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Table 2 Differential abundance methods investigated in this study

From: Normalization and microbial differential abundance strategies depend upon data characteristics

Method Description
Wilcoxon rank-sum test Also called the Mann-Whitney U test. A non-parametric rank test, which is used on the un-normalized (“None”), proportion normalized, and rarefied matrices
DESeq nbinom Test—a negative binomial model conditioned test. More conservative shrinkage estimates compared to DESeq2, resulting in stricter type I error control
DESeq2 nbinomWald Test—The negative binomial GLM is used to obtain maximum likelihood estimates for an OTU’s log-fold change between two conditions. Then Bayesian shrinkage, using a zero-centered normal distribution as a prior, is used to shrink the log-fold change towards zero for those OTUs of lower mean count and/or with higher dispersion in their count distribution. These shrunken long fold changes are then used with the Wald test for significance
edgeR exact Test—The same normalization method (in R, method = RLE) as DESeq is utilized, and for differential abundance testing also assumes the NB model. The main difference is in the estimation of the dispersion, or variance, term. DESeq estimates a higher variance than edgeR, making it more conservative in calling differentially expressed OTUs
Voom Variance modeling at the observational level—library sizes are scaled using the edgeR log counts per million (cpm) normalization factors. Then LOWESS (locally weighted regression) is applied to incorporate the mean-variance trend into precision weights for each OTU
metagenomeSeq fitZIG—a zero-inflated Gaussian (ZIG) where the count distribution is modeled as a mixture of two distributions: a point mass at zero and a normal distribution. Since OTUs are usually sparse, the zero counts are modeled with the former, and the rest of the log transformed counts are modeled as the latter distribution. The parameters for the mixture model are estimated with an expectation-maximization algorithm, which is coupled with a moderated t statistic
fitFeatureModel—a feature-specific zero-inflated lognormal model with empirical Bayes shrinkage of parameter estimates
ANCOM Analysis of composition of microbiomes—compares the log ratio of the abundance of each taxon to the abundance of all the remaining taxa one at a time. The Mann-Whitney U is then calculated on each log ratio