Targeted Sequencing for Variant Detection

Coding and flanking intronic regions were enriched using a SureSelectXT Library Prep Kit (Agilent) and a CeGaT tumor panel (Agilent), and were sequenced using an Illumina NovaSeq6000 system (CeGaT GmbH Tübingen). Illumina bcl2fastq2 was used to demultiplex sequencing reads. Adapter removal was performed with Skewer. The trimmed reads were mapped to the human reference genome (hg19) using the Burrows Wheeler Aligner (BWA-mem Version 0.7.2-cegat) [28 (link)]. Local realignment of reads in target areas was performed using ABRA [29 (link)] to achieve more accurate indel calling. In reference hg19, the pseudoautosomal regions (PAR) on chromosome Y have been masked (chrY:10001–2649520, chrY:59034050–59363566). This prevents reads that fall within this homologous region on chromosome X and Y from being assigned to more than one chromosome and, thus, being discarded during mapping. Reads mapping to more than one location with an identical mapping score were discarded. Read duplicates that likely result from PCR amplification were removed using samtools (Version 0.1.18) [28 (link)]. The remaining high-quality sequences were used to determine sequence variants (single nucleotide changes and small insertions/deletions). The variants were annotated based on several internal as well as external databases and on information from scientific literature.