Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs.

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

Accurate and complete genomes from metagenomes.

Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.

Transient Suppression of Bacterial Populations Associated with Gut Health is Critical in Success of Exclusive Enteral Nutrition for Children with Crohn's Disease.

BACKGROUND AND AIMS: Exclusive enteral nutrition [EEN] is a dietary intervention to induce clinical remission in children with active luminal Crohn's disease [CD]. While changes in the gut microbial communities have been implicated in achieving this remission, a precise understanding of the role of microbial ecology in the restoration of gut homeostasis is lacking. METHODS: Here we reconstructed genomes from the gut metagenomes of 12 paediatric subjects who were sampled before, during and after EEN. We then classified each microbial population into distinct 'phenotypes' or patterns of response based on changes in their relative abundances throughout the therapy on a per-individual basis. RESULTS: Our data show that children achieving clinical remission during therapy were enriched with microbial populations that were either suppressed or that demonstrated a transient bloom as a function of EEN. In contrast, this ecosystem-level response was not observed in cases of EEN failure. Further analysis revealed that populations that were suppressed during EEN were significantly more prevalent in healthy children and adults across the globe compared with those that bloomed ephemerally during the therapy. CONCLUSIONS: These observations taken together suggest that successful outcomes of EEN are marked by a temporary emergence of microbial populations that are rare in healthy individuals, and a concomitant reduction in microbes that are commonly associated with gut homeostasis. Our work is a first attempt to highlight individual-specific, complex environmental factors that influence microbial response in EEN. This model offers a novel, alternative viewpoint to traditional taxonomic strategies used to characterize associations with health and disease states.

Metabolic independence drives gut microbial colonization and resilience in health and disease.

BACKGROUND: Changes in microbial community composition as a function of human health and disease states have sparked remarkable interest in the human gut microbiome. However, establishing reproducible insights into the determinants of microbial succession in disease has been a formidable challenge. RESULTS: Here we use fecal microbiota transplantation (FMT) as an in natura experimental model to investigate the association between metabolic independence and resilience in stressed gut environments. Our genome-resolved metagenomics survey suggests that FMT serves as an environmental filter that favors populations with higher metabolic independence, the genomes of which encode complete metabolic modules to synthesize critical metabolites, including amino acids, nucleotides, and vitamins. Interestingly, we observe higher completion of the same biosynthetic pathways in microbes enriched in IBD patients. CONCLUSIONS: These observations suggest a general mechanism that underlies changes in diversity in perturbed gut environments and reveal taxon-independent markers of "dysbiosis" that may explain why widespread yet typically low-abundance members of healthy gut microbiomes can dominate under inflammatory conditions without any causal association with disease.

High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column-based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long-read sequencing of metagenomes.

System-wide transcriptome damage and tissue identity loss in COVID-19 patients.

The molecular mechanisms underlying the clinical manifestations of coronavirus disease 2019 (COVID-19), and what distinguishes them from common seasonal influenza virus and other lung injury states such as acute respiratory distress syndrome, remain poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match these data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue-compartment-specific damage wrought by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, evident as a function of varying viral loads during the clinical course of infection and tissue-type-specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.

Shotgun transcriptome, spatial omics, and isothermal profiling of SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions.

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection revealed in COVID-19 Autopsies and Spatial Omics Tissue Maps.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome.

INTRODUCTION: Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life. RESULTS: Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment. CONCLUSIONS: Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in interferon, ACE, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health, and new therapeutic targets.

The Wolbachia mobilome in Culex pipiens includes a putative plasmid.

Wolbachia is a genus of obligate intracellular bacteria found in nematodes and arthropods worldwide, including insect vectors that transmit dengue, West Nile, and Zika viruses. Wolbachia's unique ability to alter host reproductive behavior through its temperate bacteriophage WO has enabled the development of new vector control strategies. However, our understanding of Wolbachia's mobilome beyond its bacteriophages is incomplete. Here, we reconstruct near-complete Wolbachia genomes from individual ovary metagenomes of four wild Culex pipiens mosquitoes captured in France. In addition to viral genes missing from the Wolbachia reference genome, we identify a putative plasmid (pWCP), consisting of a 9.23-kbp circular element with 14 genes. We validate its presence in additional Culex pipiens mosquitoes using PCR, long-read sequencing, and screening of existing metagenomes. The discovery of this previously unrecognized extrachromosomal element opens additional possibilities for genetic manipulation of Wolbachia.

Author Correction: The Wolbachia mobilome in Culex pipiens includes a putative plasmid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gut microbes contribute to variation in solid organ transplant outcomes in mice.

BACKGROUND: Solid organ transplant recipients show heterogeneity in the occurrence and timing of acute rejection episodes. Understanding the factors responsible for such variability in patient outcomes may lead to improved diagnostic and therapeutic approaches. Rejection kinetics of transplanted organs mainly depends on the extent of genetic disparities between donor and recipient, but a role for environmental factors is emerging. We have recently shown that major alterations of the microbiota following broad-spectrum antibiotics, or use of germ-free animals, promoted longer skin graft survival in mice. Here, we tested whether spontaneous differences in microbial colonization between genetically similar individuals can contribute to variability in graft rejection kinetics. RESULTS: We compared rejection kinetics of minor mismatched skin grafts in C57BL/6 mice from Jackson Laboratory (Jax) and Taconic Farms (Tac), genetically similar animals colonized by different commensal microbes. Female Tac mice rejected skin grafts from vendor-matched males more quickly than Jax mice. We observed prolonged graft survival in Tac mice when they were exposed to Jax mice microbiome through co-housing or fecal microbiota transplantation (FMT) by gastric gavage. In contrast, exposure to Tac mice did not change graft rejection kinetics in Jax mice, suggesting a dominant suppressive effect of Jax microbiota. High-throughput sequencing of 16S rRNA gene amplicons from Jax and Tac mice fecal samples confirmed a convergence of microbiota composition after cohousing or fecal transfer. Our analysis of amplicon data associated members of a single bacterial genus, Alistipes, with prolonged graft survival. Consistent with this finding, members of the genus Alistipes were absent in a separate Tac cohort, in which fecal transfer from Jax mice failed to prolong graft survival. CONCLUSIONS: These results demonstrate that differences in resident microbiome in healthy individuals may translate into distinct kinetics of graft rejection, and contribute to interpersonal variability in graft outcomes. The association between Alistipes and prolonged skin graft survival in mice suggests that members of this genus might affect host physiology, including at sites distal to the gastrointestinal tract. Overall, these findings allude to a potential therapeutic role for specific gut microbes to promote graft survival through the administration of probiotics, or FMT.

Author Correction: Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes.

In the version of this Article originally published, the surname of author Sandra M. McLellan was spelt incorrectly as 'MacLellan'. This has now been corrected. In addition, Fig. 2 was mistakenly duplicated in the Supplementary Information as Supplementary Fig. 2. This has now been replaced with the correct supplementary figure (shown below).

Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes.

Nitrogen fixation in the surface ocean impacts global marine nitrogen bioavailability and thus microbial primary productivity. Until now, cyanobacterial populations have been viewed as the main suppliers of bioavailable nitrogen in this habitat. Although PCR amplicon surveys targeting the nitrogenase reductase gene have revealed the existence of diverse non-cyanobacterial diazotrophic populations, subsequent quantitative PCR surveys suggest that they generally occur in low abundance. Here, we use state-of-the-art metagenomic assembly and binning strategies to recover nearly one thousand non-redundant microbial population genomes from the TARA Oceans metagenomes. Among these, we provide the first genomic evidence for non-cyanobacterial diazotrophs inhabiting surface waters of the open ocean, which correspond to lineages within the Proteobacteria and, most strikingly, the Planctomycetes. Members of the latter phylum are prevalent in aquatic systems, but have never been linked to nitrogen fixation previously. Moreover, using genome-wide quantitative read recruitment, we demonstrate that the discovered diazotrophs were not only widespread but also remarkably abundant (up to 0.3% of metagenomic reads for a single population) in both the Pacific Ocean and the Atlantic Ocean northwest. Our results extend decades of PCR-based gene surveys, and substantiate the importance of heterotrophic bacteria in the fixation of nitrogen in the surface ocean.

Tracking microbial colonization in fecal microbiota transplantation experiments via genome-resolved metagenomics.

BACKGROUND: Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection and shows promise for treating other medical conditions associated with intestinal dysbioses. However, we lack a sufficient understanding of which microbial populations successfully colonize the recipient gut, and the widely used approaches to study the microbial ecology of FMT experiments fail to provide enough resolution to identify populations that are likely responsible for FMT-derived benefits. METHODS: We used shotgun metagenomics together with assembly and binning strategies to reconstruct metagenome-assembled genomes (MAGs) from fecal samples of a single FMT donor. We then used metagenomic mapping to track the occurrence and distribution patterns of donor MAGs in two FMT recipients. RESULTS: Our analyses revealed that 22% of the 92 highly complete bacterial MAGs that we identified from the donor successfully colonized and remained abundant in two recipients for at least 8 weeks. Most MAGs with a high colonization rate belonged to the order Bacteroidales. The vast majority of those that lacked evidence of colonization belonged to the order Clostridiales, and colonization success was negatively correlated with the number of genes related to sporulation. Our analysis of 151 publicly available gut metagenomes showed that the donor MAGs that colonized both recipients were prevalent, and the ones that colonized neither were rare across the participants of the Human Microbiome Project. Although our dataset showed a link between taxonomy and the colonization ability of a given MAG, we also identified MAGs that belong to the same taxon with different colonization properties, highlighting the importance of an appropriate level of resolution to explore the functional basis of colonization and to identify targets for cultivation, hypothesis generation, and testing in model systems. CONCLUSIONS: The analytical strategy adopted in our study can provide genomic insights into bacterial populations that may be critical to the efficacy of FMT due to their success in gut colonization and metabolic properties, and guide cultivation efforts to investigate mechanistic underpinnings of this procedure beyond associations.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.