When choosing negative controls, we prioritised mutations that were observed multiple times in clinical samples, are close to the drug binding site and do not involve a charge change or a proline residue. For the RNAP, L443F was selected since it lies within the RRDR and is close to the rifampicin binding site yet does not confer resistance and therefore is a good negative control (Fig. ). We also selected S388L and T585A which are further from the binding site and are seen in clinical samples. Finally, we choose an amino acid (Ser428) at which non-synonymous mutations are expected to confer resistance, since it lies in the RRDR, but for which no firm statistical association has been made, and choose a mutation (S428C) which minimally chemically perturbs the sidechain. We expect this to not confer resistance, since it has not been observed clinically and the sidechain points away from the drug and is therefore a good, if somewhat artificial, negative control.