Figure 1. Summary of the raw data, workflow and diagnostic plots from edgeR.
( A) Structure of the amplicons sequenced in a typical shRNA-seq screen. Each amplicon will contain sample and hairpin specific sequences at predetermined locations. In sgRNA-seq screens, the amplicon sequences have a similar structure, with the sgRNA sequence replacing the hairpin. After sequencing, the raw data is available in a fastq file. ( B) The main steps and functions used in an analysis of shRNA/sgRNA-seq screen data in edgeR are shown. ( C) Example of a multidimensional scaling (MDS) plot showing the relationships between replicate dimethyl sulfoxide (DMSO) and Nutlin treated samples (data from Sullivan et al. (2012) 4). MDS plots provide a quick display of overall variability in the screen and can highlight inconsistent samples. ( D) Plot of log 2-fold-change versus hairpin abundance (log 2CPM) for the same data. Hairpins with a false discovery rate < 0.05 from an exact test analysis in edgeR (highlighted in red) may be prioritized for further validation.