Nexus copy number 10

Somatic copy number alterations (CNAs) were analyzed using raw sequence reads (BAM files). The CNAs of each sample were defined using the ngCGH module and SNPRank segmentation statistical algorithm in Nexus Copy Number 10.0 (BioDiscovery, El Segundo, CA). Segments were classified as copy number gains and losses when the log2 ratio was >0.2 and < -0.2, respectively. Genome-wide frequencies of CNA were visualized using the Copynumber package (Nilsen et al., 2012 (link)). CNA regions with statistical differences between the two groups (Chi-square test, p < 0.001) were analyzed using the CNVruler software (Kim et al., 2012 (link)). Details are provided in the Supplementary Methods.

Tumor areas displaying >80% cancer cells without hemorrhage or necrosis were marked on the hematoxylin and eosin slides. DNA from FFPE tumor tissue samples was obtained by punching 4 to 6 tissue cylinders (diameter 0.6 mm) from each sample. DNA extraction from FFPE tissue was done as described [32 (link)]. The double-strand DNA concentration (dsDNA) was determined using the fluorescence-based Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Tumors with poor DNA quality were excluded from the study. Genome-wide DNA copy-number alterations and allelic imbalances of 33 chRCC were determined using the Affymetrix OncoScan^® CNV Assay (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA) as previously described [33 (link)]. The demographic and clinicopathological characteristics for 33 Swiss chRCCs with clinical data are summarized in Table 1. Samples were further processed by IMGM Laboratories GmbH (Martinsried, Germany) for CNV (copy number variation) determination according to the Affymetrix OncoScan CNV Assay recommended protocol. The data were analyzed by the Nexus Copy Number 10.0 (Biodiscovery, Inc., El Segundo, CA, USA) software using Affymetrix TuScan algorithm. All array data were also manually reviewed for subtle alterations not automatically called by the software.

TP53 mutational status was assessed using whole-exome sequencing (WES, n = 90) or Sanger sequencing of exons 5 to 8 (n = 5). WES data were generated using Illumina Nextera Exome enrichment (n = 89) or TWIST Human Core Exome kit (n = 1) and sequenced on an Illumina NovaSeq within the Newcastle University Genomics Core Facility or Illumina HiSeq by Eurofins Genomics (Germany). Data were analysed using the Genome Analysis Toolkit (GATK 3.7) and variants called using Mutect2. PCR products for Sanger sequencing were amplified using primers designed for TP53 (Supplementary Table 1) and sequenced by Eurofins Genomics. WES base calls were confirmed by Sanger sequencing in 39 cases, with 100% concordance between sequencing methods.
Copy number alterations (CNAs) of 17p and other chromosomes were identified using Affymetrix Cytoscan HD, Genome-wide Human SNP Array 6.0 or OncoScan arrays performed by Eurofins Genomics. Raw data were analysed and visualised in Nexus Copy Number 10.0 (BioDiscovery) to detect CNAs and copy number neutral loss of heterozygosity (CNN-LOH) in all samples.