Assessing the sensitivity and reproducibility of the screening platform. We systematically depleted subsets of hairpins by 25%, 50% or 75% within a 10k pool and compared them to a non-depleted reference set 3 days after infection of MCF7 cells (see also Additional File 1). (a) Scatter plot of log2 normalized read counts from reference and depletion sets. (b) Density plot showing the distributions of the depleted hairpin subsets: 25% depleted hairpins are plotted in red, 50% depleted in green and 75% depleted in blue. The screening methodology was capable of detecting 50% depletion in hairpin representation with high accuracy in a single experiment. (c) Scatter plot of the depletion-reference log ratio from two biological replicates, indicating a high correlation (r2 = 0.92) and thus a reproducible screening method. (d) Plot depicting the false positive rate at a fixed false negative rate of 5% in a reference depletion experiment using different numbers of PCR cycles, indicating a decrease in the false positive rate with decreased PCR cycles.
Sims et al. Genome Biology 2011 12:R104 doi:10.1186/gb-2011-12-10-r104