Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak.

Background: The 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies. Methods and Results: During the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1 + participants by RT-PCR and identified DENV1, 2, and 3 - with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018. Conclusion: These findings highlight DENV's geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

Gemykibivirus detection in acute encephalitis patients from Nepal.

Acute encephalitis syndrome (AES) causes significant morbidity and mortality worldwide. In Nepal, Japanese encephalitis virus (JEV) accounts for ~5-20% of AES cases, but ~75% of AES cases are of unknown etiology. We identified a gemykibivirus in CSF collected in 2020 from an 8-year-old male patient with AES using metagenomic next-generation sequencing. Gemykibiviruses are single stranded, circular DNA viruses in the family Genomoviridae. The complete genome of 2,211 nucleotides was sequenced, which shared 98.69% nucleotide identity to its closest relative, Human associated gemykibivirus 2 isolate SAfia-449D. Two real-time PCR assays were designed, and screening of 337 cerebrospinal fluid (CSF) and 164 serum samples from AES patients in Nepal collected in 2020 and 2022 yielded 11 CSF and 1 serum sample that were positive in both PCR assays. Complete genomes of seven of the positives were sequenced. These results identify a potential candidate etiologic agent of encephalitis in Nepal. IMPORTANCE: Viral encephalitis is a devastating disease, but unfortunately, worldwide, the causative virus in many cases is unknown. Therefore, it is important to identify viruses that could be responsible for cases of human encephalitis. Here, using metagenomic sequencing of CSF, we identified a gemykibivirus in a male child from Nepal with acute encephalitis syndrome (AES). We subsequently detected gemykibivirus DNA in CSF or serum of 12 more encephalitis patients by real-time PCR. The virus genomes we identified are highly similar to gemykibiviruses previously detected in CSF of three encephalitis patients from Sri Lanka. These results raise the possibility that gemykibivirus could be an underrecognized human pathogen.

Hecatomb: an integrated software platform for viral metagenomics.

BACKGROUND: Modern sequencing technologies offer extraordinary opportunities for virus discovery and virome analysis. Annotation of viral sequences from metagenomic data requires a complex series of steps to ensure accurate annotation of individual reads and assembled contigs. In addition, varying study designs will require project-specific statistical analyses. FINDINGS: Here we introduce Hecatomb, a bioinformatic platform coordinating commonly used tasks required for virome analysis. Hecatomb means "a great sacrifice." In this setting, Hecatomb is "sacrificing" false-positive viral annotations using extensive quality control and tiered-database searches. Hecatomb processes metagenomic data obtained from both short- and long-read sequencing technologies, providing annotations to individual sequences and assembled contigs. Results are provided in commonly used data formats useful for downstream analysis. Here we demonstrate the functionality of Hecatomb through the reanalysis of a primate enteric and a novel coral reef virome. CONCLUSION: Hecatomb provides an integrated platform to manage many commonly used steps for virome characterization, including rigorous quality control, host removal, and both read- and contig-based analysis. Each step is managed using the Snakemake workflow manager with dependency management using Conda. Hecatomb outputs several tables properly formatted for immediate use within popular data analysis and visualization tools, enabling effective data interpretation for a variety of study designs. Hecatomb is hosted on GitHub (github.com/shandley/hecatomb) and is available for installation from Bioconda and PyPI.

Gemykibivirus detection in acute encephalitis patients from Nepal.

Acute Encephalitis Syndrome (AES) causes significant morbidity and mortality worldwide. In Nepal, Japanese encephalitis virus (JEV) accounts for ~ 5-20% of AES cases, but ~75% of AES cases are of unknown etiology. We identified a gemykibivirus in CSF collected in 2020 from a male child with AES using metagenomic next-generation sequencing. Gemykibiviruses are single stranded, circular DNA viruses in the family Genomoviridae. The complete genome of 2211 nucleotides was sequenced which shared 98.69% nucleotide identity to its closest relative, Human associated gemykibivirus 2 isolate SAfia-449D. Two real-time PCR assays were designed, and screening of 337 CSF and 164 serum samples from AES patients in Nepal collected in 2020 and 2022 yielded 11 CSF and 1 serum sample that were positive in both PCR assays. Complete genomes of 7 of the positives were sequenced. These results identify a candidate etiologic agent of encephalitis in Nepal.

Draft genomes of 12 Bifidobacterium isolates from human IBD fecal samples.

Twelve Bifidobacterium strains were isolated from fecal samples of inflammatory bowel disease patients and matched "household control" individuals. These include the species Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum.

Human enteric viruses autonomously shape inflammatory bowel disease phenotype through divergent innate immunomodulation.

Altered enteric microorganisms in concert with host genetics shape inflammatory bowel disease (IBD) phenotypes. However, insight is limited to bacteria and fungi. We found that eukaryotic viruses and bacteriophages (collectively, the virome), enriched from non-IBD, noninflamed human colon resections, actively elicited atypical anti-inflammatory innate immune programs. Conversely, ulcerative colitis or Crohn's disease colon resection viromes provoked inflammation, which was successfully dampened by non-IBD viromes. The IBD colon tissue virome was perturbed, including an increase in the enterovirus B species of eukaryotic picornaviruses, not previously detected in fecal virome studies. Mice humanized with non-IBD colon tissue viromes were protected from intestinal inflammation, whereas IBD virome mice exhibited exacerbated inflammation in a nucleic acid sensing-dependent fashion. Furthermore, there were detrimental consequences for IBD patient-derived intestinal epithelial cells bearing loss-of-function mutations within virus sensor MDA5 when exposed to viromes. Our results demonstrate that innate recognition of IBD or non-IBD human viromes autonomously influences intestinal homeostasis and disease phenotypes. Thus, perturbations in the intestinal virome, or an altered ability to sense the virome due to genetic variation, contribute to the induction of IBD. Harnessing the virome may offer therapeutic and biomarker potential.

Divergent Enteroviruses from Macaques with Chronic Diarrhea.

We report the draft genome sequences of five novel members of the family Picornaviridae that were isolated from the stool of rhesus macaques (Macaca mulatta) with chronic diarrhea. The strains were named NOLA-1 through NOLA-5 because the macaques were residents of the Tulane National Primate Research Center.

Genomic atlas of the proteome from brain, CSF and plasma prioritizes proteins implicated in neurological disorders.

Understanding the tissue-specific genetic controls of protein levels is essential to uncover mechanisms of post-transcriptional gene regulation. In this study, we generated a genomic atlas of protein levels in three tissues relevant to neurological disorders (brain, cerebrospinal fluid and plasma) by profiling thousands of proteins from participants with and without Alzheimer's disease. We identified 274, 127 and 32 protein quantitative trait loci (pQTLs) for cerebrospinal fluid, plasma and brain, respectively. cis-pQTLs were more likely to be tissue shared, but trans-pQTLs tended to be tissue specific. Between 48.0% and 76.6% of pQTLs did not co-localize with expression, splicing, DNA methylation or histone acetylation QTLs. Using Mendelian randomization, we nominated proteins implicated in neurological diseases, including Alzheimer's disease, Parkinson's disease and stroke. This first multi-tissue study will be instrumental to map signals from genome-wide association studies onto functional genes, to discover pathways and to identify drug targets for neurological diseases.

Multi-Omics Analyses Show Disease, Diet, and Transcriptome Interactions With the Virome.

BACKGROUND & AIMS: The gut virome includes eukaryotic viruses and bacteriophages that can shape the gut bacterial community and elicit host responses. The virome can be implicated in diseases, such as irritable bowel syndrome (IBS), where gut bacteria play an important role in pathogenesis. We provide a comprehensive and longitudinal characterization of the virome, including DNA and RNA viruses and paired multi-omics data in a cohort of healthy subjects and patients with IBS. METHODS: We selected 2 consecutive stool samples per subject from a longitudinal study cohort and performed metagenomic sequencing on DNA and RNA viruses after enriching for viral-like particles. Viral sequence abundance was evaluated over time, as well as in the context of diet, bacterial composition and function, metabolite levels, colonic gene expression, host genetics, and IBS subsets. RESULTS: We found that the gut virome was temporally stable and correlated with the colonic transcriptome. We identified IBS-subset-specific changes in phage populations; Microviridae, Myoviridae, and Podoviridae species were elevated in diarrhea-predominant IBS, and other Microviridae and Myoviridae species were elevated in constipation-predominant IBS compared to healthy controls. We identified correlations between subsets of the virome and bacterial composition (unclassifiable "dark matter" and phages) and diet (eukaryotic viruses). CONCLUSIONS: We found that the gut virome is stable over time but varies among subsets of patients with IBS. It can be affected by diet and potentially influences host function via interactions with gut bacteria and/or altering host gene expression.

Alzheimer's disease alters oligodendrocytic glycolytic and ketolytic gene expression.

INTRODUCTION: Sporadic Alzheimer's disease (AD) is strongly correlated with impaired brain glucose metabolism, which may affect AD onset and progression. Ketolysis has been suggested as an alternative pathway to fuel the brain. METHODS: RNA-seq profiles of post mortem AD brains were used to determine whether dysfunctional AD brain metabolism can be determined by impairments in glycolytic and ketolytic gene expression. Data were obtained from the Knight Alzheimer's Disease Research Center (62 cases; 13 controls), Mount Sinai Brain Bank (110 cases; 44 controls), and the Mayo Clinic Brain Bank (80 cases; 76 controls), and were normalized to cell type: astrocytes, microglia, neurons, oligodendrocytes. RESULTS: In oligodendrocytes, both glycolytic and ketolytic pathways were significantly impaired in AD brains. Ketolytic gene expression was not significantly altered in neurons, astrocytes, and microglia. DISCUSSION: Oligodendrocytes may contribute to brain hypometabolism observed in AD. These results are suggestive of a potential link between hypometabolism and dysmyelination in disease physiology. Additionally, ketones may be therapeutic in AD due to their ability to fuel neurons despite impaired glycolytic metabolism.

The TMEM106B FTLD-protective variant, rs1990621, is also associated with increased neuronal proportion.

Apart from amyloid ß deposition and tau neurofibrillary tangles, Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neuronal loss and astrocytosis in the cerebral cortex. The goal of this study is to investigate genetic factors associated with the neuronal proportion in health and disease. To identify cell-autonomous genetic variants associated with neuronal proportion in cortical tissues, we inferred cellular population structure from bulk RNA-Seq derived from 1536 individuals. We identified the variant rs1990621 located in the TMEM106B gene region as significantly associated with neuronal proportion (p value = 6.40 × 10-07) and replicated this finding in an independent dataset (p value = 7.41 × 10-04) surpassing the genome-wide threshold in the meta-analysis (p value = 9.42 × 10-09). This variant is in high LD with the TMEM106B non-synonymous variant p.T185S (rs3173615; r2 = 0.98) which was previously identified as a protective variant for frontotemporal lobar degeneration (FTLD). We stratified the samples by disease status, and discovered that this variant modulates neuronal proportion not only in AD cases, but also several neurodegenerative diseases and in elderly cognitively healthy controls. Furthermore, we did not find a significant association in younger controls or schizophrenia patients, suggesting that this variant might increase neuronal survival or confer resilience to the neurodegenerative process. The single variant and gene-based analyses also identified an overall genetic association between neuronal proportion, AD and FTLD risk. These results suggest that common pathways are implicated in these neurodegenerative diseases, that implicate neuronal survival. In summary, we identified a protective variant in the TMEM106B gene that may have a neuronal protection effect against general aging, independent of disease status, which could help elucidate the relationship between aging and neuronal survival in the presence or absence of neurodegenerative disorders. Our findings suggest that TMEM106B could be a potential target for neuronal protection therapies to ameliorate cognitive and functional deficits.

A single-nuclei RNA sequencing study of Mendelian and sporadic AD in the human brain.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. This neurodegenerative disorder is associated with neuronal death and gliosis heavily impacting the cerebral cortex. AD has a substantial but heterogeneous genetic component, presenting both Mendelian and complex genetic architectures. Using bulk RNA-seq from the parietal lobes and deconvolution methods, we previously reported that brains exhibiting different AD genetic architecture exhibit different cellular proportions. Here, we sought to directly investigate AD brain changes in cell proportion and gene expression using single-cell resolution. METHODS: We generated unsorted single-nuclei RNA sequencing data from brain tissue. We leveraged the tissue donated from a carrier of a Mendelian genetic mutation, PSEN1 p.A79V, and two family members who suffer from sporadic AD, but do not carry any autosomal mutations. We evaluated alternative alignment approaches to maximize the titer of reads, genes, and cells with high quality. In addition, we employed distinct clustering strategies to determine the best approach to identify cell clusters that reveal neuronal and glial cell types and avoid artifacts such as sample and batch effects. We propose an approach to cluster cells that reduces biases and enable further analyses. RESULTS: We identified distinct types of neurons, both excitatory and inhibitory, and glial cells, including astrocytes, oligodendrocytes, and microglia, among others. In particular, we identified a reduced proportion of excitatory neurons in the Mendelian mutation carrier, but a similar distribution of inhibitory neurons. Furthermore, we investigated whether single-nuclei RNA-seq from the human brains recapitulate the expression profile of disease-associated microglia (DAM) discovered in mouse models. We also determined that when analyzing human single-nuclei data, it is critical to control for biases introduced by donor-specific expression profiles. CONCLUSION: We propose a collection of best practices to generate a highly detailed molecular cell atlas of highly informative frozen tissue stored in brain banks. Importantly, we have developed a new web application to make this unique single-nuclei molecular atlas publicly available.

TREM2 brain transcript-specific studies in AD and TREM2 mutation carriers.

BACKGROUND: Low frequency coding variants in TREM2 are associated with Alzheimer disease (AD) risk and cerebrospinal fluid (CSF) TREM2 protein levels are different between AD cases and controls. Similarly, TREM2 risk variant carriers also exhibit differential CSF TREM2 levels. TREM2 has three different alternative transcripts, but most of the functional studies only model the longest transcript. No studies have analyzed TREM2 expression levels or alternative splicing in brains from AD and cognitively normal individuals. We wanted to determine whether there was differential expression of TREM2 in sporadic-AD cases versus AD-TREM2 carriers vs sex- and aged-matched normal controls; and if this differential expression was due to a particular TREM2 transcript. METHODS: We analyzed RNA-Seq data from parietal lobe brain tissue from AD cases with TREM2 variants (n = 33), AD cases (n = 195) and healthy controls (n = 118), from three independent datasets using Kallisto and the R package tximport to determine the read count for each transcript and quantified transcript abundance as transcripts per million. RESULTS: The three TREM2 transcripts were expressed in brain cortex in the three datasets. We demonstrate for the first time that the transcript that lacks the transmembrane domain and encodes a soluble form of TREM2 (sTREM2) has an expression level around 60% of the canonical transcript, suggesting that around 25% of the sTREM2 protein levels could be explained by this transcript. We did not observe a difference in the overall TREM2 expression level between cases and controls. However, the isoform which lacks the 5' exon, but includes the transmembrane domain, was significantly lower in TREM2- p.R62H carriers than in AD cases (p = 0.007). CONCLUSION: Using bulk RNA-Seq data from three different cohorts, we were able to quantify the expression level of the three TREM2 transcripts, demonstrating: (1) all three transcripts of them are highly expressed in the human cortex, (2) that up to 25% of the sTREM2 may be due to the expression of a specific isoform and not TREM2 cleavage; and (3) that TREM2 risk variants do not affect expression levels, suggesting that the effect of the TREM2 variants on CSF levels occurs at post-transcriptional level.

Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens.

The potential to rapidly capture the entire microbial community structure and/or gene content makes metagenomic sequencing an attractive tool for pathogen identification and the detection of resistance/virulence genes in clinical settings. Here, we assessed the consistency between PCR from a diagnostic laboratory, quantitative PCR (qPCR) from a research laboratory, 16S rRNA gene sequencing, and metagenomic shotgun sequencing (MSS) for Clostridium difficile identification in diarrhea stool samples. Twenty-two C. difficile-positive diarrhea samples identified by PCR and qPCR and five C. difficile-negative diarrhea controls were studied. C. difficile was detected in 90.9% of C. difficile-positive samples using 16S rRNA gene sequencing, and C. difficile was detected in 86.3% of C. difficile-positive samples using MSS. CFU inferred from qPCR analysis were positively correlated with the relative abundance of C. difficile from 16S rRNA gene sequencing (r(2) = -0.60) and MSS (r(2) = -0.55). C. difficile was codetected with Clostridium perfringens, norovirus, sapovirus, parechovirus, and anellovirus in 3.7% to 27.3% of the samples. A high load of Candida spp. was found in a symptomatic control sample in which no causative agents for diarrhea were identified in routine clinical testing. Beta-lactamase and tetracycline resistance genes were the most prevalent (25.9%) antibiotic resistance genes in these samples. In summary, the proof-of-concept study demonstrated that next-generation sequencing (NGS) in pathogen detection is moderately correlated with laboratory testing and is advantageous in detecting pathogens without a priori knowledge.

Metagenomic analysis of double-stranded DNA viruses in healthy adults.

BACKGROUND: The Human Microbiome Project (HMP) was undertaken with the goal of defining microbial communities in and on the bodies of healthy individuals using high-throughput, metagenomic sequencing analysis. The viruses present in these microbial communities, the 'human virome', are an important aspect of the human microbiome that is particularly understudied in the absence of overt disease. We analyzed eukaryotic double-stranded DNA (dsDNA) viruses, together with dsDNA replicative intermediates of single-stranded DNA viruses, in metagenomic sequence data generated by the HMP. 706 samples from 102 subjects were studied, with each subject sampled at up to five major body habitats: nose, skin, mouth, vagina, and stool. Fifty-one individuals had samples taken at two or three time points 30 to 359 days apart from at least one of the body habitats. RESULTS: We detected an average of 5.5 viral genera in each individual. At least 1 virus was detected in 92% of the individuals sampled. These viruses included herpesviruses, papillomaviruses, polyomaviruses, adenoviruses, anelloviruses, parvoviruses, and circoviruses. Each individual had a distinct viral profile, demonstrating the high interpersonal diversity of the virome. Some components of the virome were stable over time. CONCLUSIONS: This study is the first to use high-throughput DNA sequencing to describe the diversity of eukaryotic dsDNA viruses in a large cohort of normal individuals who were sampled at multiple body sites. Our results show that the human virome is a complex component of the microbial flora. Some viruses establish long-term infections that may be associated with increased risk or possibly with protection from disease. A better understanding of the composition and dynamics of the virome may hold important keys to human health.

Exploration of bacterial community classes in major human habitats.

BACKGROUND: Determining bacterial abundance variation is the first step in understanding bacterial similarity between individuals. Categorization of bacterial communities into groups or community classes is the subsequent step in describing microbial distribution based on abundance patterns. Here, we present an analysis of the groupings of bacterial communities in stool, nasal, skin, vaginal and oral habitats in a healthy cohort of 236 subjects from the Human Microbiome Project. RESULTS: We identify distinct community group patterns in the anterior nares, four skin sites, and vagina at the genus level. We also confirm three enterotypes previously identified in stools. We identify two clusters with low silhouette values in most oral sites, in which bacterial communities are more homogeneous. Subjects sharing a community class in one habitat do not necessarily share a community class in another, except in the three vaginal sites and the symmetric habitats of the left and right retroauricular creases. Demographic factors, including gender, age, and ethnicity, significantly influence community composition in several habitats. Community classes in the vagina, retroauricular crease and stool are stable over approximately 200 days. CONCLUSION: The community composition, association of demographic factors with community classes, and demonstration of community stability deepen our understanding of the variability and dynamics of human microbiomes. This also has significant implications for experimental designs that seek microbial correlations with clinical phenotypes.

Nyamiviridae: proposal for a new family in the order Mononegavirales.

Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.

Biogeography of the ecosystems of the healthy human body.

BACKGROUND: Characterizing the biogeography of the microbiome of healthy humans is essential for understanding microbial associated diseases. Previous studies mainly focused on a single body habitat from a limited set of subjects. Here, we analyzed one of the largest microbiome datasets to date and generated a biogeographical map that annotates the biodiversity, spatial relationships, and temporal stability of 22 habitats from 279 healthy humans. RESULTS: We identified 929 genera from more than 24 million 16S rRNA gene sequences of 22 habitats, and we provide a baseline of inter-subject variation for healthy adults. The oral habitat has the most stable microbiota with the highest alpha diversity, while the skin and vaginal microbiota are less stable and show lower alpha diversity. The level of biodiversity in one habitat is independent of the biodiversity of other habitats in the same individual. The abundances of a given genus at a body site in which it dominates do not correlate with the abundances at body sites where it is not dominant. Additionally, we observed the human microbiota exhibit both cosmopolitan and endemic features. Finally, comparing datasets of different projects revealed a project-based clustering pattern, emphasizing the significance of standardization of metagenomic studies. CONCLUSIONS: The data presented here extend the definition of the human microbiome by providing a more complete and accurate picture of human microbiome biogeography, addressing questions best answered by a large dataset of subjects and body sites that are deeply sampled by sequencing.

Sequence analysis of the human virome in febrile and afebrile children.

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.

Novel bacterial taxa in the human microbiome.

The human gut harbors thousands of bacterial taxa. A profusion of metagenomic sequence data has been generated from human stool samples in the last few years, raising the question of whether more taxa remain to be identified. We assessed metagenomic data generated by the Human Microbiome Project Consortium to determine if novel taxa remain to be discovered in stool samples from healthy individuals. To do this, we established a rigorous bioinformatics pipeline that uses sequence data from multiple platforms (Illumina GAIIX and Roche 454 FLX Titanium) and approaches (whole-genome shotgun and 16S rDNA amplicons) to validate novel taxa. We applied this approach to stool samples from 11 healthy subjects collected as part of the Human Microbiome Project. We discovered several low-abundance, novel bacterial taxa, which span three major phyla in the bacterial tree of life. We determined that these taxa are present in a larger set of Human Microbiome Project subjects and are found in two sampling sites (Houston and St. Louis). We show that the number of false-positive novel sequences (primarily chimeric sequences) would have been two orders of magnitude higher than the true number of novel taxa without validation using multiple datasets, highlighting the importance of establishing rigorous standards for the identification of novel taxa in metagenomic data. The majority of novel sequences are related to the recently discovered genus Barnesiella, further encouraging efforts to characterize the members of this genus and to study their roles in the microbial communities of the gut. A better understanding of the effects of less-abundant bacteria is important as we seek to understand the complex gut microbiome in healthy individuals and link changes in the microbiome to disease.