The PCR was performed using a PTC-100 Programmable
Thermal Cycler (MJ Research, Watertown, MA). Ten replicates
were conducted for each sample as well as a negative control. The
PCR products were checked by 1.2% agarose gel electrophoresis. All
PCR products for each sample were pooled and purified using a PCR
clean-up kit (Axygen) according to the manufacturer's instructions.
The purified PCR product from each sample was then
sequenced on an Illumina Miseq PE250 platform by Majorbio Biopharm
Technology Co., Ltd. (Shanghai, China), and the raw
pyrosequencing data were processed as described by Zhao et al.
(2014) and Tian et al. (2015). Only sequences >150 bp in length
with an average quality score >25 and without ambiguous base
calls were included in subsequent analyses. The reads for each
sample were aligned using the fast, secondary-structure aware