Good Morning:

I’m having some problems interpreting the results of my study. I have studied the colonization of microorganisms on different substrates at the exit of two treatment plants. The whole process has gone well, until I have come to analyze beta diversity. For this I have obtained the distance_bray_curtis_matrix by:

qiime diversity core-metrics-phylogenetic

–i-phylogeny Cantoblanco/tree/cantoblancorooted-tree.qza

–i-table Cantoblanco/dada2/cantoblancotable-dada2.qza

–p-sampling-depth 70940

–m-metadata-file Cantoblanco2019-sample-metadata.tsv

–output-dir Cantoblanco/core-metrics-results3

Obtaining this graphic representation:

bray_curtis_emperor.qzv (798.0 KB)

Looking at the data, I have seen that two large groups can be distinguished depending on the place of sampling (“Place” in metadata). Taking this into account, I have resorted to making a PERMANOVA having the place as reference, by means of the following command:

qiime diversity beta-group-significance

–i-distance-matrix Cantoblanco/core-metrics-results3/bray_curtis_distance_matrix.qza

–m-metadata-file Cantoblanco2019-sample-metadata.tsv

–m-metadata-column Place

–p-method permanova

–p-pairwise

–p-permutations 9999

–o-visualization Cantoblanco/betabray/betabraypairwise

Obtaining this output:

betabraypairwisePlace.qzv (323.7 KB)

That shows that yes, the place is a significant factor. Then, I wanted to check for pairs if the same substrate, being of different place, is statistically different (logical seeing the previously obtained). Each substrate had three replicas per place to be statistically significant (I attached the command and the file):

qiime diversity beta-group-significance

–i-distance-matrix Cantoblanco/core-metrics-results3/bray_curtis_distance_matrix.qza

–m-metadata-file Cantoblanco2019-sample-metadata.tsv

–m-metadata-column Sample1

–p-method permanova

–p-pairwise --p-permutations 9999

–o-visualization Cantoblanco/betabray/betabraypairwisesample1.qzv

And I obtained that output:

betabraypairwisesample1.qzv (1.5 MB)

But I get it, although the upper graphs indicate otherwise. With everything explained I have several questions. First of all, if I am using this command correctly and correctly understanding the results, or there is a better statistical method. Also, what exactly represent the graphics? What does the value of N means and why does it oscillate between samples if the groups always consist of three replicas? Why, if I compare the groups in pairs, there are no differences and if I compare them by place if, when the graphs, those big differences are appreciated?

Thanks in advance:

Sergio