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. 2012 Mar 6;7(3):e32942. doi: 10.1371/journal.pone.0032942

Figure 3. Principal component analyses (PCA) of the microbiota profiles and Principal Coordinate of Analyses (PcoA) plot of the weighted and unweighted UniFrac average distance per site and DNA extraction method.

Figure 3

a. Principal component analyses (PCA) of the microbiota profiles of the nares and nasopharynx depicted per dilution and DNA extraction method. Depicted in colors are 16S DNA levels (blue = ≥1 pg/µl, green = <1 pg/µl). Depicted in characters are the DNA extraction methods (A = Agowa, E = Epicentre, Q = qiagen, M = Mobio). Clustering of the samples is according to DNA level and DNA extraction method. Differences in template concentration were due to differences in DNA extraction efficiency between used methods and effects of template concentration on microbiota analyses were therefore fully tied to DNA extraction effects. b.PcoA plot of the weighted UniFrac. Shown in colored circles are the DNA extraction methods (yellow = Epicentre, red = Mobio, blue = Qiagen and green = Agowa). The abbreviations represent the site of sampling (NP = nasopharynx, N = nares, OP = oropharynx, SA = saliva). Clear clustering per site of sampling was observed with saliva and oropharynx distant from nares and nasopharynx samples. For the oropharynx and saliva clusters significant sub-clustering per DNA extraction method was seen with clusters of Epicentre and Mobio, distant from Agowa and Qiagen clusters. DNA extraction method in these high density sites even introduced a larger distance in microbiota profile than origin of the sample (saliva or oropharynx). c. PCoA plot of the unweighted UniFrac as described above. Clear clustering per site of sampling was observed with saliva and oropharynx distant from nares and nasopharynx samples, and also between saliva and oropharynx. For the nares and nasopharynx clusters significant sub-clustering per DNA extraction method was seen with clusters of Agowa, Qiagen and Epicentre distant from Mobio. Both weighted and unweighted UniFrac analysis of sequence data revealed distinct clustering of saliva and oropharyngeal separate from nares and nasopharynx samples, reflecting unique differences in microbiota composition between these sites (Amova, p<0.001, Figure S4 and S5).

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