Ffpe qc kit

DNA and RNA for NGS-based mutation analysis were extracted using the Qiagen AllPrep kit for FFPE tissue and automated on the QIAcube instrument (Qiagen, Hilden, Germany). The protocol was modified with an extended proteinase K digestion (overnight) for the DNA extraction to obtain higher DNA yields. DNA from cytology slides was extracted using the QiaAmp DNA Micro kit (Qiagen, Hilden, Germany). RNA was not extracted from cytology specimens. Following extraction, DNA samples were quantified using Qubit and RNA samples were quantified using a BioAnalyzer. Then, for quality assessment DNA samples were analyzed by qPCR using the Illumina FFPE QC Kit (WG-321-1001) along with a control cell line sample with a known input mass.

DNA was extracted from cultured cells (5 million cells harvested in 200 µL PBS) using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France). Double strand DNA was quantified using a fluorometric method (Qubit dsDNA BR Assay, Thermo Fisher Scientific) and qualified by real-time PCR (FFPE QC Kit, Illumina, Paris, France). Mutation screening was performed by next generation sequencing (NGS) on a MiSeq Illumina platform using the Tumor Hotspot MASTR Plus assay (Multiplicom-Agilent, Les Ulis, France), which targets frequently occurring mutations (hotspots) in a panel of 26 genes (Supplementary Materials Table S1). Sequencing data were aligned to human genome hg19 using the BWA-MEM algorithm (Burrows–Wheeler Aligner-maximal exact matches). Variants were called using three different variant callers: VarScan, GATK HaplotypeCaller (GATK-HC), and GATK Unified Genotyper (GATK-UG). The minimum coverage per base (DP for depth) and the minimum variant allelic frequency (VAF) were fixed at 300-fold (300×) and 4% respectively, which allowed a sensitivity of 100% in the detection of mutations in the internal positive control (Tru-Q3 DNA from Horizon Diagnostics) included in each sequencing run. Reported mutations corresponded to splice site or non-synonymous exonic variants, excluding known polymorphisms, located in the genes and exons of Supplementary Materials Table S1.

For fresh-frozen tissue specimens, 10 to 20 μm serial sections were cut from each tissue specimen for manual microdissection of normal or tumour cells under morphological control, respective microscope using fine needles. DNA isolation was with the QIAamp Micro Kit, according to the manufacturer’s protocol (Qiagen, Hilden, Germany).
For FFPE tissue specimens, 5 to 10 μm serial sections were cut from each tissue specimen for manual microdissection of normal or tumour cells under morphological control, respective microscope using fine needles. From the testing and validation tissue specimens, DNA isolation was with the QIAamp FFPE DNA Kit, according to the manufacturer`s protocol (Qiagen, Hilden, Germany). For the disease course CRC tissue specimens, DNA was isolated using the Allprep DNA/RNA FFPE Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany).
For each sample, DNA concentration and purity was determined using the Nanodrop-8000 (ND1000, Peqlab, Erlangen, Germany) and the DNA quality was measured in triplicate using the “FFPE QC Kit” according to the manufacturer`s protocols (Illumina, San Diego, USA). For the latter, the average difference of Cycle-treshold (Ct) values between the DNA samples and a positive DNA control were calculated as relative “QC value”, with “<2” suggested as a valid DNA sample for library preparation by the manufacturer.

As reported by us before [69 (link)], DNA quality was measured using FFPE QC kit and libraries were prepared using the TruSeq Amplicon Cancer Panel (48 cancer related genes) (both Illumina). Quantity and quality of libraries were examined using the Bioanalyzer 2100 system (Agilent Technologies) and DNAs were pooled for sequencing on the MiSeq (Illumina). Mutations, passing the filter, were listed using VariantStudio (Illumina). Missense, stop gained and frameshift mutations displaying read depth >100, alt variant frequency >10 and occurring inside the gene were incorporated in the study.

DNA extractions from patients’ tumors, cultured cells, and stereotaxic xenograft frozen tissues (50 µm × 5) were quantified using a fluorimetric method (Qubit dsDNA BR Assay, Thermo Fisher Scientific, Illkirch, France) and qualified using real-time PCR (FFPE QC Kit, Illumina, San Diego, CA, USA). Mutation screening was performed using next generation sequencing on a MiSeq Illumina platform, using a Tumor Hotspot MASTR Plus assay (Multiplicom-Agilent, Santa Clara, CA, USA). A total of 26 gene mutations (hotspots) were screened, including H3F3A. Sequencing data were aligned to human hg19 using BWA-MEME algorithm (Burrows–Wheeler aligner-maximal exact matches). The minimum coverage per base (DP depth) and the minimum variant allelic frequency (VAF) were fixed at 500-fold and 5%, respectively. Data were visualized using Integrative Genomics Viewer (Broad Institute, USA). For BT35, BT68, BT69, and BT83, we also performed exome sequencing analyses using the SureSelect Human All Exon capture sets V4 (Agilent, Santa Clara, CA, USA), and paired-end sequenced on an Illumina HiSeq2000 with a 100 bp read length (median coverage = 100×).