text: Pyrosequencing has also been used to analyze bisulfite-treated DNA without using methylation-specific PCR.  Following PCR amplification of the region of interest, pyrosequencing is used to determine the bisulfite-converted sequence of specific CpG sites in the region.  The ratio of C-to-T at individual sites can be determined quantitatively based on the amount of C and T incorporation during the sequence extension.  The main limitation of this method is the cost of the technology.  However, Pyrosequencing does well allow for extension to high-throughput screening methods.
  A variant of this technique, described by Wong et al., uses allele-specific primers that incorporate single-nucleotide polymorphisms into the sequence of the sequencing primer, thus allowing for separate analysis of maternal and paternal alleles. This technique is of particular usefulness for genomic imprinting analysis.