Mean alignment neutrality organized by distance metrics. The line represents wildtype sequence neutrality equal to mean background neutrality. If the wildtype sequence neutrality is higher than the mean background neutrality, the sequence is considered robust. To reduce the number of points, only the mean sequence neutrality for an alignment is compared against the average of the mean background neutrality. Plotting individual sequence neutrality reveals a similar trend (Additional file 1: Figure S2). The SEN better detects mutational robustness of these sequences compared to PCC or bp-distance.