Empirical determination of a cutoff for filtering lowly-expressed genes using ERCC controls
The 92 ERCC 1 mix transcripts were added to pre-vaccination samples, and the ERCC 2 mix was added to each post-vaccination sample. ERCC spike-ins cover a range of concentrations from 0.014 to 30,000 CPM for both mixes, and the expected LFCs from mix 2 to mix 1 range from 0.25 to 2. For paired samples, the empirical fold change (based on fold change in CPM from the RNA-Seq experiment) and expected fold changes were determined for each of the 92 ERCC transcripts. Likewise, the mean LCPM of each ERCC transcript was determined by first calculating the average LCPM value for each paired sample, and then taking the mean of those averages. This results in a weighted average for each ERCC transcript across the pre- and post-vaccination samples. Based on the distribution of the mean ERCC LCPM, the 92 spike-ins were allocated into 7 bins of increasing abundance from low to high (≥13 transcripts per bin).Next, for each paired sample, the Spearman correlation between the empirical and the expected fold changes was calculated for the subset of ERCC transcripts within each bin. The mean LCPM of the transcripts within each bin was also computed for each paired sample. This produces a pair of values—the Spearman correlation and the mean LCPM—for each paired sample within each bin of ERCC transcripts. A 3The 