Genomic DNA Isolation and Next-Gen Sequencing

Genomic DNA from peripheral blood was isolated using the Maxwell 16 extractor (Promega, Madison, WI, United States) and quantified using the Quantus Fluorometer (Promega) with QuantiFluor double-stranded DNA system. Genetic screening was performed by Next Generation Sequencing (NGS) multigene panels, by using either one of the following panels: amplicon-based Illumina panel (Bartoletti-Stella et al., 2018 (link)) and probe-based Illumina panel (Truseq Neurodegeneration Illumina). Sequencing was performed on a MiSeq or NextSeq 500 sequencer using Illumina V2 reagent kit, with 2 × 150 bp paired end read cycles. Sequencing data were analyzed with an in-house bioinformatic pipeline: trimming and quality assessment of raw reads was performed with Trimmomatic (Bolger et al., 2014 (link)), mapping was performed with Burrows-Wheeler Aligner (Li and Durbin, 2009 (link)) using bwa-mem algorithm on the reference genome GRCh37/Hg19. Variant calling was performed with Strelka2 (Kim et al., 2018 (link)). Variant filtration and depth of coverage analysis were performed using Genome Analysis Toolkit (GATK) v4 (McKenna et al., 2010 (link)).