Nextseq500 mid output system

Whole-exome sequencing (WES) was performed as previously reported^{30 (link)}. In brief, after DNA extraction, exome capture was performed with the TruSeq Exome Capture Kit (Illumina), and sequencing for the three participants (patient and both parents) was conducted using the NextSeq500 mid output system (Illumina) with a 75-bp paired-end run. The sequences were aligned to the human reference genome (GRCh37), and variant calling was performed using the Genome Analysis Toolkit (GATK V3.5, Broad Institute)^{31 (link)}. Variants were first annotated by Variant Studio (V3.0, Illumina) and wANNOVAR (http://wannovar.wglab.org/)^{32 (link)}. Candidate variants were checked with ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). The pathogenicity of variants was classified according to the ACMG guidelines^{33 (link)}.

The quality and integrity of the RNA were assessed with high sensitivity D5000 Screen Tape® System (Agilent). Following the quality control, RNASeq libraries were prepared using NEBNext Ultra II Directional Poly (A) Capture Kit. RNA sequencing was carried out as paired-end sequencing with Illumina NextSeq 500 Mid Output System. RNA quality-testing, library preparation, and sequencing were performed in the Functional Genomics Unit (FUGU) at the University of Helsinki, Finland.