Advancing the microbiome research community.

The human microbiome has become a recognized factor in promoting and maintaining health. We outline opportunities in interdisciplinary research, analytical rigor, standardization, and policy development for this relatively new and rapidly developing field. Advances in these aspects of the research community may in turn advance our understanding of human microbiome biology.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

The single-cell opioid responses in the context of HIV (SCORCH) consortium.

Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.

The Neuroscience Multi-Omic Archive: a BRAIN Initiative resource for single-cell transcriptomic and epigenomic data from the mammalian brain.

Scalable technologies to sequence the transcriptomes and epigenomes of single cells are transforming our understanding of cell types and cell states. The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network (BICCN) is applying these technologies at unprecedented scale to map the cell types in the mammalian brain. In an effort to increase data FAIRness (Findable, Accessible, Interoperable, Reusable), the NIH has established repositories to make data generated by the BICCN and related BRAIN Initiative projects accessible to the broader research community. Here, we describe the Neuroscience Multi-Omic Archive (NeMO Archive; nemoarchive.org), which serves as the primary repository for genomics data from the BRAIN Initiative. Working closely with other BRAIN Initiative researchers, we have organized these data into a continually expanding, curated repository, which contains transcriptomic and epigenomic data from over 50 million brain cells, including single-cell genomic data from all of the major regions of the adult and prenatal human and mouse brains, as well as substantial single-cell genomic data from non-human primates. We make available several tools for accessing these data, including a searchable web portal, a cloud-computing interface for large-scale data processing (implemented on Terra, terra.bio), and a visualization and analysis platform, NeMO Analytics (nemoanalytics.org).

The evolution of synaptic and cognitive capacity: Insights from the nervous system transcriptome of Aplysia.

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Mutagenesis of human genomes by endogenous mobile elements on a population scale.

Several large-scale Illumina whole-genome sequencing (WGS) and whole-exome sequencing (WES) projects have emerged recently that have provided exceptional opportunities to discover mobile element insertions (MEIs) and study the impact of these MEIs on human genomes. However, these projects also have presented major challenges with respect to the scalability and computational costs associated with performing MEI discovery on tens or even hundreds of thousands of samples. To meet these challenges, we have developed a more efficient and scalable version of our mobile element locator tool (MELT) called CloudMELT. We then used MELT and CloudMELT to perform MEI discovery in 57,919 human genomes and exomes, leading to the discovery of 104,350 nonredundant MEIs. We leveraged this collection (1) to examine potentially active L1 source elements that drive the mobilization of new Alu, L1, and SVA MEIs in humans; (2) to examine the population distributions and subfamilies of these MEIs; and (3) to examine the mutagenesis of GENCODE genes, ENCODE-annotated features, and disease genes by these MEIs. Our study provides new insights on the L1 source elements that drive MEI mutagenesis and brings forth a better understanding of how this mutagenesis impacts human genomes.

Reprogramming Chromosome Ends by Functional Histone Acetylation.

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between 'stemness' and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells.

Erratum: Strains, functions and dynamics in the expanded Human Microbiome Project.

This corrects the article DOI: 10.1038/nature23889.

Strains, functions and dynamics in the expanded Human Microbiome Project.

The characterization of baseline microbial and functional diversity in the human microbiome has enabled studies of microbiome-related disease, diversity, biogeography, and molecular function. The National Institutes of Health Human Microbiome Project has provided one of the broadest such characterizations so far. Here we introduce a second wave of data from the study, comprising 1,631 new metagenomes (2,355 total) targeting diverse body sites with multiple time points in 265 individuals. We applied updated profiling and assembly methods to provide new characterizations of microbiome personalization. Strain identification revealed subspecies clades specific to body sites; it also quantified species with phylogenetic diversity under-represented in isolate genomes. Body-wide functional profiling classified pathways into universal, human-enriched, and body site-enriched subsets. Finally, temporal analysis decomposed microbial variation into rapidly variable, moderately variable, and stable subsets. This study furthers our knowledge of baseline human microbial diversity and enables an understanding of personalized microbiome function and dynamics.

HMPDACC: a Human Microbiome Project Multi-omic data resource.

The Human Microbiome Project (HMP) explored microbial communities of the human body in both healthy and disease states. Two phases of the HMP (HMP and iHMP) together generated >48TB of data (public and controlled access) from multiple, varied omics studies of both the microbiome and associated hosts. The Human Microbiome Project Data Coordination Center (HMPDACC) was established to provide a portal to access data and resources produced by the HMP. The HMPDACC provides a unified data repository, multi-faceted search functionality, analysis pipelines and standardized protocols to facilitate community use of HMP data. Recent efforts have been put toward making HMP data more findable, accessible, interoperable and reusable. HMPDACC resources are freely available at www.hmpdacc.org.

Advancing discovery in hearing research via biologist-friendly access to multi-omic data.

High-throughput cell type-specific multi-omic analyses have advanced our understanding of inner ear biology in an unprecedented way. The full benefit of these data, however, is reached from their re-use. Successful re-use of data requires identifying the natural users and ensuring proper data democratization and federation for their seamless and meaningful access. Here we discuss universal challenges in access and re-use of multi-omic data, possible solutions, and introduce the gEAR (the gene Expression Analysis Resource, umgear.org)-a tool for multi-omic data visualization, sharing and access for the ear field.

CD34 defines melanocyte stem cell subpopulations with distinct regenerative properties.

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.

Bioinformatic analysis of bacteria and host cell dual RNA-sequencing experiments.

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA sequencing technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. Several laboratory protocols have been presented that outline the collection, extraction and sequencing of total RNA for dRNA-Seq experiments, but there is relatively little guidance available for the detailed bioinformatic analyses required. This protocol outlines a typical dRNA-Seq experiment, based on a Chlamydia trachomatis-infected host cell, with a detailed description of the necessary bioinformatic analyses with currently available software tools.

Metaviz: interactive statistical and visual analysis of metagenomic data.

Large studies profiling microbial communities and their association with healthy or disease phenotypes are now commonplace. Processed data from many of these studies are publicly available but significant effort is required for users to effectively organize, explore and integrate it, limiting the utility of these rich data resources. Effective integrative and interactive visual and statistical tools to analyze many metagenomic samples can greatly increase the value of these data for researchers. We present Metaviz, a tool for interactive exploratory data analysis of annotated microbiome taxonomic community profiles derived from marker gene or whole metagenome shotgun sequencing. Metaviz is uniquely designed to address the challenge of browsing the hierarchical structure of metagenomic data features while rendering visualizations of data values that are dynamically updated in response to user navigation. We use Metaviz to provide the UMD Metagenome Browser web service, allowing users to browse and explore data for more than 7000 microbiomes from published studies. Users can also deploy Metaviz as a web service, or use it to analyze data through the metavizr package to interoperate with state-of-the-art analysis tools available through Bioconductor. Metaviz is free and open source with the code, documentation and tutorials publicly accessible.

A comparative analysis of library prep approaches for sequencing low input translatome samples.

BACKGROUND: Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. RESULTS: We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. CONCLUSION: RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.

Identification of a gene expression signature associated with the metastasis suppressor function of NME1: prognostic value in human melanoma.

Although NME1 is well known for its ability to suppress metastasis of melanoma, the molecular mechanisms underlying this activity are not completely understood. Herein, we utilized a bioinformatics approach to systematically identify genes whose expression is correlated with the metastasis suppressor function of NME1. This was accomplished through a search for genes that were regulated by NME1, but not by NME1 variants lacking metastasis suppressor activity. This approach identified a number of novel genes, such as ALDOC, CXCL11, LRP1b, and XAGE1 as well as known targets such as NETO2, which were collectively designated as an NME1-Regulated Metastasis Suppressor Signature (MSS). The MSS was associated with prolonged overall survival in a large cohort of melanoma patients in The Cancer Genome Atlas (TCGA). The median overall survival of melanoma patients with elevated expression of the MSS genes was >5.6 years longer compared with that of patients with lower expression of the MSS genes. These data demonstrate that NMEl represents a powerful tool for identifying genes whose expression is associated with metastasis and survival of melanoma patients, suggesting their potential applications as prognostic markers and therapeutic targets in advanced forms of this lethal cancer.

New signaling pathways govern the host response to C. albicans infection in various niches.

Candida albicans, the major invasive fungal pathogen of humans, can cause both debilitating mucosal infections and fatal invasive infections. Understanding the complex nature of the host-pathogen interaction in each of these contexts is essential to developing desperately needed therapies to treat fungal infections. RNA-seq enables a systems-level understanding of infection by facilitating comprehensive analysis of transcriptomes from multiple species (e.g., host and pathogen) simultaneously. We used RNA-seq to characterize the transcriptomes of both C. albicans and human endothelial cells or oral epithelial cells during in vitro infection. Network analysis of the differentially expressed genes identified the activation of several signaling pathways that have not previously been associated with the host response to fungal pathogens. Using an siRNA knockdown approach, we demonstrate that two of these pathways-platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9)-govern the host-pathogen interaction by regulating the uptake of C. albicans by host cells. Using RNA-seq analysis of a mouse model of hematogenously disseminated candidiasis (HDC) and episodes of vulvovaginal candidiasis (VVC) in humans, we found evidence that many of the same signaling pathways are activated during mucosal (VVC) and/or disseminated (HDC) infections in vivo. Our analyses have uncovered several signaling pathways at the interface between C. albicans and host cells in various contexts of infection, and suggest that PDGF BB and NEDD9 play important roles in this interaction. In addition, these data provide a valuable community resource for better understanding host-fungal pathogen interactions.