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.

核糖体分析可以对核糖体结合的 RNA 片段进行测序，揭示正在翻译的转录本以及核糖体在 mRNA 上的位置。尽管核糖体分析已应用于培养的细菌分离物，但将其应用于未培养的混合群落一直具有挑战性。我们介绍了 MetaRibo-Seq，这是一种能够将核糖体分析直接应用于人类粪便微生物组的协议。MetaRibo-Seq 是一种基准方法，包括对现有核糖体分析协议的多项修改，专门解决涉及粪便样本存储、纯度和输入要求的挑战。我们还提供用于质量控制和修剪读取的计算工作流程，从头组装具有宏基因组读取的参考宏基因组，将 MetaRibo-Seq 读取与参考对齐，并评估 MetaRibo-Seq 库质量 (https://github.com/bhattlab/bhattlab_workflows/tree/master/metariboseq)。这种 MetaRibo-Seq 协议使标准分子生物学实验室的研究人员能够在 ~5 天内研究粪便微生物组中的翻译。