Sources of bias in RNA-seq coverage. (A) RNA-seq coverage plots for IVT transcript BC003355 from simulated (black), plasmid (blue), no selection (green), rRNA-depleted (red), and polyA (orange) data. The gene model is displayed in black, below all of the coverage plots. Blocks correspond to exons and lines indicate introns. The chevrons within the intronic lines indicate the direction of transcription. (B) Distributions for coefficients of variation across data displayed above, with the addition of the QM-simulated data (gray). Note that while the graph is cutoff at a coefficient of variation of 1.3, the tails for the rRNA-depleted and polyA distributions extend out to 2.13 and 2.7, respectively. (C) Effect sizes for the differences in distribution of coefficients of variation between sequencing libraries and simulated data. Effect sizes are calculated as the per-transcript ratios of coefficients of variation between a given library and the simulated dataset. QM, quantity matched.