However, at 3% and 5% dissimilarity the rarefaction curves approximate a parallel line to the x-axis, suggesting that a reasonable coverage was obtained at the species and genus level. Using the Richard’s Seliciclib molecular weight equation we calculated that approximately 38,000 sequences would need to be sampled to identify 100% of the expected OTUs in the canine jejunum (Figure 1B). To obtain a complete coverage at 0% dissimilarity, approx. 106,000 sequences would need to be analyzed (data not shown). Figure 1 Representative rarefaction curves depicting the effect of 1%, 3%, and 5% dissimilarity
on the number of identified and maximum predicted operative taxonomical units (OTUs) in one dog. (A) This plot shows that with the average number of collected sequencing tags per dog (mean ± SD: 3188 ± 1091 sequencing tags), we underestimated the number of OTUs at 1% dissimilarity. A reasonable coverage
was obtained at 3% and 5% dissimilarity (curves approximate a parallel line to the x-axis). (B) To estimate the maximum number of OTUs at various dissimilarities, a Richards equation was fit to the rarefaction curves. The results indicate that approximately 38,000 sequences would need to be sampled to cover 100% of the expected OTUs in the canine jejunum. Table 1 Mean values for various indices. Shannon-Weaver index OTU maximum predicted OTU 1% 3% 5% 1% 3% 5% 1% 3% 5% day 0 4.55 2.88 2.03 695 218 143 950 293 169 day 14 4.58 2.84 1.87 594 149 93 789 RG-7388 purchase 197 111 day 28 3.98 2.60 1.46 542 115 72 637 136 90 Rarefaction Chao 1 ACE 1% 3% 5% 1% 3% 5% 1% 3% 5% day 0 690 217 142 984 342 197 1030 332 191 day 14 590 148 92 794 204 123 807 209 124
day 28 539 115 72 Immune system 669 150 86 660 155 92 This table shows the Shannon-Weaver bacterial diversity index, observed operative taxonomical units (OTU), the predicted maximum number of OTUs in the canine jejunum, rarefaction, and species richness estimators (ACE and Chao 1) at strain (1% dissimilarity), species (3%), and genus (5%) level across the three sampling periods. Tylosin administration led to a progressive decrease in mean indices, which were lowest on day 28 (14 days after cessation of tylosin). However, a strong individual variation was observed among all dogs (see text). On day 0, ten different bacterial phyla were identified. The major bacterial phyla were Proteobacteria (46.7% of all sequences), Firmicutes (15.0%), Actinobacteria (11.2%), Spirochaetes (14.2%), Bacteroidetes (6.2%), and Fusobacteria (5.4%). The phyla Tenericutes, Verrucomicrobia, Cyanobacteria, and Chloroflexi accounted for < 0.1% of all obtained sequencing tags each (Figure 2). Figure 2 Distributions of major bacterial groups at the phylum level. (day 0 = baseline; day 14 = after 14 days of tylosin administration; day 28 = 2 weeks after cessation of tylosin therapy).