The high background of host RNA poses a major challenge to metatranscriptome analysis of human samples. Hence, metatranscriptomics has been mainly applied to microbe-rich samples, while its application in human tissues with low ratio of microbial to host cells has yet to be explored. Since there is no computational workflow specifically designed for the taxonomic and functional analysis of this type of samples, we propose an effective metatranscriptomics strategy to accurately characterize the microbiome in human tissues with a low ratio of microbial to host content. We experimentally generated synthetic samples with well-characterized bacterial and host cell compositions, and mimicking human samples with high and low microbial loads. These synthetic samples were used for optimizing and establishing the workflow in a controlled setting. Our results show that the integration of the taxonomic analysis of optimized Kraken 2/Bracken with the functional analysis of HUMAnN 3 in samples with low microbial content, enables the accurate identification of a large number of microbial species with a low false-positive rate, while improving the detection of microbial functions. The effectiveness of our metatranscriptomics workflow was demonstrated in synthetic samples, simulated datasets, and most importantly, human gastric tissue specimens, thus providing a proof of concept for its applicability on mucosal tissues of the gastrointestinal tract. The use of an accurate and reliable metatranscriptomics approach for human tissues with low microbial content will expand our understanding of the functional activity of the mucosal microbiome, uncovering critical interactions between the microbiome and the host in health and disease.
Keywords: Microbiome; bacteria; computational biology; low microbial biomass; metatranscriptomics; mock bacterial community; mucosal tissues.