Advanced pipeline provides the same pre-processing steps and OTUs identification of the Qiime-based closed-reference analysis. Qiime 1.9.1 provides the alpha- and beta-diversity analysis, building rarefaction plots, distance matrices, UPGMA trees, 3D PCoA plots and providing statistical analysis of experimental group comparison. A partial example of the output is available here.
Advanced analysis is performed using the Qiime core_diversity_analysis with default parameters.
Multivariate analysis, including the ANOSIM and Adonis tests, is done running Qiime compare_categories. ANOSIM is a nonparametric statistical test that compares ranked beta-diversity distances between different groups and calculates a p-value and a correlation coefficient by permutation. Adonis multivariate analysis is instead included into ANOVA tests and verifies if variation within a category is smaller or greater than variation between categories. It calculates a pseudo F-value, a p-value, and a correlation coefficient (R^2). Significant p-values must be interpreted together with their R2 values to infer biological meanings from the results. Analyses are performed among the required groups and on different metrics: weighted and unweighted.
MatagenomeSeq is used to calculate specie differential abundance between experimental groups. It is a R package, implemented also in Qiime 1.9.1, performing:
- OTU table normalization
- Fisher’s test to evaluate the significance of the comparison
- Level of OTU-category correlation and its significance tests with p-value correction through False Rate Discovery (FDR).
To perform the Advanced Analysis, we need to know the experimental parameter for sample clustering in order to make statistical group comparison. The treatment column should provide at least 2 groups containing at least 5 samples. One table (mapping file) is considered as a single Advanced Analysis.
The following table is an example of what the customer should provide us by e-mail before sample shipment.
Using this mapping file, a pairwise analysis is performed among samples of the four groups: CTRL (88078-88082), HI10 (88083-88088), HI20 (88089-88094) and HI30 (88095-88098).
More information about Qiime 1.9.1 beta-diversity analysis is available here.