When the Ino18 probe (middle panel of Figure 4A ) was used with a target with 1 mmoi [i.e. (0.10) 35_1_17 (9.8)] or even 10 mmoi [(10.10) 26_10_26 (0.8)] located at the 5 0 and 3 0 ends of the target, outside the central region containing dInosines, there was no or little inhibition of hybridization at 45 C (71% of the total MFI, 4741 MFI, and 41% of the total MFI, 2773 MFI, respectively). Interestingly, when the 10 mmoi were evenly distributed within the region of 18 dInosines, as in the (0.11) 26_10_26 (9.8) target, hybridization was lost (0.9% of the total MFI, 59 MFI). The sensitivity to mmoi adjacent to dInosine was therefore investigated further. Targets with increasing numbers (2, 4 or 5) of mmoi neighboring the positions of dInosines successively reduced the hybridization signals: 2319 MFI (35% of the total) for the (10.10)_24_12_28_(0.8) target, 927 MFI (13%) for the (10.10)_22_14_30_(0.8) target, and 375 MFI (5.4%) for the (10.10)_21_15_31_(0.8) target, all at 45 C. Thus, dInosine is sensitive to neighboring mismatches. A comparison of the sensitivity of a dInosine-free probe (InflA) and a probe containing dInosine (Ino18) to neighboring mismatches showed that the dInosine-free InflA probe can hybridize to a target with 17 evenly distributed mismatches between two perfectly matching flanking sections of 9 and 8 nt, respectively [the (0.10) 35_1_17 (9.8) target, 24% of the total MFI, 1614 MFI at 45 C]. However, 7 mmoi adjacent to the dInosines completely destroyed hybridization between the Ino18 probe and the (10.10) 19_17_33 (0.8) target: 0.5% of the total MFI, 34 MFI, at 45 C. Results for 55 C are shown in Supplementary Figure S4B and Table 3 . The Ino18 probe failed to hybridize when 2 mmoi were placed next to the dInosines PAGE 11 OF . Thus, the hybridization capacity of a dInosine-containing sequence is severely reduced when the mismatch is adjacent to the dInosine. Figure 4A also shows how introduction of many dInosines affects the specificity. There was no hybridization between the influenza probe Ino18 with its 18 dInosines and the Norovirus target (70) 0_36_52 (0.8). Furthermore, although the dInosines mask 16 mismatches in the (0.11) 26_10_26 (9.8) target, the 10 mmoi that are in close proximity to the dInosines abolish hybridization (0.9% of total MFI, 59 MFI). In contrast, the other target containing 26 mismatching nt and 10 distant mmoi, (10.10) 26_10_26 (0.8), did hybridize to the Ino18 probe (41% of total MFI, 2773 MFI). Thus, the cross hybridization of a foreign (unrelated) dInosine-containing probe is dependent to a certain extent on the amount of mismatch, but is even more dependent on the distribution of mmoi. Figure 4B and Supplementary Table S6 show the origin of the nt that are not covered by the dInosines when using the Ino18 probe (MFI values from Figure 4A ). They demonstrate the number of InflA-matching nt outside the dInosine position (moi) needed for hybridization and the number of Norovirus moi causing cross hybridization. At least 37-38 InflA moi were needed to induce hybridization with the Ino18 probe but, as mentioned above, the distribution is at least as important as the actual number of matching and mismatching nt. Of the fewer than 30-31 nt that were of Norovirus origin in a target that hybridized with the Ino18 probe, 16 nt were common to both Norovirus and Influenza virus. If more than 31 nt were of Norovirus origin, hybridization to the Influenza Ino18 probe failed.