design: The starting DNA material (100-1000 ng) is quality-checked, fragmented using Covaris S220, and then processed through end repair, dA-tailing, adapter ligation, and purification. The purified DNA is size-selected, PCR-amplified to construct the library, and quantified using Qubit 3.0. Insert size and nucleic acid concentration are assessed by Agilent 2100. qPCR determines the effective concentration of each library before sequencing to ensure accuracy and data reliability