Simultaneous ribosome profiling of hundreds of microbes from the human microbiome

Ribosome profiling enables sequencing of ribosome-bound fragments of RNA, revealing which transcripts are being translated as well as the position of ribosomes along mRNAs. Although ribosome profiling has been applied to cultured bacterial isolates, its application to uncultured, mixed communities has been challenging. We present MetaRibo-Seq, a protocol that enables the application of ribosome profiling directly to the human fecal microbiome. MetaRibo-Seq is a benchmarked method that includes several modifications to existing ribosome profiling protocols, specifically addressing challenges involving fecal sample storage, purity and input requirements. We also provide a computational workflow to quality control and trim reads, de novo assemble a reference metagenome with metagenomic reads, align MetaRibo-Seq reads to the reference, and assess MetaRibo-Seq library quality ( https://github.com/bhattlab/bhattlab_workflows/tree/master/metariboseq ). This MetaRibo-Seq protocol enables researchers in standard molecular biology laboratories to study translation in the fecal microbiome in ~5 d. This protocol outlines the experimental and computational steps of MetaRibo-Seq, a modified ribosome profiling approach that allows direct interrogation of translation in uncultured bacterial communities.