A Comprehensive Analysis of DNA Methylation Profiles in Peripheral Blood Mononuclear Cells of TB patients

This study investigated the genome-wide methylation profiles in peripheral blood mononuclear cells from anti tuberculous treatment naive TB patients, diseased controls, and healthy controls (n=4 per group) using whole genome bisulfite sequencing (WGBS). The goal was to identify differentially methylated regions (DMRs) associated with TB pathophysiology.MethodsDNA quality and integrity were evaluated using the Qubit assay and agarose gel electrophoresis (Figure 16). All 12 samples passed QC, showing adequate DNA concentration and intact integrity. Libraries were prepared with 200 ng genomic DNA, followed by bisulfite treatment, adapter ligation, and purification. Quantified libraries were sequenced on an Illumina NovaSeq platform at 30X coverage, generating 2x150 bp reads per sample. The data had excellent quality metrics, with 75% of bases achieving Q30 scores >85%.FASTQ files were processed for alignment to the human reference genome and methylation calling. DMR analysis was performed for TB vs diseased controls and TB vs healthy controls.ResultsUsing a methylation cutoff of +/-30%, the following DMRs were identified:TB vs Diseased Controls:Total DMRs: 3,642 (1,756 hypermethylated, 1,886 hypomethylated).TB vs Healthy Controls:Total DMRs: 4,667 (2,464 hypermethylated, 2,203 hypomethylated).