All variants were classified according to the guidelines from the American College of Medical Genetics to pathogenic, likely pathogenic, of uncertain significance, likely benign or benign [14 (link)], and novel variants were submitted to the ClinVar Database (https://www.ncbi.nlm.nih.gov/clinvar/ ). Pathogenic and likely pathogenic variants were classified as disease causing variants.
Candidate variants found by NGS were validated using Sanger sequencing if the coverage at the variant site of the exome sequencing result was below 30x and/or the base quality score below 500, in accordance with previously published recommendations [15 (link)]. Furthermore, Sanger sequencing was employed to resolve cases with a suspected compound heterozygous combination of variants and for other familial segregation analyses. Sequencing was carried out using BigDye 3.1 sequencing chemistry (Life Technologies), followed by capillary electrophoresis on the ABI 3500 capillary sequencer (Life Technologies).
Primer sequences are available upon request.
Candidate variants found by NGS were validated using Sanger sequencing if the coverage at the variant site of the exome sequencing result was below 30x and/or the base quality score below 500, in accordance with previously published recommendations [15 (link)]. Furthermore, Sanger sequencing was employed to resolve cases with a suspected compound heterozygous combination of variants and for other familial segregation analyses. Sequencing was carried out using BigDye 3.1 sequencing chemistry (Life Technologies), followed by capillary electrophoresis on the ABI 3500 capillary sequencer (Life Technologies).
Primer sequences are available upon request.
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