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A functional readout of the gut microbiome is necessary to enable precise control of the gut microbiome’s functions, which support human health and prevent or minimize a wide range of chronic diseases. Stool metatranscriptomic analysis offers a comprehensive functional view of the gut microbiome, but despite its usefulness, it has rarely been used in clinical studies due to its complexity, cost, and bioinformatic challenges. This method has also received criticism due to potential intrasample variability, rapid changes, and RNA degradation. Here, we describe a robust and automated stool metatranscriptomic method, called Viomega, which was specifically developed for population-scale studies. Viomega includes sample collection, ambient temperature sample preservation, total RNA extraction, physical removal of ribosomal RNAs (rRNAs), preparation of directional Illumina libraries, Illumina sequencing, taxonomic classification based on a database of >110,000 microbial genomes, and quantitative microbial gene expression analysis using a database of ~100 million microbial genes. We applied this method to 10,000 human stool samples and performed several small-scale studies to demonstrate sample stability and consistency. In summary, Viomega is an inexpensive, high-throughput, automated, and accurate sample-to-result stool metatranscriptomic technology platform for large-scale studies and a wide range of applications.

A Robust Metatranscriptomic Technology for Population-Scale Studies of Diet, Gut Microbiome, and Human Health