Clc microbial genomics module v 2

Operational taxonomic unit (OTU) clustering and taxonomic analyses were performed using CLC Genomics Workbench V.10.1.1 and CLC Microbial Genomics Module V.2.5 (Qiagen). Sequences were first trimmed to remove 13 bases at the 5′ terminal position and merged considering the alignment scores as follows: mismatch cost of 2, gap cost of 2, zero maximum unaligned end mismatches and minimum score of 30.
After merging, sequences were clustered into OTUs at 97% sequence similarity level. The most abundant sequences were selected as representative of each cluster and then assigned to a taxonomy level using default values and the Greengenes Database 2013 release. Low depth samples (less than 2000 sequences per sample) were removed from the analysis. Alpha diversity indexes (Simpson, Shannon and total OTU number) were calculated. Bray-Curtis and unweighted UniFrac metric were used to calculate intersample diversity (beta diversity).
Samples were rarefied using QIIME 1.9, and multiple comparisons and statistical analyses were performed using CLC Genomics Workbench V.10.1.1 and CLC Microbial Genomics Module V.2.5 (Qiagen). A Negative Binomial GLM model was used to obtain maximum likelihood estimates for an OTU’s log-fold change between two conditions, and the Wald test was used to determine significance. False discovery rate (FDR) was performed to correct p values.

Predictions for significant differentially abundant OTUs between different groups were performed using a rarefied OTU table. Multiple comparisons, metabolic pathway analysis, and statistical analyses were performed using CLC Microbial Genomics Module v.2.5 (Qiagen) and MicrobiomeAnalyst.^{32 (link),33 (link)} The OTU table was rarefied to the minimal number of reads assigned to a sample, and a Negative Binomial GLM model was used to obtain maximum likelihood estimates for the fold change (FC) of an OTU between different groups. The Wald test was used for determination of significance, and P values were corrected using False Discovery Rate. A P value < 0.05 after controlling for False Discovery Rate of 0.05 was considered to be statistically significant. Two-tailed Spearman r correlations, statistical tests and graph construction were carried out with rarefied OTU tables using GraphPad Prism 7.02 (GraphPad Software, La Jolla, CA)^{34 (link)} and IBM SPSS Statistics Version 24. Mann–Whitney test and unpaired t test were used to compare levels of serum biomarkers between different groups. Kruskal-Wallis test was used to compared microbial diversity across all three groups. Microbial Dysbiosis Index (MD-Index), an index to measure dysbiosis (ie, the imbalance in a microbial community), was calculated during the Correlation Network analysis using MicrobiomeAnalyst.