Hiseq x ten platform

Paired-end PCR-free GS was performed by Macrogen Inc. (South-Korea) using a HiSeq X Ten platform. DNA preparation, sequencing, and sequence data processing were performed according to Macrogen’s standard protocol (coverage 30× and read length 150 bp).
The FASTQ files were transferred to the Core Unit Bioinformatics of the Berlin Institute of Health (CUBI) for variant calling. Files were further processed and securely stored in the System for Omics Data Analysis and Retrieval (SODAR) (Nieminen et al. 2020 ). GATK HC was used to call simple nucleotide variants, while structural variants were called using Delly2, PopDel, and ERDS/SV2. Afterwards, variants were processed and annotated by the VarFish platform (Holtgrewe et al. 2020 (link)). Variants were mapped according to the hg19 reference genome.

The plant material was snap-frozen in liquid nitrogen then ground using a mortar and pestle. RNA was isolated using the RNAzol reagent (MRC, Cincinnati, OH, USA) according to the manufacturer’s protocol. The RNA quality was assessed using the TapeStation system 4150 (Agilent, Santa Clara, CA, USA), software revision 3.1.1. The RNA concentration was measured using a Qubit Fluorometer and Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The MACE (Massive Analysis of cDNA Ends) libraries were generated using the Rapid MACE kit (GenXPro GmbH, Frankfurt, Germany) according to the supplier’s protocol. Library sequencing on an Illumina HiseqXTen platform (San Diego, CA, USA) was performed by Macrogen (Seoul, Republic of Korea). The MACE method used is limited to obtaining transcripts of nuclear genes and does not include the transcripts of the genes of the mitochondrial and chloroplast genomes.

Genomic DNA samples from WBC-tumor pairs from 4 patients (BC0145, BC0190, BC0025 and BC0031) ages of < 40 years were prepared at the Institute of Stem Cell and Translational Cancer Research (ISCTCR) at Chang Gung Memorial Hospital, Taiwan and stored at −80 °C according to routine laboratory practice. All genomic DNA samples were extracted by using the Gentra Puregene Blood kit (Qiagen, cat# 158445) and sequenced using the HiSeq X Ten platform (Macrogen, Inc., South Korea) with 150 × 150 bp paired-end (PE) reads.