A guide to single-cell RNA sequencing analysis using web-based tools for non-bioinformatician

Single-cell RNA sequencing (scRNA-seq) is a technique that has proven to be a powerful tool for a wide range of fields and research studies. However, scRNA-seq data analysis has been dominated by scientists highly trained in bioinformatics or those with extensive computational experience and understanding. Recently, this trend has begun to shift as more user-friendly web-based scRNA-seq analysis tools have been developed that require little computational experience to use. However, barriers persist for non-bioinformaticians in using this technique. Complex, unfamiliar language and scarce comprehensive literature guidance to provide a framework for understanding scRNA-seq analysis outputs are among the obstacles. Researchers from Vanderbilt University introduce many popular web-based tools for scRNA-seq and provides a general overview of their user interfaces and features. Then, a comprehensive start-to-finish introductory scRNA-seq analysis pipeline is described in detail, that aims to enable researchers to carry out scRNA-seq analysis, regardless of computational experience. However, as scRNA-seq continues to penetrate new fields and expand in importance, there remains a need for more literature to help overcome barriers to its use by explaining further the highly complex and advanced analyses that are introduced within this paper.

General computational scRNA-seq analysis workflow

Libraries are initially processed to remove dead cells and confounding factors such as debris and batch effects. Then, the data is plotted for visualization purposes in different mediums to check for batch effects and to visualize subpopulations. Cell types can then be identified through clustering and identification of marker gene distribution, differential gene analysis can be performed, or other advanced approaches can be pursued.

Companion video tutorials can be found at ‘EasyScRNAseqTutorials’ on YouTube (https://www.youtube.com/@scrnaseqtutorials).