Humanomni2.5 8

Genomic DNAs were extracted from peripheral blood leukocytes using QuickGene-610L DNA extraction kit (FUJIFILM Co., Tokyo, Japan). Genotyping was performed using Illumina BeadChip, both OmniExpress and HumanExome, HumanOmni2.5–8, or OmniExpress. The distortion of Hardy-Weinberg equilibrium (HWE) was not considered in this study because all samples comprised AMD cases. Stringent quality control, including minor allele frequency (MAF) ≥ 1% and genotype call rate ≥ 95% (per SNP and per individual), was performed using PLINK ver1.07 (http://pngu.mgh.harvard.edu/~purcell/plink/).

DNA was extracted according to the protocol described by Gentra^®® Puregene^®® Blood Kit Qiagen (Germantown, ML, USA), while quantification was standardized at a concentration of 50 ng/μL and identified in barcoded tubes. This step was performed using a Qubit fluorometer^® (Invitrogen, Paisley, UK) [19 (link)]. The LEPR genetic information was extracted between positions 65,886,335 and 66,103,176 on chromosome 1 and genotyping for all 149 SNVs of the LEPR gene was conducted on the HumanOmni2.5-8 platform using the BeadChip kit (Illumina, San Diego, California, USA).

Genomic DNA was prepared from peripheral blood samples according to the manufacturer’s protocol. Samples from 5299 participants who joined the Nagahama cohort from 2008 to 2009 were used for the genome-wide SNP genotyping. The analysis was performed with HumanHap610 Quad (1830 samples), HumanOmni2.5-4 (1616 samples), HumanOmni2.5-8 (378 samples), HumanOmni2.5 s (672 samples), CoreExome24 (1728 samples), and HumanExome (304 samples; Illumina, San Diego, CA, USA).
As a stringent quality control (QC), SNPs with a call rate of less than 99%, minor allele frequency of less than 1%, and significant deviation from Hardy–Weinberg equilibrium (P < 1.0 × 10⁻⁶), and samples with a call rate of <95% were excluded from the analysis. After this QC, 333 participants with estimated first- or second-degree kinship within Nagaham cohort samples (pi-hat > 0.35, PLINK ver. 1.07 [http://zzz.bwh.harvard.edu/plink/]) were also excluded.
Genotype imputation was performed using MACH software (http://www.sph.umich.edu/csg/abecasis/MACH/tour/imputation.html) with the 1000 genomes dataset (phase3 v5 release) as a reference panel. Imputed SNPs for which R² was less than 0.5 were excluded from the following association analysis. Finally, 4,710,779 SNPs from 4476 individuals were used for the discovery stage analysis. Of the 4476 samples, 14 lacked data of axial length.

DNA copy numbers were assessed using Illumina CytoSNP-12 and HumanOmni2.5-8 microarrays and read out using the iScan array scanner. Fluorescence in situ hybridization (FISH) analysis was performed for del(11)(q22.3), del(17)(p13), del(13)(q14), trisomy 12, gain(8)(q24), and gain(14)(q32). Only alterations present and absent in at least three patients were considered for analyses.

DNA copy numbers were assessed using Illumina CytoSNP-12 and HumanOmni2.5-8 microarrays (n = 169). DNA (200 ng) was processed according to the manufacturer’s instructions. Arrays were read out using the iScan array scanner. Copy number variants were verified by using the exome sequencing data (n = 107). Fluorescence in situ hybridization (FISH) analysis was performed for del11q22.3 (n = 162), del17p13 (n = 159), del13q14 (n = 155), trisomy 12 (n = 152), del6q21 (n = 132), and gain8q24 (n = 125). Information on structural variants from FISH, exome sequencing, and SNP arrays was combined into 1 table (n = 219).