Maxwell 16 mdx

WES was performed on matched tumor and non-neoplastic tissues using previously described techniques.^{19 (link),20 (link),22 (link),23 (link)} DNA was extracted from macrodissected FFPE or fresh frozen tumor tissue along with peripheral blood lymphocytes or buccal tissue using the Promega Maxwell 16 MDx. The Agilent HaloPlex kit, an enzymatic capture method, was used for library preparation. Both neuropathology review and CLONET software (OMICS_07304), a computational tool to estimate ploidy and tumor content, estimated tumor content. An Illumina HiSeq 2500 system (2 × 100 bp) was used to sequence all samples. A total of 21 522 genes were analyzed with an average coverage of 90× and 94× for tumor and non-neoplastic specimens, respectively. All bioinformatics analyses were performed according to the Clinical Laboratory Improvement Amendments (CLIA)-approved Whole-Exome Sequencing Test For Cancer-EXaCT-1-pipeline version 09.^{20 (link)}Germline variants were screened based on our most recent germline pipeline.^{19 (link),20 (link)} For the purpose of the study, we included only variants in 152 cancer-associated germline genes. All candidate pathogenic germline variants detected were manually reviewed with the Integrative Genome Viewer.^{24 (link)}

Initial identification of the fungi was performed by amplicon sequencing. Fungal biomass grown on PDA plates was collected and genomic DNA was extracted using a Maxwell-16 MDx automated system following the manufacturer’s instructions (Promega). The ITS region was amplified employing polymerase chain reaction (PCR), using primers ITS 1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) [21 (link)], and Tw13 (5′-GGT CCG TGT TTC AAG ACG-3′) [22 (link)]. PCR conditions and sample preparation steps for sequencing were described elsewhere [23 (link)]. The ITS sequences were also included in the MLST-based analysis.

Total RNA was extracted using an automated system, Maxwell 16 Mdx (Promega) and following the manufacturer’s indications. The integrity and purity of the total RNA isolated was assessed using 1% agarose gel electrophoresis and quantified in a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA). Total RNA was treated with DNase (DNA-free kit, Ambion, UK) and cDNA synthesis carried out in 20 μl reactions containing 500 ng of DNase-treated RNA, 200 ng of random hexamers (Jena Biosciences, Germany), 100 U of RevertAid (Fermentas, Thermo Fisher Scientific, USA) reverse transcriptase and 8 U of RiboLock RNase Inhibitor (Fermentas). Reactions were incubated for 10 min at 25°C and 60 min at 42°C, followed by enzyme inactivation for 10 min at 70°C, and storage at -20°C until use in the amplification of sea bass orthologue genes and RT-qPCR.

Genomic DNA was extracted from frozen OCT-embedded tumors, macrodissected FFPE tumors, and blood specimens using Promega Maxwell 16 MDx per manufacturer’s instructions (Promega, Madison, WI). DNA quality and quantity were assessed using the Agilent Tapestation 4200 (Agilent Technologies) and Qubit Fluorometer (ThermoFisher), respectively. Sample libraries were prepared with different protocols, according to their RunID (see Supplementary Table 1).

Fetal and maternal tissues were processed with RNAlater (Invitrogen, Carlsbad, CA) according to the manufacturer protocols. Viral RNA was isolated from the tissues using the Maxwell 16 LEV simplyRNA Tissue Kit (Promega, Madison, WI) on a Maxwell 16 MDx instrument (Promega, Madison, WI). A range of 20–40 mg of each tissue was homogenized using homogenization buffer from the Maxwell 16 LEV simplyRNA Tissue Kit, the TissueLyser (Qiagen, Hilden, Germany) and two 5 mm stainless steel beads (Qiagen, Hilden, Germany) in a 2 ml snapcap tube, shaking twice for 3 minutes at 20 Hz each side. The isolation was continued according to the Maxwell 16 LEV simplyRNA Tissue Kit protocol, and samples were eluted into 50 μl RNase free water.