Qiaamp dna ffpe generead kit

DNA from 4–8 sections (4–5 μm each) of formalin-fixed paraffin-embedded (FFPE) tumor samples (which were selected by a pathologist as described above) was extracted using the “QIAamp DNA FFPE GeneRead Kit” (Catalogue Reference: 180134; Qiagen, Germantown, MD, USA). Subsequently, the whole exonic region of IDH1 and IDH2 genes was then sequenced using an Illumina MiSeq device (version 3.1.0.13) following the manufacturer’s instructions, as previously described [39 (link)]. In brief, after DNA quantification from FFPE tumor samples using Qubit 1× dsDNA HS Assay Kit (Catalogue Reference: Q33230; Thermo Fisher Scientific, Waltham, MA, USA), 50–150 ng was used for mutational analysis by AmpliSeq methodology (Illumina, Inc., San Diego, CA, USA). AmpliSeq Library PLUS was used for library preparation (Catalogue Reference: 20019101; Illumina, Inc., USA), followed by the amplification of target regions and second amplification of libraries, which were diluted and denatured for bridge clonal amplification and paired-end sequencing using MiSeq Reagent kit v2 (300-cycles) (Catalogue Reference: MS-102-2002; Illumina, Inc., USA) in a MiSeq instrument (Illumina, Inc., USA). Variant calling files annotation, and the identification and classification of detected genetic variants were performed with the VariantStudio software v3.0 (Illumina, Inc., USA).

Tumor samples were obtained by open surgical resection and immediately processed by a pathologist. After macroscopic tumor selection, samples were fixed in formalin and paraffin-embedded (FFPE). The tumor region was selected and marked by a pathologist in an H&E section slide that was representative of the cellularity of samples collected from 4–8 FFPE sections that were 4–5 μm each. Tumour DNA was obtained from the areas marked by the pathologist from these 4–8 FFPE sections using the QIAamp DNA FFPE GeneRead Kit (Catalogue Reference: 180134; Qiagen, Germantown, MD, USA). DNA from plasma was purified using the QIAamp Circulating Nucleic Acid Kit (Catalogue Reference: 55114; Qiagen, Germantown, MD, USA). Tumor and plasma DNA were quantified using a QUBIT 3.0 fluorometer instrument and the Qubit 1× dsDNA HS Assay Kit (Catalogue Reference: Q33230; Thermo Fisher Scientific, Waltham, MA, USA).