Massarray analyzer

DNA was extracted from blood samples using DNeasy Blood and Tissue kit (Qiagen). The genotyping was performed using a multiplexed primer extension (SBE) chemistry of the iPLEX assay with detection of the incorporated allele by mass spectrometry with a MassARRAY analyzer from Agena Bioscience. Raw data from the mass reader was converted to genotype data using the Typer software (Agena Bioscience). Reproducibility was controlled to be 100%. The tool PLINK (version 1.07)^{64 (link)} was used for Chi-square calculations to test for association between SNV allele and phenotype. The experiment was performed twice.

Single nucleotide polymorphisms in the region of the CA6 gene were selected and genotyped on DNA isolated from whole stimulated saliva. The criteria for SNP selection were a) minor allele frequency (MAF) ≥ 5% in the CEU population b) not in complete linkage disequilibrium (r² ≤ 0.8) and c) in the 10,000 bp up-stream to 10,000 bp down-stream region of the CA6 gene. Using the NIH snptag resource (https://snpinfo.niehs.nih.gov/snpinfo/snptag.html) 30 SNPs met these criteria. Genotyping was performed using a multiplexed primer extension chemistry of the iPLEX assay with detection of the incorporated allele by mass spectrometry using a MassARRAY analyzer (Agena Bioscience, Hamburg, Germany). Raw data from the mass reader was converted to genotype data using the Typer software (Agena Bioscience)^{39 –41 (link)}. Two SNP markers had sample call rate 0%, rs2274333 and rs17032942, the reasons for the failure was low allele signals resulting in inaccurate genotype calls. An additional SNP (rs6697763) was excluded from analysis for deviation from Hardy-Weinberg equilibrium (p = 0.033), leaving 27 SNPs with valid genotype data, where the average call rate per sample was 99.4% and overall call rate was 99.8% (Table S1). Genotyping data are uploaded and available at figshare 10.6084/m9.figshare.6948020.

Each FFPE block was shaved to 75–100 µm thickness in a sterile microcentrifuge tube. The samples were then deparaffinized with xylene and absolute ethanol, respectively, before genomic DNA extraction using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. The DNA concentration from each sample was quantified using a Nanodrop Lite Spectrophotometer (Thermo Scientific, MA, USA) and kept at -20 °C until use. The DNA samples were required to have a level of quality marked by at least a 260/280 absorbance ratio between 1.5 and 2.0 and concentration ≥ 5 ng/µl, when examined using a MassARRAY analyzer (Agena Bioscience, CA, USA).

Genomic DNA was extracted from Chinese-native Jin Ling Hua (JLH) chickens (n = 384), and its quality and quantity were determined using a NanoDrop spectrometer and agarose gel electrophoresis analysis. MassARRAY detection was performed by Beijing Compass Biotechnology Co., Ltd. (Beijing, China) using the MassARRAY® analyzer (Agena Bioscience, San Diego, CA) to characterize the genotypes of candidate SNPs. Briefly, specific amplification of candidate SNPs was performed in a Veriti® 384-Well Thermal Cycler (Applied Biosystems, Foster City, CA) and the PCR products treated with alkaline phosphatase. A single-base extension reaction and resin purification were carried out, after which the PCR products were hybridized to 384 chips for mass spectrometry detection. Finally, single SNP correlation analysis of 21 SNPs in a total of 384 individuals was performed using a general linear model with PLINK.

Panel for genotyping was designed to include 62 polymorphisms that presented potential implication in patients’ treatment efficacy or drugs toxicity based on the bibliographic search performed (design named VIP-Onco). We could obtain suitable samples from 64 patients, and their genotyping was carried out at Centro Nacional de Genotipado (CEGEN, Santiago de Compostela, Spain) by mass spectrometry, employing a MassArray Analyzer (Agena Bioscience, San Diego, CA, USA).

DNA samples were transferred on PCR plates and sent to the SNP&SEQ Technology Platform, Uppsala University (National Genomics Infrastructure [NGI], SciLifeLab Sweden). The genotyping was performed using a multiplexed primer extension (SBE) chemistry of the iPLEX assay with detection of the incorporated allele by mass spectrometry with a MassARRAY analyzer from Agena Bioscience.²⁸ , ²⁹ (link), ³⁰ (link) Raw data from the mass reader were converted to genotype data using the Typer software (Agena Bioscience). Both HFE C282Y (rs1800562) and H63D (rs1799945) genotype distributions were in Hardy‐Weinberg equilibrium (Ps > .1).
Given the limited number of individual carriers of H63D or C282Y, we pooled the participants in 2 groups: non‐carriers of any of the mutations (HFE‐neg) and carriers of either the H63D and/or C282Y (HFE‐pos).