Importance Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain. Objective To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture. Design, Setting, and Patients In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain. Main Outcomes and Measures The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples. Results During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered. Conclusions and Relevance These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs. The outbreak of Shiga-toxigenic Escherichia coli (STEC), which struck Germany in May-June 2011, illustrated the effects of a bacterial epidemic on a wealthy, modern, industrialized society, with more than 3000 cases and more than 50 deaths.1 During an outbreak, rapid and accurate pathogen identification and characterization is essential for the management of individual cases and of an entire outbreak. Traditionally, clinical bacteriology has relied primarily on laboratory isolation of bacteria in pure culture as a prerequisite to identification and characterization of an outbreak strain. Often, however, in vitro culture proves slow, difficult, or even impossible, and recognition of an outbreak strain can be difficult if it does not belong to a known variety or species for which specific laboratory tests and diagnostic criteria already exist. For example, during the German outbreak, infection was caused by an unusual serotype (STEC O104:H4) that had not previously been seen in the context of epidemic disease and could not be detected easily with the standard microbiological methods in use at the start of the outbreak for diagnosing STEC infection. The term metagenomics is applied to the open-ended sequencing of nucleic acids recovered directly from samples without target-specific amplification or enrichment.2 A list of terms used in this article appear in the Box. Metagenomics has been used in a clinical diagnostic setting to identify the cause of outbreaks of viral infection.3 Drawing on examples from virology and on recent advances in sequencing technologies,4- 5 we sought to extend the scope of metagenomics as a clinical discovery platform, exploiting this approach to identify and characterize an outbreak-associated bacterial strain directly from clinical samples without the need for laboratory culture. We explored the potential of this approach on human fecal samples collected during the German STEC outbreak of 2011, performing high-throughput sequencing on 2 Illumina instruments (MiSeq and HiSeq 2500).