Fig. 2

Expression Quantification. a Read alignment and assignment rates per library preparation protocol stratified over aligner and annotation. The lighter shade represents the percentage of the total reads that could be aligned and the darker shade the percentage that also was uniquely assigned (see also Supplementary Fig. 3). For comparability, cells were downsampled to 1 million reads/cell, with the exception of 10× Genomics data that were only sequenced to on average 60,000 reads/cell. Hence, these data are farther from saturation and have a higher UMI/read ratio. b Number of genes per UMI with >1 reads for BWA and STAR alignment using the SCRB-seq data set and GENCODE annotation. Colours denote number bins of UMIs. c Number of genes detected per Library Preparation Protocol stratified over Aligner and Annotation (i.e. at least 10% nonzero expression values) (see also Supplementary Fig. 4). d Estimated mean expression, dispersion and gene dropout rates for SCRB-seq and Smart-seq2 data using STAR, BWA or kallisto alignments with GENCODE annotation (see also Supplementary Fig. 7). e Mean-dispersion fitting line applying a cubic smoothing spline with 95% variability bands for SCRB-seq and Smart-seq2 data using STAR, BWA or kallisto alignments with GENCODE annotation (see also Supplementary Fig. 8). f The effect of quantification choices on the power (TPR) to detect differential expression stratified over library preparation and aligner. The expression of 10,000 detected genes over 768 cells (384 cells per group) were simulated given the observed mean-variance relation per protocol. Five percent of the simulated genes are differentially expressed following a symmetric narrow gamma distribution. Unfiltered counts were normalised using scran. Differential expression was tested using limma-trend (see also Supplementary Fig. 9)