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Differential expression analysis in single-cell transcriptomics enables the dissection of cell-type-specific responses to perturbations such as disease, trauma, or experimental manipulations. While many statistical methods are available to identify differentially expressed genes, the principles that distinguish these methods and their performance remain unclear. Here, we show that the relative performance of these methods is contingent on their ability to account for variation between biological replicates. Methods that ignore this inevitable variation are biased and prone to false discoveries. Indeed, the most widely used methods can discover hundreds of differentially expressed genes in the absence of biological differences. To exemplify these principles, we exposed true and false discoveries of differentially expressed genes in the injured mouse spinal cord. Differential expression analysis of single-cell transcriptomics allows scientists to dissect cell-type-specific responses to biological perturbations. Here, the authors show that many commonly used methods are biased and can produce false discoveries.

Novel antibiotics are urgently needed to address the looming global crisis of antibiotic resistance. Historically, the primary source of clinically used antibiotics has been microbial secondary metabolism. Microbial genome sequencing has revealed a plethora of uncharacterized natural antibiotics that remain to be discovered. However, the isolation of these molecules is hindered by the challenge of linking sequence information to the chemical structures of the encoded molecules. Here, we present PRISM 4, a comprehensive platform for prediction of the chemical structures of genomically encoded antibiotics, including all classes of bacterial antibiotics currently in clinical use. The accuracy of chemical structure prediction enables the development of machine-learning methods to predict the likely biological activity of encoded molecules. We apply PRISM 4 to chart secondary metabolite biosynthesis in a collection of over 10,000 bacterial genomes from both cultured isolates and metagenomic datasets, revealing thousands of encoded antibiotics. PRISM 4 is freely available as an interactive web application at http://prism.adapsyn.com . Large-scale sequencing efforts have uncovered a large number of secondary metabolic pathways, but the chemicals they synthesise remain unknown. Here the authors present PRISM 4, which predicts the chemical structures encoded by microbial genome sequences, including all classes of bacterial antibiotics in clinical use.

Microbial natural products represent a rich resource of evolved chemistry that forms the basis for the majority of pharmacotherapeutics. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a particularly interesting class of natural products noted for their unique mode of biosynthesis and biological activities. Analyses of sequenced microbial genomes have revealed an enormous number of biosynthetic loci encoding RiPPs but whose products remain cryptic. In parallel, analyses of bacterial metabolomes typically assign chemical structures to only a minority of detected metabolites. Aligning these 2 disparate sources of data could provide a comprehensive strategy for natural product discovery. Here we present DeepRiPP, an integrated genomic and metabolomic platform that employs machine learning to automate the selective discovery and isolation of novel RiPPs. DeepRiPP includes 3 modules. The first, NLPPrecursor, identifies RiPPs independent of genomic context and neighboring biosynthetic genes. The second module, BARLEY, prioritizes loci that encode novel compounds, while the third, CLAMS, automates the isolation of their corresponding products from complex bacterial extracts. DeepRiPP pinpoints target metabolites using large-scale comparative metabolomics analysis across a database of 10,498 extracts generated from 463 strains. We apply the DeepRiPP platform to expand the landscape of novel RiPPs encoded within sequenced genomes and to discover 3 novel RiPPs, whose structures are exactly as predicted by our platform. By building on advances in machine learning technologies, DeepRiPP integrates genomic and metabolomic data to guide the isolation of novel RiPPs in an automated manner.

We present CFdb, a harmonized resource of interaction proteomics data from 411 co-fractionation mass spectrometry (CF-MS) datasets spanning 21,703 fractions. Meta-analysis of this resource charts protein abundance, phosphorylation, and interactions throughout the tree of life, including a reference map of the human interactome. We show how large-scale CF-MS data can enhance analyses of individual CF-MS datasets, and exemplify this strategy by mapping the honey bee interactome. Co-fractionation mass spectrometry (CF-MS) is a powerful technique for mapping protein interactions under physiological conditions. Here, the authors uniformly re-process 411 CF-MS experiments and carry out meta-analyses of protein abundance, protein-protein interactions, and phosphorylation sites in the resulting resource.

Microbial natural products are an evolved resource of bioactive small molecules, which form the foundation of many modern therapeutic regimes. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) represent a class of natural products which have attracted extensive interest for their diverse chemical structures and potent biological activities. Genome sequencing has revealed that the vast majority of genetically encoded natural products remain unknown. Many bioinformatic resources have therefore been developed to predict the chemical structures of natural products, particularly nonribosomal peptides and polyketides, from sequence data. However, the diversity and complexity of RiPPs have challenged systematic investigation of RiPP diversity, and consequently the vast majority of genetically encoded RiPPs remain chemical “dark matter.” Here, we introduce an algorithm to catalog RiPP biosynthetic gene clusters and chart genetically encoded RiPP chemical space. A global analysis of 65,421 prokaryotic genomes revealed 30,261 RiPP clusters, encoding 2,231 unique products. We further leverage the structure predictions generated by our algorithm to facilitate the genome-guided discovery of a molecule from a rare family of RiPPs. Our results provide the systematic investigation of RiPP genetic and chemical space, revealing the widespread distribution of RiPP biosynthesis throughout the prokaryotic tree of life, and provide a platform for the targeted discovery of RiPPs based on genome sequencing.

Differential accessibility (DA) analysis of single-cell epigenomics data enables the discovery of regulatory programs that establish cell type identity and steer responses to physiological and pathophysiological perturbations. While many statistical methods to identify DA regions have been developed, the principles that determine the performance of these methods remain unclear. As a result, there is no consensus on the most appropriate statistical methods for DA analysis of single-cell epigenomics data. Here, we present a systematic evaluation of statistical methods that have been applied to identify DA regions in single-cell ATAC-seq (scATAC-seq) data. We leverage a compendium of scATAC-seq experiments with matching bulk ATAC-seq or scRNA-seq in order to assess the accuracy, bias, robustness, and scalability of each statistical method. The structure of our experiments also provides the opportunity to define best practices for the analysis of scATAC-seq data beyond DA itself. We leverage this understanding to develop an R package implementing these best practices. Protocol registration The Stage 1 protocol for this Registered Report was accepted in principle on 25th October 2023. The protocol, as accepted by the journal, can be found at https://doi.org/10.6084/m9.figshare.24541816.v1 . The authors conduct a comprehensive assessment of statistical methods for identifying differentially accessible (DA) regions in scATAC-seq data, using a compendium of scATAC-seq experiments paired with bulk ATAC-seq or scRNA-seq from the same cells to evaluate accuracy, bias, robustness, and scalability.

Background An organism’s protein interactome, or complete network of protein-protein interactions, defines the protein complexes that drive cellular processes. Techniques for studying protein complexes have traditionally applied targeted strategies such as yeast two-hybrid or affinity purification-mass spectrometry to assess protein interactions. However, given the vast number of protein complexes, more scalable methods are necessary to accelerate interaction discovery and to construct whole interactomes. We recently developed a complementary technique based on the use of protein correlation profiling (PCP) and stable isotope labeling in amino acids in cell culture (SILAC) to assess chromatographic co-elution as evidence of interacting proteins. Importantly, PCP-SILAC is also capable of measuring protein interactions simultaneously under multiple biological conditions, allowing the detection of treatment-specific changes to an interactome. Given the uniqueness and high dimensionality of co-elution data, new tools are needed to compare protein elution profiles, control false discovery rates, and construct an accurate interactome. Results Here we describe a freely available bioinformatics pipeline, PrInCE, for the analysis of co-elution data. PrInCE is a modular, open-source library that is computationally inexpensive, able to use label and label-free data, and capable of detecting tens of thousands of protein-protein interactions. Using a machine learning approach, PrInCE offers greatly reduced run time, more predicted interactions at the same stringency, prediction of protein complexes, and greater ease of use over previous bioinformatics tools for co-elution data. PrInCE is implemented in Matlab (version R2017a). Source code and standalone executable programs for Windows and Mac OSX are available at https://github.com/fosterlab/PrInCE , where usage instructions can be found. An example dataset and output are also provided for testing purposes. Conclusions PrInCE is the first fast and easy-to-use data analysis pipeline that predicts interactomes and protein complexes from co-elution data. PrInCE allows researchers without bioinformatics expertise to analyze high-throughput co-elution datasets.

Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC–MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products. Microbial natural products represent a large reservoir of potential pharmaceutical agents. Here, Johnston et al . describe a computer-automated programme for connecting genome sequences with identified and isolated natural products.

Elucidating the complete network of protein-protein interactions, or interactome, is a fundamental goal of the post-genomic era, yet existing interactome maps are far from complete. To increase the throughput and resolution of interactome mapping, methods for protein-protein interaction discovery by co-migration have been introduced. However, accurate identification of interacting protein pairs within the resulting large-scale proteomic datasets is challenging. Consequently, most computational pipelines for co-migration data analysis incorporate external genomic datasets to distinguish interacting from non-interacting protein pairs. The effect of this procedure on interactome mapping is poorly understood. Here, we conduct a rigorous analysis of genomic data integration for interactome recovery across a large number of co-migration datasets, spanning diverse experimental and computational methods. We find that genomic data integration leads to an increase in the functional coherence of the resulting interactome maps, but this comes at the expense of a decrease in power to discover novel interactions. Importantly, putative novel interactions predicted by genomic data integration are no more likely to later be experimentally discovered than those predicted from co-migration data alone. Our results reveal a widespread and unappreciated limitation in a methodology that has been widely used to map the interactome of humans and model organisms.