design: To increase the sequencing throughput, a 16-bp specific barcode sequence was added to the 5' end of both the forward and reverse primers (27F: 5-AGAGTTTGATCMTGGCTCAG-3/1492R:5-TACGGYTACCTTGTTACGACTT-3), which allows for the accurate allocation of sequences to corresponding samples based on barcode information after sequencing. The third-generation sequencing technology, PacBio platform, was employed for the sequencing analysis. The PCR products were qualified by 1% agarose gel electrophoresis, followed by purification of the PCR products, library construction, and finally, loading for sequencing.