Build single-cell trajectories with the software that introduced **pseudotime**. Find out about cell fate decisions and the genes regulated as they're made. Group and classify your cells based on gene expression. Identify new cell types and states and the genes that distinguish them. Find genes that vary between cell types and states, over trajectories, or in response to perturbations using statistically robust, flexible differential analysis. In development, disease, and throughout life, cells transition from one state to another. Monocle introduced the concept of **pseudotime**, which is a measure of how far a cell has moved through biological progress. Many researchers are using single-cell RNA-Seq to discover new cell types. Monocle 3 can help you purify them or characterize them further by identifying key marker genes that you can use in follow-up experiments such as immunofluorescence or flow sorting. **Single-cell trajectory analysis** shows how cells choose between one of several possible end states. The new reconstruction algorithms introduced in Monocle 3 can robustly reveal branching trajectories, along with the genes that cells use to navigate these decisions.

Cell–cell communication mediated by ligand–receptor complexes is critical to coordinating diverse biological processes, such as development, differentiation and inflammation. To investigate how the context-dependent crosstalk of different cell types enables physiological processes to proceed, we developed CellPhoneDB, a novel repository of ligands, receptors and their interactions. In contrast to other repositories, our database takes into account the subunit architecture of both ligands and receptors, representing heteromeric complexes accurately. We integrated our resource with a statistical framework that predicts enriched cellular interactions between two cell types from single-cell transcriptomics data. Here, we outline the structure and content of our repository, provide procedures for inferring cell–cell communication networks from single-cell RNA sequencing data and present a practical step-by-step guide to help implement the protocol. CellPhoneDB v.2.0 is an updated version of our resource that incorporates additional functionalities to enable users to introduce new interacting molecules and reduces the time and resources needed to interrogate large datasets. CellPhoneDB v.2.0 is publicly available, both as code and as a user-friendly web interface; it can be used by both experts and researchers with little experience in computational genomics. In our protocol, we demonstrate how to evaluate meaningful biological interactions with CellPhoneDB v.2.0 using published datasets. This protocol typically takes ~2 h to complete, from installation to statistical analysis and visualization, for a dataset of ~10 GB, 10,000 cells and 19 cell types, and using five threads.

Tumors are complex tissues of cancerous cells surrounded by a heterogeneous cellular microenvironment with which they interact. Single-cell sequencing enables molecular characterization of single cells within the tumor. However, cell annotation—the assignment of cell type or cell state to each sequenced cell—is a challenge, especially identifying tumor cells within single-cell or spatial sequencing experiments. Here, we propose ikarus, a machine learning pipeline aimed at distinguishing tumor cells from normal cells at the single-cell level. We test ikarus on multiple single-cell datasets, showing that it achieves high sensitivity and specificity in multiple experimental contexts. **InferCNV** is a Bayesian method, which agglomerates the expression signal of genomically adjointed genes to ascertain whether there is a gain or loss of a certain larger genomic segment. We have used **inferCNV** to call copy number variations in all samples used in the manuscript.