You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Metagenomic Analysis of Human Diarrhea: Viral Detection and Discovery}
Article:
Abstract:
Worldwide, approximately 1. 8 million children die from diarrhea annually, and millions more suffer multiple episodes of nonfatal diarrhea. On average, in up to 40% of cases, no etiologic agent can be identified. The advent of metagenomic sequencing has enabled systematic and unbiased characterization of microbial populations; thus, metagenomic approaches have the potential to define the spectrum of viruses, including novel viruses, present in stool during episodes of acute diarrhea. The detection of novel or unexpected viruses would then enable investigations to assess whether these agents play a causal role in human diarrhea. In this study, we characterized the eukaryotic viral communities present in diarrhea specimens from 12 children by employing a strategy of “micro-mass sequencing” that entails minimal starting sample quantity( <100 mg stool), minimal sample purification, and limited sequencing( 384 reads per sample). Using this methodology we detected known enteric viruses as well as multiple sequences from putatively novel viruses with only limited sequence similarity to viruses in GenBank.
Introduction:
While traditional sequencing approaches are designed to characterize genomes of a single species of interest, metagenomic approaches, such as mass sequencing, transcend species boundaries allowing one to explore the makeup of microbial communities. Such methods provide a holistic look at microbial diversity within a given sample, completely bypassing the need for culturing[1]–[5]. Previous efforts in this field have explored the structure of virus communities in ecosystems as diverse as the ocean[1],[6] and the human gut[7],[8]. To date, the reported metagenomic studies of human stool have been limited to analysis of 4 specimens collected from 3 healthy patients[7],[8]. To our knowledge, no metagenomic investigation of the viral diversity found in human diarrhea has previously been described. Human diarrhea is the third leading cause of infectious deaths worldwide and is responsible for ∼1. 8 million deaths in children under age five each year[9]. Bacteria, protozoa and viruses have all been implicated as causal agents. Chief among the known etiologic agents are rotaviruses, noroviruses, astroviruses, and adenoviruses[10] However, it is estimated that on average up to 40% of diarrhea cases are of unknown etiology, suggesting that unrecognized infectious agents, including viruses, remain to be discovered[11]–[15]. Mass sequencing affords an opportunity to explore the viral diversity( including both known and novel viruses) present in stool during acute episodes of diarrhea in a systematic and unbiased fashion, thereby laying the foundation for future studies aimed at assessing whether any novel or unexpected viruses detected play a causal role in human diarrhea. In this study, mass sequencing was applied to explore specifically the viral communities present in pediatric patients suffering from diarrhea. We anticipated that the viral communities would vary significantly from specimen to specimen and that it would be desirable to sample broadly from multiple patients to obtain an overall perspective on the diversity of viruses that might be present. Toward this end, a simple yet robust experimental strategy was developed that circumvented certain technical and economic limitations of conventional mass sequencing. In both previous viral metagenomic studies of the human gut, large quantities of fecal matter( ∼500 g) were collected from adults and then extensively purified to enrich for viral particles[7],[8]. In contrast, pediatric samples provide considerably smaller volumes of stool; therefore, our strategy was designed to minimize the number of physical purification steps so that as little as 30 mg of archived fecal matter could be analyzed. Here we present data generated by performing what we refer to as ‘micro-mass sequencing’ of several hundred sequence reads per sample from 12 different patients with acute diarrhea. This analysis provides evidence for the detection of known enteric viruses, viral co-infections, and novel viruses.
Discussion:
Sequences were detected in this study from at least 9 putatively novel viruses. For 7 of these sequences, the degree of divergence observed based on phylogenetic analysis suggested that they might represent novel virus subtypes or genotypes of picobirnavirus, enterovirus, TT virus and norovirus( Fig. S2, S3, S4, and S5). Picobirnaviruses belong to an unclassified genus of double stranded RNA viruses and have been detected in fecal matter from human and other animals both with and without diarrhea[21]. Only a limited number of picobirnavirus sequences have been previously described in the literature and thus the identification of two novel picobirnaviruses significantly expands the known diversity of this taxonomic group, underscoring the unrecognized viral diversity inhabiting the human body. Sequences representing a divergent norovirus were detected in sample D06( Fig. S5). Phylogenetic analysis of individual sequence reads that mapped to the RNA polymerase and the NS4 regions of human norovirus suggested that these sequences were derived from a novel or unsequenced member of norovirus genogroup 2. In the initial screening by conventional PCR, this sample tested negative for norovirus. Upon closer examination, four mutations were observed in one of the PCR primer binding sites, which plausibly hindered the PCR screening assay[14]. In two samples, much more highly divergent sequences were detected. In D08, phylogenetic analysis of 7 unique sequence reads strongly suggested that a novel astrovirus species was present( Fig. 2). The observed sequence variation between these sequence reads and the known astrovirus genomes greatly exceeds the variation that exists between the 8 known serotypes of human astrovirus, suggesting that this virus is not simply another serotype of the known astroviruses. Astroviruses are non-enveloped, single stranded, positive sense RNA viruses that account for up to 10% of sporadic diarrhea cases[22]. Infections with astroviruses most frequently cause watery diarrhea lasting 2–4 days, and, less commonly vomiting, headache, fever, abdominal pain, and anorexia in children under the age of 2, the elderly, and immunocompromised individuals[23]. The detection of this genetically distinct astrovirus raises the question as to whether or not this is an authentic human virus, and if so, whether or not it is a causal agent of human diarrhea. Another novel sequence detected appeared by phylogenetic analysis to belong to the family Nodaviridae. Nodaviruses are small single-stranded, positive sense, bipartite RNA viruses, divided into two genera, the alphanodaviruses( insect viruses) and the betanodaviruses( fish viruses). Currently, none of the established family members are known to naturally infect mammals although experimental manipulation of the viral genome has enabled viral replication in a wide array of organisms including mammals[24]. While it is tempting to speculate that this might represent the first instance of human infection with a nodavirus, further experimentation such as serological analysis is required to definitively answer this question. Another plausible explanation is that the virus may be present simply as a result of consumption of fish infected by the virus. A prior report describing the presence of plant virus RNAs in human stool has similarly been attributed to dietary exposure[8]. Incidentally, some fish genomic sequences were detected in this particular sequence library( D09 “other” bin) supporting the possibility of dietary exposure. However, the potential piscine origin of this virus would not necessarily preclude its role as an etiologic agent of human disease. The micro-mass sequencing approach, like any other experimental methodology capable of detecting novel viruses( such as culture or degenerate PCR), cannot of course by itself determine whether the newly detected agent is pathogenic. However, this strategy can generate novel, testable hypotheses such as “Are these novel viruses involved in the etiology of human diarrhea? ” and “What is the true host of these viruses? ” that could not be asked in the absence of the knowledge that these viruses existed. 556 out of the 2013( 28%) unique high quality sequences were binned as unassigned by the BLAST criteria. Of these, 23 were identified as containing repetitive elements or low-complexity sequence by RepeatMasker[25],[26] thus explaining the lack of meaningful BLAST alignments. The origin of the remaining 533 sequences that were unassigned is uncertain, but they could be derived from unannotated host genome, novel or unsequenced microbes, or dietary sources which have not been sequenced. However, it is also possible that some of these sequences could represent viruses that have no appreciable similarity to sequences of currently known viruses. Extracting more telling information from these sequences is a challenging problem that will require the development of new computational measures capable of detecting more distant evolutionary relationships than is possible with existing methods. In addition, as more genome sequences from diverse organisms and other genomic/metagenomic projects become available, sequence similarity based methods may identify a greater fraction of these currently unassigned sequences. Our data suggest that micro-mass sequencing might be of great diagnostic utility for a number of reasons. First, viruses escaping detection in conventional assays were detected by micro-mass sequencing. In theory, the sensitivity of this strategy is limited only by the depth of sequencing. As demonstrated here, even shallow sequencing performed better than conventional diagnostics in some instances. In addition, the unbiased nature of the method enabled detection of viruses not conventionally tested for. Moreover, co-infections were detected in multiple samples. Furthermore, for multi-segmented viruses such as rotaviruses, reassortment of segments between species is a major mechanism of viral evolution that can lead to the emergence of more virulent strains[27]. Complete genome sequencing of all segments simultaneously would yield completely unambiguous identification of the viral genotype. In contrast to typical PCR or antibody based assays that target a single segment or protein, micro mass sequencing detected all 11 genomic RNA segments of rotavirus. In terms of technical practicality, samples were only minimally manipulated relative to traditional metagenomic sequencing[1],[3],[6],[8], thereby avoiding the time, labor, and use of specialized equipment required to concentrate the specimens, rendering this methodology potentially amenable to use in diagnostic laboratories. As sequencing costs diminish and efficiencies improve, mass sequencing could become a powerful diagnostic tool. In summary, we have shown that micro-mass sequencing can define the diversity of viral communities found in fecal samples from diarrhea patients. Both known viruses and novel viruses were detected by sequencing only a few hundred colonies from each sample library. These studies will serve as the springboard for further interrogations of the roles of these diverse viruses in the gastrointestinal tract. Finally, our detection of multiple novel viruses in this initial, limited exploration of a dozen samples suggests that broader sampling of patient specimens is likely to be highly fruitful in terms of identification of additional novel viruses.
Diarrhea is one of the leading infectious causes of death worldwide with an estimated 1 . 8 million deaths annually , primarily in young children in developing countries . There are many known causes of diarrhea; however , the causes of ∼40% of the cases are still unknown . One possibility is that viruses that we currently do not know about are responsible for these cases . Thus , we used an experimental strategy termed “micro-mass sequencing” to systematically identify viruses present in stool from a number of patients suffering from diarrhea . Sequences from a number of novel viruses were detected , some which differed quite significantly from any previously described virus . These new viruses may or may not be responsible for causing diarrhea . Future studies will specifically address the potential of these viruses to cause human disease . One implication of this study is that there are likely to be many more unknown viruses that can be identified in this fashion . Furthermore , by studying these viruses , we will come to a more complete understanding of the role viruses play in diarrhea . Ultimately , this may lead to the development of therapeutics and/or vaccines that decrease the disease burden of diarrhea .