Detection of Differential DNA Methylation Under Stress Conditions Using Bisulfite Sequence Analysis.

DNA methylation is the most important epigenetic change affecting gene expression in plants grown under normal as well as under stress conditions. Therefore, researchers study differential DNA methylation under distinct environmental conditions and their relationship with transcriptome abundance. Up to date, more than 25 methods and techniques are available to detect DNA methylation based on different principles. Bisulfite sequencing method is considered as a gold standard since it is able to distinguish 5-methylcytosine from cytosine using the bisulfite treatment. Therefore, it is useful for qualitative and semiquantitative measurement of DNA methylation. However, the reliability of data obtaining from this technique is mainly depending on the efficiency of bisulfite conversion and number of sequencing clones representing the target-converted sequence. Therefore, it is labor intensive and time-consuming. Revolution of next generation DNA sequencing (NGS) has allowed researches to combine conventional bisulfite sequencing methods with high-throughput Illumina sequencing in a technique called whole genome bisulfite sequencing (WGBS). This technique allows a single nucleotide resolution of 5-methylcytosine on a genome scale. WGBS technique workflow involves DNA fragmentation, processing through end blunting, terminal A(s) addition at 3' end and adaptor ligation, bisulfite treatment, PCR amplification, sequencing libraries and assembling, and finally alignment with the reference genome and data analysis. Despite the fact that WGBS is more reliable than the conventional clone-based bisulfite sequencing, it is costly, requires large amount of DNA and its output data is not easily handled.