1 SUPPLEMENTARY FIGURES 1 SUPPLEMENTARY FIGURE LEGENDS 2 Supplementary Figure 1. A) The microbial DNA is arranged into an absolute coordinate 3 space based on their multimer-frequencies. The user defined target and non-target groups are 4 shown in green and red, respectively. B) The program suggests the appropriate probes based 5 on user defined parameters in the preceding steps. Each probe’s hybridization area to the 6 target bacteria is shown and the probe sequence is colored according to its direction 7 (sense/antisense = green/red). C) The colors in the cross-labeling graph indicate the pairwise 8 compatibility of the relevant probes. The blue color between two probes means that these are 9 compatible and will not cause problems for each others. Red means that there is high 10 probability that they will hybridize to each other’s target bacteria. 11 12 Supplementary Figure 2. Evaluation of the 16S rRNA gene PCR reaction reproducibility. 13 Three replicate 16S rRNA gene PCR reactions were performed on one of the infant fecal 14 samples. To examine the signal from certain amplified target bacteria in the sample the 15 probes, 6_1_4 and 5_1_2 were chosen. The mean signal for the three individual PCR 16 products and the mean signal for a pool of the three PCR reactions in triplicate is presented. 17 The standard deviation of the triplicates and the three individual PCR reactions is indicated 18 for each mean value. The mean signal for the three individual PCR products and a pool of the 19 three PCR products were very similar and with the same or lower standard deviation 20 compared to the pooled samples. 21 22 Supplementary Figure 3. Pictures of two of the arrays illustrating the different bacterial 23 profiles of two individuals. The difference in signal for probe 6_1_4 (specific for 24
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2 SUPPLEMENTARY FIGURE LEGENDS · 7/25/2011 · 13 Supplementary Figure 2. Evaluation of the 16S rRNA gene PCR reaction reproducibility. 14 Three replicate 16S rRNA gene PCR reactions
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1
SUPPLEMENTARY FIGURES 1
SUPPLEMENTARY FIGURE LEGENDS 2
Supplementary Figure 1. A) The microbial DNA is arranged into an absolute coordinate 3
space based on their multimer-frequencies. The user defined target and non-target groups are 4
shown in green and red, respectively. B) The program suggests the appropriate probes based 5
on user defined parameters in the preceding steps. Each probe’s hybridization area to the 6
target bacteria is shown and the probe sequence is colored according to its direction 7
(sense/antisense = green/red). C) The colors in the cross-labeling graph indicate the pairwise 8
compatibility of the relevant probes. The blue color between two probes means that these are 9
compatible and will not cause problems for each others. Red means that there is high 10
probability that they will hybridize to each other’s target bacteria. 11
12
Supplementary Figure 2. Evaluation of the 16S rRNA gene PCR reaction reproducibility. 13
Three replicate 16S rRNA gene PCR reactions were performed on one of the infant fecal 14
samples. To examine the signal from certain amplified target bacteria in the sample the 15
probes, 6_1_4 and 5_1_2 were chosen. The mean signal for the three individual PCR 16
products and the mean signal for a pool of the three PCR reactions in triplicate is presented. 17
The standard deviation of the triplicates and the three individual PCR reactions is indicated 18
for each mean value. The mean signal for the three individual PCR products and a pool of the 19
three PCR products were very similar and with the same or lower standard deviation 20
compared to the pooled samples. 21
22
Supplementary Figure 3. Pictures of two of the arrays illustrating the different bacterial 23
profiles of two individuals. The difference in signal for probe 6_1_4 (specific for 24
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Bifidobacterium longum) is illustrated with white circles. Array A shows the profile for an 25
IgE sensitized one year old individual, while array B shows the profile of an IgE non-26
sensitized one year old individual. 27
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Supplementary Figure 4. Evaluation of the quantification of specific targets in constant 29
background of non-targets. In all the experiments the total amount of templates were kept 30
constant at 100 ng in the labeling reaction, while the targets for the illustrated probes were 31
diluted. Error bars represent standard deviations. 32
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Supplementary Figure 1 34
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A)
C) b)
g
ur
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Supplementary Figure 2 38
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Supplementary Figure 3 40
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Supplementary Figure 4 43
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SUPPLEMENTARY TABLES 45
Supplementary Table 1. Probe reproducibility based on 43 sample replica. 46
Probe ID
Mean %
variation R2 1
1_1 20 % 0.98
1_3_3 31 % 0.97
2_1_1 31 % 0.91
2_1_min1b 22 % 0.95
2_3_2 28 % 0.86
2_4_1 16 % 0.94
2_5_1 19 % 0.88
2_7_1 16 % 0.98
3_2 26 % 0.91
4_1 18 % 0.94
4_3_1 20 % 0.96
4_4_2 37 % 0.95
4_8_1 17 % 0.98
5_1 16 % 0.95
5_1_2 20 % 0.97
6_1_4 27 % 0.74
6_2 15 % 0.89
6_2_2 22 % 0.85
Univ 16S 12 % 0.77
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1 R
2 represents the squared regression coefficient. 48
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Supplementary Table 2. Ten probe set suggestions. 52
Theoretical bacterial target group Probe set 1 Probe set 2 Probe set 3 Probe set 4 Probe set 5 Probe set 6 Probe set 7 Probe set8 Probe set 9 Probe set 10