Snptype assay

Genomic DNA was extracted from whole blood samples stored at −80°C using DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) and set at 50 ng/μL.
Single nucleotide polymorphisms (SNPs), validated by sequencing in the 1000 Genomes project, with a minor allele frequency of ≥5% in enzymes directly involved in vitamin D metabolism (CYP2R1, CYP27A1, CYP27B1, CYP24A1, and VDR) were selected for genotyping. Of 365 SNPs that passed the aforementioned criteria, 296 SNPs passed the primer design quality control, for which SNPType assays were obtained from Fluidigm (Fluidigm Corp., South San Francisco, CA). Genotyping was performed on a 96.96 Dynamic Array IFC chip (Fluidigm, PN BMK‐M‐96.96GT) using a BioMark instrument (Fluidigm Corp.) per manufacture's specifications and protocol.
The data were analyzed using the Fluidigm Genotyping Analysis Software with Auto‐Call Analysis feature and the NTC Data Normalization Method with a Confidence Threshold set at the default of 65. There were 761 samples that were genotyped across 296 unique SNPs. Fifteen percent of the replicated samples resulted in 99% intra‐ and 97% interplate reproducibility.

Genomic DNA was extracted from the buffy coat fraction in accordance with standard procedures using a phenol-chloroform extraction method and checked for quality using Qubit (Life Technologies).
Participants were tested for three major AMD-associated SNPs: ARMS2 A69S (rs10490924), CFH I62V (rs800292), and CFH Y402H (rs1061170) using SNP Type Assays (Fluidigm, San Francisco, CA, USA). The quality control of genotyping was assessed statistically using the Hardy-Weinberg test, and P values more than 0.05 were considered that genotype distributions were in HWE. 5% random-samples were retyped by two different examiners, and those were 100% matched.

SNP genotyping was assessed using allelic discrimination with SNPType assays ordered from Fluidigm. When SNPType assays were not available, TaqMan assays (Life Technologies) were used instead. Because DNA concentration available was below supplier recommendation, a Specific Target Amplification was performed to enrich targeted SNP sequences according to the supplier’s instructions.

Tumour DNA was still available for 297 out of 378 patients from the AGO-OVAR 11 trial, from which patients with high-grade serous histology (n = 211) and complete resection (n = 96) or residual disease (n = 115) were selected for variant genotyping via SNPtype assays (Fluidigm). Assays were designed for 10 variants of interest with allele-specific primers (Fluidigm; Supplementary Table 6) and allele-specific PCR products were detected with FAM or HEX-labelled universal probes (Fluidigm). Variant genotype was then tested for association with log2 normalised DASL gene expression data for variant-gene pairs of interest under an allelic model via GraphPad Prism v9.0 using Student’s t test to compare two groups or ANOVA between three groups. A linear test for trend was performed after ANOVA as well as after a linear regression analysis to check whether the genotype was associated with the transcript levels under an allelic model. Sequences for the selected Illumina Human HT-12 WG-DASL V4.0 R2 expression bead chip assays are provided in Supplementary Table 4. eQTL analysis was also performed for patients after stratification by debulking. Variant genotypes were further tested as predictors of progression-free survival in survival analysis via GraphPad Prism v9.0.

The SNP genotyping procedure using 96.96 Dynamic Arrays™ with integrated fluidic circuits (IFCs)^{91 (link)} was conducted according to the manufacturer’s protocol for genotyping with SNPtype™ Assays (Advanced Development Protocol 34, Fluidigm corp.). Low DNA samples were pre-amplified in a modified STA for enrichment of the target loci before the SNP genotyping PCR. The pre-amplification of the target regions was conducted using 14 cycles for invasive samples and 28 cycles with extracts from non-invasive samples according to von Thaden et al.^{25 (link)}.
All experiments and sample setups included NTCs (no template controls) and STA NTCs. In all experiments NTCs and samples were replicated.