Alissa interpret software

Genomic DNA (gDNA) was extracted from peripheral blood samples of the probands and their parents using standard procedures. Exons and flanking splice junctions were captured using the Agilent SureSelect Human All Exon kit. Sequencing was performed on an Illumina platform. Reads were aligned to the human reference genome GRCh37/hg19 using BWA (http://bio-bwa.sourceforge.net/), and variants were called using the GATK haplotype caller (https://www.broadinstitute.org/gatk/). Detected variants were annotated and filtered using Alissa Interpret software (Agilent). Priority was given to rare variants (minor allele frequency < 0.1% in public databases) that fit a recessive or de novo mode of inheritance. Sanger sequencing was used to verify all identified variants and test other family members.

The MYMX genetic variant was detected through clinical whole-exome sequencing (WES) performed at the University Medical Center Utrecht Genetics department according to local standardized diagnostic procedures. WES was performed using an Illumina NovaSeq 6000 platform on exome-enriched samples (Agilent Sureselect CREv2) from DNA isolated from peripheral blood. Diagnostic variant filtering based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines (54 (link)) was performed using Agilent Alissa Interpret software. TRIBES software (v0.2.0) was used to estimate the degree of relatedness between samples (24 (link)). Analysis was performed on WES data from all family members and 7 randomly selected, unrelated in-house control samples. If no estimated degree of relatedness was detected, relatedness was manually set to 12.

DNA isolation from peripheral blood of patient and parents was performed using the Chemagic DNA Blood 4 k Kit (PerkinElmer, Waltham, MA, United States). Three micrograms of double-stranded DNA were fragmented (Covaris, Woburn, MA, United States) and exonic sequences were captured using the Agilent SureSelect Clinical Research Exome V2 (Agilent, Santa Clara, CA, United States). Paired-end sequencing was performed on a HiSeq 4,000 platform (150bp paired end) and an average coverage of at least 50X. Reads were mapped against the human reference genome GRCh37/hg19 with the Burrows-Wheeler Aligner (Li and Durbin, 2009 (link)), and variants were called using the Genome Analysis toolkit (Broad Institute, Cambridge, MA, United States). Alissa Interpret software (Agilent, Santa Clara, CA, United States) was used to filter and prioritize variants. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for the interpretation of sequence variants (Richards et al., 2015 (link)).