Supplementary Information s4

Supplementary information

To test whether sample sizes per patient were sufficiently large enough to compare enough of the bacterial diversity in the CF patient airways, we used a randomised re-sampling method using two indices of diversity (Shannon-Wiener index [H’] and Taxa richness [S*]). In essence, the diversity indices of the taxa for the taxa sampled from the 14 patients (Figure S1 – panels P1 – P14) were compared and assessed as to whether samples were large enough to calculate valid indexes (i.e. where values do not increase substantially when larger samples are taken). From each sample, 1000 random sub-samples (e.g. 5, 10, 15, 20, 25, 30, etc) up to the maximum size of the sample were taken for each measure of diversity (H’ and S*) and the sub-samples were subsequently calculated and compared. The mean index values were plotted against sample size (Figure S1). This illustrated that the diversity indices values for the bacterial taxa sampled from all CF lung samples became asymptotic or approached asymptotic status, providing stable estimates of H’ and S*. This allowed confirmation that sufficiently large samples had been collected and bacterial diversity was not under sampled. This is reflective of the low diversity in CF lung systems which favour growth of a much narrower range of bacterial taxa than is found in other habitats such as soil. As a comparison we also analysed clone libraries generated (n = 236 clones for each soil) from two soil samples (pristine and zinc contaminated) (Hill et al., 2003) and found as expected that they did not reach an asymptote and were therefore under sampled (Figure S1 - panels S1 & S2).

Hill TCJ, Walsh KA, Harris JA, Moffett BF (2003). Using ecological diversity measures with bacterial communities. FEMS Microbiol Ecol 43: 1-11. A P1 P2 P3 P4 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 P5 P6 P7 P8 80 0 50 100 150 200 25080 0 50 100 150 200 25080 0 50 100 150 200 25080 0 50 100 150 200 250 60 60 60 60

s 40 40 40 40 s

e 20 20 20 20 n

h 0 0 0 0 c i r 0 P950 100 150 200 250 0 P1050 100 150 200 250 0 P1150 100 150 200 250 0 P1250 100 150 200 250 a 80 80 80 80 x

a 60 60 60 60 T 40 40 40 40 20 20 20 20 0 0 0 0 P13 P14 S1 S2 80 0 50 100 150 200 25080 0 50 100 150 200 25080 0 50 100 150 200 25080 0 50 100 150 200 250 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 B 4.0 4.0 4.0 4.0 3.0 3.0 3.0 3.0 2.0 2.0 2.0 2.0 1.0 P11.0 P21.0 P31.0 P4 0.0 0.0 0.0 0.0 4.0 0 50 100 150 200 4.0250 0 50 100 150 200 4.0250 0 50 100 150 200 2504.0 0 50 100 150 200 250

x 3.0 3.0 3.0

e 3.0 d

n 2.0 2.0 2.0 2.0 i

e 1.0 1.0 1.0 1.0

n P5 P6 P7 P8 e

i 0.0 0.0 0.0 0.0 W - 4.0 0 50 100 150 200 2504.0 0 50 100 150 200 4.0250 0 50 100 150 200 2504.0 0 50 100 150 200 250 n o

n 3.0 3.0 3.0 3.0 n

a 2.0 2.0 2.0 2.0 h S 1.0 P91.0 P101.0 P111.0 P12 0.0 0.0 0.0 0.0 4.0 0 50 100 150 200 2504.0 0 50 100 150 200 4.0250 0 50 100 150 200 2504.0 0 50 100 150 200 250 3.0 3.0 3.0 3.0 2.0 2.0 2.0 2.0 1.0 P131.0 P141.0 S11.0 S2 0.0 0.0 0.0 0.0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Sample size

Figure S1 Taxa richness (A) and Shannon-Wiener index of diversity (B) calculated using the average of 1000 random subsets of bacterial taxa for all patients (P1 – 14) in comparison with two soil samples

(S1 & 2). Given are the 95 % confidence limits.