129 Molecular analysis for ovarian cancer detection in patient-friendly samples described before (3), using the NucleoMag 96 Tissue kit (Machery-Nagel) and a Microlab Star robotic system (Hamilton, Germany). DNA of FFPE tissue samples was isolated using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany). DNA of fresh frozen tissue samples was isolated using the DNeasy Blood & Tissue kit (Qiagen). DNA yield was quantified using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, US). Up to 250 ng of extracted DNA was subjected to bisulfite modification using the EZ DNA Methylation Kit (Zymo Research) to convert unmethylated cytosines. All procedures were performed according to manufacturer’s guidelines. Reaction conditions and instrument identifications of quantitative methylation-specific PCR Up to 50 ng of modified DNA was mixed with Epitect Multiplex PCR Mastermix (Qiagen, Venlo, Netherlands), 2.5-5.0 µM of each primer, and 5.0-10.0 µM of each hydrolysis probe in a total volume of 12.5 µl. Thermocycling conditions were: 95°C for 5 minutes, 45 cycles at 95°C for 15 seconds, 59/60/63°C for 1 minute, and 72°C for 1 minute. Quantitative methylation-specific PCR (qMSP) assays were performed using a ViiA7 real-time PCR-system (Applied Biosystems, Foster City, CA, USA) or an ABI-7500 realtime PCR-system (Applied Biosystems, Waltham, MA, US) for GHSR/SST/ZIC1. The qMSP data was analyzed with manual thresholds and automatic baseline settings using QuantStudioTM Real-Time PCR Software (v. 1.6.1) and 7500 Software (v. 2.3). Analysis of somatic copy number aberrations and cell-free DNA fragmentation patterns Processing of the sequencing data was performed by a pipeline controlled by Snakemake (v. 7.14.0). In brief, sequencing adapters and indexes were trimmed by the bbduk.sh (v. 38.79) [https://sourceforge.net/projects/bbmap/] in paired mode with parameters ‘ktrim=r k=23 mink=11 hdist=1’ and the adapter reference dataset provided with the software. Trimmed non-converted samples were mapped to the GRCh38 human genome assembly (GeneBank accession: GCA_000001405.28) using bwa mem (v. 0.7.17) [https://github.com/lh3/bwa]. Enzymatically converted reads were mapped to the same assembly using biscuit (v. 1.0.2.20220113) [https://huishenlab.github.io/biscuit/]. For both non-converted and converted samples, reads with a mapping quality lower than 5, unmapped reads, secondary mappings, chimeric and PCR duplicates were filtered using samtools (v. 1.12) [https://github.com/samtools/samtools] and sambamba markdup (v. 0.8.1) [https://lomereiter.github.io/sambamba/]. Reads passing the filtering step were submitted for somatic copy number aberrations (SCNA) analysis and tumor fraction estimation using the ichorCNA software (v. 0.3.2.0) (7) using default settings, except the use of an in-house panel-of-normals from shallow whole-genome sequencing, setting the non-tumor fraction parameter restart values to c(0.95,0.99,0.995,0.999). The tumor 5
RkJQdWJsaXNoZXIy MjY0ODMw