Panel NGS of 23 RCC Samples
The tumor mutation status was determined by targeted panel sequencing using the capture-based TruSight™ Oncology 500 panel (Illumina, Cambridge, UK). This gene panel includes all relevant putative driver genes for RCC and covers 523 genes (full exonic coverage). DNA was extracted using a Maxwell 16 Research System (Promega, Madison, WI, USA), followed by quantification using the QuBit 2.0 DNA High Sensitivity Kit (Thermo Fisher Scientific, Waltham, MA, USA). Library preparation was performed as previously described [15]. DNA integrity was assessed using the Genomic DNA ScreenTape Analysis on a 4150 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). To fragment DNA to a length of 90–250 bp, 80 ng DNA was sheared for 50–78 seconds using an ME220 Focused-Ultrasonicator (Covaris, Woburn, MA, USA). Following two target capture and purification steps, enriched libraries were amplified by 15 cycles of PCR and subsequently quality controlled using the KAPA SYBR Library Quantification Kit on a StepOnePlus qPCR system (both Thermo Fisher Scientific). Libraries were sequenced on a NextSeq 500 instrument (Illumina) to a mean coverage of ×1096 using high-output cartridge and v2 chemistry [16]. All assays were performed according to the manufacturers' protocols [17]. Targeted NGS was performed under the approval of the Ethics Committee of the University of Heidelberg School of Medicine and after written informed consent of the patient.