Allprep kit for ffpe tissue

DNA and RNA for NGS-based mutation analysis were extracted using the Qiagen AllPrep kit for FFPE tissue and automated on the QIAcube instrument (Qiagen, Hilden, Germany). The protocol was modified with an extended proteinase K digestion (overnight) for the DNA extraction to obtain higher DNA yields. DNA from cytology slides was extracted using the QiaAmp DNA Micro kit (Qiagen, Hilden, Germany). RNA was not extracted from cytology specimens. Following extraction, DNA samples were quantified using Qubit and RNA samples were quantified using a BioAnalyzer. Then, for quality assessment DNA samples were analyzed by qPCR using the Illumina FFPE QC Kit (WG-321-1001) along with a control cell line sample with a known input mass.

DNA was extracted from FFPE tissue. Extractions were performed using the Qiagen AllPrep kit for FFPE tissue (Qiagen^®, Venlo, The Netherlands). 80 ng DNA was used as input for single nucleotide polymorphism (SNP) array analysis at Eurofins Genomics Europe Genotyping A/S (Galten, Denmark, The Netherlands) using the OncoScan^® FFPE Assay Kit (Affymetrix^®, Santa Clara, CA, USA). After preprocessing using the manufacturer’s standard protocol, segmentation was performed using ASCAT (package version 2.5.2, in R version 3.6.3). With this package, we also derived copy-number profiles of tumor cells and estimates of normal cell contamination and ploidy. The fraction of the genome altered (FGA) was calculated using fraction of probe positions with a total copy-number differing from ploidy by >0.6.