Current best practices in single‐cell RNA‐seq analysis: a tutorial
Malte D Luecken
Fabian J Theis
Author Information
Mol Syst Biol (2019)15:e8746https://doi.org/10.15252/msb.20188746

Figure 1. Schematic of a typical single‐cell RNA‐seq analysis workflow
Raw sequencing data are processed and aligned to give count matrices, which represent the start of the workflow. The count data undergo pre‐processing and downstream analysis. Subplots are generated using the best‐practices workflow on intestinal epithelium data from Haber et al (2017).

Figure 5. Overview of downstream analysis methods Methods are divided into cell‐ and gene‐level analysis. Cell‐level analysis approaches are again subdivided into cluster and trajectory analysis branches, which include also gene‐level analysis methods. All methods with a blue background are gene‐level approaches.

Figure 6. Cluster analysis results of mouse intestinal epithelium dataset from Haber et al (2017)
(A) Annotated cell‐identity clusters found by Louvain clustering visualized in a UMAP representation. (B) Cell‐identity marker expression to identify stem cells (Slc12a2), enterocytes (Arg2), goblet cells (Tff3) and Paneth cells (Defa24). Corrected expression levels are visualized from low expression (grey) to high expression (red). Marker genes may be expressed also in other cell‐identity populations as shown for goblet and Paneth cells. (C) Cell‐identity composition heat maps of proximal (upper) and distal (lower) intestinal epithelium regions. High relative cell density is shown as dark red.

Figure 7. Trajectory analysis and graph abstraction of mouse intestinal epithelium data from Haber et al (2017)
(A) Distal and proximal enterocyte differentiation trajectories inferred by Slingshot. The Distal lineage is shown coloured by pseudotime from red to blue. Other cells in the dataset are grey. (B) Slingshot trajectories over clusters in PCA space. Clusters are abbreviated as follows: EP—enterocyte progenitors; Imm. Ent.—immature enterocytes; Mat. Ent.—mature enterocytes; Prox.—proximal; Dist.—distal. (C) Density over pseudotime for the distal enterocyte trajectory from Fig 7A. Colours represent the dominant cluster labels in each pseudotime bin. (D) Abstracted graph representation of the dataset projected onto a UMAP representation. Clusters are shown as coloured nodes. Clusters that appear in other trajectories are labelled for comparison. “TA” denotes transit amplifying cells. (E) Gene expression dynamics over pseudotime in a general enterocyte trajectory using the “GAM” R library.