Perator “IF” in spreadsheet application (Table S2 in File S1). Wild-type-threshold was determined according to A8:T9 ratio of wild-type reference controls A549 and wild-type HeLa cell lines. Comparing with Sanger sequencing data, three more cases were identified as BRAF mutants (Table 1). Moreover, Epigenetic Reader Domain samples of cases 17 and 29, which were only detected in part by Sanger sequencing, were all determined as mutant-positive by UBRAFV600 analysis (Table 1). These data demonstrate the higher sensitivity of pyrosequencing assay resulting in 21 BRAF-mutated cases of 29 cutaneous metastases (72.4 ).V600E, V600E2 or V600K were individually mixed together with the plasmid, 12926553 containing wild-type braf, in a proportion from 1 to 10 mutant variant and subjected to PCR amplification followed by U-BRAFV600 pyrosequencing. Analyzing only the A8:T9 ratio, 2 V600E2 can be misinterpreted either as 10 V600E or as 4 V600K (Figure 3c). In this case, the ratios A3:A5, T9:G13 and T15:C16 
should be taken into consideration in estimating the mutant-specific 
portion in signal intensities of A5, G13 or C16 (Figure 3b). In general, the presence of variant mutations beyond V600E can be determined by the difference in peak intensity values in comparison with correspondent wild-type reference peaks (Figure 2, Figure 3c). Importantly, G19 is prone to higher Autophagy background noise (Table S2 in File S1) and should therefore be excluded from the low-abundance BRAF mutation analysis.Cases with Low-abundance BRAF MutationIn case of low-copy-number BRAF- mutated samples (5 or less), the recognition patterns can be masked by background noise and, therefore, pyrograms of V600K, V600E2 or V600E;K601I could be very difficult to distinguish from V600E mutation in analyzing only the conventional A8:T9 ratio. To simulate lowabundance BRAF mutation templates, we subcloned these mutant variants as well as wild type braf exon 15. The clones containingMiSeq Ultra-deep Sequencing Validation of U-BRAFV600 DataTo prove both the sensitivity and the specificity of U-BRAFV600 assay, several FFPE samples, which yielded at least 125 ng DNA in 25 ml, were subjected to cobasH BRAF V600 Mutation Test assay. In our study, due to initially low biopsy amount, only a few FFPE samples were suitable to perform at least one cobasH BRAF V600 Mutation Test assay analysis. As expected, mutationsU-BRAFV600 State Detectionp.V600E2 (case 21), p.V600E;K601I (case 29) and p.VKS600_602.DT (case 14) were not detected by cobasH BRAF V600 Mutation Test assay, whereas both p.V600E (cases 1, 2, 3) and p.V600K (case 27) were identified as V600-mutated cases. Unfortunately, cases 15, 17, 19 and 20 with low-abundance V600E mutation were not detected by Sanger sequencing, and also not identified by cobasH 4800 15755315 BRAF V600 Mutation Test assay (Table 1). Therefore, the examined cases were further subjected to ultra-deep-sequencing analysis using MiSeq assay (Illumina). Ultra-deep sequencing of all 75 samples yielded typical coverage in the target region (exon 15 of braf) of 50,000 to 80,000fold (Submission ID: SUB157783, Sequence Read Archive (SRA), NCBI BioSample Submissions). Sequence reads were aligned with Burrows-Wheeler Aligner against the hg19 reference sequence, and variants were called using an in-house pipeline based on SAMtools/BCFtools. Variant reads at positions indicative for the studied BRAF mutations were counted and variant allele frequencies were calculated. These calculations confirm the results of the pyrose.Perator “IF” in spreadsheet application (Table S2 in File S1). Wild-type-threshold was determined according to A8:T9 ratio of wild-type reference controls A549 and wild-type HeLa cell lines. Comparing with Sanger sequencing data, three more cases were identified as BRAF mutants (Table 1). Moreover, samples of cases 17 and 29, which were only detected in part by Sanger sequencing, were all determined as mutant-positive by UBRAFV600 analysis (Table 1). These data demonstrate the higher sensitivity of pyrosequencing assay resulting in 21 BRAF-mutated cases of 29 cutaneous metastases (72.4 ).V600E, V600E2 or V600K were individually mixed together with the plasmid, 12926553 containing wild-type braf, in a proportion from 1 to 10 mutant variant and subjected to PCR amplification followed by U-BRAFV600 pyrosequencing. Analyzing only the A8:T9 ratio, 2 V600E2 can be misinterpreted either as 10 V600E or as 4 V600K (Figure 3c). In this case, the ratios A3:A5, T9:G13 and T15:C16 should be taken into consideration in estimating the mutant-specific portion in signal intensities of A5, G13 or C16 (Figure 3b). In general, the presence of variant mutations beyond V600E can be determined by the difference in peak intensity values in comparison with correspondent wild-type reference peaks (Figure 2, Figure 3c). Importantly, G19 is prone to higher background noise (Table S2 in File S1) and should therefore be excluded from the low-abundance BRAF mutation analysis.Cases with Low-abundance BRAF MutationIn case of low-copy-number BRAF- mutated samples (5 or less), the recognition patterns can be masked by background noise and, therefore, pyrograms of V600K, V600E2 or V600E;K601I could be very difficult to distinguish from V600E mutation in analyzing only the conventional A8:T9 ratio. To simulate lowabundance BRAF mutation templates, we subcloned these mutant variants as well as wild type braf exon 15. The clones containingMiSeq Ultra-deep Sequencing Validation of U-BRAFV600 DataTo prove both the sensitivity and the specificity of U-BRAFV600 assay, several FFPE samples, which yielded at least 125 ng DNA in 25 ml, were subjected to cobasH BRAF V600 Mutation Test assay. In our study, due to initially low biopsy amount, only a few FFPE samples were suitable to perform at least one cobasH BRAF V600 Mutation Test assay analysis. As expected, mutationsU-BRAFV600 State Detectionp.V600E2 (case 21), p.V600E;K601I (case 29) and p.VKS600_602.DT (case 14) were not detected by cobasH BRAF V600 Mutation Test assay, whereas both p.V600E (cases 1, 2, 3) and p.V600K (case 27) were identified as V600-mutated cases. Unfortunately, cases 15, 17, 19 and 20 with low-abundance V600E mutation were not detected by Sanger sequencing, and also not identified by cobasH 4800 15755315 BRAF V600 Mutation Test assay (Table 1). Therefore, the examined cases were further subjected to ultra-deep-sequencing analysis using MiSeq assay (Illumina). Ultra-deep sequencing of all 75 samples yielded typical coverage in the target region (exon 15 of braf) of 50,000 to 80,000fold (Submission ID: SUB157783, Sequence Read Archive (SRA), NCBI BioSample Submissions). Sequence reads were aligned with Burrows-Wheeler Aligner against the hg19 reference sequence, and variants were called using an in-house pipeline based on SAMtools/BCFtools. Variant reads at positions indicative for the studied BRAF mutations were counted and variant allele frequencies were calculated. These calculations confirm the results of the pyrose.