1. NI Assays: Troubleshooting & Analysis of Point-to-Point IC50 determination Graph 1: Standard clinical isolate – Technical Error Dark blue = first test. Pale blue = repeat test Poor replicate agreement Viewing replicate curves as a mean would have lead to high IC50 value being calculated Conclusion: If replicates are performed, they must be viewed individually 1

2. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 2: Standard clinical isolate – Technical Error (2) Dark blue = first test. Pale blue = repeat test Replicates agree fully Increasing NA activity with increasing drug concentration (implausible) High IC50 Conclusion: Technical error which would lead incorrectly to high IC50 → REPEAT 2

3. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 3: Dispensing error Failure to calculate correct IC50 Conclusion: Probably isolates with normal IC50 → REPEAT 3

4. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 4: Drug Titration Error Curves begin normally normal IC50 would be calculated S shape is not completed Conclusion: Probably normal isolates → REPEAT to check 4

5. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 5: Clinical isolate with High IC50 Good NA activity Good reproducibility of curve shape on repeat Curve does not fit shape of any other circulating strain High IC50 Conclusion: Probable resistant isolate → confirm by sequencing 5

6. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 6: Clinical isolate with high IC50 Reported as H3 (typical IC50 0.2-0.8nM) Good NA activity Shifted curve compared with subtype matched reference wild type High IC50 Curve shape match with alternate subtype (Flu B IC50 range 20- 40nM) Conclusion: Probable incorrect subtype reporting/mixed virus (H3 and FluB) → Retype by PCR 6

7. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 7: Classical Resistant Curves (1) No change in NA activity compared with wild type Shift in sigmoidal curve to left Yellow and Pink lines show low titre viruses therefore low NA activity, but still fit the pattern of wild type and resistant 7

8. NI Assays: Troubleshooting & Analysis of Point-to- Point IC50 determination Graph 8: Classical resistant curves (2) Low NA activity compared with wild type Flattened curves showing limited dose response 292K mutant almost beyond range of assay at these drug concentrations 8