Sequencing library, preparation method and use thereof

A sequencing method, comprising a process of preparing a sequencing library, wherein the process for preparing the sequencing library comprises the following steps:

• (1) a sequence to be tested is linked to a tag sequence to obtain a double-strand or single-strand linked sequence;
• (2) when the linked sequence obtained in step (1) is a double-strand sequence, the linked sequence is converted into single-strand sequences, then cyclized;
• when the linked sequence obtained in step (1) is a single-strand sequence, the linked sequence is directly cyclized;
• (3) the cyclized linked sequence obtained in step (2) is subjected to DNA amplification based on strand displacement reaction to obtain an equidirectional alternating concatemer consisting of the sequence to be tested and the tag sequence, that is, a sequencing library is prepared and obtained;
• (4) the equidirectional alternating concatemer is fragmented, and sequencing adaptors are linked to both terminals of each resulted fragment to obtain a sequencing library;
• wherein,
• the sum of the length of the sequence to be tested and the length of the tag sequence is less than half of the sequencing length of a sequencer;
• and
• the resulted fragment in step (4) has a length greater than the sequencing length of a sequencer;
• the tag sequence is linked to the 5′-terminal of the sequence to be tested;
• and
• the equidirectional alternating concatemer comprises at least two repetitive units, and each repetitive unit comprises one sequence to be tested and one tag sequence.