PCR products were electrophoresed on 2% agarose gels and purified with the AxyPrepDNA gel extraction kit (Axygen, USA) following the manufacturer's instructions. Based on the primary quantification results of AGE, the PCR products were quantified with the QuantiFluor™ -ST fluorescent quantitative system (Promega, USA) and pooled based on the sequencing proportion. Pyrosequencing was performed on the Roche Genome Sequencer FLX platform (Roche, Basel, Switzerland) (McKenna et al., 2008).