Maxwell rsc ffpe plus dna kit

DNA preparation from FFPE samples was performed by using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), the GeneRead DNA FFPE Kit (Qiagen), or the Maxwell® RSC FFPE Plus DNA Kit together with the Maxwell® RSC instrument (Promega, Mannheim, Germany). DNA extraction from frozen tissue samples was performed with the PureLink™ Genomic DNA Mini Kit (Life Technologies, Carlsbad, CA, USA) or by ultracentrifugation as described elsewhere [27 (link)]. Peripheral blood leukocyte DNA was extracted either with the PureLink™ Genomic DNA Mini Kit (Life Technologies) or with the Maxwell® RSC Blood DNA Kit. Extracted DNA was quantified by using the Quantus™ Fluorometer (Promega) or the Qubit system (ThermoFisher Scientific, Waltham, MA, USA).

All laboratory work was performed according to the respective manufacturer’s protocol available online. The preparation of formalin-fixed, paraffin-embedded (FFPE) tissue samples was performed identically for all three workflows. The purification of DNA from the FFPE tissue samples was performed using the Maxwell^® RSC Instrument (Promega Corporation) with the Maxwell^® RSC FFPE Plus DNA Kit (Promega Corporation). Therefore, the tumor containing area of a tissue section or respectively the total area of a normal tissue section was scraped off the slide, placed into a 1.5 ml tube and centrifuged at maximum speed for 15 sec. 20 μl of 20 mg/ml Proteinase K and 180 μl Incubation Buffer was added. Overnight samples were heated at 70°C. After incubation the samples were mixed with 400 μl Lysis Buffer and transferred to the Maxwell^® RSC Cartridge. Further preparation and the instrument run were performed according to manufacturer’s protocol. The concentration of DNA was measured with the Qubit 4 Fluorometer (Invitrogen). For the library preparation, panel-specific amounts of DNA were used as input: 40 ng for the GeneRead™ QIAact BRCA UMI Panel (QIAGEN), 20 ng for the Oncomine™ Comprehensive Assay v3 (Thermo Fisher) and 20 ng for the AmpliSeq™ BRCA Panel (Illumina^®).

The formalin‐fixed paraffin‐embedded (FFPE) sample of the DPH cohort was profiled with targeting sequencing (1123 gene panel) (ChosenMed, Beijing, China), and germline variation was synchronously sequenced using the paired adjacent tissues or peripheral blood samples. DNA extraction from tumor specimens was performed using the Maxwell RSC FFPE Plus DNA Kit (Promega, Cat no.AS1720), followed by shearing of genomic DNA (gDNA) into approximately 200 bp fragments using a Covaris E210 system (Covaris, Inc.). Construction of the NGS library was accomplished using the KAPA HyperPrep Kit (Roche, 07962312001) and the Agilent SureSelect XT kit (Agilent, G9702C). The library's quantity was assessed with the Qubit 3.0 Fluorometer (Life Technologies, Inc.), while its quality and fragment size were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Finally, targeted sequencing of the gDNA was conducted, employing paired‐end sequencing on an Illumina NovaSeq 6000 platform (Illumina Inc) with a 150‐bp read length.