Generead dna ffpe kit

For validation of SV events, we performed sanger sequencing assay on tumor and matched normal tissues from gDNA from FFPE sample were purified using Gene ReadTM DNA FFPE kit (QIAGEN, Germany). PCR product was analyzed by agarose gel electrophoresis. Amplified PCR products were gel purified and then sequenced via the Sanger method. The primers are listed in Supplementary Table 3.

DNA was extracted from frozen samples, FFPE specimens, and blood leukocyte samples using the ALLPrep^® DNA/RNA Micro Kit, GeneRead^TM DNA FFPE Kit, and QIAamp DNA Blood Midi/Maxi kit, respectively (Qiagen, Hilden, Germany). DNA volume was measured by Qubit HS (Qiagen). The quantity and quality of the FFPE-derived DNA was assessed by calculating normalized DNA integrity scores (ΔΔCq) using quantitative PCR with the Agilent NGS FFPE QC Kit (Agilent Technologies, San Diego, CA, USA). Genomic DNA > 60 ng with high quality was selected for library preparation.

Formalin-fixed, paraffin-embedded (FFPE) and available fresh frozen (FF) tissue samples were retrieved. Genomic DNA was extracted from FF and FFPE tumor samples containing a tumor cell fraction of more than 50%, as estimated by morphological analysis and CD20 immunostaining. The DNA extraction was performed according to the standard procedures utilizing GeneRead^TM DNA-FFPE-Kit for FFPE samples and DNeasy Blood&Tissue-Kit for FF samples (both from Qiagen, Hilden, Germany). DNA was quantified using Qubit fluorometer (Invitrogen, Eugene, OR, USA). DNA integrity was assessed by multiplex PCR assay as described [15 (link)].

Formalin-fixed paraffin-embedded (FFPE) macro-dissected specimens obtained during routine diagnostic biopsy and/or resection of primary tumor were used. Tumor cell percentage was evaluated by two pathologists (GP and AV) using hematoxylin/eosin-stained slides from adjacent sections. For all patients excluding those attaining a pathological complete response (pCR), i.e., absence of invasive cancer in breast and node surgical specimens, both pre- and post-NAC tumor samples were sequenced and matched germline DNA was obtained from negative lymph nodes or breast normal tissue. DNA was extracted using GeneRead DNA FFPE kit (Quiagen, Hilden, Germany) and analyzed using the IonAmpliseq™ Comprehensive Cancer Panel (CCP) (ThermoFisher, Waltham, MA, USA), which cover all exons of 409 cancer-related genes. Raw sequencing data were mapped to the human reference genome (hg19) using the TMAP algorithm implemented in the Torrent Suite software version 4.4.3 with default parameters. Aligned bam files for matched tumor and normal samples were analyzed for copy number alteration (CNAs) calling using ad-hoc workflow implemented on Ion Reporter version 5.10.

DNA was extracted using the QIAamp DNA Mini Kit or GeneRead DNA FFPE Kit (Quiagen, Hilden, Germany) following the manufacturer's instructions. After fragmentation of the DNA to approximately 200 bp with advanced focused acoustics (Covaris), library construction was performed with a combination of SureSelect HumanAll Exon Kit (Agilent Technology) and KAPA Hyper Prep Kit (NIPPON Genetics) according to the manufacturers' protocols. Enriched fragment libraries were then sequenced on an Illumina HiSeq 2500 in 101‐bp paired‐end mode. A robust bioinformatics pipeline validated previously was used for alignment, variant calling, and analyzing copy number variation (Supporting Information Methods).