The traditional Combined Bisulphite Restriction Analysis (COBRA) method quantifies DNA methylation at a specific gene locus using digestion with a restriction enzyme such as TaqI (TCGA). The methylation status of a particular CpG site within an enzyme recognition site can be determined, if the cytosine was methylated and remained unconverted after bisulphite treatment and amplification. Herein, we developed two genome wide methylome methods inspired by the original COBRA method.
Genome-Wide COBRA (GW-COBRA) and Linear Amplification COBRA (LA-COBRA) are reduced methylome methods which generate representative libraries for Illumina next generation sequencing in 3 to 4 days. To adapt for genome-wide analysis, linkers are ligated to the fragment ends produced after the restriction digestion. GW-COBRA uses PCR amplification whereas LA-COBRA uses in vitro reverse transcription followed by cDNA synthesis to elaborate the input DNA. Subsequently, libraries of 150-500 bp fragments containing the distinct adaptors are generated.
GW-COBRA and LA-COBRA methods require a small amount of DNA input (0.1 ug-1 ug). Both methods can be used to explore CpG-depleted as well as CpG-rich regions. Unlike the common reduced methylome methods, these can be multiplexed and tuned to reduce sequencing costs with the use of 6 base cutters instead of 4 base cutters to interrogate fewer sites. LA-COBRA has additional advantages such as reducing the PCR bias and producing full length fragments at high abundance.
In this pilot study, GW-COBRA and LA-COBRA libraries were prepared using colon carcinoma (HCT116), prostate cancer (LnCap) and epithelial (PrEC) cell lines. Libraries were sequenced with single-end HiSeq technology. I will be presenting a pairwise comparison of GW-COBRA and LA-COBRA with two popular DNA methylation methods such as Illumina 450k arrays and Reduced Representation Bisulphite Sequencing.