Maxwell 16 rsc extraction system

Referring to the genome CanFam3.1, melanoma samples were analysed for mutations in exons 2 and 3 of NRAS (ENSCAFT00000015144.4) and KRAS (ENSCAFT00000010525.4), respectively, by Sanger sequencing at the Institute of Pathology, Technische Universität München, Germany. DNA was isolated from a microdissected section of a tumour tissue block from areas in which a high tumour cell concentration (at least 60% tumour cell content, median: 80%, range: 60–95%) had been microscopically identified. DNA isolation was performed using the Maxwell 16 RSC extraction system (Promega, Madison, WI, USA). DNA quantity was measured by a QuBit 4.0 system and the QuBit high sensitivity assay (both: Thermo Fisher Scientific, Waltham, MA, USA). All exons were amplified with the primers listed in Table 1 and with 10–20 ng of DNA as input and an annealing temperature of 60 °C. Bidirectional Sanger sequencing of all PCR products was subsequently conducted on an ABI 3100 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA) according to standard protocols.

After marking of the tumor area and annotation of percentage of vital tumor tissue (≥ 50% tumor cell content) for micro-dissection, DNA was extracted using the Maxwell 16 RSC extraction system (Promega) according to the manufacturer´s protocols. DNA concentration was measured fluorometrically using the QuBit 3.0 system (Thermo Fisher Scientific) and DNA quality was determined by a qPCR assay (RNAseP assay, Thermo Fisher Scientific).

Somatic and germline whole exome sequencing data were generated through participation in the MASTER trial, led by the German Cancer Research Center and the German Cancer Consortium in Heidelberg, Germany. The detailed workflow has been described earlier (48 (link)). Briefly, following a standard protocol, unfixed tissue was flash frozen and submitted to a central sample processing laboratory for DNA isolation. Whole exome libraries were prepared from tumor DNA and from peripheral blood mononuclear cells using the Agilent SureSelect Human All Exon V6 library preparation kit. The resulting libraries were sequenced an Illumina NovaSeq 6000 Sequencer (2 × 100 paired-end) to a coverage of approximately 150 (tumor) and approximately 100 (normal) depth of coverage, respectively.
Tumor DNA for the sister of the index patient was isolated from archival FFPE blocks: 2 μm sections were prepared with a rotary and subjected to histological and IHC analysis. H&E staining was performed on deparaffinized sections according to standard protocols. Eight 10 μm sections of FFPE tumor specimens were deparaffinized and digested with Proteinase K (QIAGEN) overnight. DNA isolation was performed using the Maxwell 16 RSC extraction system (Promega).