From the Immunochip association analysis, we selected the lead marker(s) with P < 5×10−5 from 20 novel loci (3 loci from Table 2 and 17 from Supplementary Table 5 ). Markers located within about ± 100 kb from these lead markers were selected for imputation. Since IL1A-IL1B and PTPN1 loci were sparsely genotyped by the Immunochip, fine mapping was performed for these loci before imputation with iPLEX assays (TOF-MS, Agena) using the same Turkish samples. The Tagger SNP selection tool from HapMap was used to select SNPs with the intent of obtaining 100% coverage of the HapMap phase 3 SNPs with greater than 1% minor allele frequency in the CEU HapMap population with pairwise r2 > 0.8. Although already tagged, additional SNPs with r2 > 0.8 with the most significantly associated SNP of the region were also included. After combining fine mapping and Immunochip data, we imputed these loci by IMPUTE249 (link). The same loci were also imputed from Japanese GWAS data for the replication study. The 1000 Genomes Project Phase 1 integrated dataset52 (link) was used as the reference panel for imputation. Markers with info score > 0.8 and genotypes with probability > 0.9 were included in analyses. For quality control, markers with MAF < 0.01 and HWE P-value < 1×10−5 in controls were excluded.
Iplex assay
Manufactured by Agena
Sourced in United States
The IPLEX Assay is a laboratory equipment product that measures the presence and concentration of a specific analyte in a sample. It provides quantitative analysis through an immunoassay-based detection method. The core function of the IPLEX Assay is to enable accurate and reliable measurements in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Massarray system, by Agena (9 mentions)
Typer 4, by Agena (1 mentions)
Massarray assay design 3, by Agena (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Massarray, by Agena (1 mentions)
Iplex gold, by Agena (1 mentions)
Histopaque solution, by Merck Group (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Mammalian genomic dna extraction kit, by Beyotime (1 mentions)
Massarray platform, by Agena (1 mentions)
16 protocols using iplex assay
1
Immunochip Genotyping and Imputation
2
Genotyping of MMP-2 Polymorphisms
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For the present study, 500 samples with relevant clinical data and a DNA sample were available. DNA was isolated from whole blood were used the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi’an City, China) extracted. The primer is listed in Table 1 . 6 single nucleotide polymorphisms (SNPs) in MMP-2 were genotyped using the Agena iPLEX assays with allele detection by mass spectroscopy, using Sequenom MassARRAY technology (Agena Bioscience, San Diego, CA) and following the manufacturer's protocol.[20 ] In this study, we used Agena MassARRAY Assay Design 3.0 Software to design a Multiplexed SNP MassEXTEND assay. The PCR primers for each SNP are shown in Table 1 . Data management and analysis were performed using the Agena Typer 4.0 Software (Agena Bioscience, San Diego, CA).
3
Immunochip Genotyping and Imputation
4
Genotyping Cognitive and Neuropsychiatric SNPs
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Genomic DNA was extracted from the peripheral venous blood using phenol–chloroform extraction method. For this study, two multiplex panels of SNP markers that showed repeated association with cognitive performance, AD or/and schizophrenia in the GWAS were formed (see Table 2 ). Genetic markers were selected based on certain and clear criteria. First of all, we selected loci reaching a genomic value (p < 5.00 × 10−7) for the phenotype (cognitive functions, AD and/or schizophrenia in GWAS). The next requirement concerned the type of marker: only SNPs were selected. The minor allele frequency (MAF) should have been ≥ than 5% in at least one population group of the HapMap project or the 1000 Genomes project. Selected markers should form multiplexes using the Assay Design Suite v2.0 genotyping tool (https://agenacx.com/online-tools/ , accessed on 6 August 2021). All patients and controls were genotyped for 62 SNPs using iPLEX Assay following the recommended protocol by the manufacturer (Agena Bioscience™, San Diego, CA, USA). In addition to 62 SNPs, genotyped by MALDI/TOF mass spectrometry, rs7412 of APOE was genotyped by real-time PCR using the TaqMan® SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA).
5
Genotyping Study of Uric Acid SNPs
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We genotyped single nucleotide polymorphisms (SNPs) recently identified as top hits in genome-wide association studies (GWAS) of uric acid levels [17] [18] [19] . In addition, SNPs would be chosen when their minor allele frequency is >0.05 in Han Chinese according to the 1000 Genomes database. Finally, 13 SNPs in PDZK1 (rs1967017), GCKR (rs1260326), ABCG2 (rs2231142 and rs4148152), SLC2A9 (rs1014290, rs11722228, and rs3775948), SLC17A1 (rs1183201), SLC16A9 (rs2242206), SF1 (rs606458), SLC22A11 (rs17300741), SLC22A12 (rs505802), IGF1R (rs6598541) were genotyped in our study, with genotypes being determined by the MassARRAY system (Agena iPLEXassay, San Diego, USA). For all the SNPs, 12 of them passed quality control criteria (call rates >96% and duplicate concordance is 100% in the 96 samples assessed), and only one SNP (rs17300741) was not detected. None of the SNPs showed substantial deviation from Hardy-Weinberg equilibrium (P > 0.05). Individuals with>10% of the genotypes missing were excluded.
6
Assessing miRNA Target Site Polymorphisms
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Potentially functional polymorphisms at miRNA target sites were assessed using PolymiRTS database 3.0 (http://compbio.uthsc.edu/miRSNP http://cbio.mskcc.org/cancergenes
7
SNP Genotyping from Blood Samples
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Peripheral blood samples were collected from all 92 participants in our study by standard venipuncture in tubes containing ethylenediaminetetraacetic acid (EDTA) at seven in the morning, which were stored at −70°C until DNA extraction. DNA was extracted from white blood cell fractions using the Chelex-100 extraction method. Purity and concentration of the extracted DNA were determined spectrophotometrically. Extracted DNA was stored at −20°C for further analysis. SNP genotyping was performed on an Agena Bioscience MassARRAY® platform utilizing the iPLEX® assay (Agena Bioscience Inc., San Diego, CA, USA). PCR primers and single base pair extension primers were designed using ADS 2.0 [13 (link)] and are provided in Table 1 . Polymerase chain reaction (PCR) amplification and extension reactions were performed according to the manufacturer's instructions (iPLEX Gold, Agena Bioscience Inc.). Extension product sizes were determined by mass spectrometry using the MassARRAY system (Agena Bioscience Inc.). Samples were run in duplicate and positive and negative control samples were included in each plate to assess genotyping accuracy.
8
Integrated ChIP-seq and RNA-seq Analysis for SNP Selection
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We conducted an integrated analysis of ChIP-seq and RNA-seq for the SNP selection. As a result of the ChIP-seq, SNPs within H3K4me3 and H3K27ac peak regions were selected. Next, using the FuncPred utility for functional SNP prediction in the SNPinfo web server (https://snpinfo.niehs.nih.gov ), potentially functional variants were collected. And then, using RNA-seq we chose genes with a high expression level (FPKM ≥100), and SNPs within or closest to the genes were extracted. Genotyping was performed using iPLEX Assay and MassARRAY System (Agena Bioscience, San Diego, CA, USA).
9
Genetic Ancestry Estimation for Population Structure
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Genetic ancestry was estimated to investigate population structure in the cases and control cohorts (Collins‐Schramm et al., 2003 ). Genetic ancestry estimates were generated in a two‐way model of admixture, European and West African, for the neonates of each self‐reported African‐American study subject using 102 ancestry informative markers (AIMs), single nucleotide polymorphisms with large allele frequency differences between ancestral populations, (Modi et al. 2017 ). The mean allele frequency difference between ancestral populations for the AIMs panel was delta (δ) = 0.733. The AIMs panel was derived from the overlap of the WES and the Illumina African American Admixture Mapping Panel (Illumina, San Diego, CA, USA) and genotyped using a custom iPLEX assay (Agena Biosciences, San Diego, CA, USA) for study subjects who were not part of the WES discovery set (Modi et al. 2017 ). Prior allele frequencies derived from the HapMap West Africans (YRI, Yoruba in Ibadan, Nigeria) and Europeans (CEU, CEPH Utah residents with ancestry from northern and western Europe) were used to estimate individual genetic ancestry following a maximum‐likelihood approach.
10
DNA Extraction from PBMCs
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Lactase persistence and lactase non-persistence (LNP) in the study participants was determined using the iPlEX assay and MassARRAY® System (Agena Bioscience, SanDiego, USA) by Grafton Clinical Genomics (GCG, Auckland, New Zealand). Peripheral blood mononuclear cells (PBMCs) were isolated immediately from fasted whole blood collected in EDTA-containing blood collection tubes using a histopaque solution (Sigma- Aldrich, St. Louis, MO, USA) as previously described (39 (link)). The samples were stored at −80°C until DNA was extracted. All prep DNA/RNA mini kit (Qiagen, Hilden, Germany) was used to isolate genomic DNA from PBMCs as per the manufacturer's protocol.
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