Targeted NGS and Sanger Trio Testing

Genomic DNA was extracted from the peripheral blood of patients and their parents. For an NGS-based trio test, libraries were prepared, and targets were captured using various custom panels for candidate genes (gene panels and genes included in each panel are listed in Supplementary Table S1). The prepared and pooled libraries were sequenced using NextSeq 550Dx System (Illumina, San Diego, CA, United States). The sequencing data were analyzed using the custom bioinformatics pipeline as previously described (4 (link), 5 (link)). For the test, the parents underwent the same NGS panel testing that the patient received.
For a Sanger sequencing trio test, gene regions that included the target variants were amplified by target-specific primers and further sequenced using a 3,730 DNA Analyzer with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).