114 Chapter 5 cervicovaginal self-samples were collected at home and clinician-taken cervical scrapes were collected before surgery. Urine was centrifuged and separated into two fractions: the urine supernatant and the urine sediment. Both fractions and the remaining full void urine were stored for further analysis. Following DNA extraction, up to 250 ng of DNA was subjected to bisulfite modification. DNA methylation analysis by quantitative methylation-specific PCR Methylation levels of the C2CD4D (gene-ID: 100191040), CDO1 (gene-ID: 1036), GALR1 (gene-ID: 2587), GHSR (gene-ID: 2693), MAL (gene-ID: 4118), NRN1 (gene-ID: 51299), PRDM14 (gene-ID: 63978), SST (gene-ID: 6750), and ZIC1 (gene-ID: 7545) genes were measured by quantitative methylation-specific polymerase chain reactions (qMSP). Methylation markers were multiplexed to assess the methylation levels of three genes (1: GHSR/SST/ZIC1, 2: CDO1/MAL/PRDM14, 3: C2CD4D/GALR1/NRN1) and a reference gene (ACTB, gene-ID: 60) within the same reaction. Methylation analysis of CDO1, GALR1, GHSR, MAL, SST, PRDM14, and ZIC1 was performed as described previously (10, 18, 19) with a shortened amplicon size of ACTB, MAL and ZIC1 to facilitate methylation detection in fragmented urinary DNA. Assays targeting C2CD4D and NRN1 were designed based on gene loci discovered and validated by others (17, 21). Primer and probe information is provided in Supplemental Table 1. Reaction conditions, instrument identifications, and thermocycling parameters are described in the Supplemental Methods. Doublestranded gBlocks™ Gene Fragments (Integrated DNA Technologies) containing the target amplicons and H2O were taken along in each run as positive and negative control, respectively. Sample quality and sufficient input was ensured by excluding samples with a ACTB quantification cycle (Cq) ≥ 32. Methylation levels were calculated relative to ACTB levels by the comparative Cq method: 2 ^ -(Cq marker – Cq ACTB) x 100 (26). All qMSP assays were designed, multiplexed and optimized according to parameters described earlier (27). Target specificity was validated in silico (BLAST). Correct amplicon size was verified by agarose gel electrophoresis. Analytical validation was performed using a dilution series of bisulfite treated methylated DNA from the SiHa cell line (100, 50, 10, 5, 1, 0.5%) within the range of 20 to 0.1 ng (Supplemental Table 2). The discriminatory power of each assay was verified by comparing methylation marker levels in tissue samples of ovarian cancer patients with those measured in normal fallopian tube tissue. Shallow whole-genome sequencing Urine cell-free DNA (cfDNA) extracted from urine supernatant samples of ovarian cancer patients was further characterized by shallow whole-genome sequencing (~1x coverage). The cfDNA was quantified and analyzed using a Cell-free DNA ScreenTape
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