DNA was obtained using the QiaAmp DNA Mini kit (Qiagen #51304) according to the manufacturer’s instructions and concentration was determined by Nanodrop (Thermo Scientific) measurement. Array comparative genomic hybridisation (arrayCGH) was performed using 105 K (amadid#019015) or 180 K (amadid#023363) Human Genome CGH Microarray slides from Agilent Technologies (Santa Clara, CA, USA) following the manufacturer’s protocols. For shallow whole genome sequencing, DNA extraction was performed on 10 µm-thick FFPE tissue sections, using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) and protocol and deparaffinization solution. Covaris’ adaptive focused acoustics technology and M220 focused ultrasonicator (Covaris, Woburn, MA) were used to prepare fragmented DNA with fragment sizes of 200 bp. DNA libraries were constructed with the NEXTflex rapid DNA-Seq kit and protocol and NEXTflex DNA barcodes (Bioo Scientific, Austin, TX) using 200 ng of fragmented DNA as starting material, size selection, and eight PCR cycles. Cluster generation and sequencing were accomplished by, respectively, a cBot 2 and HiSeq 3000 system (Illumina, Essex, UK). The minimal number of reads (single-end; 50-cycle mode) per sample was intended to be at least 10 million (mean coverage of ~ 0.15 ×).
Copy number data were processed, analysed and visualised using VIVAR31 (link). For fluorescent in situ hybridization (FISH), four-micron-thick tissue sections were cut onto positively charged slides. The unstained slides were deparaffinized in xylene and dehydrated in graded alcohols. Cell conditioning was performed in a 1 M sodium thiocyanate water bath at 80 °C for 30 min, followed by a washing step in 2 × saline-sodium citrate (SSC) buffer and an incubation step using proteinase K (Roche, Indianapolis, IN, USA) for 20 min at 37 °C. Probes for for the SOX11 locus (CTD-2037E22) was applied, following heat block denaturation at 80 °C for 5 min, and hybridization at 37 °C for 14 to 18 h. The coverslip was removed by washing in 2 × SSC buffer. Excess probe was eliminated with 0.5 × SSC buffer stringency washes, followed by similar graded stringency washes. The digoxigenin-labeled probes were visualized using fluorescein isothiocyanate-antidigoxigenin (Roche). Using 4′,6-diamidino-2-phenylindole counterstain, nucleated cells were highlighted. A microscope, equipped with a dual-pass filter (Green/Orange; Vysis) and two single-pass filters (Green; Vysis, and Orange, Vysis), was employed to ultimately observe FISH signals. SOX11 amplifications and high-level focal gains were identified as copy number segments overlapping with the SOX11 locus with log2 ratio > = 2 and > = 0.3 respectively and a maximal size of 5 Mb.
Copy number data were processed, analysed and visualised using VIVAR31 (link). For fluorescent in situ hybridization (FISH), four-micron-thick tissue sections were cut onto positively charged slides. The unstained slides were deparaffinized in xylene and dehydrated in graded alcohols. Cell conditioning was performed in a 1 M sodium thiocyanate water bath at 80 °C for 30 min, followed by a washing step in 2 × saline-sodium citrate (SSC) buffer and an incubation step using proteinase K (Roche, Indianapolis, IN, USA) for 20 min at 37 °C. Probes for for the SOX11 locus (CTD-2037E22) was applied, following heat block denaturation at 80 °C for 5 min, and hybridization at 37 °C for 14 to 18 h. The coverslip was removed by washing in 2 × SSC buffer. Excess probe was eliminated with 0.5 × SSC buffer stringency washes, followed by similar graded stringency washes. The digoxigenin-labeled probes were visualized using fluorescein isothiocyanate-antidigoxigenin (Roche). Using 4′,6-diamidino-2-phenylindole counterstain, nucleated cells were highlighted. A microscope, equipped with a dual-pass filter (Green/Orange; Vysis) and two single-pass filters (Green; Vysis, and Orange, Vysis), was employed to ultimately observe FISH signals. SOX11 amplifications and high-level focal gains were identified as copy number segments overlapping with the SOX11 locus with log2 ratio > = 2 and > = 0.3 respectively and a maximal size of 5 Mb.
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