(A) Immunofluorescence of PCF or BSF Lister 427 cells and BSF strain Lister 427 with anti-VSG 221 antiserum or with anti-EP procyclin antiserum; top panels show the cells stained with DAPI, while the bottom panel shows the cells’ outline by DIC. Images were acquired with the Axioskop 2 imaging system and the scale bar represents 5 μm. (B) The Lister 427 bloodstream VSG expression site (BES) TAR clones sequenced by (Hertz-Fowler et al., 2008) were used to map the MFAseq data from BSF and PCF cells; note that two BES are represented by duplicate TAR clones: BES 7 (ϕ – TAR 65; ϕϕ – TAR 153), and BES 17 (ϕ – TAR 51; ϕϕ – TAR 59). The ratio between sequence coverage (read-depth) in early S phase and G2 phase cells, or late S phase and G2 phase samples, is plotted, where each point represents the median S/G2 ratio (y-axis) per 2.5 Kbp bin across the BES (x-axis). The size of each BES is shown on each x-axis in 10 Kbp intervals, and all graphs are scaled according to BES size. The y-axis scale is the same for all graphs, but the legend is only shown on the ones at the far left. BSF early S data is represented as light red, BSF late S as dark red, PCF early S as light green, and PCF late S as dark green. The red dashed box highlights BES 1. (C) The S/G2 values used to generate the graphs in (B) are shown plotted per sample (BSF early S – light red, BSF late S – dark red, PCF early S – light green, and PCF late S – dark green), rather than by genomic location, for each BES (numbered as before). Horizontal bars (black) represent the median of the S/G2 values, and error bars the interquartile range. In order to infer statistical significance, the values were analysed with the non-parametric, unmatched, Kruskal-Wallis test; statistical significance is only shown for differences between the BSF and PCF samples: (**) p-value <0.01; (***) p-value <0.001; (****) p-value <0.0001.