Additional file 1 of Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets

Additional file 1: Fig. S1 Total gene detection of all datasets compared after processing with either kallisto or Cell Ranger. The Venn diagrams show commonly detected number of genes by both pipelines and uniquely detected genes. Fig. S2 Violin-plots showing distribution of gene and UMI detection per cell of all the analyzed datasets (Table 1) run with the Cell Ranger pipeline. Fig. S3 Violin-plots showing distribution of gene and UMI detection per cell of all the analyzed datasets (Table 1) run with the kallisto pipeline. Fig. S4 Cell counts of all datasets compared after processing with either kallisto forced or Cell Ranger. The Venn diagrams show commonly detected cell barcodes by both pipelines and uniquely detected cell barcodes. Fig. S5 Alignment results of all datasets (Table 1) run with either Cell Ranger or kallisto forced against Ensembl reference. a Percent alignment rates of all reads against the reference transcriptome. b Total gene detection. c Median gene counts over all cells per dataset. d Median UMI counts over all cells per dataset. e Total cell counts of each dataset. Fig. S6 Total gene detection of all datasets compared after processing with either kallisto forced or Cell Ranger. The Venn diagrams show commonly detected number of genes by both pipelines and uniquely detected genes. Fig. S7 Violin-plots showing distribution of gene and UMI detection per cell of all the analyzed datasets (Table 1) run with the kallisto forced pipeline. Fig. S8 Violin-plots showing distribution of gene and UMI detection per cell of the dr_pineal_s2 dataset after additional filtering for downstream analysis. Run with either Cell Ranger (a), kallisto (b) or kallisto forced (c). Fig. S9 Downstream analysis of dr_pineal_s2 before cluster merging. a 2D visualization using UMAP of Cell Ranger analyzed clusters before merging, with resolution equal to 0.9. Each point represents a single cell, colored according to cell type. The cells were clustered into 21 types. b Expression profile of marker genes according to cluster [7] of (a). Clusters 0, 1, 8 and 18 are all rod-like PhRs subclusters. They expressed rod-like PhR markers (exorh, gant1, gngt1), but the expression levels differed and resulted in their separation. For simplicity, they were merged and referred as a single rod-like PhRs cluster in the main text. Similarly, cluster 7 and 12 were merged into a single Müller-glia like cluster, clusters 2, 5, 16 were merged into a single RPE-like cluster, clusters 3 and 10 were merged into a single habenula kiss1 cluster and cluster 11 and 19 were merged into a single leukocytes cluster. c. 2D visualization using UMAP of Cell Ranger analyzed clusters, with resolution equal to 2. The cells were clustered into 31 types. However, the two different cone-like PhR cell types are still not distinguished from one another. d Expression profile of marker genes according to cluster of (c). e 2D visualization using UMAP of kallisto analyzed dr_pineal_s2 clusters before merging, with resolution equal to 0.9. The cells were clustered into 24 types. f Expression profile of marker genes according to cluster of (c). Similar to the descried above, clusters 1, 2, 3, 7 and 21 were merged into a single rod-like PhRs cluster, clusters 0, 9, 17 were merged into a single RPE-like cluster, clusters 11 and 12 were merged into a single Müller-glia like cluster, clusters 4, 5 and 20 were merged into a single habenula kiss1 cluster and clusters 13 and 22 were merged into a single leukocytes cluster. g 2D visualization using UMAP of kallisto forced analyzed dr_pineal_s2 clusters, with resolution equal to 1.2. The cells were clustered into 27 types. h Expression profile of marker genes according to cluster of (g). The col14a1b gene was only detected in the kallisto and kallisto forced datasets and is the strongest DE marker within the red cone-like cluster (f, h). Fig. S10 Heatmap of genes with higher counts in kallisto pre-processed pineal data. All the UMI counts for both kallisto and Cell Ranger were summed, and the diff_ratio value was calculated ( kallisto _ counts − CellRanger _ counts kallisto _ counts + CellRanger _ counts $$\frac{\left( kallisto\_ counts- CellRanger\_ counts\right)}{\left( kallisto\_ counts+ CellRanger\_ counts\right)}$$ ) for each gene (Additional file 1: Fig. 10). The top 80 diff_ratio genes, as well as the top 20 genes uniquely identified in kallisto were plotted according to the average scaled expression per cluster. Fig. S11 Heatmap of genes with higher counts in Cell Ranger pre-processed pineal data. All the UMI counts for both kallisto and Cell Ranger were summed, and the diff_ratio value was calculated ( kallisto _ counts − CellRanger _ counts kallisto _ counts + CellRanger _ counts $$\frac{\left( kallisto\_ counts- CellRanger\_ counts\right)}{\left( kallisto\_ counts+ CellRanger\_ counts\right)}$$ ) for each gene (Additional file 1: Fig. S11). The top 80 diff_ratio genes, as well as the top 20 genes uniquely identified in Cell Ranger were plotted according to the average scaled expression per cluster.