Kapa hgdna quantification and qc kit

Tumor samples were obtained from 15 patients with TC, and histologically normal tissues from adjacent resected tissues were obtained from a subset of eight patients. DNA extraction from FFPE samples; DNA quantification and quality assessment were performed by qPCR, using the KAPA hgDNA Quantification and QC Kit (Kapa Biosystems; Wilmington, MA, USA). The generated library targets hotspot regions of 50 oncogenes and tumor suppressor genes (listed in Table S2). Details of NGS, bioinformatics analyses and validation of newly identified variants¹³, ¹⁴, ¹⁵, ¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹ are provided in Supporting Information.

DNA was isolated using Qiagen (Hilden, Germany) DNeasy Blood and Tissue Mini Kit. Samples were thawed and centrifuged at 16 000g for 15 minutes to precipitate DNA. After complete removal of Trizol, 180 μL buffer ATL and 20 μL protease was added and the tubes incubated at 56°C overnight before addition of 200 μL buffer AL. Samples were mixed well by vortexing before 200 μL ethanol was added and the samples were again mixed well by vortexing. The samples were then transferred to DNeasy Mini spin columns and further processed as per the manufacturer's instructions before DNA was eluted in 100 μL buffer AE. To improve recovery of the DNA, the elution buffer was left on the columns for 5 minutes before a final centrifugation step. For quantification and quality assessment of the DNA, quantitative polymerase chain reaction (qPCR) was performed with the KAPA hgDNA Quantification and QC Kit (KAPA Biosystems, Wilmington, MA) as per the manufacturer's instructions. Isolation of DNA from pure DCIS tumors were performed using the QIAcube system with the AllPrep DNA/RNA Universal Kit (cat.no. 80224, Qiagen, Hilden, Germany) according to protocol provided by the supplier.

FFPE-derived DNA was pre-treated to reduce the impact of potential DNA damage, before target capture. 100 ng of DNA, as quantified by the Qubit dsDNA HS kit (ThermoFisher Scientific), was digested with 1 unit of UDG enzyme (New England Biolabs (NEB)) in a 50 μL reaction in 1X of the supplied reaction buffer (NEB). The mixture was incubated at 37°C for 10 minutes, cooled to 4°C, and immediately purified with the Agencourt AmPure XP Kit at a 3:1 bead to sample volumetric ratio as per manufacturer’s instructions. Samples were eluted in 20 μL of 10 mM Tris-HCl, pH 8. Total amplifiable DNA was quantified using KAPA hgDNA Quantification and QC Kit (KAPA Biosystems) as per the manufacturer’s instructions.

DNA was extracted using QIAmp DNA Micro and AllPrep DNA/RNA Micro Kit for UMFIX/NBF-fixed and SF tumours, respectively. DNA size distribution and quality were assessed by qPCR with Illumina FFPE QC Kit and KAPA hgDNA Quantification and QC Kit, respectively.

Experiments were performed using five blood spots (3.1 mm diameter) for each patient. DNA was extracted from DBS according to the protocol recently published by St Julien and collaborators^{38 (link)}, with slight modifications. The amounts of DNA were estimated, and the quality of the retrieved material was assessed using the KAPA hgDNA Quantification and QC^® kit (Kapa Biosystems), which is designed to amplify targets of 41 base pairs (bp), 129 bp, and 305 bp within a conserved single-copy locus in the human genome. Absolute quantification is achieved using the 41 bp assay, while the longer amplicons are used to assess DNA quality. Since DNA damage has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained with the 41 bp assay. This normalization generates “Q-ratios” with values between 0 and 1, which can be used as a relative measure of DNA quality prior to NGS library construction.