NGroup-NC1
The genomic DNA of the samples was extracted by CTAB or SDS method, and then the purity and concentration of the DNA was detected by 1% agarose gel electrophoresis. Appropriate amount of sample DNA was taken in a centrifuge tube and the sample was diluted to 1ng/l with sterile water. All PCR blends were added with 15L Phusion High-Fidelity PCR Master Mix (New England Biolabs), 0.2M primers, and 10ng genomic DNA template. The first denaturation was performed at 98 C for 1 minute, followed by 30 cycles at 98 C (10s), 50 C (30s) and 72 C (30s), and finally held at 72 C for 5 minutes. The PCR products were detected by electrophoresis using 2% agarose gel. The qualified PCR products were purified by magnetic beads, quantified by enzyme labeling, and mixed according to the concentration of PCR products in the same amount. After fully mixed, the PCR products were detected by 2% agarose gel electrophoresis. The products were recovered for the target bands using the glue recovery kit provided by qiagen. The library was constructed using TruSeq DNA PCR-Free Sample Preparation Kit. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, the PE 250 computer sequencing was performed using NovaSeq 6000.