Heterogeneity of coverage at that level observed for all samples may be caused by suboptimal capture washes. A stringent wash not performed carefully enough (2 times for 5 minutes at 65 °C and not above this temperature) can result in preferential loss of the AT-rich regions over the GC rich regions, leading to a high coverage heterogeneity. The nature and the quality of the DNA samples can also lead to high coverage heterogeneity, as is frequently observed with FFPE samples.

Moreover, evaporation may occur during the O.N. hybridization. Ensure that the 0.2 ml tubes are tightly closed to avoid leakage over time.