1. Targeted Next-Generation Sequencing of 51 Genes Involved in Primary Electrical Disease 
Dorien Proost, Johan Saenen, Geert Vandeweyer, Annelies Rotthier, Maaike Alaerts, Emeline M. Van Craenenbroeck, Joachim Van Crombruggen, Geert Mortier, Wim Wuyts, Christiaan Vrints, Jurgen Del Favero, Bart Loeys, Lut Van Laer 
The Journal of Molecular Diagnostics 
Volume 19, Issue 3, Pages (May 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
For each pathology, as well as for the total of all primary electrical disease pathologies, the percentage of patients per type of variant is plotted. Patients with only pathogenic and/or likely pathogenic variants are indicated in blue, whereas patients harboring pathogenic and/or likely pathogenic variants in combination with one or more variant of unknown significance (VUS) are indicated in red. Purple bars represent patients with one or more VUS and negative patients are represented by green bars. In total 114 patients, 64 Brugada syndrome (BrS), 11 long QT syndrome (LQTS), 8 arrhythmogenic right ventricular cardiomyopathy (ARVC), 17 sudden cardiac death e causa ignota (SCD-ECI), and 14 others, were screened.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Supplemental Figure S1
The procedures leading to the sequencing of a sample. Amplification of the target regions is performed using the PED MASTR Plus assay, according to the manufacturer's instructions (Multiplicom, Niel, Belgium).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Supplemental Figure S2
Strategy for variant filtering and interpretation. First, all annotated variants derived from all patients from one MiSeq run are listed. Variants that occur more than three times are removed. Variants with an rs number from the single-nucleotide polymorphism (SNP) database (dbSNP, last accessed July 2015) are checked. Only if their nature is documented as possibly or probably pathogenic or if their minor allele frequency (MAF) is <0.005, the variants are retained within the list. All variants located further than 15 bp in the intron are excluded. The presence of the remaining variants in the Exome Variant Server (EVS, last accessed July 2015) is assessed. If they occur >15 times in the EVS, these variants are excluded. Next, the presence of the variants in the Human Gene Mutation Database (HGMD, last accessed July 2015) is checked. Afterward, the remaining variants are visually inspected with the Integrative Genomics Viewer (IGV, last accessed July 2015). At that point, obvious false-positive variants can be excluded. Subsequently, splice site predictions are performed with ALAMUT software for all remaining intronic and synonymous variants. Finally, all remaining candidate causative variants are confirmed with Sanger sequencing.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Supplemental Figure S3
Overview of the optimization and validation process. For each of the 789 target regions, primers were designed and optimized through several rounds of primer concentration adjustments and primer design, each time followed by next-generation sequencing. After optimization, 20 Human Polymorphism Study Center samples and 19 positive control samples were used to validate the panel.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Supplemental Figure S4
Detailed coverage analysis for KCNH2 exon 1 and exon 11, and TRPM4 exon 13, which shows that these are (partially) insufficiently covered. The 30× cutoff value is indicated with a red line. The cumulative normalized base-coverage plot shows that 99.5% of bases reach the 30× threshold.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Supplemental Figure S5
Coverage analysis across all regions of interest for ABCC9, AKAP9, and ANK2, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8. Supplemental Figure S6
Coverage analysis across all regions of interest for CACNA1C, CACNA2D1, and CACNB2, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

9. Supplemental Figure S7
Coverage analysis across all regions of interest for CALM1, CASQ2, CAV3, and CTNNA3, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

10. Supplemental Figure S8
Coverage analysis across all regions of interest for DES, DPP6, and DSC2, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

11. Supplemental Figure S9
Coverage analysis across all regions of interest for DSG2, DSP, GJA1, and GJA5, where the 30× cutoff value is represented by a red line. When more than one transcript exists, a union of transcripts was geneated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

12. Supplemental Figure S10
Coverage analysis across all regions of interest for GPD1L, HCN4, JUP, KCND3, KCNE1, and KCNE1L, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

13. Supplemental Figure S11
Coverage analysis across all regions of interest for KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, and LMNA, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. A completely failing exon is indicated by the red color of the bar, whereas a partially failing exon is indicated with orange. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

14. Supplemental Figure S12
Coverage analysis across all regions of interest for NKX2-5, NOS1AP, NPPA, PKP2, PLN, PRKAG2, and RANGRF, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

15. Supplemental Figure S13
Coverage analysis across all regions of interest for RYR2, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

16. Supplemental Figure S14
Coverage analysis across all regions of interest for SCN1B, SCN2B, SCN3B, SCN4B, and SCN5A, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. A completely failing exon is indicated by the red color of the bar, whereas a partially failing exon is indicated with orange. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

17. Supplemental Figure S15
Coverage analysis across all regions of interest for SLMAP, SNTA1, TGFB3, TMEM43, TRDN, and TRPM4, where the 30× cutoff value is represented by a red line. For each exon, the average coverage across the exon is shown on top of the bar. A completely failing exon is indicated by the red color of the bar. When more than one transcript exists, a union of transcripts was generated, which is indicated by (UNION) in the title of the plots.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

18. Supplemental Figure S16
Pedigree of families 2, 7, 14, 18, and 19. The index patients are indicated by the arrowheads. Family 2. It is striking that affected individual III: 6 is negative for the familial SCN5A mutation. However, the SCN5A mutation is definitively causal as this variant was previously reported and functionally tested by Hong et al49 and Rossenbacker et al,50 and this variant is a Flemish founder mutation (M.A., D.P., J.S., E.V.C., B.L., L.V.L., unpublished data). As such, within this family at least one additional causal variant remains to be identified.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

19. Supplemental Figure S17
Pedigree of families 23, 27, 28, 33, and 37. The index patients are indicated by the arrowheads.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

20. Supplemental Figure S18
Pedigree of families 38, 40, and 49. The index patients are indicated by the arrowheads.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

21. Supplemental Figure S19
Pedigree of families 62, 63, and 65. The index patients are indicated by the arrowheads.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions