0)+%*-12€¦ · Supplementary Figure 1. Richness (rarefaction curves) (a) and evenness (rank abundance curves) (b) of the three test communities (unamplified controls) as determined

Supplementary Figure 1. Richness (rarefaction curves) (a) and evenness (rank

abundance curves) (b) of the three test communities (unamplified controls) as determined

by SSU rRNA amplicon pyrosequencing. Shannon’s diversity indices for all three

environments are included in panel a.

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Supplementary Figure 2. Average number of sequence tags for each library analyzed in

this study. Error bars indicate standard deviation of three replicate samples.

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Supplementary Table 1. Experimental reproducibility estimated by pairwise

comparisons of three technical replicates for each sample.

Pearson correlation

R (avg ± s.d.) Significance

Control 0.995 ± 0.002 !0.001

GenomiPhi 1ng 0.998 ± 0.001 !0.001

GenomiPhi 10ng 0.992 ± 0.004 !0.001

GenomiPhi 100ng 0.998 ± 0.000 !0.001

NEB 1ng 0.989 ± 0.009 !0.001

NEB 10ng 0.999 ± 0.001 !0.001

NEB 100ng 0.999 ± 0.000 !0.001

RepliG 1ng 0.990 ± 0.005 !0.001

RepliG 10ng 0.985 ± 0.011 !0.001

Act

iva

ted

slu

dg

e

RepliG 100ng 0.988 ± 0.007 !0.001

Control 0.994 ± 0.003 !0.001

GenomiPhi 1ng 0.992 ± 0.000 !0.001

GenomiPhi 10ng 0.998 ± 0.001 !0.001

GenomiPhi 100ng 0.998 ± 0.002 !0.001

NEB 1ng 0.987 ± 0.006 !0.001

NEB 10ng 0.998 ± 0.001 !0.001

NEB 100ng 0.999 ± 0.001 !0.001

RepliG 1ng 0.977 ± 0.008 !0.001

RepliG 10ng 0.990 ± 0.001 !0.001 Ter

mit

e h

ind

gu

t P

3

RepliG 100ng 0.994 ± 0.002 !0.001

Control 0.955 ± 0.005 !0.001

GenomiPhi 1ng 0.861 ± 0.039 !0.001

GenomiPhi 10ng 0.940 ± 0.028 !0.001

GenomiPhi 100ng 0.876 ± 0.083 !0.001

NEB 1ng 0.064 ± 0.052 0.234

NEB 10ng 0.577 ± 0.097 !0.001

NEB 100ng 0.857 ± 0.038 !0.001

RepliG 1ng 0.771 ± 0.044 !0.001

RepliG 10ng 0.831 ± 0.042 !0.001

Ga

rden

co

mp

ost

RepliG 100ng 0.883 ± 0.035 !0.001

Control 0.990 ± 0.006 !0.001

GenomiPhi 10ng 0.999 ± 0.001 !0.001

NEB 10ng 0.998 ± 0.001 !0.001 AS

1

(7/3

/20

07

)

RepliG 10ng 0.991 ± 0.008 !0.001

Control 0.998 ± 0.001 !0.001

GenomiPhi 10ng 0.999 ± 0.001 !0.001

NEB 10ng 0.996 ± 0.002 !0.001 AS

2

(7/2

7/2

00

7)

RepliG 10ng 0.995 ± 0.004 !0.001

Control 0.996 ± 0.002 !0.001

GenomiPhi 10ng 0.986 ± 0.012 !0.001

NEB 10ng 0.993 ± 0.006 !0.001 AS

3

(10

/1/2

00

7)

RepliG 10ng 0.997 ± 0.001 !0.001


Supplementary Table 2. Proportion of OTUs detected in the unamplified controls that

were significantly skewed following MDA treatments.

% of total

OTUs skewed

% of "1%

OTUs skewed

% of < 1%

OTUs skewed

GenomiPhi 1ng 16 83 7



NEB 1ng 13 78 5

NEB 10ng 19 89 10

NEB 100ng 25 94 16

RepliG 1ng 11 61 5

RepliG 10ng 12 50 7 Act

iva

ted

slu

dg

e

RepliG 100ng 12 61 5




NEB 1ng 10 42 7

NEB 10ng 13 50 9

NEB 100ng 15 58 11

RepliG 1ng 8 25 6

RepliG 10ng 9 42 6

Ter

mit

e h

ind

gu

t P

3

RepliG 100ng 13 25 11




NEB 1ng 5 59 4

NEB 10ng 9 76 7

NEB 100ng 11 65 10

RepliG 1ng 3 24 2

RepliG 10ng 5 47 4 Ga

rden

co

mp

ost

RepliG 100ng 4 18 3


NEB 10ng 25 92 16

AS

1

RepliG 10ng 9 33 6


NEB 10ng 28 100 20

AS

2

RepliG 10ng 13 62 8


NEB 10ng 31 94 19

AS

3

RepliG 10ng 25 76 14


Supplementary Table 3. MDA-skewing of OTUs relative to controls. Significantly

over- and under-represented OTUs are highlighted in red and green respectively. Values

indicate relative (%) abundance of each OTU within a given treatment before (control)

and after amplification. Missing values indicate that OTUs were below detection in a

given sample. Phylogenetic affiliation and closest match in the greengenes database

(http://greengenes.lbl.gov/), where available, are also shown for each OTU. N/A not

available.

Termite hindgut proctodeal segment P3 OTU relative abundance (%)

GenomiPhi NEB RepliG Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Co

ntr

ol

1ng 10ng 100ng 1ng 10ng 100ng 1ng 10ng 100ng

Spirochaetes 3 217268 30.68 26.52 27.82 29.02 32.51 33.86 33.35 20.80 21.75 21.24

Spirochaetes 16 218857 10.01 8.74 8.97 8.54 7.14 6.40 6.18 10.96 12.88 11.44

Fibrobacteres 13 217282 8.75 5.69 6.56 7.44 2.96 4.00 3.73 8.99 7.69 7.53

Spirochaetes 11 217299 7.20 7.75 6.84 7.39 5.05 4.22 4.10 8.70 7.08 6.88

Spirochaetes 23 219918 7.15 7.37 8.70 8.78 5.61 6.52 6.39 11.28 10.34 10.99

Fibrobacteres 7 221373 6.79 9.96 8.05 6.55 14.38 10.71 9.84 7.24 8.21 7.89

Spirochaetes 18 221520 6.48 4.81 4.57 4.45 1.82 2.49 2.28 6.04 5.95 5.82

Spirochaetes 69 217767 2.38 2.58 2.42 2.85 2.20 2.78 2.60 1.75 1.70 2.09

Fibrobacteres 33 223487 2.12 3.84 3.16 2.93 3.65 3.21 3.23 2.94 4.15 3.94

Firmicutes 37 225919 1.00 2.01 2.21 1.99 4.65 5.13 5.71 0.59 0.69 0.92

Proteobacteria 125 221038 0.97 0.12 0.20 0.30 0.02 0.05 0.06 1.99 1.05 0.79

Fibrobacteres 382 217244 0.89 0.34 0.24 0.39 0.27 0.17 0.18 0.48 0.27 0.41

Spirochaetes 516 170322 0.83 0.57 0.57 0.74 0.75 0.70 0.88 0.47 0.77 0.71

Actinobacteria 50 113780 0.81 3.73 3.76 3.80 1.81 2.07 2.28 3.15 3.13 4.84

Spirochaetes 276 223738 0.68 0.24 0.45 0.34 0.36 0.39 0.40 0.27 0.30 0.36

Firmicutes 68 193088 0.53 0.73 0.73 0.81 1.68 2.66 2.96 0.15 0.21 0.43

Firmicutes 166 141830 0.52 0.93 0.52 0.57 0.12 0.27 0.12 0.41 0.31 0.30

Spirochaetes 82 224276 0.42 0.37 0.51 0.49 0.79 1.07 1.29 0.14 0.19 0.23

Spirochaetes 112 48891 0.31 0.28 0.59 0.55 1.03 0.61 0.65 0.36 0.46 0.47

Fibrobacteres 159 217484 0.26 0.87 0.80 0.52 0.38 0.55 0.62 0.14 0.17 0.18

Spirochaetes 7330 217359 0.25 0.22 0.22 0.20 0.24 0.29 0.32 0.10 0.09 0.14

Spirochaetes 665 141471 0.23 0.23 0.17 0.18 0.14 0.05 0.12 0.16 0.19 0.25

Firmicutes 2493 224436 0.23 0.04 0.05 0.05 0.02 0.03 0.22 0.07 0.10

Proteobacteria 525 166994 0.20 0.06 0.10 0.13 0.02 0.02 0.03 0.33 0.39 0.26

Bacteroidetes 319 219599 0.18 1.06 0.95 0.79 0.81 0.47 0.35 0.08 0.20 0.18

Spirochaetes 177 223165 0.18 0.43 0.37 0.32 2.34 1.03 1.33 0.27 0.14 0.21

Spirochaetes 7931 3984 0.17 0.07 0.05 0.06 0.11 0.09 0.14 0.08 0.05 0.04

Actinobacteria 261 114038 0.13 0.29 0.22 0.25 0.05 0.05 0.08 1.20 0.79 0.85

Bacteroidetes 462 220946 0.12 0.38 0.42 0.52 0.40 0.38 0.38 0.40 0.18

Acidobacteria 199 218159 0.11 0.12 0.13 0.12 0.05 0.08 0.10 0.78 0.87 0.72

Spirochaetes 1449 69074 0.08 0.11 0.05 0.06 0.02 0.01 0.04 0.08 0.05 0.06

Planctomycetes 656 201482 0.07 0.12 0.08 0.08 0.01 0.03 0.02 0.13 0.12 0.10

Firmicutes 355 221978 0.07 0.05 0.08 0.10 0.02 0.28 0.17 0.04 0.03 0.07

ZB3 1569 81819 0.06 0.10 0.06 0.10 0.04 0.04 0.05 0.04 0.03

Actinobacteria 1256 78074 0.06 0.24 0.21 0.17 0.06 0.10 0.16 0.26 0.21 0.38

Proteobacteria 313 217779 0.05 0.25 0.18 0.21 0.27 0.33 0.29 0.05 0.09 0.02

Acidobacteria 6877 219494 0.04 0.02 0.01 0.01

Firmicutes 346 258882 0.03 0.05 0.09 0.09 0.05 0.56 0.73 0.03 0.08

Spirochaetes 3233 225474 0.03 0.01 0.01 0.02 0.03

Firmicutes 5183 147741 0.03 0.01 0.01 0.01 0.02

Firmicutes 7006 81157 0.03 0.02

Fibrobacteres 2072 223487 0.03 0.10 0.09 0.05 0.12 0.13 0.12 0.02 0.03 0.05

Bacteroidetes 821 226064 0.03 0.38 0.19 0.23 0.03 0.15 0.08 0.07 0.06 0.08

Fibrobacteres 5753 165521 0.02 0.16 0.13 0.09 0.14 0.11 0.11 0.02 0.16 0.11

Spirochaetes 8836 220592 0.02 0.01 0.03 0.05 0.02 0.03

Spirochaetes 45899 217451 0.02 0.03 0.02 0.01 0.06 0.05 0.05

Actinobacteria 4168 113780 0.02 0.05 0.05 0.07 0.02 0.03 0.05 0.08 0.07 0.12


Activated sludge OTU relative abundance (%)

GenomiPhi NEB RepliG Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Co

ntr

ol


Proteobacteria 4 257084 22.80 3.56 6.12 12.35 0.39 0.76 2.19 19.81 20.61 21.87

Eukaryota 17 N/A 16.42 0.63 1.44 2.08 0.03 0.22 0.35 0.61 0.63 1.00

Bacteroidetes 1 335772 15.89 57.84 49.01 38.27 52.96 49.74 46.91 7.58 9.88 14.18

Actinobacteria 6 26533 6.20 1.52 3.11 5.90 0.21 0.36 0.86 26.47 24.98 21.36

Bacteroidetes 2 368794 4.75 10.45 11.56 8.93 21.05 22.76 20.81 1.01 1.75 2.28

Proteobacteria 19 345752 3.06 0.28 0.58 1.03 0.04 0.10 0.20 8.86 7.85 6.07

Proteobacteria 48 72759 3.00 0.32 0.51 0.98 0.04 0.06 0.17 1.58 1.73 1.66

Proteobacteria 61 359351 2.25 0.92 0.99 1.55 0.10 0.14 0.26 2.48 2.42 2.06

Actinobacteria 70 328815 1.89 0.10 0.21 0.48 0.01 0.04 0.11 6.67 6.91 4.94

Bacteria 62 337640 1.79 0.79 1.05 1.47 0.11 0.10 0.20 0.49 0.48 0.69

Proteobacteria 49 344892 1.75 0.39 0.62 0.97 0.04 0.05 0.17 3.74 3.42 2.69

Proteobacteria 85 141416 1.63 0.17 0.29 0.66 0.02 0.03 0.13 1.41 1.35 1.37

Proteobacteria 102 239835 1.32 0.16 0.24 0.39 0.03 0.09 1.55 1.47 1.25

Proteobacteria 44 26769 1.29 1.71 1.77 1.90 0.60 0.58 0.79 0.72 0.91 1.32

Bacteroidetes 43 362321 1.27 2.14 2.01 1.90 3.53 3.81 3.81 0.11 0.28 0.45

Bacteroidetes 25 152338 1.21 1.64 2.32 1.94 1.54 1.61 1.56 0.10 0.29 0.56

Proteobacteria 29 358750 1.10 1.61 1.47 1.55 1.45 1.57 1.80 0.14 0.26 0.40

Proteobacteria 14 32355 1.03 2.20 1.83 2.46 3.32 2.43 3.00 0.28 0.32 0.75

Bacteroidetes 116 255558 0.91 0.04 0.06 0.25 0.01 0.03 0.92 1.00 0.79

Bacteroidetes 15 306824 0.69 2.09 2.25 1.94 2.31 2.35 2.11 0.20 0.46 0.63

Bacteroidetes 67 225621 0.52 0.97 1.06 0.97 0.51 0.48 0.54 0.05 0.20 0.30

Bacteroidetes 60 65426 0.51 1.10 1.03 1.09 1.11 1.15 1.27 0.19 0.21 0.20

Proteobacteria 24 252003 0.49 1.83 1.88 1.78 1.83 1.88 1.90 0.21 0.22 0.21

Proteobacteria 34 326693 0.44 0.14 0.27 0.65 0.01 0.03 0.13 6.02 4.34 3.35

Proteobacteria 156 306864 0.33 0.09 0.12 0.29 0.02 0.03 0.95 0.75 0.83

Bacteroidetes 79 249252 0.32 0.39 0.39 0.46 0.42 0.59 0.65 0.09 0.08 0.07

Actinobacteria 626 20188 0.30 0.01 0.03 0.01 0.11 0.07 0.07

Bacteroidetes 86 146568 0.25 0.60 0.60 0.49 1.02 0.83 0.79 0.08 0.06 0.14

Eukaryota 140 N/A 0.21 0.32 0.28 0.27 0.13 0.22 0.32 0.06 0.06 0.11

Proteobacteria 347 105875 0.21 0.01 0.06 0.09 0.01 0.30 0.29 0.23

Firmicutes 122 162145 0.18 0.10 0.28 0.21 0.60 0.56 0.52 0.05 0.07

Proteobacteria 185 245343 0.15 0.28 0.26 0.30 0.53 0.38 0.49 0.02 0.07 0.09

Proteobacteria 328 355294 0.14 0.03 0.03 0.06 0.01 0.01 0.32 0.42 0.28

Actinobacteria 144 47074 0.14 0.03 0.07 0.03 1.31 1.11 1.06

Bacteroidetes 80 43204 0.13 0.56 0.59 0.29 0.37 0.57 0.60 0.02 0.06

Bacteroidetes 179 247588 0.13 0.14 0.18 0.13 0.21 0.23 0.34 0.02 0.06 0.06

Actinobacteria 4703 26533 0.10 0.03 0.06 0.04 0.01 0.02 0.36 0.30 0.17

Bacteroidetes 213 63191 0.08 0.22 0.13 0.11 0.23 0.16 0.29 0.02 0.02

Firmicutes 268 141405 0.06 0.04 0.06 0.07 0.07 0.23 0.31 0.02

Proteobacteria 1329 55851 0.06 0.02 0.03 0.01 0.02 0.07 0.04

Bacteroidetes 230 248514 0.06 0.09 0.15 0.14 0.23 0.27 0.22 0.02

Bacteroidetes 657 345293 0.05 0.04 0.02 0.02 0.01 0.04 0.04 0.03

Bacteroidetes 300 368794 0.05 0.20 0.22 0.20 0.55 0.55 0.56

Bacteroidetes 1009 368794 0.05 0.09 0.09 0.10 0.22 0.25 0.24 0.02 0.02

Bacteroidetes 2264 335772 0.05 0.28 0.24 0.17 0.23 0.27 0.27 0.03 0.04 0.07

Proteobacteria 3528 143390 0.05 0.02 0.03

Bacteria 3583 342757 0.04 0.01 0.03 0.02 0.05

Bacteroidetes 428 110915 0.04 0.23 0.11 0.16 0.32 0.32 0.35

Bacteroidetes 3523 335772 0.03 0.03 0.10 0.07 0.08 0.05 0.06

Proteobacteria 15069 26769 0.03 0.02 0.01 0.01 0.02

Bacteroidetes 232 170995 0.03 0.11 0.11 0.08 0.17 0.24 0.22 0.05 0.03

Bacteroidetes 10699 335772 0.02 0.06 0.05 0.05 0.10 0.09 0.06 0.01

Chlamydiae 674 229341 0.02 0.06 0.06 0.12 0.01 0.06 0.06 0.02 0.04


Garden compost OTU relative abundance (%)

Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Co

ntr

ol

GenomiPhi NEB RepliG


Proteobacteria 128 105985 4.29 0.94 1.32 2.57 0.05 0.22 0.73 1.94 1.93 3.26

Proteobacteria 63 75633 2.69 1.04 1.55 2.03 0.08 0.23 0.49 2.26 2.55 3.16

Bacteroidetes 35 225235 2.61 4.04 3.97 3.11 0.70 1.09 1.66 1.51 2.09 2.25

Bacteroidetes 41 141044 2.35 4.86 5.22 3.86 1.30 1.89 2.04 2.89 2.35 2.82

Actinobacteria 142 360846 1.81 0.17 0.15 0.48 0.09 0.14 0.86 1.27 0.98

Eukaryota 21 N/A 1.79 4.23 5.12 3.51 0.26 0.68 1.21 3.89 3.47 3.09

Actinobacteria 70 328815 1.64 0.10 0.25 0.63 0.08 0.13 0.96 0.88 1.03

Proteobacteria 19 345752 1.61 0.12 0.23 0.62 0.08 0.16 0.79 1.16 1.01

Proteobacteria 352 240695 1.36 0.20 0.26 0.59 0.02 0.07 0.20 1.01 0.66 0.77

Bacteroidetes 111 111136 1.25 1.75 1.82 1.40 0.32 0.40 0.55 1.15 0.71 1.02

Bacteroidetes 105 239396 1.23 1.78 1.75 1.94 0.42 0.63 0.87 1.39 1.55 1.56

Proteobacteria 32 142923 1.17 3.36 3.05 2.61 0.33 0.49 0.59 3.86 2.73 2.70

Actinobacteria 6 26533 1.17 0.11 0.17 0.40 0.06 0.16 0.71 0.59 0.58

Proteobacteria 408 83068 1.06 0.11 0.29 0.60 0.02 0.03 0.12 0.66 0.88 0.65

Proteobacteria 325 137811 1.04 0.11 0.18 0.43 0.04 0.18 0.43 0.61 0.40

Proteobacteria 4 257084 1.01 0.44 0.57 0.86 0.02 0.13 0.20 1.16 0.64 0.98

Proteobacteria 137 358990 0.98 0.46 0.58 0.99 0.04 0.06 0.20 1.37 1.04 1.15

Proteobacteria 178 217902 0.98 0.39 0.37 0.50 0.02 0.13 0.19 1.01 0.81 0.89

Proteobacteria 61 359351 0.93 0.19 0.24 0.49 0.02 0.09 0.16 0.33 0.41 0.48

Eukaryota 103 N/A 0.86 0.70 0.93 0.82 0.03 0.12 0.20 0.85 0.98 1.33

Eukaryota 277 N/A 0.80 0.44 0.59 0.74 0.04 0.10 0.11 0.16 0.34 0.40

Eukaryota 91 N/A 0.73 1.26 1.17 1.28 0.08 0.22 0.35 2.88 2.40 1.90

Eukaryota 157 N/A 0.66 0.37 0.49 0.56 0.08 0.16 0.23 0.41 0.82 0.54

Acidobacteria 315 244296 0.64 0.06 0.17 0.36 0.04 0.07 0.44 0.39 0.43

Proteobacteria 233 144759 0.62 0.05 0.16 0.38 0.06 0.09 0.49 0.63 0.45

Proteobacteria 720 216499 0.60 0.07 0.13 0.32 0.02 0.06 0.47 0.39 0.29

Bacteroidetes 305 136494 0.58 0.75 0.89 0.73 0.18 0.31 0.29 0.28 0.37 0.60

Proteobacteria 785 181525 0.55 0.06 0.16 0.32 0.04 0.08 0.26 0.52 0.37

Proteobacteria 714 101230 0.54 0.18 0.32 0.38 0.05 0.09 0.22 0.41 0.49

Proteobacteria 725 85585 0.52 0.12 0.16 0.23 0.02 0.08 0.24 0.47 0.37

Eukaryota 89 N/A 0.48 1.45 1.34 0.68 0.20 0.52 0.41 0.70 0.66 0.77

Bacteroidetes 108 159655 0.48 1.06 1.04 0.68 0.29 0.24 0.28 1.43 0.52 0.64

Verrucomicrobia 400 100737 0.48 0.35 0.21 0.28 0.03 0.04 0.13 0.19 0.14 0.30

Actinobacteria 545 275402 0.48 0.04 0.09 0.17 0.02 0.07 0.14 0.56 0.53 0.48

Proteobacteria 102 239835 0.48 0.04 0.08 0.19 0.02 0.07 0.31 0.25 0.29

Eukaryota 290 N/A 0.47 0.36 0.46 0.42 0.03 0.09 0.08 0.07 0.18 0.21

Proteobacteria 768 246920 0.47 0.17 0.11 0.26 0.02 0.04 0.21 0.31 0.39

Proteobacteria 357 137895 0.45 0.14 0.11 0.35 0.03 0.04 0.49 0.61 0.49

Eukaryota 394 N/A 0.45 0.34 0.55 0.43 0.02 0.05 0.09 0.72 0.91 0.81

Proteobacteria 425 114139 0.45 0.14 0.12 0.29 0.03 0.12 0.47 0.48 0.35

Proteobacteria 244 41721 0.45 0.51 0.46 0.55 0.03 0.07 0.15 0.44 0.52 0.53

Eukaryota 520 N/A 0.43 0.32 0.43 0.49 0.07 0.10 0.08 0.07 0.11 0.20

Proteobacteria 921 113561 0.42 0.06 0.24 0.34 0.03 0.02 0.08 0.34 0.48 0.32

Bacteroidetes 134 19106 0.41 0.86 1.00 0.63 0.18 0.18 0.34 1.02 0.91 0.89

Bacteroidetes 515 313378 0.41 0.80 0.71 0.59 0.11 0.24 0.27 0.42 0.30 0.34

Proteobacteria 118 269202 0.41 0.90 0.81 0.72 0.30 0.18 0.29 0.94 0.54 0.53

Gemmatimonadetes 358 243247 0.41 0.07 0.10 0.35 0.06 0.02 0.23 0.52 0.34

Proteobacteria 547 151468 0.41 0.08 0.09 0.27 0.02 0.05 0.26 0.30 0.41

Actinobacteria 531 214357 0.40 0.06 0.11 0.27 0.04 0.06 0.42 0.45 0.38

Actinobacteria 1021 209882 0.39 0.03 0.02 0.12 0.01 0.09 0.12 0.19

Proteobacteria 879 256455 0.39 0.03 0.05 0.12 0.02 0.04 0.52 0.36 0.25

Proteobacteria 536 67341 0.38 0.04 0.09 0.18 0.08 0.42 0.43 0.34

Actinobacteria 441 20317 0.38 0.09 0.09 0.25 0.07 0.07 0.45 0.41 0.31

Bacteroidetes 316 347504 0.37 0.40 0.72 0.67 0.05 0.12 0.27 0.38 0.48 0.32

Proteobacteria 424 142342 0.36 0.05 0.05 0.14 0.03 0.02 0.15 0.36 0.19

Actinobacteria 3094 217424 0.36 0.01 0.01 0.07 0.01 0.01 0.14 0.13 0.15

Proteobacteria 534 104318 0.35 0.22 0.26 0.28 0.02 0.04 0.08 0.53 0.36 0.37

Bacteroidetes 280 49920 0.34 0.60 0.72 0.47 0.14 0.19 0.12 0.28 0.27 0.39

Eukaryota 259 N/A 0.34 0.51 0.30 0.42 0.09 0.77 0.18 0.31 0.39 0.41

Proteobacteria 1647 240459 0.34 0.04 0.09 0.25 0.01 0.08 0.51 0.41 0.33


Garden compost (continued) OTU relative abundance (%)

Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Co

ntr

ol



Actinobacteria 730 316839 0.33 0.02 0.05 0.09 0.01 0.03 0.09 0.09 0.06

Proteobacteria 389 136796 0.33 0.16 0.15 0.20 0.04 0.25 0.20 0.26

Actinobacteria 496 12121 0.33 0.04 0.09 0.21 0.04 0.16 0.18 0.36

Acidobacteria 823 105472 0.32 0.02 0.06 0.17 0.01 0.04 0.12 0.27 0.22

Eukaryota 287 N/A 0.32 0.36 0.43 0.34 0.04 0.06 0.16 0.62 0.61 0.52

Chloroflexi 1210 134616 0.31 0.02 0.07 0.01 0.04 0.25 0.14

Proteobacteria 649 26630 0.31 0.03 0.08 0.14 0.03 0.02 0.12 0.07 0.13

Proteobacteria 1317 261579 0.30 0.09 0.06 0.16 0.02 0.01 0.19 0.11 0.20

Bacteroidetes 106 212711 0.29 1.11 1.11 0.88 0.10 0.19 0.26 0.41 0.35 0.36

Proteobacteria 381 137811 0.29 0.03 0.07 0.20 0.05 0.04 0.16 0.27 0.41

Proteobacteria 963 255866 0.29 0.02 0.11 0.08 0.01 0.03 0.07 0.16

Proteobacteria 824 151541 0.28 0.03 0.09 0.15 0.01 0.03 0.16 0.30 0.20

Eukaryota 479 N/A 0.28 0.07 0.07 0.11 0.01 0.02 0.11 0.14

Bacteroidetes 2 368794 0.27 0.71 0.69 0.53 0.16 0.20 0.31 0.38 0.27 0.31

Chloroflexi 77 257185 0.24 0.59 0.59 0.44 7.36 0.10 0.15 0.34 0.48 0.52

Actinobacteria 2489 366467 0.24 0.02 0.02 0.08 0.02 0.03 0.15 0.18 0.16

Proteobacteria 552 278121 0.24 0.19 0.15 0.19 0.02 0.05 0.11 0.16 0.29

Acidobacteria 475 199494 0.24 0.11 0.14 0.14 0.01 0.02 0.59 0.22 0.24

Eukaryota 831 N/A 0.24 0.25 0.21 0.19 0.02 0.06 0.09 0.17

Proteobacteria 1083 250313 0.23 0.02 0.10 0.02 0.03 0.17 0.21 0.20

Eukaryota 350 N/A 0.22 0.15 0.24 0.16 0.04 0.05 0.20 0.29 0.30

Eukaryota 151 N/A 0.22 0.56 0.59 0.39 0.13 0.17 0.24 0.19 0.23 0.30

Verrucomicrobia 385 246659 0.22 0.30 0.26 0.28 0.05 0.07 0.11 0.17 0.09 0.28

Proteobacteria 445 187488 0.21 0.10 0.10 0.22 0.04 0.04 0.38 0.14 0.17

Bacteroidetes 79 249252 0.21 0.54 0.37 0.32 0.04 0.16 0.17 0.18 0.34 0.21

Proteobacteria 1698 208631 0.20 0.05 0.04 0.10 0.01 0.02 0.08 0.09 0.06

Eukaryota 641 N/A 0.20 0.11 0.17 0.13 0.01 0.06 0.09 0.13 0.05 0.07

Eukaryota 383 N/A 0.20 0.25 0.45 0.34 0.04 0.09 0.10 0.25 0.35 0.34

Proteobacteria 490 37006 0.20 0.04 0.11 0.11 0.02 0.03 0.24 0.14 0.23

Proteobacteria 1513 236779 0.19 0.02 0.02 0.04 0.02 0.16 0.11 0.15

Actinobacteria 3450 312119 0.19 0.06 0.04 0.02 0.01 0.02 0.08 0.09 0.12

Bacteroidetes 286 242277 0.19 0.18 0.35 0.23 0.08 0.08 0.12 0.11 0.16 0.15

Proteobacteria 1833 364924 0.18 0.05 0.10 0.02 0.15 0.05 0.11

Proteobacteria 803 195011 0.18 0.03 0.13 0.02 0.01 0.12 0.13

Proteobacteria 1027 29077 0.18 0.01 0.01 0.08 0.02 0.11 0.07 0.10

Planctomycetes 1253 213225 0.17 0.02 0.02 0.04 0.19 0.05 0.08

Acidobacteria 1112 28670 0.17 0.02 0.01 0.03 0.26 0.13 0.12

Proteobacteria 34 326693 0.17 0.06 0.08 0.16 0.03 0.14 0.27 0.33

Proteobacteria 1114 239420 0.17 0.01 0.03 0.09 0.04 0.16 0.11

Gemmatimonadetes 845 265283 0.17 0.04 0.05 0.09 0.03 0.04 0.18 0.21 0.15

Proteobacteria 654 255452 0.16 0.04 0.05 0.09 0.01 0.02 0.23 0.23 0.18

Acidobacteria 628 277700 0.16 0.02 0.03 0.07 0.03 0.31 0.45 0.22

Bacteroidetes 198 90078 0.16 0.32 0.57 0.39 0.24 0.18 0.22 0.18 0.07 0.11

Acidobacteria 1531 199540 0.16 0.03 0.04 0.10 0.02 0.12 0.15

Bacteroidetes 317 110047 0.16 0.37 0.46 0.35 0.10 0.12 0.16 0.29 0.48 0.29

Eukaryota 73 N/A 0.15 0.51 0.62 0.43 0.06 0.11 4.85 0.50 0.16 0.37

Proteobacteria 93 8028 0.15 0.35 0.18 0.16 1.50 3.55 2.03 0.40 0.18 0.18

Proteobacteria 29 358750 0.15 0.35 0.22 0.17 0.02 0.05 0.05 0.21 0.36 0.23

Planctomycetes 1827 258352 0.14 0.01 0.03 0.08 0.11 0.09

Bacteroidetes 188 269565 0.14 0.42 0.63 0.46 0.17 0.23 0.26 0.50 0.27 0.23

Chloroflexi 301 114049 0.14 0.44 0.30 0.23 0.02 0.02 0.02 0.30 0.16 0.26

Proteobacteria 249 66625 0.14 0.54 0.36 0.24 0.04 0.09 0.08 0.13 0.07 0.15

Proteobacteria 2937 177268 0.14 0.02 0.05 0.08 0.01 0.03 0.05 0.11

Bacteroidetes 489 106339 0.14 0.26 0.10 0.16 0.04 0.10 0.19 0.11 0.07 0.12

Proteobacteria 1202 247980 0.14 0.02 0.05 0.03 0.01 0.01 0.07 0.12 0.10

Proteobacteria 1514 219413 0.14 0.04 0.02 0.06 0.01 0.09 0.06

Gemmatimonadetes 885 212075 0.14 0.02 0.03 0.07 0.13 0.13 0.12

Chloroflexi 3360 241504 0.14 0.02 0.08 0.01 0.02 0.04 0.12

Eukaryota 737 N/A 0.14 0.05 0.06 0.06 0.02 0.03 0.04 0.07 0.07

Proteobacteria 1476 239819 0.13 0.01 0.02 0.05 0.01 0.03 0.05 0.05



Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Co

ntr

ol



Eukaryota 661 N/A 0.13 0.04 0.14 0.18 0.03 0.17 0.05 0.09

Bacteroidetes 404 104934 0.13 0.25 0.20 0.18 0.04 0.04 0.05 0.18 0.18 0.14

Gemmatimonadetes 1288 250850 0.13 0.04 0.05 0.04 0.10 0.12 0.19

Eukaryota 252 N/A 0.13 0.17 0.30 0.21 0.01 0.06 0.10 0.46 0.32 0.30

Proteobacteria 293 364388 0.13 0.44 0.18 0.19 0.02 0.06 0.12 0.23 0.32 0.22

Chloroflexi 532 131339 0.13 0.24 0.10 0.08 0.01 0.23 0.14 0.15

Proteobacteria 2261 34344 0.12 0.03 0.04 0.12 0.01 0.01 0.05 0.06

Acidobacteria 2495 128220 0.12 0.03 0.03 0.01 0.09 0.07 0.02

Firmicutes 1150 249474 0.12 0.09 0.10 0.12 0.02 0.08 0.02 0.07 0.09

Proteobacteria 1175 204859 0.12 0.02 0.02 0.03 0.02 0.09 0.08

OP11 20 208492 0.12 0.28 0.27 0.71 3.53 9.31 6.19 0.09

Chloroflexi 3168 239349 0.12 0.01 0.01 0.10 0.20 0.06

Gemmatimonadetes 2348 224449 0.11 0.02 0.07 0.01 0.01 0.04 0.18 0.11

Bacteroidetes 423 114404 0.11 0.49 0.41 0.30 0.10 0.14 0.15 0.20 0.18 0.21

Eukaryota 1152 N/A 0.11 0.01 0.03 0.06 0.04 0.04 0.03

Proteobacteria 1786 112131 0.11 0.15 0.08 0.10 0.01 0.01 0.03 0.15 0.16 0.19

Bacteroidetes 245 2477 0.11 0.69 0.38 0.25 0.06 0.10 0.15 0.34 0.18 0.14

Planctomycetes 1643 84577 0.11 0.06 0.08 0.10 0.01 0.05 0.60 0.20 0.12

Firmicutes 900 364217 0.11 0.03 0.15 0.08 0.03 0.03 0.06 0.13 0.23 0.12

Proteobacteria 1615 232114 0.11 0.03 0.05 0.01 0.02

Actinobacteria 2016 157727 0.11 0.01 0.01 0.05 0.03 0.05 0.11 0.06

Proteobacteria 1948 170014 0.11 0.01 0.05 0.06 0.01 0.03 0.04

Proteobacteria 1206 236042 0.10 0.02 0.04 0.06 0.01 0.02 0.15 0.11 0.12

Acidobacteria 943 114283 0.10 0.04 0.07 0.07 0.02 0.02 0.06 0.05 0.07

Bacteroidetes 361 254871 0.09 0.10 0.18 0.15 0.08 0.07 0.07 0.27 0.16 0.08

Eukaryota 465 N/A 0.09 0.17 0.21 0.13 0.03 0.06 0.10 0.07 0.14 0.11

OP11 100 277545 0.09 0.54 0.55 0.36 0.20 1.55 0.48 0.12 0.11 0.08

Bacteroidetes 1246 240552 0.09 0.19 0.23 0.16 0.07 0.05 0.04 0.10 0.09 0.07

Proteobacteria 3369 48487 0.09 0.01 0.04 0.04 0.04

Acidobacteria 95 216666 0.09 0.69 0.14 0.16 7.00 1.74 3.71 0.08 0.07 0.05

Eukaryota 782 N/A 0.09 0.32 0.26 0.14 0.03 0.11 0.09 0.08 0.14 0.13

Verrucomicrobia 2090 91151 0.08 0.03 0.02 0.07 0.04

Proteobacteria 85 141416 0.08 0.04 0.06 0.01 0.03 0.11 0.11 0.09

Chloroflexi 1994 159189 0.08 0.06 0.03 0.05 0.02 0.03 0.10 0.07 0.05

Eukaryota 1100 N/A 0.08 0.04 0.07 0.04 0.05

Proteobacteria 8329 201499 0.08 0.03 0.01 0.03 0.01 0.04 0.03

TM7 407 109782 0.08 0.10 0.18 0.19 0.13 0.05 0.08 0.11 0.12 0.08

Proteobacteria 2458 76517 0.08 0.04 0.02 0.11 0.02 0.07 0.11 0.09

Planctomycetes 2934 252665 0.08 0.01 0.04

Planctomycetes 3195 60573 0.08 0.02 0.03 0.01 0.19 0.16 0.14

Proteobacteria 3358 220178 0.08 0.05 0.01 0.03

Proteobacteria 4459 210914 0.07 0.01 0.03 0.02 0.02 0.05 0.09 0.04

Eukaryota 1637 N/A 0.07 0.02 0.03 0.03 0.03 0.04 0.07

Chloroflexi 2274 113898 0.07 0.02 0.08 0.05 0.02 0.05 0.11 0.06

Proteobacteria 44 26769 0.07 0.03 0.06 0.01 0.06 0.07 0.03

Proteobacteria 844 168390 0.07 0.16 0.15 0.13 0.04 0.04 0.35 0.11 0.20

Proteobacteria 605 86032 0.07 0.11 0.13 0.09 0.03 0.01 0.15 0.09 0.10

Gemmatimonadetes 1927 112986 0.07 0.01 0.01 0.01 0.14 0.07

Proteobacteria 5980 158376 0.07 0.03 0.02 0.04 0.06 0.05 0.06

Eukaryota 865 N/A 0.07 0.05 0.05 0.05 0.02 0.05 0.11 0.12

Proteobacteria 2658 235868 0.07 0.01 0.04 0.06

Firmicutes 168 330001 0.07 0.21 0.05 0.13 0.03 0.03 0.11 0.83 0.43 0.06

Proteobacteria 1533 211464 0.07 0.06 0.07 0.07 0.01 0.05 0.09 0.03

Bacteroidetes 2928 279426 0.06 0.01 0.04 0.04

Proteobacteria 6034 217748 0.06 0.01 0.01 0.01 0.01 0.05

Chloroflexi 505 142371 0.06 0.29 0.15 0.13 0.04 0.05 0.11 0.16 0.07

Eukaryota 1435 N/A 0.06 0.03 0.05 0.09 0.01 0.06 0.05 0.01

Chlamydiae 996 152262 0.06 0.09 0.04 0.03 0.02 0.04 0.05 0.03

OP11 196 277545 0.06 0.20 0.22 0.21 0.15 1.04 0.32 0.26 0.09 0.08

Proteobacteria 5765 112822 0.05 0.01 0.04 0.13



Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Co

ntr

ol



Eukaryota 1103 N/A 0.05 0.15 0.16 0.10 0.08 0.10 0.09 0.09 0.11 0.08

Chloroflexi 1094 44185 0.05 0.04 0.07 0.01 0.02 0.08 0.05 0.08

Verrucomicrobia 1978 159970 0.05 0.01 0.02 0.01 0.06 0.05 0.07

Acidobacteria 2245 225577 0.05 0.03 0.04 0.07 0.01 0.02 0.03

Eukaryota 2868 N/A 0.05 0.01 0.04 0.01 0.05

Eukaryota 947 N/A 0.05 0.07 0.01 0.04 0.01 0.04 0.05 0.11 0.11

Proteobacteria 1310 218808 0.05 0.04 0.03 0.02 0.02 0.14 0.11 0.05

Eukaryota 412 N/A 0.05 0.21 0.10 0.11 0.28 0.09 0.12 0.07

Chlamydiae 96 152689 0.05 0.17 0.12 0.06 2.61 1.39 0.83 0.16 0.04 0.04

Proteobacteria 4299 152915 0.04 0.01 0.01 0.02

Proteobacteria 6758 171887 0.04 0.01 0.06 0.05

Proteobacteria 770 6721 0.04 0.01 0.03 0.01 0.02

Bacteroidetes 11726 141078 0.04 0.04 0.03 0.02 0.04 0.03 0.02

Chlamydiae 419 246786 0.04 0.11 0.10 0.09 0.20 0.82 0.69 0.04 0.03

Bacteroidetes 1628 197686 0.04 0.04 0.05 0.02 0.03 0.02 0.03

Gemmatimonadetes 1796 142105 0.04 0.02 0.06 0.07 0.01 0.01 0.07 0.12

Eukaryota 777 N/A 0.04 0.24 0.13 0.09 0.01 0.04 0.08 0.18 0.08

Actinobacteria 4807 369307 0.04 0.01 0.03 0.03

Proteobacteria 2207 155206 0.04 0.04 0.12 0.05 0.01 0.01 0.04 0.11 0.06

Eukaryota 572 N/A 0.04 0.39 0.16 0.05 0.03 0.04 0.02 0.07 0.04 0.08

Chloroflexi 559 131339 0.04 0.16 0.15 0.08 0.02 0.03 0.01 0.04 0.14 0.12

Bacteroidetes 1507 360101 0.03 0.05 0.02 0.04 0.03 0.01 0.02 0.04 0.02

Proteobacteria 3708 310427 0.03 0.02 0.01 0.01 0.07 0.04 0.02

Eukaryota 5095 N/A 0.03 0.01 0.01

Chloroflexi 1694 44260 0.03 0.04 0.03 0.07 0.06 0.16 0.07

Bacteroidetes 1273 112959 0.03 0.15 0.07 0.05 0.03 0.01 0.07 0.04

Planctomycetes 13316 210997 0.03 0.01 0.05 0.01

Bacteroidetes 852 279186 0.03 0.04 0.11 0.05 0.04 0.01 0.02 0.04 0.05

Bacteroidetes 1099 207314 0.03 0.10 0.11 0.09 0.10 0.08 0.04 0.05

Eukaryota 555 N/A 0.03 0.10 0.16 0.10 0.02 0.04 0.04 0.04 0.13 0.16

Bacteroidetes 679 141750 0.03 0.14 0.13 0.09 0.04 0.02 0.01 0.08 0.04 0.06

Bacteroidetes 933 254621 0.03 0.07 0.09 0.07 0.02 0.01 0.02 0.08 0.01

Proteobacteria 1281 134341 0.03 0.02 0.03 0.04 0.01 0.07 0.10

Chloroflexi 592 216017 0.03 0.35 0.22 0.18 0.02 0.07 0.05 0.11 0.07

Bacteroidetes 1374 68384 0.03 0.04 0.05 0.08 0.01 0.01 0.06 0.07 0.02

OP11 1599 201040 0.03 0.07 0.06 0.05 0.09 0.01 0.05 0.03

Eukaryota 2160 N/A 0.03 0.03 0.06 0.03 0.03 0.02 0.08 0.05 0.06

Proteobacteria 7400 135928 0.03 0.01 0.03

Eukaryota 3049 N/A 0.02 0.08 0.04 0.04 0.02 0.04

Bacteroidetes 1186 108542 0.02 0.09 0.04 0.06 0.02 0.07

Eukaryota 711 N/A 0.02 0.06 0.11 0.05 0.03 0.03 0.03 0.09 0.09 0.06

Proteobacteria 774 61110 0.02 0.21 0.10 0.11 0.04 0.14 0.09 0.04 0.01

Cyanobacteria 1977 46527 0.02 0.05 0.01 0.01 0.12 0.04 0.04

Firmicutes 4767 361257 0.02 0.03 0.04 0.07

Chloroflexi 3236 251145 0.02 0.02 0.01 0.04 0.01 0.04

Bacteroidetes 566 249818 0.02 0.10 0.18 0.08 0.06 0.05 0.02 0.05 0.03

Eukaryota 758 N/A 0.02 0.17 0.10 0.03 0.02 0.07 0.04 0.03


Supplementary Table 4. MDA-skewing of OTUs relative to their controls in three activated sludge samples taken from a bioreactor

at different time points: AS1 (7/3/2007), AS2 (7/27/2007) and AS3 (10/1/2007). Significantly over- and under-represented OTUs are

highlighted in red and green respectively. OTUs skewed in different directions using the same protocol are highlighted by black

boxes. Values indicate relative (%) abundance of each OTU within a given treatment before (control) and after amplification. Missing

values indicate that OTUs were below detection in a given sample. Phylogenetic affiliation and closest match in the greengenes

database (http://greengenes.lbl.gov/), where available, are also shown for each OTU. N/A not available.

OTU relative abundance (%)

Classification O

TU

ID

Closest

reference

sequence

(Greengenes

ID)

Control (10ng)

GenomiPhi (10ng)

NEB (10ng)

RepliG (10ng)

AS1 AS2 AS3 AS1 AS2 AS3 AS1 AS2 AS3 AS1 AS2 AS3

Bacteroidetes 1 335772 25.63 11.67 5.07 61.70 45.78 18.62 57.00 53.04 30.52 37.28 22.27 3.70

Proteobacteria 10 239835 2.06 1.55 7.88 0.32 0.85 6.55 0.22 0.25 1.55 1.25 1.04 9.37

Bacteroidetes 11 306824 0.11 0.41 1.12 0.38 1.62 5.18 0.31 1.84 5.71 0.19 0.48 1.42

Bacteroidetes 119 368794 0.05 0.04 0.01 0.12 0.05 0.48 0.30 0.25 0.10 0.03

Eukaryota 12 N/A 0.03 10.50 8.47 1.00 1.19 0.01 0.19 0.19 0.03 2.97 1.10

Proteobacteria 128 69403 0.15 0.39 0.15 0.17

Proteobacteria 1287 240984 0.02 0.01 0.00 0.01 0.02

Proteobacteria 13 26769 3.47 0.82 1.41 5.00 1.62 2.77 1.84 0.49 0.56 2.90 0.84 0.62

Proteobacteria 14 345752 5.12 4.00 3.27 0.80 1.35 1.61 0.41 0.35 0.31 4.43 3.93 4.93

Bacteroidetes 15 152338 0.92 0.52 0.60 0.88 1.01 2.01 1.16 0.97 2.10 0.43 0.55 0.50

Proteobacteria 16 359351 1.37 1.80 1.38 0.49 1.07 1.31 0.31 0.32 0.37 1.90 1.35 2.43

Bacteroidetes 168 43204 0.09 0.02 0.06 0.15 0.13 0.22 0.03 0.01 0.03 0.02 0.05 0.04

Bacteroidetes 17 255558 0.30 1.01 2.39 0.03 0.21 0.73 0.02 0.08 0.27 0.36 0.75 4.30

Proteobacteria 18 60760 0.53 1.07 1.47 0.50 1.42 3.34 0.14 0.34 0.36 0.43 0.83 1.27

Proteobacteria 19 252003 0.38 0.41 0.01 1.75 1.86 0.02 1.45 1.36 0.72 0.21

Bacteroidetes 199 248514 0.04 0.11 0.02 0.28 0.02 0.03

Bacteroidetes 1992 335772 0.04 0.01 0.04 0.02 0.04 0.03 0.02 0.02 0.02 0.11 0.03 0.02

Bacteroidetes 2 368794 14.99 2.82 4.47 18.03 6.05 11.20 30.91 15.97 32.46 10.44 2.86 1.81

Bacteroidetes 201 146568 0.08 0.03 0.19 0.07 0.40 0.16 0.12 0.02

Actinobacteria 2053 359997 0.03 0.13 0.15 0.06 0.02 0.03 0.02 0.02 0.12 0.36

Proteobacteria 21 355294 0.47 0.27 0.54 0.12 0.12 0.39 0.08 0.03 0.16 1.08 0.32 2.71

Actinobacteria 218 20188 0.28 0.08 0.03 0.15

Eukaryota 229 N/A 0.07 0.07 0.01 0.05 0.14 0.04 0.06 0.01 0.02 0.08

Proteobacteria 23 278055 1.33 0.48 0.39 0.19 0.22 0.29 0.11 0.07 0.06 1.07 0.61 1.25

Eukaryota 235 N/A 0.01 0.06 0.80 0.27 0.02 0.02 0.27

Proteobacteria 236 307247 0.10 0.05 0.06 0.04 0.01 0.05 0.01 0.01 0.02 0.13 0.01 0.10

Proteobacteria 24 306864 0.01 0.59 1.02 0.01 0.42 1.00 0.01 0.08 0.17 0.02 0.76 1.98

Bacteroidetes 2425 335772 0.02 0.01 0.06 0.05 0.02 0.02 0.04 0.01 0.02 0.03


Supplementary Table 4 (continued) OTU relative abundance (%)

Classification

OT

U I

D Closest

reference

sequence

(Greengenes

ID)

Control (10ng)

GenomiPhi (10ng)

NEB (10ng)

RepliG (10ng)

AS1 AS2 AS3 AS1 AS2 AS3 AS1 AS2 AS3 AS1 AS2 AS3

Proteobacteria 253 177268 0.15 0.04 0.04 0.04 0.01 0.02 0.01 0.04 0.03

Proteobacteria 2534 248590 0.01 0.01 0.02 0.04 0.02 0.05 0.09

Proteobacteria 2577 364924 0.28 0.03 0.08 0.04 0.02 0.03 0.01 0.01 0.33 0.10 0.05

Bacteroidetes 26 249252 0.07 0.14 0.16 0.12 0.25 0.44 0.27 0.69 1.11 0.05 0.04 0.08

Planctomycetes 266 2882 0.10 0.10 0.03 0.01 0.01 0.07 0.24

Bacteroidetes 267 63191 0.04 0.12 0.01 0.26 0.04

Proteobacteria 28 141416 0.67 1.56 0.24 0.12 0.59 0.19 0.11 0.22 0.05 0.93 1.14 0.52

Bacteroidetes 290 110915 0.04 0.01 0.08 0.06 0.07 0.34 0.19 0.33 0.07

Bacteroidetes 2974 335772 0.05 0.02 0.03 0.02 0.01 0.01 0.01 0.08 0.02

Proteobacteria 3 257084 4.75 31.54 20.40 0.83 12.61 12.21 0.65 4.52 3.48 4.51 23.45 25.51

Bacteroidetes 31 139593 0.02 0.43 0.04 1.20 0.06 1.35 0.02 0.33

Actinobacteria 3204 26533 0.07 0.01 0.01 0.01 0.02 0.07 0.04

Proteobacteria 33 105875 1.09 0.23 0.27 0.20 0.04 0.13 0.13 0.02 0.03 1.00 0.15 0.50

Bacteroidetes 34 277886 0.51 1.00 0.04 1.02 0.02 0.51 0.01 0.01

Proteobacteria 35 358750 0.65 0.01 1.26 0.92 0.62

Proteobacteria 38 106354 0.01 0.24 0.05 0.60 0.04 0.88 0.02 0.16

Proteobacteria 3867 257084 0.06 0.01 0.04 0.01

Proteobacteria 3875 241442 0.06 0.01 0.02 0.01

Proteobacteria 4 241442 27.79 0.10 2.14 5.99 0.09 0.05 2.21 0.03 0.03 22.32 0.06 0.24

Bacteroidetes 40 65426 0.35 0.05 0.97 0.06 1.51 0.20 0.43 0.02

Actinobacteria 43 47074 0.14 0.65 0.15 0.06 0.05 0.01 0.01 0.00 0.05 0.69 0.59

Planctomycetes 47 112330 0.01 0.31 0.09 0.03 0.03 1.30

Proteobacteria 5 344892 3.39 1.75 6.14 0.76 1.02 5.85 0.44 0.30 1.31 4.11 2.94 17.36

Bacteroidetes 52 82223 0.03 0.03 0.04 0.06 0.05 0.15 0.15 0.20 0.36 0.04 0.01

Firmicutes 538 141405 0.02 0.02 0.17 0.03

Proteobacteria 543 35018 0.06 0.02 0.04 0.02 0.15

Bacteroidetes 55 2596 0.01 0.01 0.15 0.02 0.31 0.02 0.03 0.59 0.09

Bacteroidetes 612 170995 0.01 0.01 0.06 0.12 0.05

Bacteroidetes 62 199804 0.08 0.05 0.08 0.12 0.11 0.18 0.20 0.11 0.16 0.07 0.04 0.02

Bacteroidetes 64 23809 0.06 0.15 0.15 0.01 0.73 0.05 0.02 0.13 0.04 0.02

Bacteroidetes 6857 335772 0.02 0.04 0.03 0.02 0.03 0.02 0.01 0.02 0.02

Actinobacteria 7 26533 0.84 16.96 1.85 0.18 9.21 1.12 0.08 2.39 0.27 0.98 22.46 5.06

Proteobacteria 8 32355 0.79 3.49 2.35 11.88 0.01 2.63 8.61 1.00 1.66

Actinobacteria 81 213279 0.02 0.13 0.14 0.02 0.07 0.58

Proteobacteria 9 72759 1.82 2.29 20.36 0.39 0.83 2.79 0.17 0.23 0.69 1.24 1.53 4.47

Bacteroidetes 97 362321 0.01 0.94 0.42 0.01 2.28 1.06 0.03 5.10 2.21 0.03 1.26 0.10

Proteobacteria 980 130376 0.01 0.01 0.01 0.04 0.03


SUPPLEMENTARY METHODS

Environmental sample DNA. Three 250 ml samples of activated sludge were collected

from a sequencing batch reactor performing enhanced biological phosphorus removal at

the different time points. Cells were pelleted by centrifugation at 5000 ! g for 10 min at

4°C, and the pellet was resuspended in 50 ml of 10% glycerol in phosphate buffered

saline (PBS, pH 7.4), and stored at -80°C until further processing. Termite hindgut

biomass was obtained from 50 frozen Nasutitermes corniger workers from a laboratory-

reared colony. For each worker, a hemi-transverse incision of the P3 hindgut

compartment was made with a sterile needle, and 2 µL of 100 mM PBS was mixed with

luminal contents squeezed out of the P3 compartment. P3 samples were pooled and

stored at -80˚C until DNA isolation. Genomic DNA from the sludge and termite hindgut

samples was extracted as previously described1. A 50 g sample of fresh garden compost

was collected from a suburban dwelling in San Francisco, CA, and DNA was extracted

using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA) according to

manufacturers instructions. Purified genomic DNA samples from the three environmental

sources were quantified using a Qubit fluorometer and Quant-iT dsDNA HS assay kit

(Invitrogen, Carlsbad, CA).

Multiple displacement amplification (MDA). Extracted genomic DNA from the three

environmental samples was amplified by multiple displacement amplification using the

Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Piscataway, NJ), REPLI-

g UltraFast Mini kit (Qiagen, Valencia, CA), and phi29 DNA polymerase (NEB, Ipswich,

MA). Amplifications were performed in triplicate using three different template

concentrations (1, 10, and 100 ng) for each MDA protocol. Control DNA provided with

the GenomiPhi kit and water blanks were used as positive and negative controls

respectively in each set of amplifications. All experiments were conducted with a

dedicated set of pipettors. Unopened pipette tips, tubes, tube caps, all other plasticware

used in experiments were treated with UV for 10 min in a Stratalinker (Stratagene, model

no. 1800) to minimize risk of contaminating DNA. In addition, buffers prepared for

MDA with the NEB phi29 DNA polymerase were UV-treated on ice for 10 min.

Amplifications with GenomiPhi and REPLI-g were performed at 30˚C for 1.5 hrs


according to manufacturer’s instructions. For amplifications with NEB phi29, 1 !L of

alkaline lysis buffer (400 mM KOH, 10mM EDTA, 100mM DTT) was added to 1 !L of

genomic DNA (1, 10 or 100 ng/!L dilutions) and incubated at room temperature for 15

min to denature the template DNA. The amplification reaction was performed in a 20 !L

volume containing 1X reaction buffer (40 mM Tris-Cl (pH7.5), 50 mM KCl, 10 mM

MgCl2, 5 mM (NH4)2SO4, 2 mM DTT), 0.4 mM dNTPs, 50 mM random hexamers with

two 3’ phosphorothioate bonds, and 8 U of phi29 DNA polymerase (NEB, Ipswich,

MA). The reaction was performed at 30˚C for 16 hrs, and the enzyme was inactivated by

incubation at 65˚C for 10 min.

SSU rRNA gene amplicon pyrosequencing. MDA-amplified and unamplified control

DNA samples were diluted to 10 ng/!L in nuclease free water. A ~450 bp fragment of

the SSU rRNA gene was PCR-amplified from 10 ng using the broadly conserved primer

pair 926F-1392R as described previously2. Barcoded amplicons were mixed in equal

proportions prior to emulsion PCR and sequenced on the Roche 454 GS FLX Titanium

platform. The *.sff files from SSU rRNA gene amplicon pyrosequencing have been

deposited in the GenBank Short Read Archive under submission number SRA012873.

Data analysis. Amplicon reads were analyzed with PyroTagger (http://pyrotagger.jgi-

psf.org) using LUCY end-trimming, a sequence length threshold of 220 bp and 97%

identity OTUs. Quality trimmed data consisted of a total of 801,000 sequence tags with

an average number of 6,400 (±3,400) sequences per sample ranging from 1,129 to 19,183

tags (for details see Supplementary Fig. 2). To reduce noise in statistical analysis, rare

OTUs were removed prior to analysis by eliminating all OTUs that were present in only

one of the three replicate samples. Experimental reproducibility was tested by Pearson’s

correlation between technical replicates. Similarly, simple linear regression analyses were

used to estimate biases introduced by MDA relative to the controls. Both analyses were

performed using the StatPlus package version 5.7.0 (AnalystSoft Inc., Vancouver,

Canada). To identify significantly skewed OTUs, student t-tests were performed. OTUs

that were undetectable in either the control or amplified samples were removed from the

analysis. Genomic GC content of a given OTU was estimated by finding the closest


genome-sequenced relative (BLASTn against the NCBI Genomes database) and using the

genomic GC content of the highest match as a proxy value for the OTU. Proxy OTU GC

contents were then plotted against fold skewing to detect any correlations.

1. Purkhold, U. et al. Appl. Environ. Microbiol. 66 (12), 5368-5382 (2000).

2. Kunin, V., Engelbrektson, A., Ochman, H. & Hugenholtz, P. Environ. Microbiol. 12

(1), 118-123 (2010).


Documents

0)+%*-12€¦ · Supplementary Figure 1. Richness (rarefaction curves) (a) and evenness (rank abundance curves) (b) of the three test communities (unamplified controls) as determined