Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon- Based Massive Parallel Pyrosequencing Geneviève Michils, Silke Hollants,

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Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon- Based Massive Parallel Pyrosequencing Geneviève Michils, Silke Hollants, Luc Dehaspe, Jeroen Van Houdt, Yannick Bidet, Nancy Uhrhammer, Yves-Jean Bignon, Joris R. Vermeesch, Harry Cuppens, Gert Matthijs The Journal of Molecular Diagnostics Volume 14, Issue 6, Pages 623-630 (November 2012) DOI: 10.1016/j.jmoldx.2012.05.006 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Distribution of the variant frequency (VF) of heterozygous variations versus the total coverage (n = 91). The Journal of Molecular Diagnostics 2012 14, 623-630DOI: (10.1016/j.jmoldx.2012.05.006) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Coverage distribution (up to 250×), ie, the total number of reads (forward and reverse), of each amplicon in the MASTR assay v.1.2 (n = 400 patients). For example, 6.7% of all of the amplicons had a coverage between 31× and 40×. Of the amplicons, 87% had more than 40× coverage, 0.9% of all amplicons had a coverage greater than 250×. The Journal of Molecular Diagnostics 2012 14, 623-630DOI: (10.1016/j.jmoldx.2012.05.006) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Number of reads (log2 scale) of the BRCA MASTR assay v.1.2 (n = 13 experiments and 400 samples). The bottom and top of the box are at the 25th and 75th percentile (the lower and upper quartiles, respectively), and the 50th percentile (the median) is shown in the box. The ends of the whiskers represent the lowest value within 1.5 IQR of the lower quartile, and the highest value within 1.5 IQR of the upper quartile; the outliers are marked by a diamond; 40× coverage is indicated by a dotted line. The Journal of Molecular Diagnostics 2012 14, 623-630DOI: (10.1016/j.jmoldx.2012.05.006) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Distribution of the flowgram values in and around the references at positions BRCA1 c.1010_1016, BRCA2 c.2170_2175 and BRCA2 c.9247_9253. Relative ratios (%) were calculated on basis of the number of reads of each interval versus the total amount of reads that were mapped to this position. The values were highlighted in a black-and-white scale (black indicates high, white indicates low). Depending on the stretch, the expected nominal values are 6 or 7 and in the context of a perfect interpretation (100% of reads identical to the wild-type), only black boxes should be present at the respective reference positions. Control samples and five positive controls (A: c.1016delA (2 samples) and c.1016dupA; B: c.2175dupA and C: c.9253dupA) were analyzed. These plots show that, although the distribution of the reads in or around the stretch is sometimes quite broad, the distribution of the flow values in positive controls can be clearly distinguished (see black frames). The Journal of Molecular Diagnostics 2012 14, 623-630DOI: (10.1016/j.jmoldx.2012.05.006) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions