Qiaamp tissue kittm

DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany). Then, 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA), which covers the mutation hotspots of 50 genes. The panel includes driver mutations, oncogenes, and tumor suppressor genes. By mid-2018, the gene panel was expanded using the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA), which covers genetic alterations and gene fusions. The Ampliseq cancer hotspot panel was sequenced with an Ion PGM (Thermo Fisher Scientific, Waltham, MA, USA) and the Oncomine Comprehensive Assay v3 on an Ion S5 sequencer (Thermo Fisher Scientific, Waltham, MA, USA). The generated sequencing data were analyzed afterward with the help of the Ion Reporter Software (Thermo Scientific Fisher Scientific, Waltham, MA, USA). We referred to the BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM, and 1000 genomes for variant calling and classification.

DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue Kit^TM (Qiagen, Hilden, Germany). A total of 10 ng DNA per sample was utilized for sequencing. The DNA library was generated by multiplex polymerase chain reaction (PCR) with two panels—Ion AmpliSeq Cancer Hotspot Panel v2^TM and ColonLung Panel v2^TM (both from Thermo Fisher Scientific, Waltham, MA, USA). The first panel covers mutation hotspots of 50 genes, mostly oncogenes and tumor suppressor genes, which are frequently mutated in tumors, whereas the ColonLung Panel covers the mutational hotspots of 22 genes, which are altered in colon and lung cancers. Template preparation was carried out by emulsion PCR with Ion One Touch^TM or Ion Chef^TM instruments (Thermo Fisher Scientific). Sequencing was performed with an Ion Torrent PGM^TM (Thermo Fisher Scientific).

DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany) and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA). The panel includes driver mutations, oncogenes, tumor suppressor genes, and gene fusions. See supplementary information for complete list of the gene panel. The Oncomine Comprehensive Assay v3 was optimized for sequencing on an Ion Personal Genome Machine System (Thermo Fisher Scientific). The generated sequencing data were afterwards analyzed with the help of the Ion Reporter Software (Thermo Scientific Fisher). We referred to BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM and 1000 genomes for variant calling and classification. The variants were classified according to a five-tier system comprised of the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. This classification was based on the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.