Comprehensive Evaluation of Methods for Differential Expression Analysis of Metatranscriptomics Data

Understanding the function of the human microbiome is important; however, the development of statistical methods specifically for the microbial gene expression (i.e., metatranscriptomics) is in its infancy. Many currently employed differential expression analysis methods have been designed for different data types and have not been evaluated in metatranscriptomics settings. To address this gap, we undertook a comprehensive evaluation and benchmarking of ten differential analysis methods for metatranscriptomics data. We used a combination of real and simulated data to evaluate performance (i.e., model fit, type I error, false discovery rate, and sensitivity) of the methods: log-normal (LN), logistic-beta (LB), MAST, DESeq2, metagenomeSeq, ANCOM-BC, LEfSe, ALDEx2, Kruskal-Wallis, and two-part Kruskal-Wallis. The simulation was informed by supragingival biofilm microbiome data from 300 preschool-age children enrolled in a study of early childhood caries (ECC), whereas validations were sought in two additional datasets from an ECC study and an inflammatory bowel disease (IBD) study. The LB test showed the highest sensitivity in both small and large samples and reasonably controlled type I error. Contrarily, MAST was hampered by inflated type I error. Upon application of the LN and LB tests in the ECC study, we found that genes C8PHV7 and C8PEV7, harbored by the lactate-producing Campylobacter gracilis, had the strongest association with childhood dental diseases. This comprehensive model evaluation offer practical guidance for selection of appropriate methods for rigorous analyses of differential expression in metatranscriptomics. Selection of an optimal method increases the possibility of detecting true signals while minimizing the chance of claiming false ones.