Analyses were performed in duplicates using the LightCycler 480 platform. Cisplatin cost Each run included a wild type control and a mutant, p. V600E, control for nor malization. Results were analyzed by Gene Scanning software with normalized, temperature shifted melting curves displayed as difference plot. Samples showing a melting behavior differing from the wildtype control but not that of a mutant sample were considered as border line samples. These samples were retested by direct Sanger sequencing of HRM products. Sanger sequencing Sanger sequencing was performed on the same amplicons as used for HRM analysis. 5 ul of PCR products were purified with exonuclease I and Fast AP for 15 min at 37 C and 15 min by 80 C. A sequencing reaction was set up with 1 ul of purified PCR products and the BigDye Terminator v1.

1 Cycle Sequencing Kit following the manufacturers instructions. The BigDye XTerminator Purification Kit was used for the purification Inhibitors,Modulators,Libraries of the DNA sequen cing reactions removing non incorporated BigDye terminators and salts. Solution was incubated for 30 min with agitation of 1800 rpm. Sequencing analyses were Inhibitors,Modulators,Libraries car ried out on the eight capillary 3500 Genetic Analyzer. Next generation sequencing Targeted next generation sequencing was per formed on 72 FFPE samples. Isolated DNA was amplified with an in house specified, customized Ion AmpliSeq Primer Pool. The panel com prises 102 amplicons of 14 different genes including exon 11 and 15 of the BRAF gene. PCR products were ligated to adapters and enriched for target regions using the Ion AmpliSeq PanelTM Library kit according to manufacturers instructions.

The generated libraries were equimolar pooled for amplicon sequencing to a concentration of 20 nM of each sample to counterbalance differences in sample quality. Sequencing was performed on an Illumina MiSeq benchtop sequencer. Results were visualized in the Integrative Genomics Viewer and manually analyzed. A 5% cutoff for Inhibitors,Modulators,Libraries variant calls Inhibitors,Modulators,Libraries was used and results were only interpreted if the coverage was 100. Pyrosequencing Pyrosequencing was performed with the therascreen BRAF Pyro Kit detecting certain mutations in codon 600 of the BRAF gene according to manufac turers instructions. 1 ul of each isolated DNA was ana lyzed per run. Pyrosequencing was performed on the PyroMark Q24 platform using the PyroMark Gold Q24 reagents.

Pyrograms were generated with the PyroMark Q24 software and data were analyzed Inhibitors,Modulators,Libraries manually or with a plug in tool provided by Qiagen. Sequences surrounding the site of interest served as normalization and reference peaks for quantification and quality control. Dispensation order was as follows for manual analysis. Samples with 5% mutated alleles mean or more were scored as mutation positive. Allele specific PCR For the allele specific PCR the cobas BRAF V600 test was utilized. DNA was isolated with the in house method.