A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor

Single-cell RNA sequencing (scRNA-seq) is widely used to profile the transcriptome of individual cells. This provides biological resolution that cannot be matched by bulk RNA sequencing, at the cost of increased technical noise and data complexity. The differences between scRNA-seq and bulk RNA-seq data mean that the analysis of the former cannot be performed by recycling bioinformatics pipelines for the latter. Rather, dedicated single-cell methods are required at various steps to exploit the cellular resolution while accounting for technical noise.

Here, researchers from Cancer Research UK describe a computational workflow for low-level analyses of scRNA-seq data, based primarily on software packages from the open-source Bioconductor project. It covers basic steps including quality control, data exploration and normalization, as well as more complex procedures such as cell cycle phase assignment, identification of highly variable and correlated genes, clustering into subpopulations and marker gene detection. Analyses were demonstrated on gene-level count data from several publicly available datasets involving haematopoietic stem cells, brain-derived cells, T-helper cells and mouse embryonic stem cells. This will provide a range of usage scenarios from which readers can construct their own analysis pipelines.

Heatmap of mean-centred normalized log-expression values for
correlated HVGs in the HSC dataset.

rna-seqDendrograms are formed by hierarchical clustering on the Euclidean distances between genes (row) or cells (column).

Availability – All software packages used in this workflow are publicly available from the Comprehensive R Archive Network (https://cran.r-project.org) or the Bioconductor project (http://bioconductor.org).

Lun AT, McCarthy DJ, Marioni JC. (2016) A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. F1000Res 5:2122. [article]

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