Construction method for serial sequencing libraries of rad tags

A construction method for serial sequencing libraries of RAD tags, including the following steps:

• 1) enzyme digestion:
• conducting an enzyme digestion reaction with N samples of genomic DNA using selected endonucleases to obtain N its of enzyme-digested fragments, where N is an integer greater than 2;
• 2) adaptor ligation:
• ligating N parts of enzyme-digested fragments with, adaptors, Le., N pairs of adaptor pairs are designed to obtain N parts of ligated products, and the adaptors contain restriction enzyme sites of SapI, featured base sequences for the serial ligation of RAD tags, and universal sequences for the binding of amplification primers, and the sequential ligation of N groups of enzyme-digested fragments are determined according to the added adaptors;
• 3) amplification of ligated products:
• conducting PCR amplification on the N parts of the ligated products obtained in step 2) using a different combination of biotin primers and general primers;
• collecting PCR products by gel;
• amplifying 4-8 cycles using the same method to obtain N parts of enriched PCR products;
• and equally mixing the N parts of enriched PCR products and purifying;
• 4) serial ligation of tag libraries:
• conducting enzyme digestion on the mixed and purified N parts of PCR products using the SapI enzyme to excise universal adaptor and primer sequences on both ends of each enzyme-digested fragment, and the featured base sequences form cohesive ends that enable the N parts of the PCR products to ligate in series;
• and the sequential ligation of the N parts of the tag libraries is based on the complementary pairing of the featured sequences on the adaptors.
• 5) amplification of ligated serial tags:
• purifying the long serial tags through a gel and then conducting PCR amplification using the barcode primers to construct the libraries of serial RAD tags
• 6) library sequencing:
• sequencing the libraries of serial tags on the Illumina sequencing platform.