Targeted DNA methylation analysis by high throughput sequencing in porcine peri-attachment embryos

The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx platform. The average depth of sequencing coverage was 14,611 for IVV and 17,068 for PA. Quantitative analysis of the methylation profiles of both input samples for each genomic locus showed distinct differences in methylation profiles between IVV and PA samples for six of the target loci, and subtle differences in four loci. It was concluded that high throughput sequencing technologies can be effectively applied to provide a powerful, cost-effective approach to targeted DNA methylation analysis of embryonic and other reproductive tissues.