Peripheral blood of the three experimental groups including BD and VKH disease patients and the controls was subjected to genomic DNA extraction with the QIAmp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA, USA) and the DNA was stored at −80°C. The Applied Biosystems 7500 Real-Time PCR system was utilized to genotype CCL17/rs223828 (TagMan assay ID: C_30530263_10) by the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA). Genotype identification of the other 25 SNPs was conducted with the iPLEX Gold genotyping assay and Sequenom MassARRAY (Sequenom, CA, USA). Sequenom SNP Assay Design software version 3.0 was used to design primers of iPLEX reactions. Primer sequences used were shown in Table 1 . The protocol and experimental requirements were performed strictly based on the instructions.
Iplex gold genotyping assay
Manufactured by Labcorp
Sourced in United States
The IPLEX Gold genotyping assay is a laboratory equipment product developed by Labcorp. It is a genotyping assay that can be used to analyze genetic variations. The core function of the IPLEX Gold genotyping assay is to detect and identify specific genetic markers or mutations in DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Sequenom massarray, by Labcorp (4 mentions)
Qiamp dna blood mini kit, by Qiagen (3 mentions)
Sequenom snp assay design software version 3, by Labcorp (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Massarray designer, by Labcorp (2 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Massarray, by Labcorp (1 mentions)
Goldengate assay, by Illumina (1 mentions)
Geneamp pcr system 9700 thermocycler, by Thermo Fisher Scientific (1 mentions)
Massarray platform, by Labcorp (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Massarray typer v3, by Labcorp (1 mentions)
8 protocols using iplex gold genotyping assay
1
Genotyping of CCL17 and 25 SNPs
2
Genotyping of Common Chinese Variants
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Based on the Chinese population data from HapMap database, we used HaploView 4.2 program to select the candidate tag SNPs with an r2 threshold of 0.8 and minor allele frequency (MAF) greater than 1%. Under this criteria, totally 11 tag SNPs were selected. Additionally, we added two potential functional SNPs, rs9429942 and rs6691117 18 (link), 19 (link). Therefore, we included 13 SNPs in our study, which represents common genetic variants in Chinese population.
Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Assay and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom's MassArray Designer was used to design PCR and extension primers for each SNP. The PCR primers used are available upon request. Genotyping quality control consisted of no-temple control samples for allele peaks and verifying consistencies in genotype calls of 2% randomly selected duplicate samples. In addition, two control samples were included on each plate as genotyping controls for inter-plate reproducibility. Hardy-Weinberg Equilibrium (HWE) was also evaluated in unrelated controls.
Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Assay and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom's MassArray Designer was used to design PCR and extension primers for each SNP. The PCR primers used are available upon request. Genotyping quality control consisted of no-temple control samples for allele peaks and verifying consistencies in genotype calls of 2% randomly selected duplicate samples. In addition, two control samples were included on each plate as genotyping controls for inter-plate reproducibility. Hardy-Weinberg Equilibrium (HWE) was also evaluated in unrelated controls.
3
TXNRD1 Genetic Variation Protocol
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14 SNPs covering the whole TXNRD1 genetic variability were prioritized by a tagging approach, attempting to choose those most likely to be of functional relevance (nonsynonymous SNPs, SNPs located in the 5′ and 3′ UTR regions). SNPs associated with health status and longevity in Northern Europeans [14 (link), 15 (link)] were also chosen. SNPs with a minor allele frequency (MAF) less than 5% were excluded from the analysis.
Multiplex SNP genotyping was performed using iPLEX Gold Genotyping Assay and Sequenom MassARRAY (Sequenom, San Diego, CA, USA) according to manufacturer's instructions. Sequenom's MassARRAY Designer was used to design PCR and extension primers for each of the 14 SNPs selected. However, four of them (rs10861169, rs10861197, rs10047589, and rs4964287) were skipped by the software for primers design and were not analyzed in this paper. The details for genotyped SNPs are listed inTable 1 .
Multiplex SNP genotyping was performed using iPLEX Gold Genotyping Assay and Sequenom MassARRAY (Sequenom, San Diego, CA, USA) according to manufacturer's instructions. Sequenom's MassARRAY Designer was used to design PCR and extension primers for each of the 14 SNPs selected. However, four of them (rs10861169, rs10861197, rs10047589, and rs4964287) were skipped by the software for primers design and were not analyzed in this paper. The details for genotyped SNPs are listed in
4
Genetic Profiling of AMD Patients
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Peripheral blood of AMD patients and the controls were subjected to genomic DNA extraction by using the QIAmp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA, USA) and the DNAs were stored at − 80∘C. Genotype identifications of the 39 SNPs were conducted with the iPLEX Gold genotyping assay and Sequenom MassARRAY (Sequenom, CA, USA). Sequenom SNP Assay Design software (version 3.0) was used to design the primers of iPLEX reactions [27 (link)]. Primer sequences used were shown in Additional file 1 : Table S1.
5
DNA Extraction and Genotyping of CpG-SNPs
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The two experimental groups including both BD individuals and healthy controls donated peripheral blood, which was applied to extract genomic DNA extraction with a QIAmp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA, USA) and stored in 3.2% sodium citrate-treated tubes at -80°C. The iPLEX Gold genotyping assay and Sequenom MassARRAY (Sequenom, CA, USA) were performed to identify genotype of the 10 CpG-SNPs. iPLEX reactions primers were designed by SNP Assay Design software (version 3.0) (Table 2 ). All procedures were performed according to manufacturer's instructions.
6
Genotyping Fairview Tree Samples
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A panel of 1536 SNPs was previously developed using the Illumina (San Diego, CA, USA) GoldenGate assay (Janes et al., 2014 (link)). From this GoldenGate panel, we selected 114 SNPs to develop a Sequenom panel. In total, 404 samples from the five Fairview trees (see Supplementary Table 1 ) were genotyped at McGill University and Génome Québec Innovation Centre using the Sequenom iPLEX Gold genotyping assay (Sequenom, 2008 ).
Genotyped samples and SNPs were filtered to ensure high quality and reproducibility. First, the quality of SNP and sample calls was assessed using the call rate. SNPs and samples with a call rate of ⩾80% were considered ‘good', whereas SNPs and samples with call rates <80% were removed. Second, samples were checked for reproducibility. Third, samples and SNPs with >10% missing information (that is, NN) were removed.
Genotyped samples and SNPs were filtered to ensure high quality and reproducibility. First, the quality of SNP and sample calls was assessed using the call rate. SNPs and samples with a call rate of ⩾80% were considered ‘good', whereas SNPs and samples with call rates <80% were removed. Second, samples were checked for reproducibility. Third, samples and SNPs with >10% missing information (that is, NN) were removed.
7
SNP Genotyping of Venous Blood Samples
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Venous blood samples from all subjects were collected into EDTA containing tubes and stored at −80℃. Genomic DNA was extracted with the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, California, USA) and stored at −20℃ until used. The Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, Delaware, USA) was used to qualify and quantify all DNA samples. The primers for respective SNPs were designed by MassArray Assay Design V.3.1 software (Sequenom) and stored at −20℃. The selected SNPs were genotyped by MassARRAY platform (Sequenom, San Diego, California, USA) and iPLEX Gold Genotyping Assay, and analysed by TYPER software V.4.0. The PCR was performed with the GeneAmp PCR System 9700 thermocycler (ABI, Foster City, California, USA). Because none of the primers were suitable for Sequenom MassArray, the rs27293 genotype was analysed using the TaqMan SNP Genotyping Assay (ABI, Foster City, California, USA) with the 7500 Real-Time PCR system (Applied Biosystems), and counted by TaqMan Genotyper Software. All assays were conducted in strict accordance with manufacturer’s instructions. A 90% SNP genotyping success rate was confirmed for all tested SNPs.
8
Genotyping of IP6K3 and IPMK Genes
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A panel of 17 SNPs within approximately 30 kb encompassing the entire IP6K3 gene and its 5′ and 3′ flanking regions were genotyped in all subjects included in the study and chosen based on those genotyped in the previous study by Crocco et al (2016) (link). Similarly, 14 SNPs were investigated for IPMK, mapping within and nearby the gene and prioritized by a tagging approach (De Rango et al, 2019) (link). Genotyping was performed by iPlex Gold Genotyping Assay and Sequenom MassArray (Sequenom, San Diego, CA, USA) technology, following the manufacturer's instructions. SNP assays were designed using Sequenom's MassARRAY Assay Design v3.0 Software. Spectra were analyzed using MassARRAY Typer v3.4 Software (Sequenom). For quality control, to assess the reliability of the genotype identification protocols, about 10% of the samples were reanalyzed and the concordance rate of the genotypes was higher than 99%. For additional quality control, genotypes were excluded if Hardy-Weinberg equilibrium among controls p < 0.05 or call rates < 90%.
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