FXR1 rs496250 and GSK3β rs12630592 genotypes in Sample 1 were ascertained using an Illumina HumanOmni2.5-8 v1 BeadChip platform (Illumina, Inc., San Diego, CA, USA). More in detail, approximately 200-ng DNA was used for genotyping analysis. DNA was concentrated at 50 ng/ml (diluted in 10-mM Tris/1-mM EDTA) with a Nanodrop Spectrophotometer (ND-1000). Each sample was whole-genome amplified, fragmented, precipitated, and resuspended in appropriate concentrations of hybridization buffer. Denatured samples were hybridized on the prepared Illumina HumanOmni2.5-8 v1 BeadChip. After hybridization, the BeadChip oligonucleotides were extended by a single labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader. Normalized bead intensity data obtained for each sample were loaded into the Illumina GenomeStudio (Illumina, v.2010.1) with cluster position files provided by Illumina, and fluorescence intensities were converted into SNP genotypes. After genotypes were called and the pedigree file was assembled, we removed SNPs showing minor allele frequency (MAF) < 1%, genotype missing rate > 5%, or deviation from Hardy–Weinberg equilibrium (p < 0.0001). Individuals were also removed if their overall genotyping rate was below 97%. Sample duplications and cryptic relatedness were ruled out through identity-by-state analysis of genotype data.
Humanomni2.5 8 v1 beadchip
Manufactured by Illumina
The Illumina HumanOmni2.5-8 v1 BeadChip is a high-throughput genotyping array designed to analyze genetic variation across the human genome. It features over 2.5 million markers to provide comprehensive genome coverage.
Automatically generated - may contain errors
4 protocols using humanomni2.5 8 v1 beadchip
1
Genotyping of FXR1 and GSK3β variants
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FXR1 rs496250 and GSK3β rs12630592 genotypes in Sample 1 were ascertained using an Illumina HumanOmni2.5-8 v1 BeadChip platform (Illumina, Inc., San Diego, CA, USA). More in detail, approximately 200-ng DNA was used for genotyping analysis. DNA was concentrated at 50 ng/ml (diluted in 10-mM Tris/1-mM EDTA) with a Nanodrop Spectrophotometer (ND-1000). Each sample was whole-genome amplified, fragmented, precipitated, and resuspended in appropriate concentrations of hybridization buffer. Denatured samples were hybridized on the prepared Illumina HumanOmni2.5-8 v1 BeadChip. After hybridization, the BeadChip oligonucleotides were extended by a single labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader. Normalized bead intensity data obtained for each sample were loaded into the Illumina GenomeStudio (Illumina, v.2010.1) with cluster position files provided by Illumina, and fluorescence intensities were converted into SNP genotypes. After genotypes were called and the pedigree file was assembled, we removed SNPs showing minor allele frequency (MAF) < 1%, genotype missing rate > 5%, or deviation from Hardy–Weinberg equilibrium (p < 0.0001). Individuals were also removed if their overall genotyping rate was below 97%. Sample duplications and cryptic relatedness were ruled out through identity-by-state analysis of genotype data.
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FXR1 rs496250 and GSK3β rs12630592 genotypes in Sample 1 were ascertained using an Illumina HumanOmni2.5-8 v1 BeadChip platform (Illumina, Inc., San Diego, CA, USA). More in detail, approximately 200-ng DNA was used for genotyping analysis. DNA was concentrated at 50 ng/ml (diluted in 10-mM Tris/1-mM EDTA) with a Nanodrop Spectrophotometer (ND-1000). Each sample was whole-genome amplified, fragmented, precipitated, and resuspended in appropriate concentrations of hybridization buffer. Denatured samples were hybridized on the prepared Illumina HumanOmni2.5-8 v1 BeadChip. After hybridization, the BeadChip oligonucleotides were extended by a single labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader. Normalized bead intensity data obtained for each sample were loaded into the Illumina GenomeStudio (Illumina, v.2010.1) with cluster position files provided by Illumina, and fluorescence intensities were converted into SNP genotypes. After genotypes were called and the pedigree file was assembled, we removed SNPs showing minor allele frequency (MAF) < 1%, genotype missing rate > 5%, or deviation from Hardy–Weinberg equilibrium (p < 0.0001). Individuals were also removed if their overall genotyping rate was below 97%. Sample duplications and cryptic relatedness were ruled out through identity-by-state analysis of genotype data.
2
Genome-Wide Genotyping of Blood Samples
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Genomic DNA was extracted from frozen whole blood using the QIAamp DNA Blood Mini kit (Qiagen, Inc., Valencia, CA), and the DNA genotype was screened for ∼ 2,500,000 haplotype tagging SNPs using an Illumina HumanOmni2.5-8v1 BeadChip (Illumina, San Diego, CA) according to Illumina protocols at the University of Miami Hussman Institute for Human Genomics Genotyping Core. Both genotype clustering and calling were performed using Illumina’s GenomeStudio V2011.1 software. The genotyping quality control/assurance included (i) four internal controls in each plate, (ii) randomly assigned case and reference samples in each plate to avoid any biases between plates, and (iii) the Hardy-Weinberg equilibrium (HWE) test to identify problematic SNPs. SNPs were excluded from the analysis if they had no genotype for > 5% of individuals, were not in HWE within a reference group (using threshold p < 1.0 × 10−6) or had minor allele frequency < 5%. Subjects were also excluded if they had > 5% of all variants missing. The final dataset contained 1,344,832 SNPs with a genotype call rate of 99.8%. All the quality control procedures were conducted using PLINK (v1.09) (http://zzz.bwh.harvard.edu/plink/ ) [18 (link)].
3
GWAS of Severe Malaria Anemia in Children
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A pilot GWAS case-control study was performed in children infected with falciparum malaria to identify novel genes/gene pathways involved in the pathogenesis of SMA. Children were stratified into polarized phenotypes: SMA (cases, avg. Hb = 4.1 g/dL, n = 70) and UM (controls, avg. Hb = 10.8, n = 74) to enrich the signals in the tails (extremes) of the disease distribution. Selected individuals were from a cohort of children infected with P. falciparum (n = 1218), and matched according to age, sex, and peripheral parasite density, excluding children with HIV-1, bacteremia, sickle-cell disease (SSD), and G6PD deficiency. The GWAS was performed using the Illumina® Infinium® HD Super Assay in conjunction with Illumina’s® Human Omni2.5-8v1 BeadChip (with > 2.45 M markers). Data were analyzed using single SNP logistic regression analysis with an additive model of inheritance, while controlling for covariates. Analysis was performed using SNP & Variation Suite v8.6 (Golden Helix, Inc., Bozeman, MT, http://www.goldenhelix.com ).
4
Genome-wide Genotyping and Population Stratification
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Genome-wide genotyping of the study subjects' DNA was performed using Illumina HumanOmni2.5-8v1BeadChip. To test their genetic background, principal component analysis (PCA)-based population stratification was then conducted using these data and genotype data from the HapMap Project (http://hapmap.ncbi.nlm.nih.gov/ ) (Supplementary Figure S1).
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