Overall 40 samples were at least analyzed with each of the six evaluated methods. DNA isolation All samples were fixed in neutral buffered formalin prior to paraffin embedding. On a haematoxylin eosin stained slide tumor areas were this research selected by a patholo gist and DNA was extracted from corresponding unstained 10 um thick slides by manual micro dissection. The DNA was isolated by automated extraction using the BioRobot M48 following the manu facturers protocols. Quality and quantity of isolated DNA was assessed by agarose gel electrophoresis, by a Nanodrop 2000c spectrophotometer or in the case of next generation sequencing with the Qubit Fluorometer. High resolution melting analysis High resolution melting analysis was set up using 10 ng of genomic DNA, 3.

5 mM MgCl2, 1�� Light Cycler 480 High Resolution Melting Master and 200 nM of each primer in a final reaction volume with an annealing temperature of 59 C. Analyses were performed in duplicates using the LightCycler 480 platform. Each run included a wild Inhibitors,Modulators,Libraries type control and a mutant, p. V600E, control for nor malization. Results were analyzed by Gene Scanning software Inhibitors,Modulators,Libraries 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 of the DNA sequen cing reactions removing non incorporated BigDye Inhibitors,Modulators,Libraries terminators and salts. Solution was Inhibitors,Modulators,Libraries incubated for 30 min Inhibitors,Modulators,Libraries with agitation of 1800 rpm. Sequencing analyses were 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 never quality. Sequencing was performed on an Illumina MiSeq benchtop sequencer. Results were visualized in the Integrative Genomics Viewer and manually analyzed.