Computational protocol: Structural Alteration of Gut Microbiota during the Amelioration of Human Type 2 Diabetes with Hyperlipidemia by Metformin and a Traditional Chinese Herbal Formula: a Multicenter, Randomized, Open Label Clinical Trial

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[…] High-quality sequencing data extraction was performed as follows (). (i) Both ends of the forward and reverse reads were truncated at the base, where the Phred quality score was no more than 20. (ii) If the forward and reverse reads had a minimum 50-bp-length overlap, they could be merged into a complete read. (iii) These reads were not kept unless they were longer than 399 bp and the expected errors were less than 0.5. Operational taxonomic units (OTUs) were delineated at the cutoff of 97% using the USEARCH v.7.0.1090 pipeline. The protocol can be found on the website http://drive5.com/usearch/manual/uparse_pipeline.html. The detailed procures were stated in our previous publication (). α- and β-diversity analyses were performed using QIIME v1.8.0 (). To normalize read depth, all samples were randomly subsampled to 5,000 reads/sample. Shannon’s index, Simpson’s index, rarefaction, and Chao1 estimates were evaluated. A normalized and log10-transformed OTU abundance table was used for the β-diversity analysis, including principal-component analysis (PCA) and principal-coordinate analysis (PCoA) based on Bray-Curtis, UniFrac, and Jaccard distances. The taxonomy of each OTU was assigned online by RDP Classifier (RDP Naive Bayesian rRNA Classifier version 2.10) with a bootstrap cutoff of 80%. [...] In ecologic systems, species with similar requirements and functions would constitute an ecologic guild to adapt to the environments. Species from the same guild were shown to be coabundant. In this study, coabundant network analysis was used to find the key phylotypes associated with the improvements in glucose and lipid homeostasis. The correlations between the core OTUs (which existed in more than 20% samples) were calculated with the SparCC algorithm (bootstrap value, 100) (). The 214 core OTUs were clustered into 24 coabundant groups (CAGs) by using the ward algorithm and PERMANOVA (999 permutations; P < 0.001) based on the SparCC correlation coefficient matrix with R version 3.1.3. The 21 CAGs with an R coefficient higher than 0.4 were visualized into a network with cytoscape v3.1.1 (http://www.cytoscape.org/). The abundance of each CAG was the sum of relative abundances of the OTUs in this CAG. […]

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