Bhat Smitha, Mallya Sandeep, Varghese Vinay Koshy, Jayaram Pradyumna, Chakrabarty Sanjiban, Joshi Kalpana S, Nesari Tanuja M, Satyamoorthy Kapaettu
Department of Biotechnology, School of Life Sciences, Manipal University, Manipal 576104, Karnataka, India.
Department of Biotechnology, Sinhgad College of Engineering, S. P. University of Pune, Pune, Maharashtra, India.
Gene. 2016 Dec 15;594(2):259-267. doi: 10.1016/j.gene.2016.09.019. Epub 2016 Sep 13.
With a purpose of accurate and simultaneous determination of DNA methylation from multiple loci in multiple samples, here, we are demonstrating a method to aid rapid DNA methylation detection of genomic sequences. Using genomic DNA of peripheral blood from 14 healthy individuals, DNA methylation in 465 CpG sites from 12 loci of genes (ADAM22, ATF2, BCR, CD83, CREBBP, IL12B, IL17RA, MAP2K2, RBM38, TGFBR2, TGFBR3, and WNT5A) was analysed by targeted next generation bisulfite sequencing. Analysed region for three genes, BCR, IL17RA and RBM38 showed an absolute mean DNA methylation of 25.6%, 89.2% and 38.9% respectively. Other nine gene loci were unmethylated and exhibited <10% absolute mean DNA methylation. Two genes, IL17RA and RBM38 were technically validated using direct capillary sequencing and results were comparable with positive correlation (P=0.0088 & P<0.0001 respectively) in the CpG sites for DNA methylation. All CpG sites analysed from RBM38 genes locus displayed 95% limits of agreement for DNA methylation measurements from the two methods. The present approach provides a fast and reliable DNA methylation quantitative data at single base resolution with good coverage of the CpG sites under analysis in multiple loci and samples simultaneously. Use of targeted next generation bisulfite sequencing may provide an opportunity to explore genes in the discovery panel for biomarker identification and facilitate functional validation.
为了准确、同时地测定多个样本中多个位点的DNA甲基化，在此，我们展示了一种有助于快速检测基因组序列DNA甲基化的方法。使用14名健康个体的外周血基因组DNA，通过靶向新一代亚硫酸氢盐测序分析了12个基因位点（ADAM22、ATF2、BCR、CD83、CREBBP、IL12B、IL17RA、MAP2K2、RBM38、TGFBR2、TGFBR3和WNT5A）的465个CpG位点的DNA甲基化情况。对三个基因BCR、IL17RA和RBM38的分析区域显示绝对平均DNA甲基化分别为25.6%、89.2%和38.9%。其他九个基因位点未甲基化，绝对平均DNA甲基化低于10%。使用直接毛细管测序对两个基因IL17RA和RBM38进行了技术验证，结果在DNA甲基化的CpG位点具有正相关性（分别为P = 0.0088和P < 0.0001）。从RBM38基因位点分析的所有CpG位点在两种方法的DNA甲基化测量中显示出95%的一致性界限。本方法在单碱基分辨率下提供了快速可靠的DNA甲基化定量数据，同时对多个位点和样本中分析的CpG位点具有良好的覆盖。使用靶向新一代亚硫酸氢盐测序可能为在发现组中探索基因以进行生物标志物鉴定和促进功能验证提供机会。
