4.3. Genetic Analyses

The entire coding regions of the 50 candidate genes were analyzed by amplicon-based target enrichment and Next-Generation Sequencing (NGS) of the exons and exon-intron boundaries on a Illumina^® MiSeq platform (Illumina, San Diego, CA, USA). NGS analysis was performed on n = 65 AD; n = 102 DLB, n = 58 FTLD and n = 75 CTRL samples. The quality assessment of gDNA was performed on a 0.8% agarose gel and gDNA was quantified with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). A total of 200 ng of gDNA was used for library preparation with a Nextera Flex for Enrichment kit (Illumina, Inc., USA). gDNA was tagmented, amplified and purified with AMPure XP Beads (Beckman Coulter, Inc., Brea, CA, USA). The size, quality and quantity of libraries was assessed with a High Sensitivity DNA kit on a Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). A 12 pM sample of the pooled library was loaded on a MiSeq reagent cartridge v3 and sequenced on an Illumina MiSeq platform. Whole exome sequencing (WES) was performed on a NextSeq 500 platform (Illumina, USA) on n = 217 AD, n = 12 DLB, n = 243 FTLD samples. The exons were captured by a SeqCap^® EZ Human Exome Probes v3.0 (Roche, Basel, Switzerland) kit with a paired-end read length of 250 bp. Whole genome sequencing (WGS) was performed on a HiSeq X platform (Illumina, USA) on n = 176 CTRL samples as described before [69]. Sanger sequencing was performed on a SeqStudio Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) and on an AB3730 DNA analyzer (Life Technologies, Waltham, MA, USA) to confirm selected variants with a low coverage. The chromatograms were viewed through a CLC Main Workbench 20.0.4 (QIAGEN, Copenhagen, Denmark).