Discrimination of pseudogene and parental gene DNA methylation using allelic bisulfite sequencing

Determining the methylation status of genes with pseudogenes can be technically challenging due to sequence homology. High sequence homology can result in the amplification of both pseudogene and parental gene alleles, potentially leading to data misinterpretation. Allelic bisulfite sequencing allows for detection of the methylation status of individual alleles at nucleotide resolution and represents the most reliable method for discriminating pseudogene and parental gene sequences. Here, we discuss important points that should be considered when investigating pseudogene and parental gene methylation status and we describe the method of allelic bisulfite sequencing, including assay design.