Current metagenome assembled human gut phage catalogs contained mostly fragmented genomes. Here, comprehensive gut virome detection procedure is developed involving virus-like particle (VLP) enrichment from ≈500 g feces and combined sequencing of short- and long-read. Applied to 135 samples, a Chinese Gut Virome Catalog (CHGV) is assembled consisting of 21,499 non-redundant viral operational taxonomic units (vOTUs) that are significantly longer than those obtained by short-read sequencing and contained ≈35% (7675) complete genomes, which is ≈nine times more than those in the Gut Virome Database (GVD, ≈4%, 1,443). Interestingly, the majority (≈60%, 13,356) of the CHGV vOTUs are obtained by either long-read or hybrid assemblies, with little overlap with those assembled from only the short-read data. With this dataset, vast diversity of the gut virome is elucidated, including the identification of 32% (6,962) novel vOTUs compare to public gut virome databases, dozens of phages that are more prevalent than the crAssphages and/or Gubaphages, and several viral clades that are more diverse than the two. Finally, the functional capacities are also characterized of the CHGV encoded proteins and constructed a viral-host interaction network to facilitate future research and applications.

Small open reading frames (smORFs) shorter than 100 codons are widespread and perform essential roles in microorganisms, where they encode proteins active in several cell functions, including signal pathways, stress response, and antibacterial activities. However, the ecology, distribution and role of small proteins in the global microbiome remain unknown. Here, we construct a global microbial smORFs catalog (GMSC) derived from 63,410 publicly available metagenomes across 75 distinct habitats and 87,920 high-quality isolate genomes. GMSC contains 965 million non-redundant smORFs with comprehensive annotations. We find that archaea harbor more smORFs proportionally than bacteria. We moreover provide a tool called GMSC-mapper to identify and annotate small proteins from microbial (meta)genomes. Overall, this publicly-available resource demonstrates the immense and underexplored diversity of small proteins.

Genetic variations are instrumental for unraveling phage evolution and deciphering their functional implications. Here, we explore the underlying fine-scale genetic variations in the gut phageome, especially structural variations (SVs). By using virome-enriched long-read metagenomic sequencing across 91 individuals, we identified a total of 14,438 nonredundant phage SVs and revealed their prevalence within the human gut phageome. These SVs are mainly enriched in genes involved in recombination, DNA methylation, and antibiotic resistance. Notably, a substantial fraction of phage SV sequences share close homology with bacterial fragments, with most SVs enriched for horizontal gene transfer (HGT) mechanism. Further investigations showed that these SV sequences were genetic exchanged between specific phage-bacteria pairs, particularly between phages and their respective bacterial hosts. Temperate phages exhibit a higher frequency of genetic exchange with bacterial chromosomes and then virulent phages. Collectively, our findings provide insights into the genetic landscape of the human gut phageome.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs) within the global microbiome and leverage a vast dataset of 63,410 metagenomes and 87,920 prokaryotic genomes from environmental and host-associated habitats to create the AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, few of which match existing databases. AMPSphere provides insights into the evolutionary origins of peptides, including by duplication or gene truncation of longer sequences, and we observed that AMP production varies by habitat. To validate our predictions, we synthesized and tested 100 AMPs against clinically relevant drug-resistant pathogens and human gut commensals both in vitro and in vivo. A total of 79 peptides were active, with 63 targeting pathogens. These active AMPs exhibited antibacterial activity by disrupting bacterial membranes. In conclusion, our approach identified nearly one million prokaryotic AMP sequences, an open-access resource for antibiotic discovery.

The fungal component of the human gut microbiome, also known as the mycobiome, plays a vital role in intestinal ecology and human health. However, the overall structure of the gut mycobiome as well as the inter-individual variations in fungal composition remains largely unknown. In this study, we collected a total of 3363 fungal sequencing samples from 16 cohorts across three continents, including 572 newly profiled samples from China.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine learning-based approach to predict prokaryotic antimicrobial peptides (AMPs) by leveraging a vast dataset of 63,410 metagenomes and 87,920 microbial genomes. This led to the creation of AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, the majority of which were previously unknown. We observed that AMP production varies by habitat, with animal-associated samples displaying the highest proportion of AMPs compared to other habitats. Furthermore, within different human-associated microbiota, strain-level differences were evident. To validate our predictions, we synthesized and experimentally tested 50 AMPs, demonstrating their efficacy against clinically relevant drug-resistant pathogens both in vitro and in vivo. These AMPs exhibited antibacterial activity by targeting the bacterial membrane. Additionally, AMPSphere provides valuable insights into the evolutionary origins of peptides. In conclusion, our approach identified AMP sequences within prokaryotic microbiomes, opening up new avenues for the discovery of antibiotics.

DNA methylation plays a crucial role in the survival of bacteriophages (phages), yet the understanding of their genome methylation remains limited. In this study, DNA methylation patterns are analyzed in 8848 metagenome-assembled high-quality phages from 104 fecal samples using single-molecule real-time sequencing. The results demonstrate that 97.60% of gut phages exhibit methylation, with certain factors correlating with methylation densities. Phages with higher methylation densities appear to have potential viability advantages. Strikingly, more than one-third of the phages possess their own DNA methyltransferases (MTases). Increased MTase copies are associated with higher genome methylation densities, specific methylation motifs, and elevated prevalence of certain phage groups. Notably, the majority of these MTases share close homology with those encoded by gut bacteria, suggesting their exchange during phage-bacterium interactions. Furthermore, these MTases can be employed to accurately predict phage-host relationships. Overall, the findings indicate the widespread utilization of DNA methylation by gut DNA phages as an evasion mechanism against host defense systems, with a substantial contribution from phage-encoded MTases.

Microbial genes encode the majority of the functional repertoire of life on earth. However, despite increasing efforts in metagenomic sequencing of various habitats, little is known about the distribution of genes across the global biosphere, with implications for human and planetary health. Here we constructed a non-redundant gene catalogue of 303 million species-level genes (clustered at 95% nucleotide identity) from 13,174 publicly available metagenomes across 14 major habitats and use it to show that most genes are specific to a single habitat. The small fraction of genes found in multiple habitats is enriched in antibiotic-resistance genes and markers for mobile genetic elements. By further clustering these species-level genes into 32 million protein families, we observed that a small fraction of these families contain the majority of the genes (0.6% of families account for 50% of the genes). The majority of species-level genes and protein families are rare. Furthermore, species-level genes, and in particular the rare ones, show low rates of positive (adaptive) selection, supporting a model in which most genetic variability observed within each protein family is neutral or nearly neutral.

OGEE is an Online GEne Essentiality database. Gene essentiality is not a static and binary property, rather a context-dependent and evolvable property in all forms of life. In OGEE we collect not only experimentally tested essential and non-essential genes, but also associated gene properties that contributes to gene essentiality. We tagged conditionally essential genes that show variable essentiality statuses across datasets to highlight complex interplays between gene functions and environmental/experimental perturbations. OGEE v3 contains gene essentiality datasets for 91 species; almost doubled from 48 species in previous version. To accommodate recent advances on human cancer essential genes (as known as tumor dependency genes) that could serve as targets for cancer treatment and/or drug development, we expanded the collection of human essential genes from 16 cell lines in previous to 581. These human cancer cell lines were tested with high-throughput experiments such as CRISPR-Cas9 and RNAi; in total, 150 of which were tested by both techniques. We also included factors known to contribute to gene essentiality for these cell lines, such as genomic mutation, methylation and gene expression, along with extensive graphical visualizations for ease of understanding of these factors. OGEE v3 can be accessible freely at https://v3.ogee.info.

Phages invade microbes, accomplish host lysis and are of vital importance in shaping the community structure of environmental microbiota. More importantly, most phages have very specific hosts; they are thus ideal tools to manipulate environmental microbiota at species-resolution. The main purpose of MVP (Microbe Versus Phage) is to provide a comprehensive catalog of phage-microbe interactions and assist users to select phage(s) that can target (and potentially to manipulate) specific microbes of interest. We first collected 50 782 viral sequences from various sources and clustered them into 33 097 unique viral clusters based on sequence similarity. We then identified 26 572 interactions between 18 608 viral clusters and 9245 prokaryotes (i.e. bacteria and archaea); we established these interactions based on 30 321 evidence entries that we collected from published datasets, public databases and re-analysis of genomic and metagenomic sequences. Based on these interactions, we calculated the host range for each of the phage clusters and accordingly grouped them into subgroups such as 'species-', 'genus-' and 'family-' specific phage clusters. MVP is equipped with a modern, responsive and intuitive interface, and is freely available at: http://mvp.medgenius.info.

OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info.

Drug resistance is becoming a serious problem that leads to the failure of standard treatments, which is generally developed because of genetic mutations of certain molecules. Here, we present GEAR (A database of Genomic Elements Associated with drug Resistance) that aims to provide comprehensive information about genomic elements (including genes, single-nucleotide polymorphisms and microRNAs) that are responsible for drug resistance. Right now, GEAR contains 1631 associations between 201 human drugs and 758 genes, 106 associations between 29 human drugs and 66 miRNAs, and 44 associations between 17 human drugs and 22 SNPs. These relationships are firstly extracted from primary literature with text mining and then manually curated. The drug resistome deposited in GEAR provides insights into the genetic factors underlying drug resistance. In addition, new indications and potential drug combinations can be identified based on the resistome. The GEAR database can be freely accessed through http://gear.comp-sysbio.org.

Phosphorylation and proteolysis are among the most common post-translational modifications (PTMs), and play critical roles in various biological processes. More recent discoveries imply that the crosstalks between these two PTMs are involved in many diseases. In this work, we construct a post-translational regulatory network (PTRN) consists of phosphorylation and proteolysis processes, which enables us to investigate the regulatory interplays between these two PTMs. With the PTRN, we identify some functional network motifs that are significantly enriched with drug targets, some of which are further found to contain multiple proteins targeted by combinatorial drugs. These findings imply that the network motifs may be used to predict targets when designing new drugs. Inspired by this, we propose a novel computational approach called NetTar for predicting drug targets using the identified network motifs. Benchmarking results on real data indicate that our approach can be used for accurate prediction of novel proteins targeted by known drugs.

Mendelian disorders are often caused by mutations in genes that are not lethal but induce functional distortions leading to diseases. Here we study the extent of gene duplicates that might compensate genes causing monogenic diseases. We provide evidence for pervasive functional redundancy of human monogenic disease genes (MDs) by duplicates by manifesting 1) genes involved in human genetic disorders are enriched in duplicates and 2) duplicated disease genes tend to have higher functional similarities with their closest paralogs in contrast to duplicated non-disease genes of similar age. We propose that functional compensation by duplication of genes masks the phenotypic effects of deleterious mutations and reduces the probability of purging the defective genes from the human population; this functional compensation could be further enhanced by higher purification selection between disease genes and their duplicates as well as their orthologous counterpart compared to non-disease genes. However, due to the intrinsic expression stochasticity among individuals, the deleterious mutations could still be present as genetic diseases in some subpopulations where the duplicate copies are expressed at low abundances. Consequently the defective genes are linked to genetic disorders while they continue propagating within the population. Our results provide insight into the molecular basis underlying the spreading of duplicated disease genes.

The increased availability of large-scale open-access resources on bioactivities of small molecules has a significant impact on pharmacology facilitated mainly by computational approaches that digest the vast amounts of data. We discuss here how computational data integration enables systemic views on a drug's action and allows to tackle complex problems such as the large-scale prediction of drug targets, drug repurposing, the molecular mechanisms, cellular responses or side effects. We particularly focus on computational methods that leverage various cell-based transcriptional, proteomic and phenotypic profiles of drug response in order to gain a systemic view of drug action at the molecular, cellular and whole-organism scale.

Combinatorial therapy is a promising strategy for combating complex disorders due to improved efficacy and reduced side effects. However, screening new drug combinations exhaustively is impractical considering all possible combinations between drugs. Here, we present a novel computational approach to predict drug combinations by integrating molecular and pharmacological data. Specifically, drugs are represented by a set of their properties, such as their targets or indications. By integrating several of these features, we show that feature patterns enriched in approved drug combinations are not only predictive for new drug combinations but also provide insights into mechanisms underlying combinatorial therapy. Further analysis confirmed that among our top ranked predictions of effective combinations, 69% are supported by literature, while the others represent novel potential drug combinations. We believe that our proposed approach can help to limit the search space of drug combinations and provide a new way to effectively utilize existing drugs for new purposes.