Massarray iplex gold

TagSNP genotyping was performed using MassARRAY iPLEX Gold technology (Sequenom, San Diego, CA) based on multiplexed amplification followed by mass-spectrometric product separation. This technique was carried-out by the Unidade de Genómica/Serviço de Genotipagem do Instituto Gulbenkian de Ciência.All polymorphisms not included in the tagSNPs analysis were characterized through allelic discrimination (Real-Time Polymerase Chain Reaction) using validated TaqMan® SNP genotyping assays (C___2517145_20, C___7550203_10, C___15909858_20, C___16038735_10 for the rs689466, rs5275, rs2612656 and rs2555639, respectively) with the exception of the polymorphism −765G>C (rs20417) which was custom designed (Applied Biosystems, Foster City, California USA). Allelic discrimination was performed by measuring end-point fluorescence using ABI PRISM Sequence Detection System (Applied Biosystems, Foster City, California USA).

CHEK2:p.I157T was first genotyped by independent studies using various methods including MassARRAY iPLEX Gold (Sequenom, San Diego, CA, USA), TaqMan (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) and Fluidigm (Fluidigm, San Francisco, CA, USA) as listed in Additional file 1: Table S1. Quality control was implemented as follows: each study performed duplicate measurements of at least two samples from each sample plate and genotyped 93 CEPH control DNAs (HAPMAPPT01, Coriell Institute for Medical Research, Cambden, NJ, USA). If a study reported more than two discordant genotyping results of the CEPH DNAs, all genotype data from that study was excluded. Later, p.I157T was genotyped centrally using a custom Illumina iSelect genotyping array for the Collaborative Oncological Gene-environment Study (COGS) [18 (link)]. Discordant genotyping results were clarified with Sanger sequencing. CHEK2:c.1100delC was genotyped by independent studies using mainly TaqMan (Additional file 1: Table S1), as described earlier [17 (link)].

The iPLEX Gold (Sequenom, San Diego, CA, USA) or TaqMan (Applied Biosystem, foster City, CA, USA) methods following manufacturer’s protocol were employed to genotype selected variants in the entire sample sets of 623 NHWs and 788 ABs. Whole genome amplified DNAs in 384-well plates were used both genotyping methods. The ABI Prism 7900HT Sequence Detection Systems was used for end-point fluorescence reading for TaqMan genotyping and genotype calls were analyzed by using the SDSv2.4.1 and TaqMan Genotyper software. The MassARRAY iPLEX Gold (Sequenom, San Diego, CA) genotyping technique was applied in Genomics and Proteomics Core laboratories of the University of Pittsburgh. In addition to random replicates included in the genotyping process for quality control (QC) assessment, the subsets of samples used in both sequencing and genotyping steps allowed us to also evaluate the concordance between the sequencing and genotyping results. QC filters used for genotyped variants included extensive missing data (>15%) and/or deviation from Hardy-Weinberg Equilibrium (HWE) (P<0.01).

DNA was isolated from whole blood extracted at baseline from all participants where blood samples were available, in total 2870 participants. Using a saturation approach representing HapMap SNPs and HaploView scoring, a total of 11 SNPs covering 100 kb of the genetic region surrounding the CYP2R1 gene including the 3’ and 5’ untranslated regions (UTRs) were selected. Genotyping was performed using the Sequenom Mass ARRAY iPLEX Gold technology (Sequenom Inc., Newton, MA) by single base primer extension and MALDI TOF Mass Spectrometry. Successful genotyping was obtained from 8 SNPs (rs10766197, rs11023374, rs10741657, rs10832313, rs16930609, rs16930625, rs11023371 and rs7936142) with overall call rate of 97.8%. Allele frequencies were calculated and found to be in Hardy-Weinberg (HW) equilibrium in the cohort for all SNPs. Haploview software version 4.2 was used to calculate linkage disequilibrium (LD) values, generate haplotype blocks and diagrams, as well as suggesting tagging SNPs using the tagger algorithm [21 (link)]. The preselected SNPs and haplotypes computed using the Arlequine population genetic data analysis program, were analyzed for associations between vitamin D values and other biochemical parameters (calcium, phosphate, FGF-23 and PTH), as well as markers of bone mineral density (femoral neck, lumbar spine and total hip).

TagSNP genotyping was performed using MassARRAY iPLEX Gold technology (Sequenom) based on multiplexed amplification followed by mass-spectrometric product separation. This technique was carried-out by the Genomic Unit, Genotyping Service, Gulbenkien Science Institute.