Methylation assay and data analysis
Genomic DNA was also extracted from nasal mucosal tissues by using the RNA-seq using Illumina Infinium Methylation EPIC Bead Chip kits (Illumina, Inc.), and arrays were scanned by the Standard Illumina procedures using an Illumina iScan scanner. Each methylation data point is represented by fluorescent signals from the M (methylated) and U (unmethylated) alleles. Background intensity computed from a set of negative controls was subtracted from each analytical data point. The ratio of fluorescent signals was then computed from the 2 alleles ß = (max (M, 0))/(|U| + |M| + 100). The ß-value reflects the methylation level of each CpG site. A ß-value of 0-1 was reported signifying percent methylation, from 0% to 100%, for each CpG site.
After methylation assay, array data export processing and analysis were performed using Illumina GenomeStudio v2011.1 (Methylatioin Module v1.9.0) and R 3.0.2 (http://www.r-project.org). Differentially methylated probes (DMP) and regions were detected through the in house python scripts using the numpy and scipy package14 and ChAMP15 with a cutoff threshold of P value < 0.001.