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Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. The protocol comprises three major steps: definition of a gene list from omics data, determination of statistically enriched pathways, and visualization and interpretation of the results. We describe how to use this protocol with published examples of differentially expressed genes and mutated cancer genes; however, the principles can be applied to diverse types of omics data. The protocol describes innovative visualization techniques, provides comprehensive background and troubleshooting guidelines, and uses freely available and frequently updated software, including g:Profiler, Gene Set Enrichment Analysis (GSEA), Cytoscape and EnrichmentMap. The complete protocol can be performed in ~4.5 h and is designed for use by biologists with no prior bioinformatics training. This protocol describes pathway enrichment analysis of gene lists from RNA-seq and other genomics experiments using g:Profiler, GSEA, Cytoscape and EnrichmentMap software.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Microsporidia are thought to reproduce mostly asexually, although most are likely diploid based on genomic heterozygosity and conservation of meiotic genes [2–5]. Because of the intimate relationship between microsporidia and their hosts, they are heavily dependent on host resources and have undergone extensive genomic reduction [6,7]. The proteins that have been retained function in essential core cellular processes such as DNA replication, transcription, and translation [6,8,9]. The microsporidia Vavraia culicis was shown to have mistranslation error rates of 5.9% at positions that coded for leucine—although valine, which has a corresponding tRNA synthetases with an intact editing domain, has a mistranslation rate of 7.5%, suggesting that there are other factors responsible for the high levels of mistranslated proteins [12].

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

Microsporidia are a large phylum of intracellular parasites that can infect most types of animals. Species in the Nematocida genus can infect nematodes including Caenorhabditis elegans , which has become an important model to study mechanisms of microsporidia infection. To understand the genomic properties and evolution of nematode-infecting microsporidia, we sequenced the genomes of nine species of microsporidia, including two genera, Enteropsectra and Pancytospora , without any previously sequenced genomes. Core cellular processes, including metabolic pathways, are mostly conserved across genera of nematode-infecting microsporidia. Each species encodes unique proteins belonging to large gene families that are likely used to interact with host cells. Most strikingly, we observed one such family, NemLGF1, is present in both Nematocida and Pancytospora species, but not any other microsporidia. To understand how Nematocida phenotypic traits evolved, we measured the host range, tissue specificity, spore size, and polar tube length of several species in the genus. Our phylogenetic analysis shows that Nematocida is composed of two groups of species with distinct traits and that species with longer polar tubes infect multiple tissues. Together, our work details both genomic and trait evolution between related microsporidia species and provides a useful resource for further understanding microsporidia evolution and infection mechanisms.

Medulloblastoma is the most common malignant brain tumor of childhood. Group 3 medulloblastoma, the most aggressive molecular subtype, frequently disseminates through the leptomeningeal cerebral spinal fluid (CSF) spaces in the brain and spinal cord. The mechanism of dissemination through the CSF remains poorly understood, and the molecular pathways involved in medulloblastoma metastasis and self-renewal are largely unknown. Here we show that NOTCH1 signaling pathway regulates both the initiation of metastasis and the self-renewal of medulloblastoma. We identify a mechanism in which NOTCH1 activates BMI1 through the activation of TWIST1. NOTCH1 expression and activity are directly related to medulloblastoma metastasis and decreased survival rate of tumor-bearing mice. Finally, medulloblastoma-bearing mice intrathecally treated with anti-NRR1, a NOTCH1 blocking antibody, present lower frequency of spinal metastasis and higher survival rate. These findings identify NOTCH1 as a pivotal driver of Group 3 medulloblastoma metastasis and self-renewal, supporting the development of therapies targeting this pathway. Group 3 medulloblastoma is an aggressive pediatric brain tumour that disseminates through the leptomeningeal cerebral spinal fluid. Here, the authors show that in Group 3 medulloblastoma NOTCH1 activates BMI1 through the activation of TWIST1, driving metastasis and self-renewal, and in mouse models a NOTCH1 blocking antibody decreased spinal metastases.

The original version of this Article omitted Suzana A. Kahn, Siddhartha S. Mitra & Samuel H. Cheshier as jointly supervising authors. This has now been corrected in both the PDF and HTML versions of the Article.

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode C. elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work we systematically analyzed every C. elegans AGO, using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

Microsporidia are a large phylum of intracellular parasites that can infect most types of animals. Species in the Nematocida genus can infect nematodes including Caenorhabditis elegans, which has become an important model to study mechanisms of microsporidia infection. To understand the genomic properties and evolution of nematode-infecting microsporidia, we sequenced the genomes of nine species of microsporidia, including two genera, Enteropsectra and Pancytospora, without any previously sequenced genomes. Core cellular processes, including metabolic pathways, are mostly conserved across genera of nematode-infecting microsporidia. Each species encodes unique proteins belonging to large gene families that are likely used to interact with host cells. Most strikingly, we observed one such family, NemLGF1, is present in both Nematocida and Pancytospora species, suggesting horizontal gene transfer between species from different genera. To understand how Nematocida phenotypic traits evolved, we measured the host range, tissue specificity, spore size, and polar tube length of several species in the genus. Our phylogenetic analysis shows that Nematocida is composed of two groups of species with distinct traits and that species with longer polar tubes infect multiple tissues. Together, our work details both genomic and trait evolution between related microsporidia species and provides a useful resource for further understanding microsporidia evolution and infection mechanisms.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We have made revisions to the manuscript to improve the clarity of our study. We have also added some additional analysis in figure S12.