Exome panel

Next generation sequencing experiments, comprising DNA extraction and samples quality control, were performed by Genomix4life S.R.L. (Baronissi, Salerno, Italy). DNA was extracted from FFPE sections using AllPrep DNA/RNA FFPE Kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s instructions. Indexed libraries were prepared from 100 ng/ea purified DNA with DNA Prep for Enrichment Kit with Illumina Exome Panel (45 M size) according to the manufacturer’s instructions. Libraries were quantified using the TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) and Invitrogen Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). They were then pooled such that each index-tagged sample was present in equimolar amounts, with a final concentration of the pooled samples of 2 nM. The pooled samples were subject to cluster generation and sequencing using an Illumina NextSeq 550 System (Illumina, San Diego, CA, USA) in a 2 × 150 paired-end format. Paired-end reads were aligned to the NCBI reference sequence (GRCh37/hg19) using the Borrows–Wheeler Algorithm (BWA) and variant calls were made using the Genomic Analysis Tool kit (GATK). With Picard tools in Java that work with next-generation sequencing data in BAM format, mean coverage information for every target can also be computed. The means coverage of at least 50× were obtained.

Pre- and post-treatment sequencing libraries were prepared from 100 ng of DNA using the Illumina DNA Prep with Enrichment kit and the Illumina Exome Panel. During library preparation, DNA was amplified with IDT for Illumina DNA Unique Dual Indexes, resulting in a pool of twelve dual-indexed paired-end libraries that were sequenced on Illumina’s NovaSeq 6000 (2 × 100) version 1.5.

Genomic DNA was extracted from peripheral blood samples using a Wizard® Genomic DNA Purification Kit (Promega). All samples were processed using two different WES enrichment platforms: Nextera Flex for Enrichment, an Exome panel (Illumina), and an Accel-NGSR 2S Hyb DNA Library Kit (Swift). In total, 50–1000 ng of DNA was sheared into short fragments using enzymatically (Illumina) and Covaris M220 Focused-ultrasonicator (Swift). Size selection, sequence capturing, enrichment, and elution were performed according to the manufacturer’s instructions. The size distribution of DNA libraries was then measured using 4150 TapeStation (Agilent). Finally, the validated DNA libraries were sequenced on an Illumina sequencing platform (NovaSeq 6000) with 150 bp paired-end reads. The read pair data (fastq) of each sample were generated from the sequencing system. The resulting reads were then aligned to the reference human genome sequence (GRCh37/hg19), and nucleotide variant calling was performed using the DRAGEN Enrichment app (ver 3.6.3) in the Illumina basespace sequence hub. The VCF files from the DRAGEN Enrichment app were annotated and analyzed in the CLC Genomics Workbench (ver 20) software with default parameters.