Overview of location, extraction method, quantitation method and mutation detection method for each laboratory
| ID | Country | Extraction method | Quantitation method | EGFR detection method | BRAF detection method |
|---|---|---|---|---|---|
| Lab A | USA | Manual silica matrix solid-phase purification (Qiagen QIAamp) | Nanodrop | Sanger sequencing (in-house assay) | qPCR (in-house assay) |
| Lab B | UK | Automated silica matrix solid-phase purification (Qiagen EZ1) | Nanodrop | Pyrosequencing (in-house assay) | Pyrosequencing (in-house assay) |
| Lab C | USA | Automated silica matrix solid-phase purification (Qiagen EZ1) | Nanodrop | Fragment analysis (in-house assay) | qPCR (in-house assay) |
| Lab D | UK | Manual silica matrix solid-phase purification (LifeTech RecoverAll) | Nanodrop | NT | Sanger sequencing (in-house assay) |
| Lab E | USA | Manual silica matrix solid-phase purification (Qiagen DNeasy) | Nanodrop | NT | NT |
| Lab F | UK | Manual silica matrix solid-phase purification (Roche COBAS) | Nanodrop | qPCR (Roche COBAS) | qPCR (Roche COBAS) |
| Lab G | USA | Manual silica matrix solid-phase purification (Qiagen QIAamp) | Nanodrop | SNaPshot sequencing (in-house assay) | qPCR (in-house assay) |
| Lab H | USA | Manual silica matrix solid-phase purification (Qiagen QIAamp) | Nanodrop | Sanger sequencing (in-house assay) | Sanger sequencing (in-house assay) |
| Lab I | USA | Manual silica matrix solid-phase purification (Qiagen QIAamp) | Nanodrop | qPCR (Qiagen Therascreen) | Pyrosequencing |
| Lab J | USA | Manual silica matrix solid-phase purification (LifeTech RecoverAll w/mods) | Nanodrop | Massively paralleled next-generation sequencing (Ion Torrent PGM) | Massively paralleled next generation sequencing (Ion Torrent PGM) |
| Lab K | UK | Manual silica matrix solid-phase purification (Qiagen DNeasy) | Nanodrop | NT | Pyrosequencing |
| Lab L | UK | Automated silica matrix solid-phase purification (Qiagen EZ1) | Nanodrop | Sanger sequencing | Pyrosequencing |
| Lab M | UK | Manual silica matrix solid-phase purification (Qiagen QIAamp) | Qubit | Sanger sequencing | Sanger sequencing |
‘NT’ indicates the mutation in question was not routinely tested. Five laboratories were based in the UK and seven in the USA. The majority (12/13) were molecular pathology laboratories associated with university hospitals. Laboratory J was a private laboratory based in the USA. Laboratory E did not perform routine epidermal growth factor receptor (EGFR) or BRAF mutation testing but did perform DNA extraction from formalin-fixed paraffin-embedded. Laboratories D and K routinely screened for BRAF mutations but not EGFR mutations. Laboratories C and G did not include the EGFR G719S mutation in their EGFR testing panel. The remaining laboratories routinely tested for both EGFR and BRAF mutations. DNA extraction method varied though Qiagen products were favoured. Notably, DNA quantitation by Nanodrop spectrophotometry was used by all laboratories except for laboratory M, which used Qubit Fluorometry. Mutation analysis was performed using a range of methods, of which Sanger sequencing and Pyrosequencing were the most common.