Functional and evolutionary significance of unknown genes from uncultivated taxa.

Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies1. Here we analysed 149,842 environmental genomes from multiple habitats2-6 and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.

eggNOG 6.0: enabling comparative genomics across 12 535 organisms.

The eggNOG (evolutionary gene genealogy Non-supervised Orthologous Groups) database is a bioinformatics resource providing orthology data and comprehensive functional information for organisms from all domains of life. Here, we present a major update of the database and website (version 6.0), which increases the number of covered organisms to 12 535 reference species, expands functional annotations, and implements new functionality. In total, eggNOG 6.0 provides a hierarchy of over 17M orthologous groups (OGs) computed at 1601 taxonomic levels, spanning 10 756 bacterial, 457 archaeal and 1322 eukaryotic organisms. OGs have been thoroughly annotated using recent knowledge from functional databases, including KEGG, Gene Ontology, UniProtKB, BiGG, CAZy, CARD, PFAM and SMART. eggNOG also offers phylogenetic trees for all OGs, maximising utility and versatility for end users while allowing researchers to investigate the evolutionary history of speciation and duplication events as well as the phylogenetic distribution of functional terms within each OG. Furthermore, the eggNOG 6.0 website contains new functionality to mine orthology and functional data with ease, including the possibility of generating phylogenetic profiles for multiple OGs across species or identifying single-copy OGs at custom taxonomic levels. eggNOG 6.0 is available at http://eggnog6.embl.de.

PhyloCloud: an online platform for making sense of phylogenomic data.

Phylogenomics data have grown exponentially over the last decades. It is currently common for genome-wide projects to generate hundreds or even thousands of phylogenetic trees and multiple sequence alignments, which may also be very large in size. However, the analysis and interpretation of such data still depends on custom bioinformatic and visualisation workflows that are largely unattainable for non-expert users. Here, we present PhyloCloud, an online platform aimed at hosting, indexing and exploring large phylogenetic tree collections, providing also seamless access to common analyses and operations, such as node annotation, searching, topology editing, automatic tree rooting, orthology detection and more. In addition, PhyloCloud provides quick access to tools that allow users to build their own phylogenies using fast predefined workflows, graphically compare tree topologies, or query taxonomic databases such as NBCI or GTDB. Finally, PhyloCloud offers a novel tree visualisation system based on ETE Toolkit v4.0, which can be used to explore very large trees and enhance them with custom annotations and multiple sequence alignments. The platform allows for sharing tree collections and specific tree views via private links, or make them fully public, serving also as a repository of phylogenomic data. PhyloCloud is available at https://phylocloud.cgmlab.org.

GeCoViz: genomic context visualisation of prokaryotic genes from a functional and evolutionary perspective.

Synteny conservation analysis is a well-established methodology to investigate the potential functional role of unknown prokaryotic genes. However, bioinformatic tools to reconstruct and visualise genomic contexts usually depend on slow computations, are restricted to narrow taxonomic ranges, and/or do not allow for the functional and interactive exploration of neighbouring genes across different species. Here, we present GeCoViz, an online resource built upon 12 221 reference prokaryotic genomes that provides fast and interactive visualisation of custom genomic regions anchored by any target gene, which can be sought by either name, orthologous group (KEGGs, eggNOGs), protein domain (PFAM) or sequence. To facilitate functional and evolutionary interpretation, GeCoViz allows to customise the taxonomic scope of each analysis and provides comprehensive annotations of the neighbouring genes. Interactive visualisation options include, among others, the scaled representations of gene lengths and genomic distances, and on the fly calculation of synteny conservation of neighbouring genes, which can be highlighted based on custom thresholds. The resulting plots can be downloaded as high-quality images for publishing purposes. Overall, GeCoViz offers an easy-to-use, comprehensive, fast and interactive web-based tool for investigating the genomic context of prokaryotic genes, and is freely available at https://gecoviz.cgmlab.org.