Fig S1 – RNA-seq data quality, related to Fig 1, Supplemental Experimental Procedures (A) Spearman correlation between all pairs of all samples for relative normalized TPM abundances. Neighboring samples are as correlated as replicates. (B) Scatter plots of all TPM abundances for all samples. Color indicates density of points. Horizontal and vertical strips indicate measurements only present in one sample. (C) RNA standard performance. Scatter plots compare RNA standard TPM abundances between all pairs of datasets. Color indicates spike-in species. Red boxes mark ArrayControl spikes present only in Clutch B PolyA+. Although, we find small numbers of reads that align uniquely to ArrayControl sequences when ArrayControl spikes are not present in the sample. We note that this is not true for ERCC spikes, we never find ERCC reads when ERCC spikes are not present (data not shown). ERCC PolyA+ and rdRNA comparisons lie off the diagonal indicating poor PolyA+ performance. ERCC-00116 has the worst PolyA+ performance and is not used in absolute normalization. (D) Identification of poorly performing libraries. Normalized leave-one-out score (Supplemental Experimental Procedures), higher scores indicate a poorly performing sample. Samples adjacent to a bad sample can also receive high scores. Red and blue samples in Clutch A PolyA+ are selected for repeat library construction and sequencing (E). Red samples excluded from all analysis. (E) Technical replicates in Clutch A PolyA+ of poorly performing samples reveal that the poor performance is highly reproducible indicating that performance issues are not due to library construction and sequencing. Red boxes marked those discarded, black boxes marked those retained. (F) Projections onto the first two principle components for Clutch A PolyA+ (left), Clutch B PolyA+ (center), and Clutch A rdRNA (right). Note strong temporal correlation between samples, major changes in the transcriptome are visible.
Image published in: Owens ND et al. (2016)
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