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Figure S1. Calculation of the Total ESRseq Score Change for a given exonic variant. The analysis of BRCA2 c.617C>G is presented as an example. As shown, any single substitution modifies 6 overlapping hexamers (numbered 1 to 6) covering a stretch of 11 nucleotides centered on the position of the sequence change (underlined). The ESRseq scores of each hexamer, both in the wild-type (WT) and the variant (VAR) contexts, as indicated, were retrieved from Ke et al. (2011). Then, we calculated Total ESRseq Score for WT and VAR by adding the scores of the corresponding individual hexamers. Finally, the Change in Total ESRseq Score was computed by subtracting the Total ESRseq Score of WT from that of VAR. E, positive ESRseq score; S, negative ESRseq score; N, null ESRseq score.

Figure S2. Comparison of results from the ESRseq score-based analysis of BRCA2 exon 7 with data obtained in the pcDNA-Dup assay. The ESRseq scores of each RNA hexamer are plotted as in Figure 3A, with positive and negative scores indicating potential ESEs and ESSs, respectively. The nucleotides below the graph indicate the sequence taken into account in the ESRseq score-based analysis; i.e. BRCA2 exon 7 (upper case letters) and 6 nucleotides of flanking intronic sequences (lower case letters). The horizontal bars under the sequence of the exon represent the tiled 30 bp-long exonic fragments (r1 to r7) analyzed in the pcDNA-Dup assay as described in Figure 2B. The colors of the fragments illustrate the results shown in Figure 2C relative to middle exon splicing: white, no inclusion; grey, partial/weak inclusion; black, total/strong inclusion.

Figure S3. ESRseq Scores for variants located outside splice sites (other than c.520C>T, c.581G>A, and c.617C>G) that increased exon skipping in the pCAS2-BRCA2-exon 7 minigene splicing assay. ESRseq scores are shown for each hexamer sequence overlapping the positions of the variants, as indicated, both in the wild-type (WT) and variant (VAR) contexts. Hexamer numbering (#) refers to the relative position of each hexamer within the 11-nucleotide stretch taken into account in this analysis, as described under Materials and Methods. According to results from the QUEPASA method, exonic hexamers can be classified for their impact on splicing either as potential ESEs (positive ESRseq), potential ESSs (negative ESRseq) or Neutral (null ESRseq) (Ke et al., 2011). The Total ESRseq Scores indicated above each graph for WT and VAR were calculated as described under Materials and Methods.

Figure S4. Comparison of experimental data derived from the splicing minigene reporter assay with fold changes in ESS/ESE ratio calculated by using EX-SKIP for each BRCA2 exon 7 variant. The dataset on the left refers to variants that increased exon skipping whereas the one on the right refers to variants that did not increase exon skipping in the minigene assay. The identity of the variants of each dataset is indicated below the graph. Supp. Table S1. Number of hexamers taken into consideration for the calculation of the Total ESRseq Score for each variant (VAR) as compared to wild-type (WT)

Table S2. Bioinformatics predictions of 5’ splice site alterations for BRCA2 variants c.631G>A and c.631G>C

Table S3. Fold changes in ESS/ESE ratio calculated for BRCA2 exon 7 variants by using EX-SKIP and comparison with experimental data

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