4440066.3 28395 M Fish 82,442 20,635 147

71 Healthy fish gut 4440055.3 28389 M Fish 51,498 16,377 63



72 Morbid fish gut 4440056.3 28391 M Fish 60,311 17,996 91

73 Healthy fish slime 4440065.3 28401 V Fish 61,476 9,051

74 Morbid fish slime 4440064.3 28403 V Fish 60,111 13,826

75 Cow rumens pool

plankton 4440357.3

28611

M Terrestrial

Animals

236,830 38,626 313

76 Cow rumens 80F6 4440356.3

28605

M Terrestrial

Animals

178,713 29,989 240

77 Cow rumens 640F6 4440355.3

28607

M Terrestrial

Animals

264,849 39,775 386

78 Cow rumens 710 F 4440387.3

28609

M Terrestrial

Animals

345,317 130,089 757

79 Lean Mice 4440324.3

17401

M Terrestrial

Animals

49,074 8,688 42

80 Obese Mice 4440325.3

17401

M Terrestrial

Animals

35,053 9,161 37

81 Chicken cecum NCTC 4440367.3 28599 M Terrestrial 237,940 49,256 451



Animals

82 Chicken cecum

Uninfected 4440368.3

28597

M Terrestrial

Animals

294,682 83,912 533

83 Lung sputum Cystic

fibrosis patient 4440441.3

28441

V Terrestrial

Animals

92,223 7,946

84 Lung sputum

Healthy 4440442.4

28439

V Terrestrial

Animals

39,807 3,292

85 Mosquito

Oceanside Ca 4440052.3

28413

V Mosquito 340,098 97,269

86 Mosquito San Diego 4440053.3 28467 V Mosquito 657,204 232,886

87 Mosquito Mission Valley

Ca 4440054.3

28469

V Mosquito 615,576 112,761



Table S2. The percent of phage and prophage sequences in the microbial and viral metagenomes. Ns = no sample.

Type Microbial metagenomes Viral metagenomes

Sample

number

Percent

phage

Percent

prophage

Sample

number

Percent

phage

Percent

prophage

Subterranean 1 1.879 3.802 ns ns

Subterranean 2 1.838 3.638 ns ns

Hyper-saline 3 0.983 3.802 12 3.922 5.456

Hyper-saline 4 0.000 3.595 13 8.861 3.927

Hyper-saline 5 0.375 3.638 14 25.517 3.744

Hyper-saline 6 0.557 3.802 15 14.463 3.554

Hyper-saline 7 0.000 1.238 16 29.762 3.578

Hyper-saline 8 1.695 2.779 17 34.884 4.940

Hyper-saline 9 4.918 3.802 18 17.647 3.263



Hyper-saline 10 1.286 3.802 19 4.545 4.341

Hyper-saline 11 1.961 3.638 20 1.056 4.777

Hyper-saline ns ns 21 3.198 3.667



Marine 24 0.589 3.638 32 1.051 3.474

Marine 25 3.797 3.580 33 2.171 3.523

Marine 26 1.073 3.762 34 0.351 3.802

Marine 27 0.763 3.146 35 15.764 3.803

Marine 28 0.727 3.720 36 3.243 2.655

Marine 29 1.342 3.299 37 0.531 3.802

Marine 30 0.478 3.746 38 11.189 3.864

Marine 31 1.370 3.415 39 7.563 3.921

Marine ns ns 40 30.469 3.855



Marine ns ns 41 8.824 4.352

Freshwater 42 6.759 3.802 46 41.176 3.185

Freshwater 43 3.204 3.809 47 68.182 5.143

Freshwater 44 3.472 4.032 48 50.000 4.628

Freshwater 45 0.321 3.802 49 58.301 3.723

Coral 50 5.797 3.575 57 2.602 3.503

Coral 51 0.000 2.839 58 9.385 4.047

Coral 52 30.864 3.786 59 2.871 3.903

Coral 53 2.222 3.385 60 11.765 4.357

Coral 54 2.941 4.504 61 4.348 3.602

Coral 55 0.000 3.807 62 2.985 3.205

Coral 56 0.472 3.712 ns ns

Microbialites 63 3.162 3.536 66 11.712 3.214

Microbialites 64 9.063 3.192 67 92.548 4.178



Microbialites 65 0.591 3.802 68 0.000 6.258

Fish 69 1.467 3.645 73 0.628 3.707

Fish 70 3.101 3.638 74 0.922 3.489

Fish 71 0.949 3.638 ns ns

Fish 72 0.833 3.675 ns ns

Terrestrial

animals

75 4.245 3.802 83 0.000 4.486

Terrestrial

animals

76 4.504 3.802 84 0.000 3.579

Terrestrial

animals

77 1.380 3.802 ns ns

Terrestrial

animals

78 3.229 3.802 ns ns

Terrestrial

animals

79 4.195 3.802 ns ns

Terrestrial 80 3.624 3.802 ns ns



animals

Terrestrial

animals

81 5.481 3.802 ns ns

Terrestrial

animals

82 5.472 3.802 ns ns

Mosquito ns ns 85 11.995 3.638





Table S3. The thirty most abundant motility and chemotaxis protein sequences found within the metagenomes, ordered with respect to the microbial metagenomes.

Motility proteins Microbial

metagenomes

Viral

metagenomes

Twitching motility protein PilT 0.033 0.023

Methyl-accepting chemotaxis protein I 0.029 0.033

Flagellar biosynthesis protein flhA 0.025 0.089

Chemotaxis protein CheA 0.018 0.059

Dipeptide-binding ABC transporter 0.018 0.064

Type II secretory pathway 0.017 0.008

Chemotaxis protein methyltransferase CheR 0.016 0.026

GldJ 0.015 0.005

Acetylornithine deacetylases 0.015 0.076

Flagellum-specific ATP synthase fliI 0.014 0.032



Flagellar motor rotation protein motB 0.014 0.021

Flagellar hook-length control protein fliK 0.013 0.033

Flagellar hook protein flgE 0.010 0.014

Flagellar basal-body rod protein flgG 0.010 0.027

Chemoreceptor signals to flagelllar motor CheY 0.010 0.012

type 4 fimbrial biogenesis protein PilY1 0.010 0.022

Flagellar regulatory protein fleQ 0.010 0.011

General secretion pathway protein E /ATPase PilB 0.010 0.002

Flagellar motor rotation protein motA 0.009 0.018

lagellin protein flaA 0.009 0.009

Chemotaxis response regulator CheB 0.009 0.051

Aerotaxis sensor receptor protein 0.008 0.016

Flagellar motor switch protein fliG 0.008 0.014

Flagellar biosynthesis protein flhB 0.008 0.030



Cell division protein ftsX 0.007 0.008

Chemotaxis protein CheV 0.007 0.012

Flagellar motor switch protein fliM 0.007 0.015

Flagellar motor switch protein fliG 0.007 0.009

Flagellar biosynthesis protein fliP 0.006 0.015

Maltose/maltodextrin ABC transporter MalE 0.006 0.042



Table S4. The variation for each metabolism identified for the microbial and viral communities across the nine biomes, using three statistical tests. The table includes the F value and P value and where possible the biome that was identified as showing differences for the particular metabolism.

Microbial metagenomes Viral metagenomes

Metabolism ANOVA Krus/wal Median Duncan ANOVA Krus/wal Medium Duncan

Amino Acids

F=5.655

P<0.001

F=22.01

P=0.003

F=13.15

P=0.012

Coral

F=1.743

P=0.132

F=9.919

P=0.193

F=10.84

P=0.064

Carbohydrates

F=4.965

P<0.001

F=12.56

P=0.083

F=18.35

P=0.226

Coral

F=5.335

P<0.001

F=20.17

P=0.005

F=14.80

P=0.012

Multiple

Cell Division &

Cell Cycle

F=12.55

P<0.001

F=29.79

P<0.001

F=1.865

P=0.002

Coral,

Terrestrial

animals.

Microbialite

F=3.040

P=0.014

F=17.47

P=0.015

F=1.754

P=0.023

Multiple



Cell Wall and

Capsule

F=9.929

P<0.001

F=34.78

P<0.001

F=3.171

P<0.001

Coral,

Hyper-saline

Marine

F=0.875

P=0.536

F=6.260

P=0.510

F=3.562

P=0.339

Cofactors,

Vitamins, etc

F=8.950

P<0.001

F=26.66

P<0.001

F=5.593

P<0.001

Coral

F=1.266

P=0.296

F=9.063

P=0.248

F=6.147

P=0.692

DNA Metabolism

F=16.20

P<0.001

F=35.33

P<0.001

F=4.138

P<0.001

Multiple

F=6.236

P<0.001

F=26.70

P<0.001

F=5.453

P=0.002

Microbialite

Freshwater

Fatty Acids and

Lipids

F=2.765

P=0.020

F=18.101

P=0.012

F=3.063

P=0.040 Multiple

F=1.514

P=0.196

F=10.75

P=0.150

F=3.006

P=0.151

Membrane

Transport

F=15.92

P<0.001

F=29.99

P<0.001

F=2.551

P<0.001

Multiple

F=4.494

P=0.001

F=14.95

P=0.037

F=2.435

P=0.204

Fish

mosquito

Aromatic

Compounds

F=8.464

P<0.001

F=22.43

P=0.002

F=2.137

P=0.017

Fish

F=2.225

P=0.056

F=16.28

P=0.023

F=1.834

P=0.020

None obvious

Motility and F=3.517 F=19.27 F=0.858 Fish F=3.692 F=15.26 F=0.833 Multiple



Chemotaxis P=0.005 P=0.007 P=0.007 Subterranean P=0.005 P=0.033 P=0.047

Nitrogen

Metabolism

F=8.887

P<0.001

F=26.28

P<0.001

F=1.613

P=0.003 Coral

F=2.252

P=0.054

F=12.79

P=0.077

F=1.137

P=0.057

Nucleosides,

Nucleotides

F=6.949

P<0.001

F=18.87

P=0.009

F=3.424

P=0.014 Coral

F=2.022

P=0.081

F=17.58

P=0.014

F=6.701

P=0.012

None obvious

Phosphorus

Metabolism

F=1.498

P=0.198

F=15.65

P=0.029

F=0.809

P=0.020

F=1.904

P=0.099

F=11.50

P=0.118

F=1.033

P=0.532

Photosynthesis

F=10.46

P<0.001

F=29.49

P<0.001

F=0.049

P=0.001 Coral

F=1.722

P=0.137

F=13.53

P=0.060

F=0.050

P=0.074

Potassium

metabolism

F=4.720

P=0.001

F=20.37

P=0.005

F=0.791

P=0.009 Multiple

F=4.634

P=0.001

F=17.35

P=0.015

F=0.680

P=0.103

Protein

Metabolism

F=6.814

P<0.001

F=23.93

P=0.001

F=9.316

P<0.001 Multiple

F=1.631

P=0.160

F=14.17

P=0.048

F=8.448

P=0.074

Cell signaling F=4.701 F=21.06 F=0.717 Microbialite F=2.346 F=12.89 F=0.734



P=0.001 P=0.004 P=0.012 P=0.046 P=0.075 P=0.115

Respiration

F=5.158

P<0.001

F=26.00

P=0.001

F=4.607

P=0.003

Coral

F=3.633

P=0.005

F=14.70

P=0.040

F=3.669

P=0.052

Multiple

RNA Metabolism

F=2.740

P=0.021

F=19.41

P=0.007

F=3.858

P=0.144

F=1.348

P=0.259

F=8.769

P=0.270

F=3.721

P=0.122

Secondary

Metabolism

F=1.366

P=0.249

F=13.47

P=0.061

F=0.131

P=0.116

F=1.200

P=0.329

F=10.65

P=0.154

F=0.093

P=0.230

Stress

Response

F=6.162

F<0.001

F=23.40

P=0.001

F=2.616

P=0.018

Coral

Fish

Freshwater

F=1.878

P=0.104

F=16.23

P=0.023

F=3.133

P=0.033

Sulfur

Metabolism

F=12.05

P<0.001

F=28.86

P<0.001

F=1.084

P=0.005 Fish

F=2.290

P=0.050

F=10.06

P=0.185

F=1.079

P=0.327

Virulence

F=5.150

P<0.001

F=30.79

P<0.001

F=9.698

F=0.002

Coral

Marine

F=3.953

P=0.003

F=13.67

P=0.057

F=10.65

P=0.208

Microbialite



Equator

Tropic of Cancer

Arctic Circle

Pacific Ocean

Atlantic Ocean

2

0�

20�

40�

60�

80�

Subterranean MarineHyper-salineFreshwaterCoralMicrobialiteFishTerrestrial AnimalsMosquito

66

2

65

242

44

4912

3

Figure S1. The sampling location of the metagenomes, circles indicate

microbial and squares viral metagenomes. The number of metagenomes

collected at each site is given, except where only one metagenome per site was

taken.



Figure S2. Functional diversity of the a) microbial and b) viral metagenomes

quantified as a function of sequence number, suggesting high functional

diversity is gained at low sequence number. Note the different scale on the x-

axis.



Figure S3. Comparison of mean (± s.e.m.) functional diversity and evenness

between microbial and viral metagenomes and all sequenced bacterial

genomes. Note the different scale on the y-axis.



Figure S4. The percent of sequences found within the sulfur metabolism

pathways within the microbial metagenomes. The overrepresentation of the a)

alkanesulfonates assimilation, b) alkanesulfonates utilization and c) taurine

utilization subsystem suggests the addition of an organic source of the sulfur,

most likely taurine, whereas the subsystems involved with the utilization of

inorganic sulfur (d) were not overrepresented.



Figure S5. Canonical discriminant analysis of the a) microbial and b) viral

metagenomes on a reduced set of biomes (subterranean, fish and mosquito

metagenomes removed) to demonstrate the stability of the analysis and

variations in the influence of the potential metabolic processes between

environments.



Figure S6. Canonical discriminant analysis of the a) microbial and b) viral

metagenomes on a reduced set of biomes (hyper-saline biomes removed) to

demonstrate the stability of the analysis and variations in the influence of the

potential metabolic processes between environments.



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