Maxwell 16 mdx instrument

The samples were processed applying the standard laboratory protocol in order to perform the comparative analysis. The Genomic DNA extraction required a manual lysis phase followed by automated extraction through “Maxwell® 16 MDx Instrument” (Promega, WI, USA) in association with the kit “DNA IQ™ Casework Pro Kit for Maxwell® 16” (Promega, WI, USA) according to the manufacturer’s instruction. Quantifiler® Trio DNA Quantification Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate the concentration and the quality of the purified DNA followed by analysis through the software “HID Real-Time PCR Analysis Software” according to the instructions provided by the kit. The Quantifiler™ Trio DNA Quantification Kit simultaneously assesses DNA concentration and degradation by detecting two human multicopy autosomal targets: a small amplicon of 80 bp in length and a large amplicon of 214 bp size. Using the ratio between the concentrations of both amplicons (short/large) the level of degradation could be measured. Thus, the larger target is more likely to be affected by degradation of the DNA template than the shorter autosomal target. When DNA is degraded, the concentration of DNA detected with the degradation target is less than the concentration detected with the autosomal target.

DNA was extracted on a Maxwell® 16 MDx instrument (Promega) using 5 × 10 μm scrolls prepared from the diagnostic formalin‐fixed, paraffin embedded tissue block. Then, EBV was detected by a qPCR assay targeting the BamH1W segment of EBV, as previously described.21 Cases were considered positive if the fluorescence signal crossed the critical threshold in fewer than 40 cycles.

Bone marrow buffy coats were collected from the patients; the median lymphoid cell percentage was 85.75% (range, 41.60–99.00%). CD19-positive B cells were collected from five healthy donors using magnetic bead sorting (EasySep^TM; STEMCELL Technologies, Inc., Vancouver, Canada). Purity was confirmed using flow cytometry analysis (>95.0%). Genomic DNA was isolated using the Promega Maxwell® 16 MDx Instrument. Genomic DNA (1 µg) was sheared to 200–400 bp using a Covaris LE220 sonicator; the fragments were then subject to methyl-CpG enrichment using the Invitrogen MethylMiner Methylated DNA Enrichment Kit, which uses a recombinant form of human MBD protein-2. The enriched methylated DNA fragments were eluted as a single enriched population with a 2,000 mM NaCl elution buffer. The eluted DNA was then used to generate libraries according to the standard Illumina protocol. Briefly, the DNA fragments were subject to end repair, A-tailing of the 3′ end, Illumina adapter ligation, size selection (aiming for 300–500 bp), PCR amplification, and validation using an Agilent Bioanalyzer. The libraries were then sequenced on the Illumina HiSeq 2000 platforms.

Formalin-fixed paraffin-embedded tissue blocks from 206 patients were used to prepare 10-μm-thick sections. Genomic tumor DNA was extracted using the Maxwell 16 MDx Instrument (Promega, Madison, WI, USA), in accordance with the manufacturer's instructions. The tumor tissues were macro-dissected, in accordance with a previously described method (13 (link)). IDH1 and IDH2 mutations were analyzed using genomic DNA from 195 patients with CC and 11 patients with BilIN. The extracted genomic DNA was subjected to mutation analysis using the PNAClamp™ IDH Mutation detection kit (Panagene, Daejeon, Korea), as previously described (14 (link)). The mutation spots of IDH1 and IDH2 that are detected with the PNAClamp™ kit are listed in Supplementary Table 1. The PNA clamping probes were complementary to the wild-type sequence and competitively inhibited the binding of the DNA primers. Consequently, they preferentially amplified the mutant alleles. The reported sensitivity of the PNAClamp™ kit for IDH1/2 mutation testing is 1% with cloned DNA, according to the manufacturer's verification. Real-time PCR reactions were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).