Tools, simulations and trees for scRNA-seq
The past few years have seen an explosion in the development of single-cell RNA-sequencing technology and it has quickly become a commonly used tool for interrogating complex tissues. Access to this new type of data has lead to a corresponding surge in the production of statistical and computational tools to analyse it. We are cataloguing in the scRNA-tools database (www.scRNA-tools.org). Deciding which of these tools to use for a specific task is difficult and comprehensive evaluations and comparisons are required. One way to demonstrate how well tools perform at their selected task is by testing them on simulated data. To make this easier we developed Splatter, a Bioconductor R package that provides a consistent, easy-to-use interface for multiple models for simulating scRNA-seq data (https://bioconductor.org/packages/splatter). Providing independent simulation software avoids relying on simulations that are not reproducible, match the tools assumptions and do not demonstrate similarity to real datasets.
Even the most effective methods usually have parameters that affect how they perform. For scRNA-seq data one of the analysis tasks that has received the most attention is defining groups of similar cells, usually through unsupervised clustering. Most clustering methods have parameters which, directly or indirectly, affect the number of clusters produced. The clustering resolution that is chosen can have a profound effect on further analysis and interpretation but it is unclear how to make this choice. To aid analysts in deciding which clustering resolution to use we have developed clustering trees, a visualisation that shows how clusters form and change as the resolution increases. These trees can be produced using the clustree R package (http://cran.r-project.org/package=clustree) and are applicable to any clustering method. Clustering trees highlight instability that may indicate over clustering and help choose which resolution to use, particularly when combined with existing domain knowledge such as the expression of marker genes. This presentation will demonstrate our methods and tools using an scRNA-seq dataset we have generated to explore the cell type composition of kidney organoids.