Single-chain connector, method for detecting nucleic acid terminal, kit and application of single-chain connector

Abstract

The invention relates to a single-chain connector, a method for detecting the tail end of a nucleic acid fragment, a kit and application of the single-chain connector. The single-chain connector disclosed by the invention can be simultaneously connected with the same-side tail end of a to-be-detected double-chain nucleic acid fragment, a protruding tail end sequence of double-chain nucleic acid can be efficiently and accurately captured, and the motif and position information of the tail end of the double-chain nucleic acid can be favorably and accurately identified through sequencing analysis.

Description

Technical Field

[0001] This application belongs to the field of DNA detection and library construction technology, and relates to a method for detecting nucleic acid ends, specifically a single-linked head, a method for detecting nucleic acid ends, a reagent kit, and their applications. Background Technology

[0002] Circulating free DNA (cfDNA) is a fragmented mixture of DNA in blood plasma, derived from apoptotic and necrotic cells with different topological domains. Effectively enriching diagnostically valuable cfDNA fragments remains a significant challenge for clinical applications. The characteristics of cfDNA fragmentomics include fragment length, fragment size distribution, terminal motifs, preferred end coordinates, and breakpoint motifs. Recent evidence suggests that the characteristics of cfDNA fragmentomics differ between individuals and tissues during growth and development, and between healthy individuals and diseased individuals, making it a promising new biomarker. Unlike fragment length, the sequence characteristics of cfDNA have only recently gained attention. The single-stranded ends carried by double-stranded cfDNA molecules are called jagged ends. Because library construction targeting cfDNA fragments typically involves end repair steps, these jagged ends are often masked during this process, thus their presence has been overlooked for many years. Recent research shows that in hepatocellular carcinoma patients, tumor-derived DNA molecules exhibit more jagged ends compared to non-tumor DNA. Furthermore, the degree of serration varies with the size of plasma DNA fragments and is related to nucleosome patterns. Detecting and analyzing the serrated ends of cfDNA will facilitate more sensitive cancer detection.

[0003] To explore the serrated end characteristics of cfDNA, methodological changes are needed. In 2020, researchers developed Jag-seq technology for accurately identifying the 5' serrated ends of cfDNA, the first method to detect 5' serrated ends in double-stranded cfDNA molecules. This technology utilizes DNA end repair to introduce differential methylation signals between the original sequence and the serrated ends, and determines the specific length of the serrated ends through sequencing analysis. Results showed that tumor-derived cfDNA exhibited more serrated ends compared to non-tumor cfDNA, indicating the diagnostic potential of serrated ends in cfDNA for detecting cancer patients. Specifically, based on the introduced differential methylation signals, Jag-seq technology can be divided into unmethylated Jag-seq and methylated Jag-seq methods. The unmethylated Jag-seq method uses cfDNA containing highly methylated CpG sites as a template, and constructs blunt ends by extending the serrated ends of the DNA strand using Taq polymerase and dNTPs with unmethylated C. Subsequently, the DNA is treated with bisulfite to convert all unmethylated cytosine C to uracil U, while methylated C remains unchanged. Because highly methylated CpG sites are fragmented more frequently than low-methylated CpG sites, sequencing analysis shows that if the original cfDNA molecule contains serrated ends, the methylation level of CpG sites near the ends will be lower than that further away. Since CpG sites are not common in human cfDNA, researchers developed a methylation Jag-seq method. This method uses methylated C to fill DNA molecules with 5' protruding ends. The resulting blunt ends are then treated with bisulfite, resulting in a higher methylation level compared to the original cfDNA fragment with serrated ends. By comparing changes in methylation levels, the location of the serrated ends can be inferred.

[0004] Jag-seq technology has established a method for resolving the 5' serrated ends of cfDNA based on methylation, but this method cannot detect the 3' serrated ends of cfDNA. Furthermore, for the 5' serrated ends of cfDNA, if the 3' end base is not C or the first base added by the polymerase is not C, the starting position of the serrated end cannot be accurately identified.

[0005] Furthermore, XACTLY, a simple ligation-based NGS library method, provides comprehensive information on the native state of fragmented DNA ends. By omitting the conventional DNA end repair step, XACTLY technology encodes the break type at each molecule's end using custom sequencing adapters, providing information on cfDNA fragmentation without needing to know the expected cut pattern. The technique first phosphorylates the cfDNA but does not passivate the ends, preserving the intact native sticky or blunt ends. Then, XACTLY adapter sets with random bases of varying lengths are hybridized and ligated to the cfDNA fragments, followed by a second round of phosphorylation, sealing any gaps due to 5' dephosphorylation of the adapters. Finally, the library is amplified by PCR using Illumina-compatible universal primers. Compared to Jag-seq technology, XACTLY is able to detect different types of serrated ends in cfDNA, as well as the relative abundance of the length and type of each cfDNA single-stranded overhang. Overall detection accuracy is between 80-90%. Additionally, in the detection of ends with a 1-base overhang at the 5' end, an increased cytosine read count was observed, which may originate from deviations in the synthesis of custom oligonucleotides.

[0006] Therefore, providing more accurate detection methods for cfDNA serrated ends and providing technical support for more sensitive analysis of the relationship between cfDNA fragmentomics characteristics and cellular processes or disease development remains an urgent problem to be solved. Summary of the Invention

[0007] This application aims to at least address one of the aforementioned technical problems existing in the prior art. In view of this, this application provides a single-linked header, a method for detecting nucleic acid ends, a reagent kit, and their applications.

[0008] According to one aspect of this application, a single linker is provided, wherein both the 5' and 3' ends of the single linker are phosphate groups. In some embodiments, the single linker is a synthetic single linker. In some preferred embodiments, the single linker includes a tag sequence. In some embodiments, the number of bases in the single linker can be 5-100 nt. Preferably, the number of bases in the single linker can be 5-30 nt. More preferably, the number of bases in the single linker can be 6-25 nt. In some preferred embodiments, the last base of the sequence of the single linker, from the 5' to the 3' end, is at least one ribonucleotide base (rNTP).

[0009] According to some embodiments of this application, the single-linker provided can simultaneously connect to the same-side ends of the double-stranded nucleic acid fragment to be tested, efficiently and accurately capturing the protruding end sequences of the double-stranded nucleic acid. Sequencing analysis allows for the advantageous and precise identification of the length, type, start, and termination positions of the double-stranded nucleic acid ends based on the sequence of the single-linker. The specific sequence of the single-linker can be modified according to the embodiments provided in various aspects of this application while still achieving equivalent technical effects. The single-linker provided in this application can be used not only to detect serrated ends of cfDNA fragments but also to detect double-stranded breaks in nucleic acids within cells.

[0010] According to another embodiment of this application, a method for detecting nucleic acid ends is provided, comprising the following steps:

[0011] Provide a dephosphorylated nucleic acid fragment, wherein the 5' end and 3' end of the dephosphorylated nucleic acid fragment are both hydroxyl groups;

[0012] A single linker is provided, wherein both the 5' and 3' ends of the single linker are phosphate groups;

[0013] The dephosphorylated nucleic acid fragment is ligated to the single linker to obtain a nucleic acid fragment ligation product, and the nucleic acid fragment ligation product is detected.

[0014] According to some embodiments of this application, the single-linker provided herein connects the positive and negative strands of a nucleic acid fragment that has undergone dephosphorylation at both ends, advantageously enabling the detection of different types of double-stranded nucleic acid ends, including 5' serrated ends, 3' serrated ends, or types with either blunt or serrated ends. Furthermore, by using the single-linker provided herein to connect the positive and negative strands of cfDNA, sequencing analysis can accurately identify the start and end positions of the serrated ends.

[0015] According to some embodiments of this application, a dephosphorylated nucleic acid fragment is obtained by removing the phosphate groups at the 5' and 3' ends of the nucleic acid fragment to be tested. According to some embodiments of this application, the nucleic acid fragment to be tested comprises a double-stranded nucleic acid fragment. According to some embodiments of this application, the nucleic acid fragment to be tested comprises a DNA fragment. According to some embodiments of this application, the nucleic acid fragment to be tested comprises a cfDNA fragment. According to some embodiments of this application, the cfDNA fragment is obtained from circulating peripheral blood. According to some embodiments of this application, the cfDNA fragment comprises any free DNA fragment in circulating peripheral blood. According to some embodiments of this application, the cfDNA fragment comprises any DNA fragment present in extracellular fluid. According to some embodiments of this application, the cfDNA fragment comprises fragmented endogenous DNA free outside cells in circulating peripheral blood.

[0016] According to some embodiments of this application, the nucleic acid fragment to be tested includes ends having different types. According to some embodiments of this application, the nucleic acid fragment to be tested may have a 5' protruding end, a 3' protruding end, or a blunt end at either end or both ends.

[0017] According to some embodiments of this application, the nucleic acid fragment to be tested may have a 5' phosphate group, a 3' phosphate group, a 5' hydroxyl group, or a 3' hydroxyl group at either end or both ends. According to some embodiments of this application, by removing the 5' and 3' phosphate groups from the nucleic acid fragment, hydroxyl groups are formed at both ends of the nucleic acid fragment. This not only helps to avoid undesirable self-ligation between nucleic acid fragments, but also helps to improve the ligation efficiency with single-linked heads in subsequent steps.

[0018] According to some embodiments of this application, phosphatases are used to remove the phosphate groups at the 5' and 3' ends of nucleic acid fragments to obtain dephosphorylated nucleic acid fragments. To remove the phosphate groups at the 5' and 3' ends of nucleic acid fragments, those skilled in the art can rationally select existing known phosphatases, or extract enzymes with completely identical or similar functions as substitutes. For example, phosphatases with catalytic 5' dephosphorylation activity or 3' dephosphorylation activity can be selected to sequentially or simultaneously remove the phosphate groups at the 5' and 3' ends of the nucleic acid fragment. Alternatively, phosphatases possessing both catalytic 5' dephosphorylation activity and 3' dephosphorylation activity can be selected to simultaneously remove the phosphate groups at the 5' and 3' ends of the nucleic acid fragment.

[0019] In some embodiments of this application, the phosphatase may be T4 polynucleotide kinase (T4 PNK). T4 PNK is a polynucleotide 5'-hydroxykinase capable of catalyzing the transfer of the γ-phosphate group of ATP to the 5'-hydroxy terminus of oligonucleotide chains (double-stranded or single-stranded DNA or RNA) and to 3'-monophosphate nucleosides. However, T4 PNK can also catalyze the reverse reaction of phosphorylation, exhibiting 3'-terminal phosphatase activity, capable of catalyzing the hydrolysis of 3'-phosphate groups from the 3'-phosphate terminus of oligonucleotides, deoxy3'-monophosphate nucleosides, and deoxy3'-diphosphate nucleosides. When ADP is present, T4 PNK also exhibits 5'-terminal phosphatase activity, catalyzing the exchange of 5'-P-oligonucleotides and the 5'-terminal phosphate group of ATP.

[0020] In some embodiments of this application, the phosphatase may be an acid phosphatase or an alkaline phosphatase. Acid phosphatases are known to include 20 isoenzymes, which can be broadly classified into two categories: prostatic acid phosphatases and non-prostatic acid phosphatases. Compared to acid phosphatases, alkaline phosphatases (APs) have a wider range of applications in fields such as medicine and molecular biology. Preferably, the alkaline phosphatase is a nonspecific phosphomonoesterase. Nonspecific phosphomonoesterases can catalyze the hydrolysis of the 5'-phosphate and 3'-phosphate groups of almost all phosphate monoesters.

[0021] In some preferred embodiments, the phosphatase may be at least one selected from bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), and secretory alkaline phosphatase (SEAP). In some more preferred embodiments, the phosphatase may be a wild-type enzyme or a recombinant enzyme. Compared to wild-type enzymes, recombinant enzymes obtained through recombinant expression do not contain affinity tags or other modifications found in wild-type or natural enzymes.

[0022] In some preferred embodiments, the phosphatase may be selected from at least one of bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), secretory alkaline phosphatase (SEAP), and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are relatively heat-sensitive alkaline phosphatases, easily inactivated at around 65°C; bacterial alkaline phosphatase (BAP) and placental alkaline phosphatase (PLAP) are relatively heat-resistant, and can be heated at 65°C or above for one hour or longer without losing activity. Bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), and placental alkaline phosphatase (PLAP) can all non-specifically catalyze the dephosphorylation of 5' and 3' terminal phosphomonyl esters of DNA and RNA, acting on the 5' or 3' protruding ends and blunt ends, and preventing self-ligation of linear nucleic acid fragments by dephosphorylation.

[0023] In some further preferred embodiments, the phosphatase may be at least one of shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP), and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are heat-sensitive alkaline phosphatases, which can be completely and irreversibly inactivated by heating at 65°C or above for several minutes, thus eliminating the need for purification before the ligation or end-labeling steps. Those skilled in the art can select appropriate buffers, coenzyme factors, and salt ions, and adjust the reaction temperature and time, as well as the pH and ion concentration of the reaction system, according to the specific type of phosphatase used and the desired effect. For example, a commercially available phosphatase kit can be used, and the phosphatase and buffer in the kit can be used according to the manufacturer's instructions to perform the dephosphorylation reaction.

[0024] According to some embodiments of this application, the single linker has phosphate groups at both its 5' and 3' ends. According to some embodiments of this application, the single linker is a synthetic single linker. In some embodiments, the number of bases in the single linker can be 5-100 nt. To ensure efficient linking of the single linker to the positive and negative strands at the ends of the nucleic acid fragment, preferably, the number of bases in the single linker can be 5-30 nt. More preferably, the number of bases in the single linker can be 6-25 nt. In some preferred embodiments, at least one base in the last position of the sequence of the single linker, from the 5' to the 3' end, is a ribonucleotide base (rNTP).

[0025] In some implementations, tag sequences can be introduced into the single linker to facilitate subsequent library construction and detection analysis. In some preferred implementations, the tag sequences introduced into the single linker include, but are not limited to, unique molecular tag (UMI) sequences and sample tag sequences. Unique molecular tag (UMI) sequences are used to count the copy number of nucleic acid molecules in a sample. Sample tag sequences are used to distinguish different samples for subsequent multi-sample pooling sequencing. For example, sample tag sequences can be barcode sequences or index sequences.

[0026] According to some embodiments of this application, a dephosphorylated nucleic acid fragment is ligated to the single linker. By providing a single linker with phosphate groups at both the 5' and 3' ends, it is advantageous to link to the hydroxyl groups on the positive and negative strands of the nucleic acid fragment that has been dephosphorylated at both ends in the previous step. By ligating the dephosphorylated nucleic acid fragment to the single linker, with the 5' and 3' ends of each nucleic acid fragment connected by the same single linker, the constructed nucleic acid fragment ligation product forms a dumbbell-shaped structure, as shown in... Figure 1As shown, given a known single-linker sequence, the type of the original nucleic acid terminology and the start and end positions of the protruding ends can be accurately identified by detecting and analyzing the ligation products of nucleic acid fragments.

[0027] According to some embodiments of this application, a dephosphorylated nucleic acid fragment is ligated to the single linker by a nucleic acid ligase. In some embodiments, the nucleic acid ligase may include a DNA ligase or an RNA ligase. In some preferred embodiments, the nucleic acid ligase includes a nucleic acid ligase with activity catalyzing the formation of a phosphodiester bond (5'PO4 to 3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide, and a nucleic acid ligase with activity catalyzing the formation of a phosphodiester bond (3'PO4 to 5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide. Those skilled in the art can reasonably select existing known nucleic acid ligases, or can extract enzymes with completely identical or similar functions for substitution. For example, a nucleic acid ligase with catalyzing 5'PO4→3'OH linkage activity or catalyzing 3'PO4→5'OH linkage activity can be selected to catalyze the 5'PO4→3'OH linkage and the 3'PO4→5'OH linkage sequentially or simultaneously. Alternatively, a nuclease with both catalytic 5'PO4→3'OH and catalytic 3'PO4→5'OH linkage activities can be selected, which can simultaneously catalyze the 5'PO4→3'OH and 3'PO4→5'OH linkages.

[0028] In some embodiments of this application, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (5'PO4→3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide includes at least one of ATP-dependent nucleic acid ligases and NAD+-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 5'PO4→3'OH linkage includes, but is not limited to, at least one of T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV. For T4 RNA ligase, the coenzyme factors include ATP and magnesium ions (Mg²⁺). 2+ ).

[0029] In some embodiments of this application, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (3'PO4→5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide includes GTP-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 3'PO4→5'OH linkage is not limited to RtcB ligase. For RtcB ligase, the coenzyme factors include GTP and manganese ions (Mn). 2+ ).

[0030] Those skilled in the art can select appropriate buffers, coenzyme factors, and salt ions based on the specific type of nucleic acid ligase used and the desired effect, and adjust the reaction temperature, time, pH value, and ion concentration of the reaction system. For example, by using a commercially available nucleic acid ligation kit and following the manufacturer's instructions to use the nucleic acid ligase and buffer in the kit, a ligation reaction procedure can be performed to connect nucleic acid fragments to single-linked heads.

[0031] According to some embodiments of this application, the method further includes a purification step. According to some embodiments of this application, the purification step includes purifying the nucleic acid fragment ligation product.

[0032] According to some embodiments of this application, the method further includes a digestion step. According to some embodiments of this application, the digestion step includes digesting unconnected nucleic acid fragments.

[0033] In some preferred embodiments, unligated nucleic acid fragments are digested using nucleases. In some more preferred embodiments, the nucleases include exonucleases. Unlike nucleic acid fragment ligation products that have closed structures at both ends, nucleic acid fragments unligated at both ends or ligated at only one end are readily hydrolyzed from the open ends by exonucleases through sequential hydrolysis of phosphodiester bonds. Those skilled in the art can reasonably select existing known nucleases, or extract enzymes with completely identical or similar functions for substitution. In some more preferred embodiments, the exonucleases include, but are not limited to, at least one of exonuclease I (ExoI), exonuclease III (ExoIII), and exonuclease IV (ExoIV). Those skilled in the art can also select appropriate buffers, coenzyme factors, and salt ions, and adjust the reaction temperature and time, as well as the pH and ion concentration of the reaction system, according to the specific type of exonuclease used and the desired effect. For example, a commercially available nucleic acid digestion enzyme kit can be used, and the nuclease and buffer in the kit can be used according to the manufacturer's instructions to perform the nucleic acid digestion reaction.

[0034] Those skilled in the art are familiar with many methods for purifying target nucleic acids, including but not limited to the phenol / chloroform method, centrifugal column method, magnetic bead method, etc., and can rationally select the appropriate nucleic acid purification method according to the specific desired effect and the characteristics of the sample.

[0035] The phenol / chloroform method is one of the most common DNA purification methods. This method utilizes the density difference between phenol and chloroform to separate DNA molecules from other impurities. An equal volume of phenol / chloroform mixture is added to a solution containing DNA molecules, mixed thoroughly, and then centrifuged to separate the layers. The upper DNA layer is then removed, and an equal volume of isopropanol is added to precipitate the DNA molecules.

[0036] The centrifugation column method is also a rapid and simple DNA purification method. This method utilizes the special structure of a centrifugation column to separate DNA molecules from other impurities. By adding a DNA solution to the centrifugation column and centrifuging to separate DNA molecules from other impurities, the DNA molecules in the column are then washed and eluted to obtain purified DNA molecules.

[0037] Magnetic bead purification is a highly efficient and automated method for DNA purification. It utilizes the specific binding of affinity molecules on the surface of magnetic beads to DNA molecules, separating DNA molecules from other impurities. The specific steps are as follows: First, cells are lysed to release DNA molecules into the solution; then, magnetic beads are added to bind to the DNA molecules; finally, magnetic force is used to separate the magnetic beads from the DNA molecules, yielding purified DNA molecules.

[0038] In some preferred embodiments, magnetic beads can be used to purify nucleic acid fragment ligation products. Various magnetic bead purification kits are commercially available to those skilled in the art. These magnetic bead purification methods are primarily based on solid-phase reversible immobilization technology. The surface of the magnetic beads is specifically modified, and the adsorption and elution of DNA are achieved through interactions such as electrostatic, hydrophilic, and hydrophobic interactions between the magnetic beads and DNA molecules. Some magnetic bead purification kits work by using magnetic beads with silanol or carboxyl functional groups modified on their outer surface. In a purification buffer system containing PEG, high salt ions, etc., DNA is adsorbed by forming ion bridges between DNA, salt ions, and carboxyl groups. Simple magnetic field treatment can separate the DNA-adsorbed magnetic beads from impurities in other supernatants. In a buffer solution free of PEG and salt ions, the ion bridges between DNA and the magnetic beads are broken, thereby reversibly desorbing the target DNA from the magnetic beads. In some more preferred embodiments, the magnetic bead method may include, for example, AMPure XP magnetic beads or DNA Clean Beads. Those skilled in the art can rationally select appropriate magnetic bead purification kits based on the desired specific effects and the characteristics of the sample.

[0039] According to some embodiments of this application, the method further includes a sequencing step. Given that the single linker is a synthetic single linker, the sequence of the single linker is known. Therefore, those skilled in the art can design primers accordingly for the sequence of the linker portion in the nucleic acid fragment ligation product. For example, amplification primers can be designed to amplify the nucleic acid fragment ligation product; or sequencing primers can be designed to sequence the nucleic acid fragment ligation product or a library of further amplified nucleic acid fragment ligation products.

[0040] In some embodiments of this application, the method further includes an amplification step to obtain a nucleic acid fragment ligation product library. In some embodiments, the amplification step includes using the nucleic acid fragment ligation product as a template to perform amplification to obtain a nucleic acid fragment ligation product library.

[0041] In some embodiments, the amplification includes a strand substitution amplification reaction. In some embodiments, the nucleic acid fragment ligation product library is a multi-copy nucleic acid fragment ligation product library containing the sense and antisense strands of the nucleic acid to be tested.

[0042] In some implementations, the amplification step may employ PCR amplification or PCR-free amplification.

[0043] In some embodiments, PCR amplification is performed using the nucleic acid fragment ligation product as a template. In some embodiments, the primers for PCR amplification are designed to target the linker sequence in the nucleic acid fragment ligation product. In some embodiments, whether the single linker contains a tag sequence or not, the primers can be designed to include the tag sequence. Given that the sequence of the single linker is known, or a known tag sequence is introduced through the single linker and amplification primers, those skilled in the art can reasonably select appropriate sequencing technologies to detect the nucleic acid fragment ligation product library.

[0044] In some implementations, PCR-free amplification is performed using nucleic acid fragment ligation products as templates. PCR-free amplification, or PCR-free library preparation, as the name suggests, refers to a library preparation process that does not require PCR amplification. Its advantage is that it avoids errors introduced by PCR amplification throughout the entire process from library preparation to sequencing. PCR-free amplification technology involves binding a processed DNA sample to a DNA bridging connector. A DNA bridging connector is a short DNA molecule with a specific sequence that can attach to the end of a DNA sample, providing a sequence that can be analyzed by next-generation sequencing technology. After DNA bridging, two adjacent DNA molecules in the DNA sample are linked together by the connector. Then, DNA amplification methods in PCR-free technology, such as rolling circle amplification (RCA), can be used to amplify the ligated DNA sequence. RCA is an amplification technique that does not require PCR cycling and can generate a large number of DNA copies. Through the above steps, the PCR-free method can amplify DNA samples and generate sufficient DNA for sequencing analysis without PCR cycling, avoiding the errors and selective amplification problems caused by PCR cycling in traditional PCR methods, and saving time and costs.

[0045] In some embodiments, the sequencing step includes sequencing a library of ligated nucleic acid fragments to obtain the sequence information of the nucleic acid to be tested. In some embodiments, the sequencing step includes sequencing a library of ligated multi-copy nucleic acid fragments to obtain the sequence information of the sense and antisense strands of the nucleic acid to be tested, and obtaining the terminal information of the nucleic acid to be tested by comparing the sequence information of the sense and antisense strands.

[0046] According to some embodiments of this application, a strand displacement amplification reaction is performed by adding extension primers to the ligation product to generate a multi-copy nucleic acid library with the sense and antisense strands of the nucleic acid to be tested.

[0047] According to some embodiments of this application, sequencing reaction is performed based on a multi-copy nucleic acid library to obtain the nucleic acid sequence information of the positive and negative strands of the nucleic acid to be tested. The nucleic acid information of the positive and negative strands of the nucleic acid to be tested is compared to obtain the end result of the nucleic acid to be tested.

[0048] Those skilled in the art can reasonably select existing known sequencing technologies or design other suitable sequencing technologies. Furthermore, they can reasonably select amplification methods compatible with the chosen or designed sequencing technology. For example, commercially available sequencing pretreatment kits and sequencing platforms can be selected, and nucleic acid fragment ligation products or nucleic acid fragment ligation product libraries can be pretreated according to the manufacturer's instructions. Then, the pretreated samples can be sequenced using the sequencing platform.

[0049] In some preferred embodiments, the sequencing step includes repeating sequencing of the nucleic acid fragment ligation product as the same template. In some preferred embodiments, the sequencing methods include, but are not limited to, MGI's DNBseq sequencing technology or PacBio's SMRT sequencing technology.

[0050] In some specific implementations, DNBseq sequencing technology is used to sequence nucleic acid fragment ligation products or libraries of nucleic acid fragment ligation products. MGI's DNBseq technology consists of three stages: library circularization; amplification of DNBs (DNA nanoballs) via rolling circle amplification (RCA); and loading the DNBs onto a regular array of a sequencing chip for sequencing. Because the nucleic acid fragment ligation products obtained by the method in this application have a unique dumbbell-shaped structure, this structure undergoes denaturation, breaking the hydrogen bonds between the nucleic acid double strands, resulting in single-stranded circular nucleic acid molecules. Therefore, the library circularization step can be conveniently completed. Furthermore, based on rolling circle amplification of single-stranded circular nucleic acid molecules, DNBseq technology not only effectively increases the copy number of the target nucleic acid, greatly enhancing signal intensity, but also avoids the accumulation of errors during PCR amplification by performing rolling circle replication on the same template, effectively improving sequencing accuracy. After circularization amplification, the DNBs are loaded onto the sequencing chip array. DNB (Digital Briggs-Absorber) hybridizes complementaryly with the adapters on the array. Under the catalysis of polymerase, the sequencing template binds to fluorescently labeled probes in the sequencing reagents. This excitation of fluorescent groups produces light signals, which are then collected by the instrument's camera. These signals are processed and converted into digital signals, which are then further processed to ultimately obtain the base sequence information of the sample. By increasing the copy number of the nucleic acid sample and referencing the original template for each amplification, DNB enhances both signal strength and sequencing accuracy.

[0051] In some specific implementations, SMRT sequencing technology is used to sequence nucleic acid fragment ligation products or libraries of nucleic acid fragment ligation products. PacBio's PacBio SMRT sequencing technology employs single-molecular real-time (SMRT) sequencing. Its principle involves ligating the target nucleic acid fragment to a special DNA polymerase, forming a DNA polymerase-DNA complex. This DNA polymerase-DNA complex is then immobilized on a sequencing chip, and nucleic acid fragments are sequenced one by one using a laser, achieving high-throughput sequencing of nucleic acid sequences. The wells of the SMRT sequencing chip contain a special DNA polymerase. When the DNA molecule to be tested binds to this DNA polymerase, DNA strand synthesis occurs simultaneously under the catalysis of the polymerase. Fluorescently labeled dNTPs are used; the fluorescent group emits light during synthesis, and the bases are read by detecting the light. Furthermore, PacBio sequencing offers ultra-long read lengths, and because it involves "natural" strand synthesis, abnormal GC regions have no impact on its sequencing. PacBio's circular consensus sequencing (CCS) mode, based on the PacBio Sequel system, derives a shared sequence from multiple detections of a single template molecule, thereby achieving high base accuracy and producing high-fidelity reads (HiFi reads).

[0052] According to some embodiments of this application, sequencing technology that repeatedly sequences the same template can be used to correct read sequences, thereby improving sequencing accuracy and making the identification of serrated end sequences more accurate.

[0053] According to another embodiment of this application, a kit for detecting the ends of nucleic acid fragments is provided, comprising: a nucleic acid dephosphorylation kit; and a adapter ligation kit.

[0054] In some embodiments, the nucleic acid dephosphorylation kit includes a phosphatase. In some embodiments, the phosphatase includes a phosphatase having 5' dephosphorylation activity and a phosphatase having 3' dephosphorylation activity. In some embodiments, the phosphatase has both 5' dephosphorylation and 3' dephosphorylation activities. Those skilled in the art can reasonably select existing known phosphatases, or can extract enzymes with completely identical or similar functions as substitutes.

[0055] In some embodiments, the phosphatase includes T4 polynucleotide kinase (T4 PNK). T4 PNK is a polynucleotide 5'-hydroxykinase capable of catalyzing the transfer of the γ-phosphate group of ATP to the 5'-hydroxy terminus of an oligonucleotide chain (double-stranded or single-stranded DNA or RNA) and to a 3'-monophosphate nucleoside. However, T4 PNK can also catalyze the reverse reaction of phosphorylation, exhibiting 3'-terminal phosphatase activity, capable of catalyzing the hydrolysis of the 3'-phosphate group from the 3'-phosphate terminus of the oligonucleotide, deoxy3'-monophosphate nucleoside, and deoxy3'-diphosphate nucleoside. When ADP is present, T4 PNK also possesses 5'-terminal phosphatase activity, catalyzing the exchange of the 5'-phosphate group between the 5'-P-oligonucleotide / polynucleotide and the 5'-terminal ATP.

[0056] In some embodiments, the phosphatase includes acid phosphatase or alkaline phosphatase. In some embodiments, the acid phosphatase includes prostatic acid phosphatase and non-prostatic acid phosphatase. In some embodiments, the alkaline phosphatase is a nonspecific phosphomonoesterase. Nonspecific phosphomonoesterases can catalyze the hydrolysis of the 5'-phosphate and 3'-phosphate groups of almost all phosphate monoesters.

[0057] In some preferred embodiments, the phosphatase comprises an alkaline phosphatase. In some preferred embodiments, the alkaline phosphatase includes, but is not limited to, at least one of bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), and secretory alkaline phosphatase (SEAP). In some more preferred embodiments, the phosphatase may be a wild-type enzyme or a recombinant enzyme. Compared to wild-type enzymes, recombinant enzymes obtained through recombinant expression do not contain affinity tags or other modifications found in wild-type or natural enzymes.

[0058] In some preferred embodiments, the phosphatase includes, but is not limited to, at least one of bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), secretory alkaline phosphatase (SEAP), and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are relatively heat-sensitive alkaline phosphatases, easily inactivated at around 65°C; bacterial alkaline phosphatase (BAP) and placental alkaline phosphatase (PLAP) are relatively heat-resistant, and can be heated at 65°C or above for one hour or longer without losing activity. Bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), and placental alkaline phosphatase (PLAP) can all non-specifically catalyze the dephosphorylation of 5' and 3' terminal phosphomonyl esters of DNA and RNA, acting on the 5' or 3' protruding ends and blunt ends, preventing self-ligation of linear nucleic acid fragments through dephosphorylation.

[0059] In some further preferred embodiments, the phosphatase includes at least one of shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP), and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are heat-sensitive alkaline phosphatases that can be completely and irreversibly inactivated by heating at 65°C or above for several minutes, thus eliminating the need for purification before the ligation or end-labeling step.

[0060] In some embodiments, the nucleic acid dephosphorylation kit further includes a buffer solution. In some preferred embodiments, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer, and Tris hydrochloride buffer. For alkaline phosphatases, the buffer solution should provide an alkaline reaction system. For acidic phosphatases, the buffer solution should provide an acidic reaction environment.

[0061] In some embodiments, the nucleic acid dephosphorylation kit further includes a coenzyme factor. For alkaline phosphatase, the coenzyme factor includes zinc ions (Zn). 2+ ) or magnesium ions (Mg 2+ ); and ADP or AMP. Preferably, zinc ions are provided in the form of ZnCl2; magnesium ions are provided in the form of MgCl2.

[0062] Those skilled in the art can provide appropriate buffer solutions, coenzyme factors, and salt ions based on the specific type of phosphatase used, so that the nucleic acid dephosphorylation reaction can be carried out under appropriate reaction system, pH value, coenzyme factor concentration, and salt ion concentration.

[0063] In some embodiments, the linker assembly includes a single linker having phosphate groups at both its 5' and 3' ends. By providing a single linker with phosphate groups at both its 5' and 3' ends, it is advantageous to link to hydroxyl groups on the positive and negative strands of a dephosphorylated nucleic acid fragment. By linking the dephosphorylated nucleic acid fragment to the single linker, with the 5' and 3' ends of each end of the nucleic acid fragment connected by the same single linker, the constructed nucleic acid fragment ligation product forms a dumbbell-shaped structure, as shown in the image. Figure 1 As shown.

[0064] In some embodiments, the single linker is a synthetic single linker. Given the known single linker sequence, the type of the original nucleic acid fragment and the start and end positions of the protruding ends can be accurately identified by detecting and analyzing the nucleic acid fragment ligation product.

[0065] In some embodiments, the number of bases in the single linker can be 5-100 nt. Preferably, the number of bases in the single linker can be 5-30 nt. More preferably, the number of bases in the single linker can be 6-25 nt. In some preferred embodiments, at least one base in the last position of the sequence of the single linker is a ribonucleotide base (rNTP) in the 5' to 3' direction. In some preferred embodiments, the single linker includes a tag sequence, which facilitates subsequent library construction and detection analysis. In some preferred embodiments, the tag sequence of the single linker includes, but is not limited to, a unique molecular tag (UMI) sequence and a sample tag sequence. The unique molecular tag (UMI) sequence is used to count the copy number of nucleic acid molecules in the sample. The sample tag sequence is used to distinguish different samples for subsequent multi-sample pooling sequencing. For example, the sample tag sequence can be a barcode sequence or an index sequence.

[0066] In some embodiments, the adapter ligation kit further includes a nucleic acid ligase. In some embodiments, the nucleic acid ligase may include a DNA ligase or an RNA ligase. In some preferred embodiments, the nucleic acid ligase includes a nucleic acid ligase having activity in catalyzing the formation of a phosphodiester bond (5'PO4 to 3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide, and a nucleic acid ligase having activity in catalyzing the formation of a phosphodiester bond (3'PO4 to 5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide. In some embodiments, the nucleic acid ligase is a nucleic acid ligase possessing both catalytic 5'PO4 to 3'OH linkage activity and catalytic 3'PO4 to 5'OH linkage activity. Those skilled in the art can reasonably provide existing known nucleic acid ligases, or can provide enzymes with completely identical or similar functionality as alternatives.

[0067] By providing nucleoligases with either catalytic 5'PO4→3'OH or catalytic 3'PO4→5'OH ligation activity, the ligation of 5'PO4→3'OH and 3'PO4→5'OH can be catalyzed sequentially or simultaneously. Alternatively, by providing nucleoligases with both catalytic 5'PO4→3'OH and 3'PO4→5'OH ligation activities, the ligation of 5'PO4→3'OH and 3'PO4→5'OH can be catalyzed simultaneously.

[0068] In some embodiments, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (5'PO4→3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide includes at least one of ATP-dependent nucleic acid ligases and NAD+-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 5'PO4→3'OH linkage includes, but is not limited to, at least one of T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV.

[0069] In some embodiments, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (3'PO4→5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide includes GTP-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 3'PO4→5'OH linkage is not limited to RtcB ligase.

[0070] In some embodiments, the connector kit further includes a buffer solution. In some preferred embodiments, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer, and Tris hydrochloride buffer.

[0071] In some embodiments, the adapter ligation kit further includes coenzyme factors. For T4 RNA ligase, coenzyme factors include ATP and magnesium ions (Mg²⁺). 2+ For RtcB ligases, coenzyme factors include GTP and manganese ions (Mn). 2+ Preferably, magnesium ions are provided in the form of MgCl2; manganese ions are provided in the form of MnCl2. Those skilled in the art can provide suitable buffer solutions, coenzyme factors, and salt ions according to the specific type of nucleic acid ligase used, to ligate dephosphorylated nucleic acid fragments to the single linker under suitable reaction systems, pH values, coenzyme factor concentrations, and salt ion concentrations, thereby efficiently constructing nucleic acid fragment ligation products.

[0072] In some embodiments of this application, the kit further includes a digestion kit. The digestion kit is used to digest unconnected nucleic acid fragments.

[0073] In some embodiments, the digestion kit includes a nuclease. In some preferred embodiments, the nuclease includes an exonuclease. Unlike nucleic acid fragment ligation products that have closed structures at both ends, nucleic acid fragments unligated at both ends or ligated at only one end are readily hydrolyzed from their open ends by an exonuclease through sequential hydrolysis of phosphodiester bonds. Those skilled in the art can reasonably provide existing known nucleases, or can provide enzymes with completely identical or similar functionality as substitutes. In some more preferred embodiments, the exonuclease includes, but is not limited to, at least one of exonuclease I (ExoI), exonuclease III (ExoIII), and exonuclease IV (ExoIV).

[0074] In some embodiments, the digestion kit further includes a buffer solution. In some embodiments, the digestion kit further includes a coenzyme factor. In some embodiments, the coenzyme factor includes ATP. In some embodiments, the coenzyme factor includes magnesium ions (Mg²⁺). 2+ Those skilled in the art can provide appropriate buffer solutions, coenzyme factors, and salt ions based on the specific type of nuclease used, so that the unligated nucleic acid fragments are digested and degraded while retaining the nucleic acid fragment ligation products to the maximum extent.

[0075] In some embodiments of this application, the kit further includes a purification kit. The purification kit is used to purify nucleic acid fragment ligation products. Those skilled in the art are familiar with many reagents for purifying target nucleic acids, including but not limited to kits based on methods such as phenol / chloroform, centrifugation column, and magnetic bead methods. Those skilled in the art can reasonably provide suitable nucleic acid purification kits according to the specific desired effect and the characteristics of the sample.

[0076] In some preferred embodiments, the purification kit includes a magnetic bead-based purification kit. Magnetic bead purification is primarily based on solid-phase reversible immobilization technology. The magnetic beads are specifically modified, and the adsorption and elution of DNA are achieved through interactions such as electrostatic, hydrophilic, and hydrophobic interactions between the magnetic beads and DNA molecules. Some magnetic bead purification kits work by using magnetic beads with silanol or carboxyl functional groups modified on their outer surface. In a purification buffer system containing PEG and high-salt ions, DNA is adsorbed by forming ion bridges between DNA, salt ions, and carboxyl groups. Simple magnetic field treatment separates the DNA-adsorbed magnetic beads from impurities in the supernatant. In a buffer solution free of PEG and salt ions, the ion bridges between DNA and the magnetic beads are broken, thereby reversibly desorbing the target DNA from the magnetic beads. In some more preferred embodiments, the purification kit may include, but is not limited to, AMPure XP magnetic beads and DNA Clean Beads. Those skilled in the art can reasonably provide suitable magnetic bead purification kits as purification solutions based on the desired specific effects and sample characteristics.

[0077] In some embodiments of this application, the kit further includes an amplification kit. The amplification kit is used to amplify nucleic acid fragment ligation products to obtain a nucleic acid fragment ligation product library.

[0078] In some embodiments, the amplification kit includes a nucleic acid polymerase and amplification primers. In some preferred embodiments, the nucleic acid polymerase has strand displacement activity. A nucleic acid polymerase with strand displacement activity is capable of dissociating the hydrogen bonds of a single strand already bound to a double-stranded nucleic acid template, and then synthesizing a new single strand. In some preferred embodiments, the nucleic acid polymerase is a DNA-dependent DNA polymerase. In some more preferred embodiments, the DNA-dependent DNA polymerase includes at least one of T4 DNA polymerase, DNA polymerase I (Klenow) large fragment (Klenow fragment), T7 DNA polymerase, DNA polymerase I, Taq DNA polymerase, Bst DNA polymerase, and Phi29 DNA polymerase.

[0079] In some embodiments, the amplification kit includes a PCR amplification kit. In some embodiments, the PCR amplification kit includes PCR amplification primers designed for the sequence of the single linker. In some embodiments, the PCR amplification primers contain a tag sequence. Given that the sequence of the single linker is known, appropriate primers can be readily designed to perform PCR amplification using the DNA fragment ligation product as a template. Where the single linker or the PCR amplification primers contain a tag sequence, those skilled in the art can advantageously utilize suitable sequencing techniques to detect the amplified library of the DNA fragment ligation product.

[0080] In some embodiments, the PCR amplification kit further includes: DNA polymerase; PCR buffer; magnesium ions; and dNTPs, including dATP, dGTP, dTTP, and dCTP. Those skilled in the art can rationally provide suitable amplification kits according to the specific desired effect and the characteristics of the sample, and can provide suitable PCR reaction systems based on the specific type of DNA polymerase used.

[0081] In some embodiments, the amplification kit includes a PCR-free amplification kit. Those skilled in the art can select a library preparation kit that does not require PCR amplification based on relevant publicly available techniques. In some embodiments, the PCR-free amplification kit includes a rolling circle amplification (RCA) kit. For example, those skilled in the art can prepare DNA nanospheres (DNB) according to relevant publicly available techniques, or DNB preparation kits are commercially available. DNA nanospheres can be prepared using a suitable DNB preparation kit. For example, the DNBseq one-step DNB preparation kit from MGI Tech.

[0082] In some embodiments of this application, the kit further includes a sequencing kit. Those skilled in the art can reasonably provide sequencing kits based on existing known sequencing technologies or other suitable sequencing technologies. Simultaneously, those skilled in the art can also provide compatible amplification kits and sequencing kits. For example, a pretreatment kit or amplification kit compatible with the provided sequencing technology can be provided to pretreatment or amplify nucleic acid fragment ligation products or nucleic acid fragment ligation product libraries, and then the pretreated or amplified samples can be sequenced using the sequencing kit. Generally, sequencing kits include, but are not limited to, sequencing chips, sequencers, or read length analyzers.

[0083] In some preferred embodiments, the sequencing kit includes, but is not limited to, sequencing kits based on DNBseq sequencing technology from MGI (Mega-Tech International) or SMRT sequencing technology from PacBio (PacBio Corporation). According to some embodiments of this application, sequencing technology that repeatedly sequences the same template can correct read sequences, thereby improving sequencing accuracy and making the identification of serrated-end sequences more accurate.

[0084] Those skilled in the art will understand that there may be no clear boundaries between the various kits distinguished by the processing or reaction stage of the test sample, and the kits and their reagent components are not entirely independent. For example, compatible buffer solutions or compatible salt ion mixtures may be provided for use in one or more reaction stages. Those skilled in the art are capable of reasonably determining which reagent components are suitable for use in one or more reaction stages, thereby providing only a sufficient number of portions in the kit. Those skilled in the art will understand that the aforementioned kits or packages will include reagents, instruments or consumables, instructions, etc.

[0085] According to another embodiment of this application, a nucleic acid library comprising a sense strand and a negative sense strand of a nucleic acid to be tested is provided, characterized in that: the nucleic acid library has a partially double-stranded closed loop dumbbell-shaped structure, wherein the double-stranded portion is the nucleic acid to be tested portion, the dumbbell-shaped portion is a single linker located at both ends of the nucleic acid to be tested, wherein the single linker includes a tag sequence, and wherein the 3' end of the single linker contains at least one ribonucleotide base.

[0086] According to another embodiment of this application, the application of the single-linked header of the above-described embodiment in the detection of nucleic acid ends is provided.

[0087] According to another embodiment of this application, the application of the single-linked header of the above-described embodiment in the detection of double-strand breaks in nucleic acids is provided.

[0088] According to another embodiment of this application, the application of the single-linked head of the above-described embodiments in the preparation of a kit for detecting nucleic acid ends is provided.

[0089] According to another embodiment of this application, the application of the single-linked head of the above-described embodiments in the preparation of a kit for detecting double-strand breaks in nucleic acids is provided.

[0090] According to another embodiment of this application, the application of the single linker, method for detecting nucleic acid ends, and kit for detecting nucleic acid ends of the above-described embodiments in the analysis of cfDNA fragmentomics characteristics is provided.

[0091] In some embodiments, characterizing cfDNA fragments includes analyzing the serrated ends of cfDNA fragments. In some embodiments, the application of characterizing cfDNA fragments or analyzing the serrated ends of cfDNA fragments is for non-therapeutic or diagnostic purposes, such as for scientific research purposes. Characterizing cfDNA fragments for scientific research purposes and analyzing the serrated ends of cfDNA fragments are well known to those skilled in the art.

[0092] In some implementations, characterizing cfDNA fragmentomics includes identifying the type of cfDNA ends.

[0093] In some implementations, characterizing cfDNA fragmentomics includes identifying the relative abundance of cfDNA end types.

[0094] In some implementations, analyzing the serrated ends of cfDNA fragments includes determining the length of the serrated ends of the cfDNA.

[0095] In some implementations, analyzing the serrated ends of cfDNA fragments includes identifying the location of the serrated ends of the cfDNA.

[0096] In some implementations, analyzing the serrated ends of cfDNA fragments includes identifying the starting position of the serrated ends of the cfDNA.

[0097] In some implementations, analyzing the serrated ends of cfDNA fragments includes identifying the termination position of the serrated ends of the cfDNA.

[0098] In some implementations, analyzing cfDNA fragment omics characteristics or analyzing the serrated ends of cfDNA fragments includes transient or long-term analysis of individuals or populations of humans or animals.

[0099] In some implementations, the applications include analyzing cfDNA fragment omics characteristics or the association between cfDNA fragment serrated ends and organism development.

[0100] In some implementations, the applications include analyzing cfDNA fragment omics characteristics or the association between cfDNA fragment serrated ends and disease occurrence.

[0101] In some implementations, the applications include analyzing cfDNA fragment omics characteristics or the link between cfDNA fragment serrated ends and cancer development.

[0102] In some implementations, the applications include analyzing cfDNA fragment omics characteristics or the association between cfDNA fragment serrated ends and drug resistance.

[0103] According to the embodiments provided in various aspects of this application, this application provides a technical solution for detecting nucleic acid ends, particularly serrated ends of cfDNA. By dephosphorylating plasma DNA, the 3' and 5' ends of the adapter are ligated to the 5' and 3' ends of the DNA on the same side using a ligase, respectively, circularizing and sealing the DNA ends to form a dumbbell-shaped structure. The ligation product or further amplified library is sequenced, and based on the known adapter sequence, detailed information about the protruding DNA ends can be obtained through data analysis.

[0104] By using the single-linker provided in this application, the positive and negative strands of a nucleic acid fragment with both ends dephosphorylated can be linked, advantageously enabling the detection of different types of double-stranded nucleic acid ends, including 5' serrated ends, 3' serrated ends, or either blunt or serrated ends. Simultaneously, by using the single-linker provided in this application to link the positive and negative strands of cfDNA, sequencing analysis can accurately identify the start and end positions of the serrated ends. With the single-linker sequence known, detection and analysis of the cfDNA fragment ligation product can accurately identify the end type of the original cfDNA fragment to be tested, as well as the start and end positions of the serrated ends.

[0105] The single-linker provided in this application can simultaneously connect to the same-side ends of the double-stranded nucleic acid fragment to be tested, efficiently and accurately capturing the protruding end sequences of the double-stranded nucleic acid. Sequencing analysis allows for the precise identification of the length, type, start, and termination positions of the double-stranded nucleic acid ends based on the sequence of the single-linker. According to the embodiments provided in various aspects of this application, the specific sequence of the single-linker can be changed while still achieving equivalent technical effects. The single-linker provided in this application can be used not only to detect serrated ends of cfDNA fragments but also to detect double-stranded breaks in nucleic acids within cells.

[0106] Furthermore, sequencing technology that uses repeated sequencing of the same template can correct read sequences, thereby improving sequencing accuracy and making the identification of serrated end sequences more accurate. Attached Figure Description

[0107] Figure 1 A schematic diagram of the structure of a nucleic acid fragment ligation product according to an embodiment of this application is shown.

[0108] Figure 2 A schematic flowchart of a detection method according to an embodiment of this application is shown.

[0109] Figure 3 A schematic flowchart of a detection method according to an embodiment of this application is shown.

[0110] Figure 4 A gel electrophoresis diagram of the ligation product according to one embodiment of this application is shown.

[0111] Figure 5 The sequencing analysis results of the serrated ends of a cfDNA fragment according to one embodiment of this application are shown.

[0112] Figure 6 The sequencing analysis results of the serrated ends of a cfDNA fragment according to one embodiment of this application are shown. Detailed Implementation

[0113] The following detailed description of specific embodiments further illustrates the content of this application. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples of this application can be obtained from conventional commercial sources or through existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0114] Unless otherwise stated, those skilled in the art will understand that the chemical terms used above and throughout this specification have their common meaning in the art. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its common meaning. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0115] Unless otherwise indicated or defined, all terms used herein have their usual meaning in the art, which will be clear to those skilled in the art. For example, refer to standard manuals such as Molecular Cloning: A Laboratory Manual (Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press, 2012); Lewin's Genes XI (Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, 2017); and Modern Molecular Biology (Zhu Yuxian et al., Modern Molecular Biology (5th Edition), Higher Education Press, 2019).

[0116] The subject matter of this application and the claims specifically relates to artificial products or methods of using or producing such artificial products, which may be variants of natural (wild-type) products. Although there may be a degree of sequence identity with natural structures, it is understood that the materials, methods, and uses of this application (e.g., specifically isolated nucleic acid sequences) are “artificial” or synthetic and should therefore not be considered as a result of “natural laws”.

[0117] The terms “first” and “second” as used herein are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as “first” or “second” may explicitly or implicitly include at least one of that feature. In the description herein, “multiple” means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0118] The terms “comprising,” “including,” “having,” and “containing” as used herein can be used as synonyms and should be understood as open-ended definitions that allow for the presence of other components, parts, or elements. “Comprising of…” is considered the most closed definition, containing no other elements besides those defining the constituent features. Therefore, “comprising” is broader and encompasses the definition of “comprising of…”.

[0119] As used herein, the term “about” means the same value or a value that differs from a given value by + / - 5%, specifically by + / - 2%, and more specifically by + / - 1%.

[0120] As used herein and in the claims, the singular form, such as "an / a," includes the plural unless the context clearly indicates otherwise.

[0121] In this article, "high-throughput sequencing," also known as "next-generation sequencing" (NGS), is a sequencing technology distinct from the traditional dideoxy sequencing (Sanger) method. It is a technique that enables the parallel sequencing of a large number of nucleic acid molecules in a single run, typically producing at least 100 Mb of sequencing data per reaction. Based on differences in development history, influence, sequencing principles, and techniques, the main types include: Massively Parallel Signature Sequencing (MPSS), Polony Sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, ABI SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, and combinatorial probe-anchor synthesis (cPAS). High-throughput sequencing technology allows for the sequencing of hundreds of thousands to millions of DNA molecules in a single run, making it possible to perform detailed and comprehensive analyses of the transcriptome and genome of a species.

[0122] In this article, the term "ATP" refers to adenine triphosphate, the term "TTP" refers to thymidine triphosphate, the term "CTP" refers to cytosine triphosphate, the term "GTP" refers to guanine triphosphate, and the term "UTP" refers to uracil triphosphate.

[0123] In this article, the term "dNTP" refers to deoxyribonucleoside triphosphate, including deoxyATP (dATP), deoxyTTP (dTTP), deoxyCTP (dCTP), and deoxyGTP (dGTP).

[0124] In this article, the term "rNTP" refers to ribonucleoside triphosphate, including ribose ATP (rATP), ribose UTP (UTP), ribose CTP (rCTP), and ribose GTP (rGTP).

[0125] According to one aspect of this application, a single linker is provided, wherein both the 5' and 3' ends of the single linker are phosphate groups. In some embodiments, the single linker is a synthetic single linker.

[0126] In some preferred embodiments, the single linker includes a tag sequence. In some embodiments, the number of bases in the single linker can be 5-100 nt. Preferably, the number of bases in the single linker can be 5-30 nt. More preferably, the number of bases in the single linker can be 6-25 nt. Even more preferably, the number of bases in the single linker can be 8-20 nt. In some preferred embodiments, at least one base in the last position of the sequence of the single linker is a ribonucleotide base (rNTP) in the 5' to 3' direction.

[0127] According to another embodiment of this application, a method for detecting nucleic acid ends is provided, comprising the following steps:

[0128] Provide a dephosphorylated nucleic acid fragment, wherein the 5' end and 3' end of the dephosphorylated nucleic acid fragment are both hydroxyl groups;

[0129] A single linker is provided, wherein both the 5' and 3' ends of the single linker are phosphate groups;

[0130] The dephosphorylated nucleic acid fragment is ligated to the single linker to obtain a nucleic acid fragment ligation product, and the nucleic acid fragment ligation product is detected.

[0131] In this paper, the term "cfDNA" encompasses any fragmented DNA circulating in the bloodstream of humans or animals, free outside cells, originating from apoptotic and necrotic cells with different topological domains. Characteristics of cfDNA fragmentomics include fragment length, fragment size distribution, terminal motifs, preferred end coordinates, and breakpoint motifs. Recent evidence suggests that the characteristics of cfDNA fragmentomics differ between individuals and tissues during growth and development, and between healthy and diseased individuals, making it a highly promising new biomarker.

[0132] In this article, when used for cfDNA, the term "jagged end" refers to the single-stranded protruding end carried by a double-stranded cfDNA molecule.

[0133] According to some embodiments of this application, a dephosphorylated nucleic acid fragment is obtained by removing the phosphate groups at the 5' and 3' ends of the nucleic acid fragment to be tested. According to some embodiments of this application, the nucleic acid fragment to be tested comprises a double-stranded nucleic acid fragment. According to some embodiments of this application, the nucleic acid fragment to be tested comprises a DNA fragment. According to some embodiments of this application, the nucleic acid fragment to be tested comprises a cfDNA fragment. According to some embodiments of this application, the cfDNA fragment is obtained from circulating peripheral blood. According to some embodiments of this application, the cfDNA fragment comprises any free DNA fragment in circulating peripheral blood. According to some embodiments of this application, the cfDNA fragment comprises any DNA fragment present in extracellular fluid. According to some embodiments of this application, the cfDNA fragment comprises fragmented endogenous DNA free outside cells in circulating peripheral blood.

[0134] According to some embodiments of this application, the nucleic acid fragment to be tested may include ends of different types. According to some embodiments of this application, the nucleic acid fragment to be tested may have a 5' protruding end, a 3' protruding end, or a blunt end at either end or both ends.

[0135] According to some embodiments of this application, the nucleic acid fragment to be tested may have a 5' phosphate group, a 3' phosphate group, a 5' hydroxyl group, or a 3' hydroxyl group at either end or both ends. According to some embodiments of this application, by removing the 5' and 3' phosphate groups from the nucleic acid fragment, hydroxyl groups are formed at both ends of the nucleic acid fragment. This not only helps to avoid undesirable self-ligation between nucleic acid fragments, but also helps to improve the ligation efficiency with single-linked heads in subsequent steps.

[0136] According to some embodiments of this application, dephosphorylated nucleic acid fragments are obtained by removing the phosphate groups at the 5' and 3' ends of nucleic acid fragments using phosphatases. In some embodiments, the phosphatases include phosphatases with catalytic 5' dephosphorylation activity and phosphatases with 3' dephosphorylation activity. In some embodiments, the phosphatases have both catalytic 5' dephosphorylation activity and 3' dephosphorylation activity. To remove the phosphate groups at the 5' and 3' ends of nucleic acid fragments, those skilled in the art can rationally select existing known phosphatases, or can extract enzymes with completely identical or similar functions for substitution.

[0137] In some embodiments, the phosphatase may include T4 polynucleotide kinase (T4 PNK). T4 PNK is a polynucleotide 5'-hydroxykinase capable of catalyzing the transfer of the γ-phosphate group of ATP to the 5'-hydroxy terminus of an oligonucleotide chain (double-stranded or single-stranded DNA or RNA) and to a 3'-monophosphate nucleoside. However, T4 PNK can also catalyze the reverse reaction of phosphorylation, exhibiting 3'-terminal phosphatase activity, capable of catalyzing the hydrolysis of the 3'-phosphate group from the 3'-phosphate terminus of the oligonucleotide, deoxy3'-monophosphate nucleoside, and deoxy3'-diphosphate nucleoside. When ADP is present, T4 PNK also possesses 5'-terminal phosphatase activity, catalyzing the exchange of the 5'-phosphate group between the 5'-P-oligonucleotide / polynucleotide and the 5'-terminal ATP.

[0138] In some embodiments, the phosphatase may include an acid phosphatase or an alkaline phosphatase. In some embodiments, the acid phosphatase includes prostatic acid phosphatase and non-prostatic acid phosphatase. In some preferred embodiments, the alkaline phosphatase is a nonspecific phosphomonoesterase. Nonspecific phosphomonoesterases can catalyze the hydrolysis of the 5'-phosphate and 3'-phosphate groups of almost all phosphate monoesters.

[0139] In some preferred embodiments, the phosphatase may include at least one of bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), and secretory alkaline phosphatase (SEAP). In some more preferred embodiments, the phosphatase may be a wild-type enzyme or a recombinant enzyme. Compared to wild-type enzymes, recombinant enzymes obtained through recombinant expression do not contain affinity tags or other modifications found in wild-type or natural enzymes.

[0140] In some preferred embodiments, the phosphatase may be selected from at least one of bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), secretory alkaline phosphatase (SEAP), and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are relatively heat-sensitive alkaline phosphatases, easily inactivated at around 65°C; bacterial alkaline phosphatase (BAP) and placental alkaline phosphatase (PLAP) are relatively heat-resistant, and can be heated at 65°C or above for one hour or longer without losing activity. Bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), and placental alkaline phosphatase (PLAP) can all non-specifically catalyze the dephosphorylation of 5' and 3' terminal phosphomonyl esters of DNA and RNA, acting on the 5' or 3' protruding ends and blunt ends, and preventing self-ligation of linear nucleic acid fragments by dephosphorylation.

[0141] In some preferred embodiments, the phosphatase may be at least one of shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP), and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are heat-sensitive alkaline phosphatases that can be completely and irreversibly inactivated by heating at 65°C or above for several minutes, thus eliminating the need for purification before the ligation or end-labeling steps.

[0142] According to some embodiments of this application, the dephosphorylation reaction system for removing the 5' and 3' phosphate groups from nucleic acid fragments includes a buffer solution. In some preferred embodiments, the buffer solution includes citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer, and Tris hydrochloride buffer. For alkaline phosphatase, the buffer solution should provide an alkaline reaction system. In some preferred embodiments, the pH of the alkaline reaction system is 9-13; more preferably, the pH of the alkaline reaction system is 10-11.

[0143] In some preferred embodiments, the dephosphorylation reaction system includes a coenzyme factor. For alkaline phosphatase, the coenzyme factor includes zinc ions (Zn). 2+ ) or magnesium ions (Mg 2+ ); and ADP or AMP. In some preferred embodiments, zinc ions are provided in the form of ZnCl2; magnesium ions are provided in the form of MgCl2. In some preferred embodiments, zinc ions (ZnCl2) are provided in the form of ZnCl2; and magnesium ions are provided in the form of MgCl2. 2+ ) and / or magnesium ions (Mg 2+ The concentration of ADP and / or AMP is 0.01-5 mmol / L; more preferably, the concentration of zinc ions and / or magnesium ions is 0.05-2 mmol / L; even more preferably, the concentration of zinc ions and / or magnesium ions is 0.1-1 mmol / L. In some preferred embodiments, the concentration of ADP and / or AMP is 0.1-100 mmol / L; more preferably, the concentration of ADP and / or AMP is 1-50 mmol / L; even more preferably, the concentration of ADP and / or AMP is 5-25 mmol / L.

[0144] In some embodiments, the dephosphorylation reaction, which removes the 5' and 3' phosphate groups from the nucleic acid fragment, is carried out at 16-50°C for 5-60 min. More preferably, the dephosphorylation reaction is carried out at 25-40°C for 10-40 min. Even more preferably, the dephosphorylation reaction is carried out at 30-40°C for 20-35 min. Those skilled in the art can select appropriate buffers, coenzyme factors, and salt ions, and adjust the reaction temperature and time, as well as the pH and ion concentration of the reaction system, according to the specific type of phosphatase used and the desired effect. For example, a commercially available phosphatase kit can be used, and the phosphatase and buffer in the kit can be used according to the manufacturer's instructions to perform the dephosphorylation reaction.

[0145] According to some embodiments of this application, the 5' and 3' ends of the single linker both have phosphate groups. According to some embodiments of this application, the single linker is a synthetic single linker. Given the single linker sequence, the type of the original nucleic acid to be tested and the start and end positions of the protruding ends can be accurately identified by detecting and analyzing the nucleic acid fragment ligation product.

[0146] In some embodiments, the number of bases in the single linker can be 5-100 nt. To ensure efficient ligation between the single linker and the positive and negative strands at the ends of the nucleic acid fragment, preferably, the number of bases in the single linker can be 5-30 nt. More preferably, the number of bases in the single linker can be 6-25 nt. Even more preferably, the number of bases in the single linker can be 8-20 nt. In some preferred embodiments, at least one base in the last position of the single linker sequence, from the 5' to the 3' end, is a ribonucleotide base (rNTP).

[0147] In some preferred embodiments, the single linker includes a tag sequence, which facilitates subsequent library construction and detection analysis. In some preferred embodiments, the tag sequence of the single linker includes, but is not limited to, a unique molecular tag (UMI) sequence and a sample tag sequence. The unique molecular tag (UMI) sequence is used to count the copy number of nucleic acid molecules in a sample. The sample tag sequence is used to distinguish different samples for subsequent multi-sample pooling sequencing. For example, the sample tag sequence may include a barcode sequence or an index sequence.

[0148] In this article, the term "Unique Molecular Identifier" (UMI) refers to molecular barcoding or molecular tagging technology, which involves attaching a unique tag sequence to each nucleic acid fragment, followed by PCR amplification and sequencing. The unique molecular tag (UMI) sequence is primarily used to distinguish different fragments within the same sample. In subsequent data analysis, these tags help eliminate errors introduced by DNA polymerase, amplification, and sequencing processes. Molecular tags typically consist of a random sequence of approximately 10 nt (e.g., NNNNNNNNN) or degenerate bases (NNNRNYN).

[0149] In this article, "barcode sequence" or "barcode sequence" refers to the use of a specific sequence tag to distinguish different samples in high-throughput sequencing. This tag is used to differentiate samples within the same flow channel, allowing for the separation of different sample data during subsequent analysis.

[0150] In this article, "index sequence" or "index sequence" refers to a special nucleotide sequence in sequencing technology, similar to a barcode sequence, used to separate data from different samples in high-throughput sequencing. This sequence is typically ligated to both ends of the target nucleic acid fragment and hybridized with a known sequence on the sequencing chip during sequencing, thereby binding the library to the chip and initiating sequencing.

[0151] According to some embodiments of this application, a dephosphorylated nucleic acid fragment is ligated to the single linker. By providing a single linker with phosphate groups at both the 5' and 3' ends, it is advantageous to link to the hydroxyl groups on the positive and negative strands of the nucleic acid fragment that has been dephosphorylated at both ends in the previous step. By ligating the dephosphorylated nucleic acid fragment to the single linker, with the 5' and 3' ends of each nucleic acid fragment connected by the same single linker, the constructed nucleic acid fragment ligation product forms a dumbbell-shaped structure, as shown in... Figure 1 As shown, given a known single-linker sequence, the type of the original nucleic acid terminology and the start and end positions of the protruding ends can be accurately identified by detecting and analyzing the ligation products of nucleic acid fragments.

[0152] According to some embodiments of this application, a nucleic acid ligase is used to ligate a dephosphorylated nucleic acid fragment to the single linker. In some embodiments, the nucleic acid ligase may include a DNA ligase or an RNA ligase. In some preferred embodiments, the nucleic acid ligase may include a nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (5'PO4 to 3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide, and a nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (3'PO4 to 5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide. In some embodiments, the nucleic acid ligase is a nucleic acid ligase that has both catalyzing 5'PO4 to 3'OH linkage and catalyzing 3'PO4 to 5'OH linkage activities. Those skilled in the art can reasonably provide existing known nucleic acid ligases, or can provide enzymes with completely identical or similar functions as substitutes.

[0153] By providing nucleoligases with either catalytic 5'PO4→3'OH or catalytic 3'PO4→5'OH ligation activity, the ligation of 5'PO4→3'OH and 3'PO4→5'OH can be catalyzed sequentially or simultaneously. Alternatively, by providing nucleoligases with both catalytic 5'PO4→3'OH and 3'PO4→5'OH ligation activities, the ligation of 5'PO4→3'OH and 3'PO4→5'OH can be catalyzed simultaneously.

[0154] In some embodiments, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (5'PO4→3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide includes at least one of ATP-dependent nucleic acid ligases and NAD+-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 5'PO4→3'OH linkage includes, but is not limited to, at least one of T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV.

[0155] In some embodiments, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (3'PO4→5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide includes GTP-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 3'PO4→5'OH linkage is not limited to RtcB ligase.

[0156] In some embodiments, the ligation reaction system for linking the dephosphorylated nucleic acid fragment to the single linker includes a buffer solution. In some preferred embodiments, the buffer solution includes citrate buffer, MES buffer, phosphate buffer, Bis-tris buffer, and Tris hydrochloride buffer. In some preferred embodiments, the pH of the ligation reaction system is 6-9. More preferably, the pH of the ligation reaction system is 6.5-8.5. Even more preferably, the pH of the ligation reaction system is 7-8.

[0157] In some preferred embodiments, the ligation reaction system includes coenzyme factors. For T4 RNA ligase, the coenzyme factors include ATP and magnesium ions (Mg²⁺). 2+ For RtcB ligases, coenzyme factors include GTP and manganese ions (Mn). 2+ In some preferred embodiments, magnesium ions are provided in the form of MgCl2; manganese ions are provided in the form of MnCl2. In some preferred embodiments, magnesium ions (MgCl2) are provided in the form of MgCl2. 2+ ) and / or manganese ions (Mn 2+ The concentration of ATP or GTP is 0.1-50 mmol / L. More preferably, the concentration of magnesium ions and / or manganese ions is 1-25 mmol / L. Even more preferably, the concentration of magnesium ions and / or manganese ions is 5-20 mmol / L. In some preferred embodiments, the concentration of ATP or GTP is 0.01-50 mmol / L; more preferably, the concentration of ATP or GTP is 0.1-10 mmol / L; even more preferably, the concentration of ATP or GTP is 1-5 mmol / L.

[0158] In some embodiments, the ligation reaction of connecting the dephosphorylated nucleic acid fragment to the single linker is carried out at 16-50°C for 5-60 min. More preferably, the ligation reaction is carried out at 25-40°C for 10-40 min. More preferably, the ligation reaction is carried out at 30-40°C for 20-35 min. Those skilled in the art can select appropriate buffers, coenzyme factors, and salt ions, and adjust the reaction temperature and time, as well as the pH and ion concentration of the reaction system, according to the specific type of nucleic acid ligase used and the desired effect. For example, a commercially available nucleic acid ligation kit can be used, and the nucleic acid ligase and buffer in the kit can be used according to the manufacturer's instructions to carry out the ligation reaction of the nucleic acid fragment to the single linker.

[0159] According to some embodiments of this application, the method further includes a purification step. According to some embodiments of this application, the purification step includes purifying the nucleic acid fragment ligation product. According to some embodiments of this application, the method further includes a digestion step. According to some embodiments of this application, the digestion step includes digesting unligated nucleic acid fragments.

[0160] In some embodiments, unligated nucleic acid fragments are digested using nucleases. In some preferred embodiments, the nucleases include exonucleases. Unlike nucleic acid fragment ligation products that have closed structures at both ends, nucleic acid fragments unligated at both ends or ligated at only one end are readily hydrolyzed from their open ends by exonucleases through sequential hydrolysis of phosphodiester bonds. In some more preferred embodiments, the exonucleases include, but are not limited to, at least one of exonuclease I (ExoI), exonuclease III (ExoIII), and exonuclease IV (ExoIV). Those skilled in the art can reasonably select existing known nucleases, or can extract enzymes with completely identical or similar functions for substitution.

[0161] In some embodiments, the digestion reaction system for digesting unligated nucleic acid fragments includes a buffer solution. In some preferred embodiments, the buffer solution includes citrate buffer, MES buffer, phosphate buffer, Bis-tris buffer, and Tris hydrochloride buffer. In some preferred embodiments, the pH of the ligation reaction system is 6-9. More preferably, the pH of the ligation reaction system is 6.5-8.5. Even more preferably, the pH of the ligation reaction system is 7-8.

[0162] In some preferred embodiments, the digestion reaction system includes a coenzyme factor. In some embodiments, the coenzyme factor includes ATP. In some embodiments, the coenzyme factor includes magnesium ions (Mg). 2+ ATP or magnesium ions (Mg) 2+ Whether magnesium ions are needed depends on the specific nuclease chosen. In some preferred embodiments, magnesium ions are provided in the form of MgCl2. In some preferred embodiments, magnesium ions (MgCl2) are provided in the form of MgCl2. 2+ The concentration of ATP is 0.1-50 mmol / L. More preferably, the concentration of magnesium ions is 1-25 mmol / L. Even more preferably, the concentration of magnesium ions is 5-20 mmol / L. In some preferred embodiments, the concentration of ATP is 0.01-50 mmol / L; more preferably, the concentration of ATP is 0.1-10 mmol / L; even more preferably, the concentration of ATP is 1-5 mmol / L.

[0163] In some embodiments, the digestion reaction of unligated nucleic acid fragments is carried out at 16-50°C for 5-60 min. More preferably, the digestion reaction is carried out at 25-40°C for 10-40 min. More preferably, the digestion reaction is carried out at 30-40°C for 20-35 min. Those skilled in the art can select appropriate buffer solutions, coenzyme factors, and salt ions, and adjust the reaction temperature and time, as well as the pH and ion concentration of the reaction system, according to the specific type of nuclease used and the desired effect. For example, a commercially available nucleic acid digestion enzyme kit can be used, and the nuclease and buffer solution in the kit can be used according to the manufacturer's instructions to perform the nucleic acid digestion reaction.

[0164] Those skilled in the art are familiar with many methods and reagents for purifying target nucleic acids, including but not limited to the phenol / chloroform method, centrifugal column method, magnetic bead method, etc. Those skilled in the art can rationally select appropriate nucleic acid purification methods and nucleic acid purification kits according to the specific effects required and the characteristics of the sample.

[0165] The phenol / chloroform method is one of the most common DNA purification methods. This method utilizes the density difference between phenol and chloroform to separate DNA molecules from other impurities. An equal volume of phenol / chloroform mixture is added to a solution containing DNA molecules, mixed thoroughly, and then centrifuged to separate the layers. The upper DNA layer is then removed, and an equal volume of isopropanol is added to precipitate the DNA molecules.

[0166] The centrifugation column method is also a rapid and simple DNA purification method. This method utilizes the special structure of a centrifugation column to separate DNA molecules from other impurities. By adding a DNA solution to the centrifugation column and centrifuging to separate DNA molecules from other impurities, the DNA molecules in the column are then washed and eluted to obtain purified DNA molecules.

[0167] Magnetic bead purification is a highly efficient and automated method for DNA purification. It utilizes the specific binding of affinity molecules on the surface of magnetic beads to DNA molecules, separating DNA molecules from other impurities. The specific steps are as follows: First, cells are lysed to release DNA molecules into the solution; then, magnetic beads are added to bind to the DNA molecules; finally, magnetic force is used to separate the magnetic beads from the DNA molecules, yielding purified DNA molecules.

[0168] In some preferred embodiments, nucleic acid fragment ligation products are purified using magnetic beads. Various magnetic bead purification kits are commercially available to those skilled in the art, and these magnetic bead purification methods are primarily based on solid-phase reversible immobilization technology. The surface of the magnetic beads is specifically modified, and DNA adsorption and elution are achieved through interactions such as electrostatic, hydrophilic, and hydrophobic interactions between the magnetic beads and DNA molecules. Some magnetic bead purification kits work by using magnetic beads with silanol or carboxyl functional groups modified on their outer surface. In a purification buffer system containing PEG, high salt ions, etc., DNA is adsorbed by forming ion bridges between DNA, salt ions, and carboxyl groups. Simple magnetic field treatment separates the DNA-adsorbed magnetic beads from impurities in the supernatant. In a buffer solution free of PEG and salt ions, the ion bridges between DNA and the magnetic beads are broken, thereby reversibly desorbing the target DNA from the magnetic beads. In some more preferred embodiments, the magnetic bead method may include, for example, AMPure XP magnetic beads or DNA CleanBeads. Those skilled in the art can rationally select a suitable magnetic bead purification kit based on the desired specific effect and the characteristics of the sample.

[0169] According to some embodiments of this application, the method further includes a sequencing step. Given that the single linker is a synthetic single linker and its sequence is known, those skilled in the art can design primers accordingly for the sequence of the linker portion in the nucleic acid fragment ligation product. For example, amplification primers can be designed to amplify the nucleic acid fragment ligation product; or sequencing primers can be designed to sequence the DNA fragment ligation product or a library of further amplified nucleic acid fragment ligation products.

[0170] In some embodiments, the method further includes an amplification step before the sequencing step to obtain a nucleic acid fragment ligation product library. In some embodiments, the amplification step includes amplification using the nucleic acid fragment ligation product as a template to obtain a nucleic acid fragment ligation product library. In some embodiments, the amplification includes a strand substitution amplification reaction. In some embodiments, the nucleic acid fragment ligation product library is a multi-copy nucleic acid fragment ligation product library containing the sense and antisense strands of the nucleic acid to be tested. In some embodiments, the amplification step can employ PCR amplification or PCR-free amplification.

[0171] In some implementations, nucleic acid polymerases with strand displacement amplification are used to amplify nucleic acid fragment ligation products. In this document, the term "strand displacement activity" refers to a property of nucleic acid polymerases that allows DNA polymerases to "displace" one strand of an existing DNA double helix during nucleic acid (such as DNA) replication or synthesis, thereby enabling the successful synthesis of a new DNA single strand.

[0172] In some embodiments, PCR amplification is performed using the nucleic acid fragment ligation product as a template. In some embodiments, the primers for PCR amplification are designed to target the linker sequence in the nucleic acid fragment ligation product, i.e., to target the sequence of the single linker. In some embodiments, whether the single linker contains a tag sequence or not, the tag sequence can be incorporated into the PCR amplification primers by designing the tagged sequence. Given that the sequence of the single linker is known, or a known tag sequence is introduced through the single linker and the amplification primers, those skilled in the art can reasonably select appropriate sequencing technologies to detect the nucleic acid fragment ligation product or the nucleic acid fragment ligation product library obtained through further amplification.

[0173] In some implementations, PCR-free amplification is performed using nucleic acid fragment ligation products as templates. In this document, the terms "PCR-free amplification" or "PCR-free library preparation" refer to a library preparation process that does not require PCR amplification. Its advantage lies in avoiding errors introduced by PCR amplification throughout the entire process from library preparation to sequencing. PCR-free amplification technology involves binding a processed DNA sample to a DNA bridging connector. A DNA bridging connector is a short DNA molecule with a specific sequence that can attach to the ends of a DNA sample, providing a sequence that can be analyzed by next-generation sequencing technology. After DNA bridging, two adjacent DNA molecules in the DNA sample are linked together by the connector, and then DNA amplification methods in PCR-free technology, such as rolling circle amplification (RCA), can be used to amplify the ligated DNA sequence.

[0174] In some implementations, the PCR-free amplification includes rolling circle amplification (RCA). Hereinafter, the term "rolling circle amplification" (RCA) refers to an amplification technique that does not require PCR cycling and can produce a large number of DNA copies. PCR-free methods can amplify DNA samples and produce sufficient DNA for sequencing analysis without PCR cycling, avoiding the errors and selective amplification problems associated with PCR cycling in traditional PCR methods, and saving time and cost.

[0175] In some embodiments, the sequencing step includes sequencing a library of ligated nucleic acid fragments to obtain the sequence information of the nucleic acid to be tested. In some embodiments, the sequencing step includes sequencing a library of ligated multi-copy nucleic acid fragments to obtain the sequence information of the sense and antisense strands of the nucleic acid to be tested, and obtaining the terminal information of the nucleic acid to be tested by comparing the sequence information of the sense and antisense strands.

[0176] According to some embodiments of this application, a strand displacement amplification reaction is performed by adding extension primers to the ligation product to generate a multi-copy nucleic acid library with the sense and antisense strands of the nucleic acid to be tested.

[0177] According to some embodiments of this application, sequencing reaction is performed based on a multi-copy nucleic acid library to obtain the nucleic acid sequence information of the positive and negative strands of the nucleic acid to be tested. The nucleic acid information of the positive and negative strands of the nucleic acid to be tested is compared to obtain the end result of the nucleic acid to be tested.

[0178] For the sequencing step, those skilled in the art can reasonably select existing known sequencing technologies or design other suitable sequencing technologies. Simultaneously, they can also reasonably select amplification methods compatible with the selected or designed sequencing technology. For example, commercially available sequencing pretreatment kits and sequencing platforms can be selected, and nucleic acid fragment ligation products or nucleic acid fragment ligation product libraries can be pretreated according to the manufacturer's instructions. Then, the pretreated samples can be sequenced using the sequencing platform.

[0179] According to some embodiments of this application, the sequencing method includes sequencing technologies that repeatedly sequence nucleic acid fragment ligation products using the same template. In some preferred embodiments, the sequencing method includes, but is not limited to, the DNBseq sequencing technology from MGI Tech or the SMRT sequencing technology from PacBio.

[0180] In some specific implementations, DNBseq sequencing technology is used to sequence nucleic acid fragment ligation products or libraries of nucleic acid fragment ligation products. MGI's DNBseq technology consists of three stages: library circularization; amplification of DNBs (DNA nanoballs) via rolling circle amplification (RCA); and loading the DNBs onto a regular array of a sequencing chip for sequencing. Because the nucleic acid fragment ligation products obtained by the method in this application have a unique dumbbell-shaped structure, these products undergo heat denaturation, resulting in the breakage of hydrogen bonds between the nucleic acid double strands, thus forming single-stranded circular molecules. Therefore, the library circularization step can be conveniently completed. Furthermore, based on rolling circle amplification of single-stranded circular molecules, DNBseq technology not only effectively increases the copy number of the DNA to be tested, greatly enhancing the signal intensity, but also avoids the accumulation of errors during PCR amplification by performing rolling circle replication on the same template, effectively improving sequencing accuracy. After circularization amplification, the DNBs are loaded onto the sequencing chip array. DNB (Digital Briggs-Absorber) hybridizes complementaryly with the adapters on the array. Under the catalysis of polymerase, the sequencing template binds to fluorescently labeled probes in the sequencing reagents. This excitation of fluorescent groups produces light signals, which are then collected by the instrument's camera. These signals are processed and converted into digital signals, which are then further processed to ultimately obtain the base sequence information of the sample. By increasing the copy number of the nucleic acid sample and referencing the original template for each amplification, DNB enhances both signal strength and sequencing accuracy.

[0181] In some specific implementations, SMRT sequencing technology is used to sequence nucleic acid fragment ligation products or libraries of nucleic acid fragment ligation products. PacBio's PacBio SMRT sequencing technology employs single-molecular real-time (SMRT) sequencing. Its principle involves ligating the target DNA fragment to a special DNA polymerase, forming a DNA polymerase-DNA complex. This DNA polymerase-DNA complex is then immobilized on a sequencing chip, and DNA fragments are sequenced one by one using a laser, achieving high-throughput sequencing of the DNA sequence. The wells of the SMRT sequencing chip contain a special DNA polymerase. When the DNA molecule to be tested binds to this DNA polymerase, DNA strand synthesis occurs simultaneously under the catalysis of the polymerase. Fluorescently labeled dNTPs are used; the fluorescent group emits light during synthesis, and the bases are read by detecting the light. Furthermore, PacBio sequencing offers ultra-long read lengths, and because it involves "natural" strand synthesis, abnormal GC regions have no impact on its sequencing. PacBio's circular consensus sequencing (CCS) mode, based on the PacBio Sequel system, derives a shared sequence from multiple detections of a single template molecule, thereby achieving high base accuracy and producing high-fidelity reads (HiFi reads).

[0182] According to another embodiment of this application, a reagent kit for detecting nucleic acid ends is provided, comprising: a nucleic acid dephosphorylation kit; and a adapter connection kit.

[0183] In some embodiments, the nucleic acid dephosphorylation kit includes a phosphatase. In some embodiments, the phosphatase includes a phosphatase having 5' dephosphorylation activity and a phosphatase having 3' dephosphorylation activity. In some embodiments, the phosphatase has both 5' dephosphorylation and 3' dephosphorylation activities. Those skilled in the art can reasonably select existing known phosphatases, or can extract enzymes with completely identical or similar functions as substitutes.

[0184] In some embodiments, the phosphatase comprises an acid phosphatase or an alkaline phosphatase. In some embodiments, the acid phosphatase comprises prostatic acid phosphatase and non-prostatic acid phosphatase. In some preferred embodiments, the phosphatase comprises an alkaline phosphatase. In some embodiments, the alkaline phosphatase is a nonspecific phosphomonoesterase. Nonspecific phosphomonoesterases can catalyze the hydrolysis of the 5'-phosphate and 3'-phosphate groups of almost all phosphate monoesters.

[0185] In some preferred embodiments, the phosphatase includes, but is not limited to, at least one of bacterial alkaline phosphatase (BAP), shrimp alkaline phosphatase (SAP), calf intestinal alkaline phosphatase (CIAP), placental alkaline phosphatase (PLAP), secretory alkaline phosphatase (SEAP), and their recombinant enzymes. Compared to wild-type enzymes, the recombinant enzymes obtained through recombinant expression do not contain affinity tags or other modifications found in wild-type or natural enzymes. In some more preferred embodiments, the phosphatase includes at least one of shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) and their recombinant enzymes. Shrimp alkaline phosphatase (SAP) and calf intestinal alkaline phosphatase (CIAP) are heat-sensitive alkaline phosphatases that can be completely and irreversibly inactivated by heating at 65°C or above for several minutes, thus eliminating the need for purification before ligation or end-labeling steps.

[0186] In some embodiments, the nucleic acid dephosphorylation kit further includes a buffer solution. In some embodiments, the nucleic acid dephosphorylation kit further includes a coenzyme factor. Those skilled in the art can provide suitable buffer solutions, coenzyme factors, and salt ions according to the specific type of phosphatase used, so that the nucleic acid dephosphorylation reaction is carried out under suitable reaction system, pH value, coenzyme factor concentration, and salt ion concentration.

[0187] In some embodiments, the linker ligation kit includes a single linker having phosphate groups at both its 5' and 3' ends. By providing a single linker with phosphate groups at both its 5' and 3' ends, it is advantageous to link to hydroxyl groups on the positive and negative strands of the dephosphorylated nucleic acid fragment. In some embodiments, the single linker is a synthetic single linker. When the single linker sequence is known, the type of ends of the original test nucleic acid and the start and end positions of the protruding ends can be accurately identified by detecting and analyzing the nucleic acid fragment ligation product.

[0188] In some embodiments, the number of bases in the single linker can be 5-100 nt. Preferably, the number of bases in the single linker can be 5-30 nt. More preferably, the number of bases in the single linker can be 6-25 nt. Even more preferably, the number of bases in the single linker can be 8-20 nt. In some preferred embodiments, at least one base in the last position of the sequence of the single linker is a ribonucleotide base (rNTP) in the 5' to 3' direction. In some preferred embodiments, the single linker includes a tag sequence, which facilitates subsequent library construction and detection analysis. In some preferred embodiments, the tag sequence of the single linker includes, but is not limited to, a unique molecular tag (UMI) sequence and a sample tag sequence. The unique molecular tag (UMI) sequence is used to count the copy number of nucleic acid molecules in the sample. The sample tag sequence is used to distinguish different samples for subsequent multi-sample pooling sequencing. For example, the sample tag sequence can be a barcode sequence or an index sequence.

[0189] In some embodiments, the connector kit further includes a nucleic acid ligase. In some preferred embodiments, the nucleic acid ligase includes a DNA ligase or an RNA ligase. In some preferred embodiments, the nucleic acid ligase includes a nucleic acid ligase having activity in catalyzing the formation of a phosphodiester bond (5'PO4 to 3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide, and a nucleic acid ligase having activity in catalyzing the formation of a phosphodiester bond (3'PO4 to 5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide. In some embodiments, the nucleic acid ligase is a nucleic acid ligase that has both catalyzing 5'PO4 to 3'OH linkage activity and catalyzing 3'PO4 to 5'OH linkage activity. Those skilled in the art can reasonably provide existing known nucleic acid ligases, or can provide enzymes with completely identical or similar functions as alternatives.

[0190] In some embodiments, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (5'PO4→3'OH linkage) between the 5' phosphate group of a nucleic acid chain or mononucleotide and the 3' hydroxyl group of another nucleic acid chain or mononucleotide includes at least one of ATP-dependent nucleic acid ligases and NAD+-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 5'PO4→3'OH linkage includes, but is not limited to, at least one of T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV.

[0191] In some embodiments, the nucleic acid ligase having the activity of catalyzing the formation of a phosphodiester bond (3'PO4→5'OH linkage) between the 3' phosphate group of a nucleic acid chain or mononucleotide and the 5' hydroxyl group of another nucleic acid chain or mononucleotide includes GTP-dependent nucleic acid ligases. In some more preferred embodiments, the nucleic acid ligase having the activity of catalyzing 3'PO4→5'OH linkage is not limited to RtcB ligase.

[0192] In some embodiments, the adapter ligation kit further includes a buffer solution. In some embodiments, the adapter ligation kit further includes a coenzyme factor. Those skilled in the art can provide suitable buffer solutions, coenzyme factors, and salt ions, depending on the specific type of nucleic acid ligase used, to ligate dephosphorylated nucleic acid fragments to the single-linker under suitable reaction systems, pH values, coenzyme factor concentrations, and salt ion concentrations, thereby efficiently constructing nucleic acid fragment ligation products.

[0193] In some embodiments of this application, the kit further includes a digestion kit. The digestion kit is used to digest unconnected nucleic acid fragments.

[0194] In some embodiments, the digestion kit includes a nuclease. In some preferred embodiments, the nuclease includes an exonuclease. In some more preferred embodiments, the exonuclease includes, but is not limited to, at least one of exonuclease I (ExoI), exonuclease III (ExoIII), and exonuclease IV (ExoIV). Those skilled in the art can reasonably provide existing known nucleases, or can provide enzymes with completely identical or similar functions as substitutes.

[0195] In some embodiments, the digestion kit further includes a buffer solution. In some embodiments, the digestion kit further includes a coenzyme factor. In some embodiments, the coenzyme factor includes ATP. Those skilled in the art can provide appropriate buffer solutions, coenzyme factors, and salt ions according to the specific type of nuclease used, so that the unligated nucleic acid fragments are digested and degraded while maximizing the retention of nucleic acid fragment ligation products.

[0196] In some embodiments of this application, the kit further includes a purification kit. The purification kit is used to purify nucleic acid fragment ligation products. Those skilled in the art are familiar with many reagents for purifying target nucleic acids, including but not limited to kits based on methods such as phenol / chloroform, centrifugation column, and magnetic bead methods. Those skilled in the art can reasonably provide suitable nucleic acid purification kits according to the specific desired effect and the characteristics of the sample.

[0197] In some preferred embodiments, the purification kit includes a magnetic bead-based purification kit. In some more preferred embodiments, the purification kit may include, but is not limited to, AMPure XP magnetic beads and DNA Clean Beads. Those skilled in the art can reasonably provide suitable magnetic bead purification kits as purification kits based on the specific desired effect and the characteristics of the sample.

[0198] In some embodiments of this application, the kit further includes an amplification kit. The amplification kit is used to amplify nucleic acid fragment ligation products to obtain a nucleic acid fragment ligation product library.

[0199] In some embodiments, the amplification kit includes a nucleic acid polymerase and amplification primers. In some preferred embodiments, the nucleic acid polymerase has strand displacement activity. A nucleic acid polymerase with strand displacement activity is capable of dissociating the hydrogen bonds of a single strand already bound to a double-stranded nucleic acid template, and then synthesizing a new single strand. In some preferred embodiments, the nucleic acid polymerase is a DNA-dependent DNA polymerase. In some more preferred embodiments, the DNA-dependent DNA polymerase includes at least one of T4 DNA polymerase, DNA polymerase I (Klenow) large fragment (Klenow fragment), T7 DNA polymerase, DNA polymerase I, Taq DNA polymerase, Bst DNA polymerase, and Phi29 DNA polymerase.

[0200] In some embodiments, the amplification kit includes a PCR amplification kit. In some embodiments, the PCR amplification kit includes PCR amplification primers designed for the sequence of the single linker. In some embodiments, the PCR amplification primers contain a tag sequence. Given that the sequence of the single linker is known, appropriate primers can be readily designed to perform PCR amplification using the nucleic acid fragment ligation product as a template. When the single linker or the PCR amplification primers contain a tag sequence, those skilled in the art can advantageously utilize suitable sequencing techniques to detect the amplified library of the nucleic acid fragment ligation product.

[0201] In some embodiments, the PCR amplification kit further includes: DNA polymerase; PCR buffer; magnesium ions; and dNTPs, including dATP, dGTP, dTTP, and dCTP. In some more preferred embodiments, the DNA polymerase includes at least one of T4 DNA polymerase, DNA polymerase I (Klenow) large fragment (Klenow fragment), T7 DNA polymerase, DNA polymerase I, Taq DNA polymerase, Bst DNA polymerase, and Phi29 DNA polymerase. Those skilled in the art can reasonably provide suitable amplification kits according to the specific effects required and the characteristics of the sample, and can provide suitable PCR reaction systems according to the specific type of DNA polymerase used.

[0202] In some embodiments, the amplification kit includes a PCR-free amplification kit. Those skilled in the art can select library preparation kits that do not require PCR amplification based on relevant disclosed technologies. In some embodiments, the PCR-free amplification kit includes a rolling circle amplification (RCA) kit. For example, the DNBseq one-step DNB preparation kit from MGI Tech.

[0203] In some embodiments of this application, the kit further includes a sequencing kit. Those skilled in the art can reasonably provide sequencing kits based on existing known sequencing technologies or other suitable sequencing technologies. Simultaneously, those skilled in the art can also provide compatible amplification kits and sequencing kits. For example, a pretreatment kit or amplification kit compatible with the provided sequencing technology can be provided to pretreatment or amplify nucleic acid fragment ligation products or nucleic acid fragment ligation product libraries, and then the pretreated or amplified samples are sequenced using the sequencing kit. In some embodiments, the sequencing kit includes, but is not limited to, a sequencing chip, a sequencer, or a read length analyzer.

[0204] In some preferred embodiments, the sequencing kit includes, but is not limited to, sequencing kits based on DNBseq sequencing technology from MGI (Mega-Tech International) or SMRT sequencing technology from PacBio (PacBio Corporation). According to some embodiments of this application, sequencing technology that repeatedly sequences the same template can correct read sequences, thereby improving sequencing accuracy and making the identification of serrated-end sequences more accurate.

[0205] Those skilled in the art will understand that there may be no clear boundaries between the various kits distinguished by the processing or reaction stage of the test sample, and the kits and their reagent components are not entirely independent. For example, compatible buffers or compatible salt ion mixtures may be provided for use in one or more reaction stages. Those skilled in the art are capable of reasonably determining which reagent components are suitable for use in one or more reaction stages, thereby providing only a sufficient number of portions in the kit.

[0206] According to another embodiment of this application, a nucleic acid library comprising a sense strand and a negative sense strand of a nucleic acid to be tested is provided, characterized in that: the nucleic acid library has a partially double-stranded closed loop dumbbell-shaped structure, wherein the double-stranded portion is the nucleic acid to be tested portion, the dumbbell-shaped portion is a single linker located at both ends of the nucleic acid to be tested, wherein the single linker includes a tag sequence, and wherein the 3' end of the single linker contains at least one ribonucleotide base.

[0207] According to another embodiment of this application, the application of the single-linked header of the above-described embodiments in the detection of nucleic acid ends is provided. According to another embodiment of this application, the application of the single-linked header of the above-described embodiments in the detection of DNA double-strand breaks is provided.

[0208] According to another embodiment of this application, the application of the single-linked header from the above-described embodiments in the preparation of a kit for detecting nucleic acid ends is provided. According to another embodiment of this application, the application of the single-linked header from the above-described embodiments in the preparation of a kit for detecting DNA double-strand breaks is provided.

[0209] According to another embodiment of this application, the application of the single linker, method for detecting nucleic acid ends, and kit for detecting nucleic acid ends of the above-described embodiments in the analysis of cfDNA fragmentomics characteristics is provided.

[0210] In some embodiments, characterizing cfDNA fragments includes analyzing the serrated ends of cfDNA fragments. In some embodiments, the application of characterizing cfDNA fragments or analyzing the serrated ends of cfDNA fragments is for non-therapeutic or diagnostic purposes, such as for scientific research purposes. Characterizing cfDNA fragments for scientific research purposes and analyzing the serrated ends of cfDNA fragments are well known to those skilled in the art.

[0211] In some implementations, characterizing cfDNA fragmentomics includes identifying the type of cfDNA ends. In other implementations, characterizing cfDNA fragmentomics includes identifying the relative abundance of different cfDNA end types.

[0212] In some embodiments, analyzing the serrated ends of a cfDNA fragment includes identifying the length of the serrated ends. In some embodiments, analyzing the serrated ends of a cfDNA fragment includes identifying the location of the serrated ends.

[0213] In some embodiments, analyzing the serrated ends of the cfDNA fragment includes identifying the start position of the serrated ends of the cfDNA. In some embodiments, analyzing the serrated ends of the cfDNA fragment includes identifying the end position of the serrated ends of the cfDNA.

[0214] In some implementations, analyzing cfDNA fragment omics characteristics or analyzing the serrated ends of cfDNA fragments includes transient or long-term analysis of individuals or populations of humans or animals.

[0215] In some embodiments, the application includes analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and organism development. In some embodiments, the application includes analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and disease occurrence. In some embodiments, the application includes analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and cancer development. In some embodiments, the application includes analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and drug resistance.

[0216] Example

[0217] The embodiments described herein are illustrative examples of the subject matter of this application and are not intended to limit the scope of this application. Many modifications and variations can be made to the technical solutions described and illustrated herein without departing from the scope of this application. Therefore, it should be understood that these embodiments are merely illustrative and do not limit the scope of this application.

[0218] The materials used in the general methods disclosed herein for detecting cfDNA fragment ends are obtainable through standard, well-known methods, and many materials are commercially available. Even if not commercially available, they can be prepared and extracted using standard techniques known to those skilled in the art. The detection methods provided herein comprise multiple reaction steps, each of which can be performed independently and may or may not have any one or more preceding or subsequent steps. Therefore, this application aims to protect each individual reaction step of the detection methods separately provided herein.

[0219] According to some specific embodiments of this application, a universal detection method for cfDNA fragment ends based on MGI's DNBseq sequencing technology is provided. See [link to relevant documentation]. Figure 2 The flowchart includes the following steps:

[0220] 1. Dephosphorylation: After extracting plasma cfDNA fragments, alkaline phosphatase is added for dephosphorylation treatment, so that hydroxyl groups are formed at both the 5' and 3' ends of the cfDNA fragments.

[0221] 2. Adapter ligation: A single-linker with phosphate groups at both the 5' and 3' ends is added to the dephosphorylated cfDNA fragment. T4 RNA ligase and RtcB ligase are then added to initiate the ligation reaction. T4 RNA ligase ligates the 5' end of the single-linker to the 3' end of the cfDNA fragment, while RtcB ligase ligates the 3' end of the single-linker to the 5' end of the cfDNA fragment. The single-linker circularizes and closes the positive and negative strand ends on both sides of the cfDNA fragment, forming a dumbbell-shaped cfDNA fragment ligation product.

[0222] 3. Digestion and purification: After the above ligation reaction is completed, linear DNA digestion enzyme is added to remove the non-circularized cfDNA fragments at the ends, and the cfDNA fragment ligation product is purified by magnetic bead method.

[0223] 4. DNA single-strand circularization: The purified cfDNA fragment ligation product is denatured in an alkaline reaction system or under high temperature heating to obtain circular single-stranded DNA.

[0224] 5. DNB Preparation and Sequencing: Using circular single-stranded DNA as a template, rolling circle amplification is performed under the action of DNA polymerase, amplifying the circular single-stranded DNA to 100-1000 copies. The amplified product is DNB. After passing quality control, sequencing is performed using a sequencer (e.g., PE150 or SE400). Finally, information about the serrated ends of the cfDNA is obtained through data analysis.

[0225] According to some specific embodiments of this application, a universal detection method for cfDNA fragment ends based on PacBio's SMRT sequencing technology is provided. See [link to relevant documentation]. Figure 3 The flowchart includes the following steps:

[0226] 1. Dephosphorylation: After extracting plasma cfDNA fragments, alkaline phosphatase is added for dephosphorylation treatment, so that hydroxyl groups are formed at both the 5' and 3' ends of the cfDNA fragments.

[0227] 2. Adapter ligation: A single-linker with phosphate groups at both the 5' and 3' ends is added to the dephosphorylated cfDNA fragment. T4 RNA ligase and RtcB ligase are then added to initiate the ligation reaction. T4 RNA ligase ligates the 5' end of the single-linker to the 3' end of the cfDNA fragment, while RtcB ligase ligates the 3' end of the single-linker to the 5' end of the cfDNA fragment. The single-linker circularizes and closes the positive and negative strand ends on both sides of the cfDNA fragment, forming a dumbbell-shaped cfDNA fragment ligation product.

[0228] 3. Digestion and purification: After the above ligation reaction is completed, linear DNA digestion enzyme is added to remove the non-circularized cfDNA fragments at the ends, and the cfDNA fragment ligation product is purified by magnetic bead method.

[0229] 4. Preparation of DNA polymerase complex: The cfDNA fragment ligation product is sequentially combined with universal sequencing primers and DNA polymerase (with strand substitution and biotin modification) to form a primer-DNA-polymerase complex (theoretically, each end of a DNA fragment binds one sequencing primer and one DNA polymerase).

[0230] 5. Sequencing: The prepared sequencing complex is loaded onto the SMRT chip to immobilize the sequencing complex, and single-molecule sequencing can be performed. Finally, the information of the cfDNA serrated ends is obtained through data analysis.

[0231] The technical solution of this application can accurately identify the ends of cfDNA fragments, especially the precise information such as the type, length, start and end positions of the serrated ends, facilitating subsequent research to reveal the link between cfDNA fragment omics characteristics and organism development, disease occurrence, cancer development, and drug resistance. By adding only a single linker to the cfDNA end in situ for ligation, the specificity of the data results is improved. The addition of the linker also facilitates subsequent data analysis, achieving precise localization of the serrated ends of the cfDNA. Using T4 RNA ligase and RtcB ligase simultaneously for the ligation reaction, the positive and negative strand ends on the same side of the cfDNA are connected through the linker, which is simple to operate, highly efficient, and better reveals the information of the serrated ends of the cfDNA.

[0232] Example 1: Testing the ligation efficiency of RtcB ligase and T4 RNA ligase for different types of ends at the same side of the 3' and 5' ends of a DNA fragment.

[0233] I. Experimental Materials

[0234] Provide DNA fragments with the following sequences and different types of ends.

[0235] Class A1: DNA fragments with 3' overhangs.

[0236] F1: 5'OH-GATAGCTGTGGAGTCTTAAGCCCAGTGAAGAATCGTCCATTTCCAGAATC AATGAGAAGTAAAGCTGAAAATCATTCAGTTCAGTCTGTGGC-3'OH (SEQ ID NO: 1);

[0237] R1: 5'OH-GAACTGAATGATTTTCAGCTTTACTTCTCATTGATTCTGGAAATGGACGA TTCTTCACTGGGCTTAAGACTCCACAGCTATCTCAGTACGCT-3'OH (SEQ ID NO: 2).

[0238] Class A2: DNA fragments with 5' overhangs.

[0239] F2: 5'OH-GATAGCTGTGGAGTCTTAAGCCCAGTGAAGAATCGTCCATTTCCAGAATC AATGAGAAGTAAAGCTGAAAATCATTCAGTTC-3'OH (SEQ ID NO: 3);

[0240] R2: 5'OH-CTCTGATCGAGAACTGAATGATTTTCAGCTTTACTTCTCATTGATTCTGG AAATGGACGATTCTTCACTGGGCTTAAGACTC-3'OH (SEQ ID NO: 4).

[0241] A3 type: DNA sequences with blunt ends on one side and protruding ends at the 3' end on the other side.

[0242] F3: 5'OH-GATAGCTGTGGAGTCTTAAGCCCAGTGAAGAATCGTCCATTCCAGAAT CAATGAGAAGTAAAGCTGAAAATCATTCAGTTCAGTCTGTGGC-3'OH (SEQ ID NO: 5);

[0243] R3: 5'OH-GAACTGAATGATTTTCAGCTTTACTTCTCATTGATTCTGGAAATGGACGA TTCTTCACTGGGCTTAAGACTCCACAGCTATC-3'OH (SEQ ID NO: 6).

[0244] A4 type: DNA sequences with blunt ends on one side and protruding ends at the 5' end on the other side.

[0245] F4: 5'OH-GATAGCTGTGGAGTCTTAAGCCCAGTGAAGAATCGTCCATTTCCAGAATC AATGAGAAGTAAAGCTGAAAATCATTCAGTTC-3'OH (SEQ ID NO: 7);

[0246] R4: 5'OH-GAACTGAATGATTTTCAGCTTTACTTCTCATTGATTCTGGAAATGGACGATTCTTCACTGGGCTTAAGACTC-3'OH (SEQ ID NO: 8).

[0247] Provide a single link header with the following sequence:

[0248] 5'P-GAACGACATGGCTArC-3'P (SEQ ID NO:9, where rC represents cytosine ribonucleotide).

[0249] II. Experimental Procedure

[0250] 1. Connector connection

[0251] Prepare 5 ng of A1 / A2 / A3 / A4 type DNA fragments and add them to PCR tubes. Prepare 50 μL of different ligation reaction systems as shown in Table 1 below, then add each to a PCR tube and incubate at 37°C for 1 h to obtain the ligation products. T4 RNA ligase and RtcB ligase were both purchased from NEB.

[0252] Table 1

[0253] Components Formula 1 Formula 2 Connector (100pm) 1μL 1μL 10×RtcB reaction buffer 5μL 5μL 20 mmol / L GTP 0.5μL 0.5μL 70 mmol / L MgCl2 5μL 5μL 100mmol / L MnCl2 0.5μL 0.5μL 100 mmol / L ATP 0.5μL 0.5μL 50% PEG8000 12.5μL 12.5μL T4 RNA ligase (NEB, M0204S) 2.5μL 0μL RtcB ligase (NEB, M0458) 2.5μL 0μL RNAse inhibitors 1μL 1μL Enzyme-free water Add 50μL Add 50μL

[0254] 2. Gel electrophoresis

[0255] Prepare a 12% denaturing polyacrylamide (PAGE) gel, and perform denaturing PAGE gel electrophoresis on the ligation products obtained under different ligation reaction conditions at 120V.

[0256] III. Experimental Results

[0257] The fragment size of the ligation products was found to be in line with expectations by 12% denaturing polyacrylamide gel electrophoresis. Figure 4 Quantitative analysis of the electrophoretic bands was performed using Bio-Bad Image Lab gel analysis software. The system in formulation 1 showed a ligation efficiency of 25% for DNA fragments with A1-type ends, and over 95% for DNA fragments with A2, A3, and A4-type ends. Subsequent experiments revealed that increasing the enzyme amount or extending the reaction time could further improve the overall ligation efficiency.

[0258] Example 2: Detection of serrated ends of human plasma cfDNA

[0259] I. Experimental Materials

[0260] Human plasma cfDNA was used as the sample.

[0261] II. Experimental Procedure

[0262] 1. Dephosphorylation of cfDNA

[0263] Human plasma cfDNA was extracted, and 5 ng of cfDNA was added to a PCR tube as a sample. 1 μL of recombinant shrimp alkaline phosphatase (rSAP, purchased from NEB, catalog number M0371L) and 1 μL of 10×rSAP buffer (purchased from NEB, catalog number M0371L) were added, and enzyme-free water (NF) was added to bring the total volume to 10 μL. After vortexing and briefly centrifuging, the PCR tube was placed in a PCR instrument and incubated at 37°C for 30 min. The reaction was terminated by treating at 65°C for 5 min.

[0264] 2. Connector connection

[0265] After the dephosphorylation reaction, remove the PCR tube and centrifuge briefly. Prepare a 50 μL ligation reaction mixture as shown in Table 2, then add it to the PCR tube and incubate at 37°C for 1 h to obtain the ligation product. T4 RNA ligase and RtcB ligase were both purchased from NEB.

[0266] Linker: 5'P-TCCTCCTCCTCCGTU-3'P (SEQ ID NO:10, where U represents uracil ribonucleotide).

[0267] Table 2

[0268]

[0269]

[0270] 3. Digestion and purification

[0271] (1) Add 2.5 μL of digestion enzyme (Digestion Enzyme, purchased from MGI, catalog number 1000005431) to the ligation product, vortex to mix, and then centrifuge briefly. Place the PCR tube in the PCR instrument and incubate at 37°C for 30 min to digest and remove linear DNA.

[0272] (2) After the digestion reaction is complete, briefly centrifuge to collect the reaction solution to the bottom of the tube. Add 170 μL of DNA Clean Beads equilibrated to room temperature to the digestion product, vortex to mix, and incubate at room temperature for 5 min. Place the centrifuge tube on a magnetic rack and let it stand for 5 min until the liquid is clear. Gently aspirate the supernatant with a pipette and discard it. Keeping the centrifuge tube on the magnetic rack, add 500 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls twice. Open the centrifuge tube cap and dry at room temperature until the surface of the magnetic beads is no longer reflective. Remove the centrifuge tube from the magnetic rack, add 22 μL of TE Buffer for DNA elution, and transfer 20 μL of the supernatant to a new PCR tube.

[0273] 4. Preparation of sequencing complex

[0274] The Revio polymerase kit (purchased from PacBio, catalog number 102-817-600) was used. Following the kit instructions, the purified DNA product was annealed with universal sequencing primers, and then DNA polymerase was used to form a sequencing complex. The sequencing complex was then purified using magnetic beads.

[0275] Sequencing primers: 5'OH-AACGGAGGAGGAGGA-3'OH (SEQ ID NO:11).

[0276] 5. PacBio sequencing

[0277] Prepare a Sequel II binding kit 3.2 (purchased from PacBio, 102-333-300), load the obtained sequencing complex onto an SMRT chip, fix the sequencing complex, and perform sequencing on a PacBio Sequel II system, with at least 10× sequencing coverage per sample.

[0278] III. Experimental Results

[0279] A cfDNA ligation product library was constructed using plasma cfDNA. The resulting DNA product library was successfully used to prepare sequencing complexes and subjected to PacBio sequencing. The sequencing data were preprocessed to remove adapter sequences and low-quality bases. The proportion mapped to the hg19 human reference genome was statistically analyzed, and the identity sequences of individual nucleic acids were determined. Based on the adapter sequences, the base information of the positive and negative strands of cfDNA was statistically analyzed, further determining the length and base information of the serrated ends. The length distribution of the serrated ends of the cfDNA fragments conformed to expectations. Figure 5 ).

[0280] According to the experimental protocol of this embodiment, plasma DNA from both the fetus and mother is used as samples. Analysis of the sequencing data reveals that fetal DNA molecules have longer serrated ends than maternal DNA molecules. Furthermore, according to the experimental protocol of this embodiment, plasma cfDNA from patients infected with chronic hepatitis B virus (HBV), hepatocellular carcinoma (HCC) patients, and healthy individuals (subjects without HBV or HCC) is used as samples. Analysis of the sequencing data shows that the serrated index of cfDNA from HBV-infected patients and HCC patients is higher than that from healthy individuals. The technical solution of this application can accurately identify cfDNA fragment ends, and cfDNA end detection opens up new possibilities in fields such as non-invasive prenatal testing and tumor detection.

[0281] Example 3: Detection of serrated ends of human plasma cfDNA

[0282] I. Experimental Materials

[0283] Human plasma cfDNA was used as the sample.

[0284] II. Experimental Procedure

[0285] 1. Dephosphorylation of cfDNA

[0286] Human plasma cfDNA was extracted, and 5 ng of cfDNA was added to a PCR tube as a sample. 1 μL of recombinant shrimp alkaline phosphatase (rSAP, purchased from NEB, catalog number M0371L) and 1 μL of 10×rSAP buffer (purchased from NEB, catalog number M0371L) were added, and enzyme-free water (NF) was added to bring the total volume to 10 μL. After vortexing and briefly centrifuging, the PCR tube was placed in a PCR instrument and incubated at 37°C for 30 min. The reaction was terminated by treating at 65°C for 5 min.

[0287] 2. Connector connection

[0288] After the dephosphorylation reaction, remove the PCR tube and centrifuge briefly. Prepare a 50 μL ligation reaction mixture as shown in Table 3 below, then add it to the PCR tube and incubate at 37°C for 1 h to obtain the ligation product. T4 RNA ligase and RtcB ligase were both purchased from NEB.

[0289] Linker: 5'P-TCCTCCTCCTCCGTU-3'P (SEQ ID NO:10, where U represents uracil ribonucleotide).

[0290] Table 3

[0291] Components volume Connector (100pm) 1μL 10×RtcB reaction buffer 5μL 20 mmol / L GTP 0.5μL 70 mmol / L MgCl2 5μL 100mmol / L MnCl2 0.5μL 100 mmol / L ATP 0.5μL 50% PEG8000 12.5μL RtcB ligase (NEB, M0458) 2.5μL T4 RNA ligase (NEB, M0204S) 2.5μL RNAse inhibitors 1μL Enzyme-free water Add 50μL

[0292] 3. Digestion and purification

[0293] (1) Add 2.5 μL of digestion enzyme (Digestion Enzyme, purchased from MGI, catalog number 1000005431) to the ligation product, vortex to mix, and then centrifuge briefly. Place the PCR tube in the PCR instrument and incubate at 37°C for 30 min to digest and remove linear DNA.

[0294] (2) After the digestion reaction is complete, briefly centrifuge to collect the reaction solution to the bottom of the tube. Add 170 μL of DNA Clean Beads equilibrated to room temperature to the digestion product, vortex to mix, and incubate at room temperature for 5 min. Place the centrifuge tube on a magnetic rack and let it stand for 5 min until the liquid is clear. Gently aspirate the supernatant with a pipette and discard it. Keeping the centrifuge tube on the magnetic rack, add 500 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls twice. Open the centrifuge tube cap and dry at room temperature until the surface of the magnetic beads is no longer reflective. Remove the centrifuge tube from the magnetic rack, add 22 μL of TE Buffer for DNA elution, and transfer 20 μL of the supernatant to a new PCR tube.

[0295] 4. MGIDNBseq sequencing

[0296] Prepare the DNBseq one-step DNB preparation kit V2.0 (OS-SB)(4RXN)(MGI,940-000035-00). Prepare DNA nanospheres according to the kit instructions. After the concentration is verified to be qualified, use the MGISEQ-2000RS high-throughput sequencing reagent kit (SE400) (MGI,1000013853) on the MGISEQ-2000 sequencing platform to perform SE400 sequencing, with at least 10× sequencing coverage for each sample.

[0297] III. Experimental Results

[0298] A cfDNA ligation product library was constructed using plasma cfDNA. The resulting DNA product library was then processed into DNA buffer (DNB) and sequenced using an MGISEQ-2000 scanner. The sequencing data were preprocessed to remove adapter sequences and low-quality bases. The proportion mapped to the hg19 human reference genome was statistically analyzed, and the identity sequence of individual nucleic acids was determined. Based on the adapter sequences, the base information of the positive and negative strands of the cfDNA was statistically analyzed to further determine the length and base information of the serrated ends. In this embodiment, the length distribution of the serrated ends of the obtained cfDNA fragments conformed to expectations. Figure 6 The sequencing results were basically consistent with those of the same sample in Example 2, with no significant differences.

[0299] The subject matter of this application has been fully described through the above embodiments. Those skilled in the art will understand that the implementation of this application is not limited to the above embodiments, and the same solution can be implemented within a broad equivalent scope without affecting the scope of the subject matter or specific aspects described herein. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and are included within the protection scope of this application.

Claims

1. A single chain linker, characterized in that, The single linker has phosphate groups at both its 5' and 3' ends.

2. The single link head according to claim 1, characterized in that: The single link header is a composite single link header; And / or, the single link header contains a sequence of tags; And / or, the number of bases in the single linker is 5-100 nt; And / or, in the 5' to 3' direction, the last base of the single-link head nucleic acid sequence is at least a ribonucleotide base.

3. A method of detecting the end of a nucleic acid, characterized in that, Includes the following steps: Provide a dephosphorylated nucleic acid fragment, wherein the 5' and 3' ends of the dephosphorylated nucleic acid fragment are both hydroxyl groups; A single linker is provided, wherein both the 5' and 3' ends of the single linker are phosphate groups; The dephosphorylated nucleic acid fragment is ligated to the single linker to obtain a nucleic acid fragment ligation product, and the nucleic acid fragment ligation product is detected.

4. The method for detecting nucleic acid ends according to claim 3, characterized in that: The dephosphorylated nucleic acid fragment was ligated to the single linker using a nucleic acid ligase. Preferably, the nucleic acid ligase includes a DNA ligase or an RNA ligase; Preferably, the nucleic acid ligase comprises a nucleic acid ligase having catalytic activity for 5'PO4 to 3'OH ligation and a nucleic acid ligase having catalytic activity for 3'PO4 to 5'OH ligation; more preferably, the nucleic acid ligase is a nucleic acid ligase having both catalytic activity for 5'PO4 to 3'OH ligation and catalytic activity for 3'PO4 to 5'OH ligation. Preferably, the nucleic acid ligase includes at least one of T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, RtcB ligase, DNA ligase I, DNA ligase III, and DNA ligase IV; more preferably, the nucleic acid ligase includes at least one of T4 RNA ligase and RtcB ligase.

5. The method for detecting nucleic acid ends according to claim 3, characterized in that: The method further includes an amplification step; preferably, the amplification step includes amplification using nucleic acid fragment ligation products as templates to obtain a nucleic acid fragment ligation product library. Preferably, the amplification includes a chain displacement amplification reaction; Preferably, the nucleic acid fragment ligation product library is a multi-copy nucleic acid fragment ligation product library containing the sense and antisense strands of the nucleic acid to be tested.

6. The method for detecting nucleic acid according to claim 5, characterized in that: The method further includes a sequencing step; preferably, the sequencing step includes sequencing a nucleic acid fragment ligation product library to obtain the sequence information of the nucleic acid to be tested; Preferably, the sequencing step includes sequencing a multi-copy nucleic acid fragment ligation product library to obtain the sequence information of the sense and antisense strands of the nucleic acid to be tested, and obtaining the terminal information of the nucleic acid to be tested by comparing the sequence information of the sense and antisense strands of the nucleic acid to be tested.

7. A kit of parts for detecting the ends of a nucleic acid, characterised in that, include: Nucleic acid dephosphorylation kit; and Connector connection kit; Preferably, the nucleic acid dephosphorylation kit includes: phosphatase; More preferably, the phosphatase includes a phosphatase having catalytic 5' dephosphorylation activity and a phosphatase having 3' dephosphorylation activity; more preferably, the phosphatase has both catalytic 5' dephosphorylation activity and 3' dephosphorylation activity. More preferably, the phosphatase is a nonspecific phosphomonoesterase; More preferably, the phosphatase includes acid phosphatase or alkaline phosphatase; more preferably, the phosphatase includes at least one of bacterial alkaline phosphatase, shrimp alkaline phosphatase, calf intestinal alkaline phosphatase, placental alkaline phosphatase, secretory alkaline phosphatase, and their recombinant enzymes. Preferably, the connector kit includes: a single connector; and a nucleic acid ligase; More preferably, both the 5' and 3' ends of the single linker have phosphate groups; More preferably, the single link header contains a tag sequence; More preferably, the number of bases in the single linker is 5-100 nt; More preferably, in the 5' to 3' direction, the last base of the sequence of the single linker is at least a ribonucleotide base; Preferably, the nucleic acid ligase includes a DNA ligase or an RNA ligase; More preferably, the nucleic acid ligase includes a nucleic acid ligase having catalytic activity for 5'PO4 to 3'OH ligation and a nucleic acid ligase having catalytic activity for 3'PO4 to 5'OH ligation; more preferably, the nucleic acid ligase is a nucleic acid ligase having both catalytic activity for 5'PO4 to 3'OH ligation and catalytic activity for 3'PO4 to 5'OH ligation. More preferably, the nucleic acid ligase includes at least one of T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, RtcB ligase, DNA ligase I, DNA ligase III, and DNA ligase IV; more preferably, the nucleic acid ligase includes at least one of T4 RNA ligase and RtcB ligase.

8. The reagent kit according to claim 7, characterized in that: The kit further includes: a digestion kit for digesting unligated nucleic acid fragments; preferably, the digestion kit includes a nuclease; more preferably, the nuclease includes an exonuclease; even more preferably, the exonuclease includes at least one of exonuclease I, exonuclease III, and exonuclease IV; and / or Preferably, the kit further includes: an amplification kit for amplifying nucleic acid fragment ligation products; preferably, the amplification kit includes a nucleic acid polymerase and amplification primers; more preferably, the nucleic acid polymerase has strand displacement activity.

9. A nucleic acid library comprising a nucleic acid sense strand and an antisense strand to be tested, characterized in that: The nucleic acid library has a partially double-stranded closed circular dumbbell-shaped structure, wherein the double-stranded portion is the nucleic acid to be tested, and the dumbbell-shaped portion is a single linker located at both ends of the nucleic acid to be tested, wherein the single linker includes a tag sequence, and wherein the 3' end of the single linker contains at least one ribonucleotide base.

10. The application of the single linker as described in claim 1 or 2, the method for detecting the ends of nucleic acid fragments as described in any one of claims 3-6, and the kit as described in claim 7 or 8 in the analysis of cfDNA fragmentomics characteristics; Preferably, the feature of the cfDNA fragment omics analysis includes the analysis of the serrated ends of the cfDNA fragments; Preferably, the analysis of cfDNA fragment omics characteristics or the analysis of the serrated ends of the cfDNA fragments are for non-therapeutic or diagnostic purposes; more preferably, the analysis of cfDNA fragment omics characteristics or the analysis of the serrated ends of the cfDNA fragments are for scientific research purposes. Preferably, the analysis of cfDNA fragment omics characteristics or the analysis of the serrated ends of the cfDNA fragments includes transient or long-term analysis of individuals or groups of humans or animals; Preferably, the features of the cfDNA fragment omics analysis include identifying the type of cfDNA ends, identifying the relative abundance of cfDNA end types, identifying the length of cfDNA serrated ends, or identifying the location of cfDNA serrated ends; Preferably, the applications include analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and organism development, analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and disease occurrence, analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and cancer development, or analyzing the association between cfDNA fragment omics characteristics or cfDNA fragment serrated ends and drug resistance.

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