Abstract: Provided is a PCR-free library construction and sequencing method. A PCR-free high-throughput sequencing method is provided, including the following steps: obtaining a DNA fragment of target size by performing or not performing, based on a size of a nucleic acid sample, fragmentation on the nucleic acid sample; performing end repair and an A-tailing reaction; ligating an adapter containing a barcode; obtaining DNA nanoballs by performing single-strand cyclization and rolling circle replication; and loading and sequencing.

Abstract: Provided are a hooked probe, a method for ligating a nucleic acid and a method for constructing a sequencing library. The hooked probe comprises a target specific area and a hooked area ligated thereto; the target specific area comprises a sequence complementarily paired with at least part of the single chain of the nucleic acid fragment to be ligated; the hooked area comprises a sequence unpaired with the nucleic acid fragment; the end of the hooked area is a ligatable end; and the ligatable end can ligate the end of the single chain of the nucleic acid fragment.

Abstract: Provided are a hooked probe, a method for ligating a nucleic acid and a method for constructing a sequencing library. The hooked probe comprises a target specific area and a hooked area ligated thereto; the target specific area comprises a sequence complementarily paired with at least part of the single chain of the nucleic acid fragment to be ligated; the hooked area comprises a sequence unpaired with the nucleic acid fragment; the end of the hooked area is a ligatable end; and the ligatable end can ligate the end of the single chain of the nucleic acid fragment.