PDD-mediated rRNA depletion. A: 18S rRNA profile of read 5′ end-density (PDD-treated as a percentage of that of untreated) from the total RNA library. Reads were divided into the insert length ranges indicated on the right and normalized to the number of reads mapped to mRNA ORFs within each size category. Annealing positions of depletion probes are indicated (red bars). B: Pile-up of 5′ read densities (PDD-treated as a percentage of untreated) flanking all probes targeting 18S and 25S rRNA (moving average with 9 nt window, x-coordinate is relative to the probe 3′ end) in four different size-ranges of library inserts. Black lines: average read ratio of smoothed (moving average, 7 nt window) individual profiles. Probe lengths are indicated by line colours. C: Trade-off between library insert size and probe spacing from the total RNA library. For each pair of adjacent probes along rRNA, reads falling between probes (+10 nt extending into each probe) were counted, and used to calculate a normalized read ratio of each inter-probe segment of rRNA (PDD-treated as a percentage of untreated; y-axis). This was compared to the intervening distance between probes (x-axis, see Additional file 2: Figure S1B for a schematic). Four different library insert size ranges were analysed and the minimum insert size for each group is indicated on the x-axis (coloured arrowheads).