DNM/iSNP calling and DNM validation
Sequence reads were aligned to the Human Genome Reference Assembly GCRh37 using Burrows-Wheeler Alignment (BWA) version 0.7.17 (Li, 2013) and indexed using SAMtools (Li et al., 2009) and Picard version 1.6 (PicardToolkit, 2019). SNVs and indels were subsequently called by the Genome Analysis Toolkit (GATK)) HaplotypeCaller v4.1.4.1 (McKenna et al., 2010). De novo analysis was performed using custom-designed in-house analysis pipelines and validation of DNMs in the proband and parents was performed using standard Sanger Sequencing (Oud et al., 2022). iSNP calling was also performed using an in-house pipeline, which called proband and parent variants using Clair v2 (Luo et al., 2020), proband variants were selected based on unique parental inheritance with a minimum parental coverage of 5x. A single iSNP is selected for parent-of-origin assignment, see section ‘Bioinformatics’