Proportion of reads that contribute to pile-ups as a function of the minimum size of a pile-up. Along Y-axes we have plotted the ratio between the number of reads mapping to pile-ups and the total number of all mapped reads (pile-up-to-read ratio). Along X-axes we have plotted the minimum number of reads that map to the same location to be considered a pile-up. The pile-up-to-read ratio is 1 when the minimum number of reads per pile-up is 1 since in this case all reads are considered to be part of a pile-up. As the minimum number of reads per pile-up is increased, the ratio declines. The magnitude of the decline is a measure of the extent to which the set of reads has a tendency to form pile-ups (faster decline means fewer pile-ups). (a) Genome-wide pile-up-to-read ratios of 5’ read mappings. (b) and (c) pile-up-to-read ratios for the GABAB1 gene and the ACTB gene, respectively. For both the genome-wide and the gene-specific cases, the pile-up-to-read ratios decrease in the order Ctl, Heat, and Heat + OptiK. Thus, Ctl has the highest tendency to form pile-ups, while Heat + OptiK has the lowest tendency.