Library construction method based on single-tube approach and use thereof

Abstract

Provided are a library construction method based on a single-tube approach and the use thereof. The method effectively solves the problem of complex library construction procedures for large-scale samples in existing detection methods, simplifies conventional library preparation processes, and can realize fragmentation, end repair, and 3'-end A-tailing reaction in the same system on the basis of nucleic acid molecules of different sizes, and by means of optimization, allows a fragmentation and end-repair system to be compatible with a ligation system, such that adapter ligation can be performed directly in the original system without further purification, thereby effectively reducing cost and time required for large-scale sequencing.

Description

A Library Construction Method Based on the One-Pipe Approach and Its Application Technical Field

[0001] This invention relates to the field of gene library construction technology, and in particular to a one-tube library construction method and its application. Background Technology

[0002] With the rapid development of next-generation sequencing (NGS) technology, its application potential in clinical molecular diagnostics, especially in gene testing related to precision medicine, has gained widespread industry recognition. Compared to traditional testing technologies, NGS technology can simultaneously detect multiple gene loci and various gene variants, offering high throughput and broad coverage. Given the increasing demand for multi-gene testing in clinical settings, NGS has extremely high application prospects. Current development of NGS technology primarily focuses on achieving higher throughput, lower cost, and faster speed.

[0003] Whole-genome sequencing (WGS) generally refers to the determination or sequencing of the complete genome sequence of an organism using sequencing technology to obtain complete genomic information. Its purpose is to accurately detect the set of variations in the genome of each sample (including humans), that is, those DNA sequences that differ between individuals. Next-generation sequencing (NGS) has significant advantages such as high throughput, low cost, and large scale, and is therefore widely used in whole-genome sequencing. Typically, NGS-based whole-genome sequencing mainly includes: nucleic acid extraction (DNA or RNA), library preparation (also known as library construction), sequencing, and data analysis. Library construction has a significant impact on whole-genome sequencing. Library preparation is a crucial step before whole-genome sequencing, directly affecting the accuracy and reliability of the sequencing data. Different library preparation methods can lead to significant differences in sequencing results, including the accuracy of genomic and functional predictions. Existing technologies demonstrate that using different library preparation methods (such as Illumina Nextera XT, Illumina TruSeq DNA PCR-free kit, KAPA Biosystems Hyper Prep PCR and PCR-free systems) for metagenomic sequencing can lead to taxonomic differences and even drastically different conclusions in microbiome research (see Jones, Marcus B, et al. "Library preparation methodology can influence genomic and functional predictions in human microbiome research." Proceedings of the National Academy of Sciences 112.45(2015):14024-14029.). Therefore, choosing an appropriate library preparation method is crucial for obtaining accurate whole-genome sequencing results. Furthermore, each step in the library preparation process, such as genomic DNA fragmentation, adapter addition, and PCR amplification (if applicable), requires precise control to ensure the accuracy of the sequencing data. Errors or improper operation at any stage can lead to a decrease in library concentration, thereby affecting the quantity and quality of sequencing data. Therefore, library construction is not only a key step in whole-genome sequencing technology, but also an important link in ensuring the accuracy of sequencing results.

[0004] Currently, commercially available whole-genome sequencing library preparation kits can be mainly divided into two categories: methods based on whole-genome sequencing library preparation with PCR amplification and methods based on whole-genome sequencing library preparation without PCR amplification (i.e., PCR-free). Each method has its advantages and disadvantages. Specifically, the main advantage of whole-genome sequencing library preparation methods with PCR amplification is that the sample input requirement is relatively low. However, the disadvantages are that the operation is complex and there is a PCR amplification bias. This can lead to amplification errors and uneven coverage of high-GC and low-GC regions. On the other hand, the main advantage of whole-genome sequencing library preparation methods without PCR amplification is that the operation is simpler than methods with PCR amplification, and it avoids the bias and amplification errors introduced by PCR amplification, thus achieving better sequence coverage. However, the disadvantage is that it requires more sample than PCR-amplified whole-genome sequencing library preparation methods, which imposes certain limitations on the application conditions and scenarios of these methods. While conventional PCR-free library preparation methods based on platforms (such as the MGI high-throughput sequencing platform) are simple to operate, requiring only genomic DNA fragmentation (i.e., breaking), end repair and A addition, adapter ligation, single-strand circularization, and rolling circle replication to form DNA nanospheres, existing platforms still suffer from excessive costs in terms of library construction time and manpower for large-scale whole-genome sequencing projects, thus limiting their universality in this area. More specifically, conventional whole-genome library preparation methods mainly include: fragmentation, purification, end repair and A addition, adapter ligation, optional PC amplification, purification, single-strand circularization, and DNA nanosphere formation—multiple independent steps. The fragmentation step and the two purification steps need to be performed independently (i.e., transferred to new tubes or systems because the existing fragmentation and end repair systems are incompatible with the ligation systems, requiring purification before adapter ligation. Similarly, since the ligation reaction needs to be inactivated after adapter ligation, individual samples also need to be purified before subsequent mixing), which significantly increases the overall construction time and reagent costs. Moreover, in conventional whole-genome library preparation, each sample needs to undergo independent library construction and sequencing processes as much as possible. This means that for multi-sample testing, multiple tests are required to complete the sequencing of all samples, which further increases the cost of testing.

[0005] Therefore, developing a method that simplifies current library preparation steps and enables mixed or simultaneous detection of multiple samples will greatly improve the availability and universality of NGS in whole-genome sequencing, thereby further promoting the efficient application of NGS and whole-genome sequencing in the fields of diagnosis and treatment.

[0006] Summary of the Invention

[0007] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide a one-tube library construction method and its application. The one-tube library construction method of this invention effectively solves the problem of complex library construction processes for large-scale samples in existing detection methods. It simplifies the fragmentation, purification, and 3' end repair and 3' end addition processes in conventional library preparation (for rapid library preparation processes, it simplifies fragmentation, end repair, 3' end addition, and purification). It can achieve fragmentation, end repair, and 3' end addition reactions in the same system based on nucleic acid molecules of different sizes. Through optimization, the fragmentation and end repair system and the ligation system are compatible, meaning that adapter ligation can be performed directly in the original system without further purification, thereby effectively reducing the cost and time required for large-scale sequencing.

[0008] Furthermore, in this invention, a high-salt solution is used to terminate the connection reaction after the connector connection step, thereby overcoming the defect that multiple samples cannot be mixed in conventional library preparation processes. This allows for the construction of libraries for a large number of samples in a single library construction process, which greatly improves the throughput for large-scale sample library construction.

[0009] A first aspect of the present invention provides a method for constructing a nucleic acid library, comprising:

[0010] a. Fragment the nucleic acid to be tested, repair the ends, and add an A to the 3' end of the nucleic acid fragment.

[0011] b. Terminate or delay the fragmentation reaction, and add a linker and ligation reagent to initiate the ligation reaction.

[0012] c. Add a ligation termination reagent to terminate or delay the ligation reaction.

[0013] d. Mix the multiple ligation products from step c, and purify the mixed products to obtain a nucleic acid library.

[0014] In some embodiments of the present invention, the reactions described in steps a, b, and c are all carried out in the same tube.

[0015] In this invention, the phrase "same tube" refers to a reaction carried out in the same reaction vessel. No limitation is made to the reaction vessel in this invention. Those skilled in the art can select a suitable reaction vessel based on the actual reaction requirements, including but not limited to: PCR tubes, test tubes, or flow cells. Generally, the same tube is also considered as the same reaction system, meaning that all parts and components of the system are under the same external conditions (such as temperature, solvent, etc.). Generally, reactions in the same tube occur synchronously or simultaneously and eventually reach equilibrium.

[0016] In this invention, the term "fragmentation" refers to the process of cutting or cleaving nucleic acid molecules by means of enzymatic methods or other chemical or physical methods known in the art, such as ultrasound.

[0017] In this invention, the term "end repair" refers to the process of repairing the ends of fragments of target nucleic acid molecules during nucleic acid library preparation to make them suitable for subsequent sequencing reactions. Generally, end repair includes operations such as trimming, cutting, and / or adding specific sequences (such as adding poly-A tails) to the ends of fragments of target nucleic acid molecules to facilitate the smooth execution of subsequent sequencing reactions.

[0018] In this invention, the phrase "adding an A" or "adding a poly-A tail" refers to the process of adding a prominent A base to the end (typically the 3' end) of a fragment of a target nucleic acid molecule after the end-patch is closed. The main purpose of adding the A is to generate sticky ends so that adapter primers can be added in the next step. In this process, one or more enzymes may participate synergistically, including but not limited to T4 DNA polymerase, T4 polynucleotide kinase, and Taq DNA polymerase.

[0019] In some embodiments of the present invention, step d further includes an amplification reaction or an extension reaction after purification.

[0020] In this invention, the term "amplification reaction" refers to a reaction in the art in which a specific nucleic acid fragment is increased in quantity by repeatedly replicating it. In some embodiments of this invention, this increase in quantity can be proportional or exponential, or disproportionate or non-exponential.

[0021] In this invention, the term "extension reaction" refers to a reaction in which a base is linked from one end (preferably the 3' end) of another nucleic acid molecule that is bound to the target nucleic acid molecule (base pairing or partial pairing) via enzymatic methods or other methods known in the art, thereby synthesizing the complementary strand of the target nucleic acid molecule. In some embodiments of this invention, the direction of synthesis is from the 5' end to the 3' end.

[0022] In some embodiments of the present invention, the amplification reaction includes an isothermal amplification reaction or a PCR amplification reaction.

[0023] In some embodiments of the present invention, the isothermal amplification reaction includes rolling circle amplification, strand displacement amplification, etc. Of course, those skilled in the art can also rationally use other isothermal amplification reactions to achieve the amplification of the target nucleic acid molecule according to actual usage needs, including but not limited to the above-mentioned rolling circle amplification and strand displacement amplification.

[0024] In some embodiments of the present invention, step b further includes a cyclization reaction, the step of which includes: further adding oligonucleotide fragments in step b, so that the nucleic acid to be tested with adapter sequences at both ends in the reaction process of step b undergoes a single-strand cyclization reaction.

[0025] In some embodiments of the present invention, step d is further followed by a cyclization reaction, the steps of which include: adding oligonucleotide fragments and ligase to the purified product obtained in step d, and performing a single-strand cyclization reaction on the test nucleic acid with adapter sequences at both ends.

[0026] In some embodiments of the present invention, the termination or delay of the fragmentation reaction in step b includes high-temperature treatment of the system obtained in step a.

[0027] In some embodiments of the present invention, the temperature of the high-temperature treatment is 60°C-120°C.

[0028] In some embodiments of the present invention, the temperature is adjusted based on a method for terminating or delaying the fragmentation reaction, including but not limited to enzymatic methods or other methods known in the art. When using an enzymatic method, the temperature is adaptively adjusted to a range that inactivates the enzyme, based on the selection of the enzyme.

[0029] In some embodiments of the present invention, the construction method uses a non-thermosensitive cleavage enzyme for fragmentation.

[0030] In some embodiments of the present invention, the temperature of the high-temperature treatment is 60°C-75°C.

[0031] In some embodiments of the present invention, the temperature of the high-temperature treatment is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75°C.

[0032] In some embodiments of the present invention, the high-temperature treatment time is 10 min to 60 min.

[0033] In some embodiments of the present invention, the high-temperature treatment time is 10-55 min, 10-50 min, 10-45 min, 10-40 min, 10-35 min, or 10-30 min. Of course, those skilled in the art can reasonably adjust the treatment time based on changes in other conditions (such as temperature), including but not limited to the above time ranges.

[0034] In some embodiments of the present invention, the number of ligation product samples is greater than or equal to two. In some embodiments of the present invention, the number of ligation product samples is 2-1000, 2-800, 2-500, 2-200, 5-200, 10-200, 50-200, or 50-100, or greater than 1000. It is understood that the ligation product is the ligation product obtained after processing the nucleic acid to be tested in the sample through the aforementioned steps. Of course, it should be understood that, in the present invention, those skilled in the art can adjust the number of ligation products mixed according to factors such as the size (volume) of the reaction vessel used and the amount of reagents used, including but not limited to the above ranges.

[0035] In some embodiments of the present invention, step d, the purification process includes nucleic acid purification using methods such as membrane filtration, nuclease treatment, organic solvent extraction, centrifugation, magnetic beading, and silicate separation. It should be understood that the method of nucleic acid purification is not limited in the present invention, and those skilled in the art can use any technique or means with nucleic acid purification effect to purify nucleic acid molecules, including but not limited to the above-described nucleic acid purification methods.

[0036] In some embodiments of the present invention, the purification process is performed using magnetic beads.

[0037] In some embodiments of the present invention, the construction method may not include library amplification. In the present invention, the library construction method and the sequencing method based on the library may be PCR-free (i.e., PCR-free).

[0038] In some embodiments of the present invention, the amplification includes PCR amplification and bacterial culture amplification.

[0039] In some embodiments of the present invention, the nucleic acid to be tested is a full-length genomic nucleic acid molecule or a fragment thereof.

[0040] In some embodiments of the present invention, the genomic nucleic acid molecule (full length) or a fragment thereof may be obtained by any of the following methods, including but not limited to: extracting chromosomal nucleic acid molecules from donor cells, chemical synthesis, PCR amplification, or mechanical cutting and digestion with nucleic acid restriction endonucleases.

[0041] In some embodiments of the present invention, the loading amount of the nucleic acid to be tested is 1-1000 ng.

[0042] In some embodiments of the present invention, the loading amount of the nucleic acid to be tested is 50-500 ng.

[0043] In some embodiments of the present invention, the loading amount of the nucleic acid to be tested is 100-300 ng.

[0044] In some embodiments of the present invention, the loading amount of the nucleic acid to be tested is 0.1-1000 μg. In the present invention, the loading amount of the nucleic acid to be tested can be adjusted according to factors such as its length, reaction vessel capacity, upper limit of reactor loading, and amount of other reagents in the reaction system, including but not limited to the loading amount range mentioned above.

[0045] In some embodiments of the present invention, the reaction system in step a includes: 5'-hydroxytransferase, polymerase or its active fragment.

[0046] In some embodiments of the present invention, the 5' hydroxytransferase includes T4 polynucleotide kinase (T4 PNK).

[0047] In some embodiments of the present invention, the polymerase or its active fragment includes DNA polymerase or its active fragment.

[0048] In some embodiments of the present invention, the DNA polymerase or its active fragment includes the Klenow fragment, recombinant Taq DNA polymerase, and T4 DNA polymerase.

[0049] Of course, those skilled in the art can also reasonably adjust the specific selection of the above enzymes according to actual usage needs, and use enzymes with completely consistent or similar functions to replace them, including but not limited to the above-mentioned T4 PNK, Klenow fragment, recombinant Taq DNA polymerase and T4 DNA polymerase.

[0050] In some embodiments of the present invention, the reaction system in step a further includes a nuclease.

[0051] In some embodiments of the present invention, the nuclease includes deoxyribonuclease.

[0052] In some embodiments of the present invention, the reaction system in step a further includes at least one of a buffer solution, a metal salt, and a nucleoside triphosphate.

[0053] In some embodiments of the present invention, the buffer solution includes, but is not limited to, Tris-HCl buffer, phosphate buffer (PBS), MOPS buffer, HEPES buffer, etc.

[0054] In some embodiments of the present invention, the metal salts include, but are not limited to, calcium (Ca) salts, potassium (K) salts, sodium (Na) salts, magnesium (Mg) salts, iron (Fe) salts, zinc (Zn) salts, molybdenum (Mo) salts, chromium (Cr) salts, and cobalt (Co) salts.

[0055] In some embodiments of the present invention, the metal salt is a magnesium salt and a calcium salt.

[0056] In some embodiments of the present invention, the magnesium salt may be any soluble salt of magnesium or its oxide, including but not limited to magnesium chloride, magnesium sulfate, magnesium citrate, magnesium glycine, magnesium oxide, magnesium malate, etc.

[0057] In some embodiments of the present invention, the calcium salt may be any soluble salt of calcium or its oxide, including but not limited to calcium gluconate, calcium hydrogen phosphate, calcium lactate and calcium chloride.

[0058] In some embodiments of the present invention, the nucleoside triphosphate includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), and uridine triphosphate (UTP), etc.

[0059] In some embodiments of the present invention, the reaction system in step a includes or is composed of the following components: Tris-HCl, dATP, dNTP Mix, magnesium chloride, calcium chloride, T4 PNK, Klenow fragment, rTaq DNA polymerase, T4 DNA polymerase, DNase I (RNase-free), glycerol, and water.

[0060] In some embodiments of the present invention, the reaction system in step a includes or is composed of the following components:

[0061] In some embodiments of the present invention, the concentration of dATP is 1-500mM, 1-200mM, 5-200mM, 10-200mM, 50-200mM, 50-150mM or 50-100mM.

[0062] In some embodiments of the present invention, the concentration of magnesium chloride is 0.1-10M, 0.1-5M, 0.5-5M or 1-5M.

[0063] In some embodiments of the present invention, the concentration of calcium chloride is 0.1-10M, 0.1-5M, 0.5-5M or 1-5M.

[0064] In some embodiments of the present invention, the activity of the T4 PNK is 0.1-100 U / μL, 0.5-100 U / μL, 1-100 U / μL, 1-50 U / μL, 5-50 U / μL, or 5-10 U / μL.

[0065] In some embodiments of the present invention, the activity of the Klenow fragment is 0.1-100 U / μL, 0.5-100 U / μL, 1-100 U / μL, 1-50 U / μL, 5-50 U / μL, or 1-5 U / μL.

[0066] In some embodiments of the present invention, the activity of the rTaq DNA polymerase is 0.1-100 U / μL, 0.5-100 U / μL, 1-100 U / μL, 1-50 U / μL, 5-50 U / μL, or 1-5 U / μL.

[0067] In some embodiments of the present invention, the activity of DNase I is 0.1-100 U / μL, 0.5-100 U / μL, 1-100 U / μL, 1-50 U / μL, 5-50 U / μL, or 1-5 U / μL.

[0068] In some embodiments of the present invention, the mixing ratio (volume ratio) of the nucleic acid to be tested and the reaction system in step a is 1-100:0.1-50, 1-50:1-10, 1-20:1-10, 5-20:1-10, 10-20:1-10, 15-20:1-10, or 15-20:5-10.

[0069] In some embodiments of the present invention, the nucleic acid to be tested is homogenized before being mixed with the reaction system in step a.

[0070] In this invention, the term "homogenization" is also called "uniformization," which refers to correcting differences between different samples using specific technical means to obtain comparable and accurate results. In this art, homogenization methods include, but are not limited to, homogenizing concentration, mass, or volume (through dilution or addition). In this embodiment of the invention, the homogenization of the nucleic acid to be tested employs concentration homogenization.

[0071] In some embodiments of the present invention, the end in step a includes a flat end and a sticky end.

[0072] In this invention, the term "flat end" refers to a type of end structure in which the nucleic acid molecule breaks at the center of a symmetrical structure under the action of restriction endonucleases, forming a flat end structure.

[0073] In this invention, the term "sticky end" refers to a single-stranded nucleic acid sequence (also called a protrusion or protrusion) of a certain length at the end of a nucleic acid molecule, which can pair with the sticky end of another nucleic acid molecule or its own other end to form a connection.

[0074] In some embodiments of the present invention, the connector in step b comprises or consists of a universal primer sequence and a tag sequence.

[0075] In some embodiments of the present invention, the label sequence may be a barcode sequence or an index sequence.

[0076] In this invention, universal primer sequences can be obtained according to the target detection object through conventional design (according to technical manuals or textbooks in the field), commercial purchase, or any conventional method.

[0077] In some embodiments of the present invention, the 5' end of the connector is modified with a blocking group, the modifying group comprising a phosphate group.

[0078] In some embodiments of the present invention, the modification group is selected from at least one of the following modifications: phosphorylation modification (including 5' phosphorylation modification and / or 3' phosphorylation modification), thiothiol modification.

[0079] In some embodiments of the present invention, multiple connectors are added in step b.

[0080] In some embodiments of the present invention, the number of connector types added in step b is greater than or equal to 2.

[0081] In some embodiments of the present invention, the type of connector added in step b is 2.

[0082] In some embodiments of the present invention, different connectors have the same or different blocking group modifications.

[0083] In some embodiments of the present invention, the sealing groups between different connectors are modified in the same way.

[0084] In some embodiments of the present invention, when using multiple connectors, the label sequences in different connectors are different from each other.

[0085] In some embodiments of the present invention, the ligation reaction reagent in step b includes: nucleic acid ligase.

[0086] In this invention, the term "nucleic acid ligase," also known as ligase or synthase, generally refers to an enzyme capable of catalyzing the binding of two large molecules together with a novel chemical bond. In this invention, it specifically refers to an enzyme capable of ligating nucleic acid fragments.

[0087] In some embodiments of the present invention, the nucleic acid ligase includes a DNA ligase.

[0088] In some embodiments of the present invention, the DNA ligase includes, but is not limited to, ATP-dependent DNA ligases and NAD+-dependent DNA ligases.

[0089] In some embodiments of the present invention, the DNA ligase includes, but is not limited to, T4 DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV.

[0090] Of course, those skilled in the art can also reasonably adjust the specific selection of the above enzymes according to actual usage needs, and use enzymes with completely consistent or similar functions to replace them, including but not limited to the above-mentioned T4 DNA ligase, DNA ligase I, DNA ligase III and DNA ligase IV.

[0091] In some embodiments of the present invention, the ligation reaction reagent in step b further includes a salt ion buffer.

[0092] In some embodiments of the present invention, the salt includes metal salts and non-metal salts; the metal salts include, but are not limited to, salts formed by metals with organic bases, salts formed with organic acids, salts formed with inorganic acids, salts formed with basic or acidic amino acids, etc. Examples of metal salts include, but are not limited to, calcium (Ca) salts, potassium (K) salts, sodium (Na) salts, magnesium (Mg) salts, iron (Fe) salts, zinc (Zn) salts, molybdenum (Mo) salts, chromium (Cr) salts, cobalt (Co) salts, etc. The organic bases may include: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. The inorganic acids may include: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Organic acids can include: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Basic amino acids can include: arginine, lysine, ornithine, etc. Acidic amino acids can include: aspartic acid, glutamic acid, etc.

[0093] In some embodiments of the present invention, the salt ion buffer includes, but is not limited to, PNK buffer, Tris-HCl, and H2PO4-HPO4. 2- Buffer pairs are equivalent to NH3·H2O-NH4Cl buffer pairs.

[0094] In some embodiments of the present invention, the concentration of the salt ion buffer is greater than or equal to 0.05 mol / L.

[0095] In some embodiments of the present invention, the concentration of the salt ion buffer is 0.5 to 5 mol / L.

[0096] In some embodiments of the present invention, the linking reaction reagent in step b further includes a coagulant and / or nucleoside triphosphate.

[0097] In some embodiments of the present invention, the coagulant includes polyethylene glycol and hexaamminecobalt chloride.

[0098] In some embodiments of the present invention, the polyethylene glycol includes polyethylene glycols of different molecular weights, including polyethylene glycols with molecular weights of 400, 600, 800, 1000, 1450, 1500, 2000, 3000, 3350, 4000, 6000 or 8000.

[0099] In some embodiments of the present invention, the ligation reaction reagent in step b includes or is composed of the following components: PNK buffer, ATP, PEG8000, T4 DNA ligase and water.

[0100] In some embodiments of the present invention, the linkage reaction system in step b includes or is composed of the following components:

[0101] In some embodiments of the present invention, the connector is two different connectors.

[0102] In some embodiments of the present invention, the connector is as shown in SEQ ID NO: 1 and 2.

[0103] In some embodiments of the present invention, the mixing ratio (volume ratio) between the two types of connectors is 0.1-2:0.1:2.

[0104] In some embodiments of the invention, the connector is pre-diluted with a conversion buffer.

[0105] In some embodiments of the present invention, the conversion buffer comprises or is composed of the following components: Tris-HCl, EDTA, and NaCl.

[0106] In some embodiments of the present invention, the concentration of Tris-HCl in the conversion buffer is 10-200 mM, 10-100 mM, 20-100 mM, or 20-50 mM.

[0107] In some embodiments of the present invention, the EDTA concentration in the conversion buffer is 0.01-10mM, 0.05-10mM, 0.05-5mM, 0.05-1mM, 0.05-0.5mM, or 0.05-0.1mM.

[0108] In some embodiments of the present invention, the NaCl concentration in the conversion buffer is 10-200mM, 10-100mM, 20-100mM, or 20-50mM.

[0109] In some embodiments of the present invention, the mixing ratio (volume ratio) of the adapter and the conversion buffer in the conversion buffer is 1:0.5-10, 1:0.5-5, 1:0.5-2 or 1:1-2.

[0110] In some embodiments of the present invention, the reaction vessel is preheated before the connector is connected.

[0111] In some embodiments of the present invention, preheating is performed using a PCR instrument (hot-lid operation). Of course, those skilled in the art can also use other preheating methods, including but not limited to using a PCR instrument for hot-lid operation. In this invention, the term "hot-lid" refers to preheating an empty reaction vessel in a PCR experiment or an experiment using a PCR instrument to prevent evaporation of the contents. In this invention, the method of hot-lid operation is not limited and may include, but is not limited to, using the PCR instrument itself or its included programs for hot-lid operation, or using other equipment for preheating.

[0112] In some embodiments of the present invention, the connection termination reaction reagent in step c is a high-salt reaction reagent.

[0113] The salts include both metal salts and non-metal salts; the metal salts include, but are not limited to, salts formed by metals with organic bases, salts formed with organic acids, salts formed with inorganic acids, salts formed with basic or acidic amino acids, etc. Examples of metal salts include, but are not limited to, calcium (Ca) salts, potassium (K) salts, sodium (Na) salts, magnesium (Mg) salts, iron (Fe) salts, zinc (Zn) salts, molybdenum (Mo) salts, chromium (Cr) salts, cobalt (Co) salts, etc. Organic bases may include: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. Inorganic acids may include: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Organic acids can include: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Basic amino acids can include: arginine, lysine, ornithine, etc. Acidic amino acids can include: aspartic acid, glutamic acid, etc.

[0114] In some embodiments of the present invention, the salt is an inorganic salt.

[0115] In some embodiments of the present invention, the salt content in the connection termination reaction reagent is greater than or equal to 1 mol / L.

[0116] In some embodiments of the present invention, the salt content in the connection termination reaction reagent is 1-20 mol / L.

[0117] In some embodiments of the present invention, the salt content in the connection termination reaction reagent is 1-10 mol / L.

[0118] In some embodiments of the present invention, the salt content in the connection termination reaction reagent is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mol / L.

[0119] In some embodiments of the present invention, the inorganic salt includes any soluble inorganic salt capable of affecting the linkage of nucleic acid molecules, including but not limited to the above-mentioned metal inorganic salts, such as NaCl, KaCl, etc.

[0120] In some embodiments of the present invention, the connection termination reaction reagent further comprises a buffer solution and a chelating agent.

[0121] In some embodiments of the present invention, the chelating agent includes amino acid compounds, hydroxy acid compounds, phosphates, and carboxylic acid compounds.

[0122] In some embodiments of the present invention, the amino acid compound includes amino acids or their derivatives.

[0123] In some embodiments of the present invention, the amino acids or their derivatives include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), glutamic acid, aspartic acid, leucine, etc.

[0124] In some embodiments of the present invention, the hydroxy acid compounds include, but are not limited to, hydroxyethylidene diphosphonic acid (HEDTA) and octanoyl hydroxamic acid (CHA).

[0125] In some embodiments of the present invention, the phosphate includes, but is not limited to, sodium tripolyphosphate, sodium polyphosphate, sodium hexametaphosphate, sodium pyrophosphate, etc.

[0126] In some embodiments of the present invention, the carboxylic acid compounds include, but are not limited to, citric acid, oxalic acid, salicylic acid, tartaric acid, and gluconic acid.

[0127] In some embodiments of the present invention, the buffer solution includes, but is not limited to, TE buffer (i.e., Tris-EDTA), Tris, and water.

[0128] In some embodiments of the present invention, the ligation termination reaction reagent includes or is composed of the following components: EDTA, NaCl, and TE buffer.

[0129] In some embodiments of the present invention, the concentration of EDTA in the ligation termination reaction reagent is 0.1-10M, 0.1-5M, 0.5-5M, or 0.5-1M.

[0130] In some embodiments of the present invention, the connection termination reaction reagent comprises or is composed of the following components:

[0131] In some embodiments of the present invention, the mixing ratio (volume ratio) of the ligation termination reaction reagent to the reaction system is 1-20:30-50, 5-20:40-50, 10-20:40-50, or 15-20:40-45.

[0132] In some embodiments of the present invention, in step c, multiple linkage products are mixed in equal amounts.

[0133] In some embodiments of the present invention, the equal quantities include at least one of equal volume, equal mass, and equal concentration.

[0134] In some embodiments of the present invention, the nucleic acid to be tested is derived from at least one of DNA, RNA, or DNA-RNA hybrid chains.

[0135] In some embodiments of the present invention, the DNA includes, but is not limited to, cDNA, cfDNA, dsDNA, and ssDNA.

[0136] In this invention, the term "DNA" refers to deoxyribonucleic acid, and "cDNA, cfDNA, dsDNA, and ssDNA" correspond to complementary (sometimes called copy) DNA (a DNA strand that is complementary to RNA after reverse transcription in vitro), circulating free DNA, double-stranded DNA (a DNA molecule composed of two single-stranded DNA molecules through base complementarity), and single-stranded DNA, respectively.

[0137] In some embodiments of the present invention, the RNA includes, but is not limited to, mRNA, lncRNA, and miRNA.

[0138] In this invention, the term "RNA" refers to ribonucleic acid, and "mRNA, lncRNA, and miRNA" correspond to messenger RNA, long non-coding RNA, and microRNA, respectively.

[0139] In some embodiments of the present invention, when the nucleic acid to be tested does not belong to the nucleic acid molecules involved in the above aspects, it can be further processed using conventional techniques in the art to transform it into a nucleic acid molecule suitable for library construction and sequencing by the methods in the embodiments of the present invention. The processing includes, but is not limited to: reverse transcription, chemical modification, and denaturation / renaturation.

[0140] In some embodiments of the present invention, the nucleic acid to be tested is derived from the following biological samples: cells, fresh tissues, fresh organs, decaying tissues, formalin-fixed tissues, paraffin-embedded tissues, forensic samples, paleontological fossils, and biological materials containing cfDNA or cfRNA.

[0141] In some embodiments of the present invention, the biological materials include, but are not limited to: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph, bronchoalveolar lavage fluid, amniotic fluid, blastocyst cavity fluid, cell culture medium, embryo culture medium, microbial culture medium, soil leachate, and bone meal leachate.

[0142] In some embodiments of the present invention, the total reaction time of steps a to d is less than or equal to 3 hours.

[0143] In some embodiments of the present invention, the total reaction time for steps a to d is 50 to 150 minutes.

[0144] In this invention, the term "total reaction time" broadly refers to the total time consumed from the start of step a to the end of step d without additional operations, as understood by those skilled in the art. It can also be understood as the time required for spontaneous or non-spontaneous reactions that substantially affect or change the system or its components, or the time required when combined with human interventions or operations that are not feasible for this invention.

[0145] In a second aspect, the present invention provides a nucleic acid library construction kit or apparatus, the nucleic acid library construction kit or apparatus comprising a tube-type reaction premix, a adapter, a ligation reaction reagent, and a ligation termination reaction reagent.

[0146] In some embodiments of the present invention, the one-tube reaction premixed reagent includes: a fragmented enzyme and a fragmented reaction reagent.

[0147] In some embodiments of the present invention, the fragmentation enzyme includes a nuclease.

[0148] In some embodiments of the present invention, the nuclease includes deoxyribonuclease.

[0149] In some embodiments of the present invention, the one-tube reaction premixed reagent further comprises: 5'-hydroxytransferase and polymerase or their active fragments.

[0150] In some embodiments of the present invention, the 5' hydroxytransferase includes T4 polynucleotide kinase (T4 PNK).

[0151] In some embodiments of the present invention, the polymerase or its active fragment includes DNA polymerase or its active fragment.

[0152] In some embodiments of the present invention, the DNA polymerase or its active fragment includes the Klenow fragment, recombinant Taq DNA polymerase, and T4 DNA polymerase.

[0153] In some embodiments of the present invention, the linking reaction reagent is the same as the linking reaction reagent described in the above aspects.

[0154] In some embodiments of the present invention, the connection termination reaction reagent contains a high-salt reaction reagent.

[0155] In some embodiments of the present invention, the salt is an inorganic salt.

[0156] In some embodiments of the present invention, the salt content in the connection termination reaction reagent is greater than or equal to 1 mol / L.

[0157] In some embodiments of the present invention, the salt content in the ligation termination reaction reagent is 1-20 mol / L or 1-10 mol / L.

[0158] In some embodiments of the present invention, the inorganic salt includes any soluble inorganic salt capable of affecting the linkage of nucleic acid molecules, including but not limited to the metal inorganic salts described above, such as NaCl, KaCl, etc.

[0159] In some embodiments of the present invention, the one-tube reaction premixed reagent further comprises at least one of a buffer solution, a metal salt, and a nucleoside triphosphate.

[0160] In some embodiments of the present invention, the buffer solution includes, but is not limited to, Tris-HCl buffer, phosphate buffer (PBS), MOPS buffer, HEPES buffer, etc.

[0161] In some embodiments of the present invention, the metal salts include, but are not limited to, calcium salts, potassium salts, sodium salts, magnesium salts, iron salts, zinc salts, molybdenum salts, chromium salts, and cobalt salts.

[0162] In some embodiments of the present invention, the magnesium salt may be any soluble salt of magnesium or its oxide, including but not limited to magnesium chloride, magnesium sulfate, magnesium citrate, magnesium glycine, magnesium oxide, magnesium malate, etc.

[0163] In some embodiments of the present invention, the calcium salt may be any soluble salt of calcium or its oxide, including but not limited to calcium gluconate, calcium hydrogen phosphate, calcium lactate and calcium chloride.

[0164] In some embodiments of the present invention, the nucleoside triphosphate includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), and uridine triphosphate (UTP), etc.

[0165] In some embodiments of the present invention, the one-tube reaction premixed reagent comprises or is composed of the following components: Tris-HCl, dATP, dNTP Mix, magnesium chloride, calcium chloride, T4 PNK, Klenow fragment, rTaq DNA polymerase, T4 DNA polymerase, DNase I (RNase-free), glycerol, and water.

[0166] In some embodiments of the present invention, the connection termination reaction solution further comprises a buffer solution and a chelating agent.

[0167] In some embodiments of the present invention, the chelating agent includes amino acid compounds, hydroxy acid compounds, phosphates, and carboxylic acid compounds.

[0168] In some embodiments of the present invention, the amino acid compound includes amino acids or their derivatives.

[0169] In some embodiments of the present invention, the amino acids or their derivatives include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), glutamic acid, aspartic acid, leucine, etc.

[0170] In some embodiments of the present invention, the hydroxy acid compounds include, but are not limited to, hydroxyethylidene diphosphonic acid (HEDTA) and octanoyl hydroxamic acid (CHA).

[0171] In some embodiments of the present invention, the phosphate includes, but is not limited to, sodium tripolyphosphate, sodium polyphosphate, sodium hexametaphosphate, sodium pyrophosphate, etc.

[0172] In some embodiments of the present invention, the carboxylic acid compounds include, but are not limited to, citric acid, oxalic acid, salicylic acid, tartaric acid, and gluconic acid.

[0173] In some embodiments of the present invention, the buffer solution includes, but is not limited to, TE buffer (i.e., Tris-EDTA), Tris, and water.

[0174] In some embodiments of the present invention, the adapter includes a universal primer sequence and a tag sequence.

[0175] In some embodiments of the present invention, the label sequence may be a barcode sequence or an index sequence.

[0176] In this invention, universal primer sequences can be obtained according to the target detection object through conventional design (according to technical manuals or textbooks in the field), commercial purchase, or any conventional method.

[0177] In some embodiments of the present invention, the type of the connector is greater than or equal to 2.

[0178] In some embodiments of the invention, the connector is pre-diluted with a conversion buffer.

[0179] In some embodiments of the present invention, the conversion buffer comprises or is composed of the following components: Tris-HCl, EDTA, and NaCl.

[0180] In some embodiments of the present invention, different connectors have the same or different blocking group modifications.

[0181] In some embodiments of the present invention, the sealing groups between different connectors are modified in the same way.

[0182] In some embodiments of the present invention, the nucleic acid library construction kit or apparatus may further include a chip or beads as a carrier. The beads are selected from agarose gel beads, agarose beads, magnetic beads, protein A conjugated beads, protein G conjugated beads, protein L conjugated beads, oligomeric (dT) conjugated beads, silica beads, hydrogel beads, silica sample beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof.

[0183] In some embodiments of the present invention, the chip is a chip suitable for sequencing.

[0184] A third aspect of the present invention provides the application of the construction method or nucleic acid library construction kit or assembly described above in gene sequencing.

[0185] In some embodiments of the present invention, the gene sequencing is not used for disease diagnosis.

[0186] In some embodiments of the present invention, the gene sequencing targets a full-length genomic nucleic acid molecule or a fragment thereof.

[0187] In a fourth aspect, the present invention provides a nucleic acid library, which is constructed using the construction method described above or a nucleic acid library construction kit or assembly.

[0188] In some embodiments of the present invention, the nucleic acid library includes a library of full-length genomic nucleic acid molecules or fragments thereof.

[0189] In some embodiments of the present invention, the nucleic acid library is a genomic nucleic acid molecular library.

[0190] In some embodiments of the present invention, the nucleic acid library is derived from multiple ligation product samples.

[0191] In some embodiments of the present invention, the number of the ligation product samples is greater than or equal to 2.

[0192] In some embodiments of the present invention, the number of the ligation product samples is 2-1000.

[0193] In some embodiments of the present invention, the number of the ligation product samples is 2-500.

[0194] In some embodiments of the present invention, the ligation product sample may be incorporated into the nucleic acid library once or multiple times.

[0195] A fifth aspect of the present invention provides a sequencing method comprising the following steps: constructing a library using the construction method described above or a nucleic acid library construction kit or assembly, and sequencing the library.

[0196] In some embodiments of the present invention, the library is further prepared into nanospheres before sequencing.

[0197] In some embodiments of the present invention, the library is further prepared into a cluster by bridging amplification before sequencing.

[0198] In some embodiments of the present invention, the library is cyclized before it is prepared into nanospheres.

[0199] In some embodiments of the present invention, the cyclization treatment, nanosphere preparation, or bridging amplification can be performed using conventional techniques in the art or commercially available kits. The present invention does not limit the methods or means of cyclization treatment, nanosphere preparation, and bridging amplification selected.

[0200] In some embodiments of the present invention, sequencing can be performed using any method known in the art, including but not limited to using conventional sequencing platforms, instruments or equipment.

[0201] In some embodiments of the present invention, sequencing is performed using a sequencer.

[0202] In some embodiments of the present invention, the sequencer is a DNBSEQ-T7, and the sequencing depth per sample is 1×. Of course, those skilled in the art can adjust the sequencing parameters appropriately based on the selected sequencing method to achieve the sequencing objective.

[0203] In some embodiments of the present invention, the sequencing method further includes analyzing the sequencing data.

[0204] In some embodiments of the present invention, the analysis includes analyzing the data to obtain at least one of the following: number of reads, Q30 of the reads, barcode splitting rate, GC content, CV of a single sample, average sequencing depth of a single sample, and 1× coverage. Of course, those skilled in the art can increase or decrease the obtained analysis results according to their needs and purposes.

[0205] In some embodiments of the present invention, the sequencing is directed at a full-length genomic nucleic acid molecule or a fragment thereof.

[0206] In some embodiments of the present invention, the sequencing is performed on a genomic nucleic acid molecular library.

[0207] In some embodiments of the present invention, the sequencing method may further include quality control of the library before sequencing.

[0208] A sixth aspect of the present invention provides a sequencing product comprising the nucleic acid library construction kit or apparatus described in the above aspects, and sequencing or sequencing aids.

[0209] In some embodiments of the present invention, the sequencing or sequencing aid product includes at least one of a sequencer, a nucleic acid extraction product, a reverse transcription product, and a nucleic acid purification product.

[0210] In some embodiments of the present invention, the nucleic acid extraction product, reverse transcription product, and nucleic acid purification product can be used to process the nucleic acid to be tested for library construction or sequencing.

[0211] In some embodiments of the present invention, the sequencing product includes reagents, kits, or chips. Of course, those skilled in the art can also reasonably construct the products of the present invention in other product forms according to usage requirements, including but not limited to the reagents, kits, or chips described above.

[0212] In some embodiments of the present invention, the chip is a chip suitable for sequencing.

[0213] The beneficial effects of this invention are:

[0214] 1. This invention develops a highly efficient library preparation method for large-scale samples, which is simpler than conventional PCR-free library construction techniques or rapid PCR-free library construction techniques. This method can simultaneously perform fragmentation, end repair, and 3' end A addition reaction based on nucleic acid molecules of different sizes, and can directly perform adapter ligation without further purification, realizing all processes before circularization in one tube, thereby effectively improving construction efficiency and greatly saving time and reagent costs.

[0215] 2. The present invention further provides a connector connection method using a dual-label connector connection + a high-salt connector reaction termination solution, thereby overcoming the defects of conventional methods that require additional purification steps, thus effectively simplifying the library construction process and providing a technical basis and support for achieving single-tube connection.

[0216] 3. The library construction method in this invention introduces the addition of salt solution during the fragmentation process, end repair, and 3' end A addition reaction. Therefore, the amount of salt added to the ligation system is effectively reduced during subsequent linker ligation, thereby further controlling the amount of other related solutions added, saving reagent costs, and effectively controlling the total volume of the system.

[0217] 4. The library preparation method in this invention can perform co-purification of multiple samples after ligation according to the sequencing throughput and single-sample sequencing depth requirements, thereby avoiding the inefficient detection requirement of one sample per test, greatly improving the library preparation throughput, and thus enabling rapid detection of large-scale samples. Attached Figure Description

[0218] Figure 1 is a flowchart of the method for constructing a large-scale whole genome library in an embodiment of the present invention.

[0219] Figure 2 shows the length detection result of a randomly selected pig ear-derived gDNA nucleic acid sample after being fragmented using the method in this embodiment of the invention.

[0220] Figure 3 shows the sequencing results (unfiltered Q30 results) based on the large-scale whole-genome library construction method and the rapid PCR-free library construction method in the embodiments of the present invention.

[0221] Figure 4 shows the sequencing results (base distribution) of the large-scale whole genome library construction method and the rapid PCR-free library construction method based on the embodiments of the present invention.

[0222] Figure 5 shows the sequencing results (GC content distribution) of the large-scale whole genome library construction method and the rapid PCR-free library construction method based on the embodiments of the present invention. Detailed Implementation

[0223] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0224] Materials and Definitions

[0225] In the following embodiments, some of the materials are sourced as follows:

[0226] In the following embodiments, the abbreviations and their corresponding full names are as follows:

[0227] Example 1

[0228] In this embodiment, a method for constructing a large-scale whole-genome library is provided, and the construction process is shown in Figure 1.

[0229] In this embodiment, porcine gDNA (extracted from 384 commercially available porcine ear samples using conventional methods in the art) was used as the detection sample, and a whole-genome library was prepared according to the following method (200 ng / sample). Simultaneously, an existing Fast PCR-free library construction method (based on the commercially available MGIEAsy_Fast_PCR-FREE restriction enzyme digestion library preparation kit; see its instruction manual for specific library construction methods) was used as a control. The control method differs from the method in this embodiment in that it uses a different fragmentation system reagent formulation (see the table below) and a different library construction procedure (the control method also includes a purification step after fragmentation).

[0230] Table 1. Composition of the interruption system reagents in the control method.

[0231] The prepared whole-genome library was sequenced on a DNBSEQ-T7 sequencer using the PE150 sequencing type, with a sequencing depth of 1× per sample. The sequencing data were then analyzed. This analysis included parameters such as the volume fraction (CV) of a single sample, the proportion of clean reads, and the distribution of inserted fragments, to verify the effectiveness of the sequencing method for the library constructed using the whole-genome library construction method described in this embodiment of the invention.

[0232] The specific method is as follows:

[0233] (1) Sample acquisition:

[0234] 384 fresh pig ears were purchased from a market, and their gDNA was obtained using a commercially available kit as test samples. 200 ng of each test sample was homogenized to ensure a consistent concentration for subsequent steps. In this step, the volume of each test sample after homogenization was 18 μL.

[0235] (2) Construction of the interruption-end repair plus A (ployA) mixed reaction system:

[0236] Construct a mixed reaction system of interruption-end repair plus A according to the table below.

[0237] Table 2. Components and content of the interruption-end repair plus A mixed reaction system

[0238] (3) Add the prepared break-end repair plus A mixed reaction system from step (2) to 18 μL of homogenized test sample for reaction. The resulting mixed system is 25 μL. The incubation conditions are: 30℃ for 8.5 min, 72℃ for 20 min. After the reaction, the reaction product can be stored at 4℃ for later use, or directly used in subsequent steps. Incubation can be performed using a PCR instrument or other conventional temperature-controlled incubation devices in the field. Before using PCR, a hot cap should be applied at a temperature of 80℃.

[0239] (4) Connector connection:

[0240] In this embodiment, the connector sequence used is as follows:

[0241] Phosphorylated linker B chain:

[0242] 5'- / Phos / -TCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO: 1).

[0243] Phosphorylated linker T-chain:

[0244] 5'- / Phos / -AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCTTGATAAGGTCGCCATGCC-3' (SEQ ID NO: 2).

[0245] In the above linker sequence, " / / " represents a modifying group, such as / X / indicating the presence of a modifying group X; "Phos" indicates phosphorylation modification; and N represents any one of A, T, C, or G.

[0246] It should be understood that the connector sequence in this embodiment is only shown as an example. Those skilled in the art can reasonably adjust the design of the connector sequence according to the actual detection object, so as to use it for library construction of the corresponding object, including but not limited to SEQ ID NO:1 and 2 mentioned above.

[0247] Preparation of the connector solution:

[0248] 20 μL of the phosphorylated B-chain and 20 μL of the phosphorylated T-chain of the above-mentioned adapter were mixed thoroughly to obtain a 25 μM adapter stock solution. The adapter buffer was composed of 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, and 50 mM NaCl, based on the final concentration. The 25 μM adapter stock solution was incubated at room temperature for at least half an hour, and then further diluted for subsequent steps or stored at -20°C for later use. In this embodiment, the 25 μM adapter stock solution was further diluted with TE buffer to a 6 μM adapter solution.

[0249] Add the diluted adapter solution (6 μM) to the reaction product obtained in step (3) above and mix thoroughly. Then add the ligation reaction reagent. Vortex mix and then briefly centrifuge. Then place the centrifuged product in a PCR instrument for reaction under the following conditions: 25℃ for 15 min. After the reaction, the reaction product can be stored at 4℃ for later use or used directly in subsequent steps. Before using PCR, a hot capping is required at a temperature of 30℃. In this step, a total of 42 μL of reaction product was obtained.

[0250] The composition of the connecting reaction reagents is shown in the table below.

[0251] Table 3. Components and content of the reagents used in the ligation reaction

[0252] (5) Add the ligation termination reaction reagent to the 42 μL of reaction product obtained in step (4) above, vortex mix and then perform instantaneous centrifugation to terminate the ligation reaction.

[0253] The composition of the reagent used to terminate the reaction is shown in the table below.

[0254] Table 4. Components and content of reagents for terminating the ligation reaction.

[0255] (6) Pooling:

[0256] The ligation products obtained in the above steps (a total of 384) were randomly divided into 4 tubes of 96 each and mixed in equal volumes (pooling). The amount of each ligation product used was 10 μL.

[0257] Take 120 μL of the mixture from each tube, purify it with magnetic beads, and then transfer it to a new 0.2 mL PCR tube. The specific procedure is as follows: Add 30 μL of TE buffer to each 120 μL mixture, then add 36 μL of magnetic beads. Vortex to mix, incubate at room temperature for 5 minutes, then place on a magnetic rack to incubate for 2 minutes (until the liquid is clear). Carefully aspirate the supernatant into a new 0.2 mL PCR tube (only the supernatant is retained in this step). Add another 15 μL of magnetic beads to the supernatant for further purification, vortex to mix, incubate at room temperature for 5 minutes, then place on a magnetic rack to incubate for 2 minutes (until the liquid is clear), and discard the supernatant. Keep the PCR tube on the magnetic rack, add 200 μL of 80% ethanol to the 0.2 mL PCR tube, let stand for 30 seconds, and discard the supernatant. Wash once more with 200 μL of 80% ethanol, discarding as much residual ethanol as possible using a small-range pipette, and air dry at room temperature. Resuspend the magnetic beads in 32 μL of TE buffer, vortex to mix, bind at room temperature for 10 minutes, place on a magnetic rack to bind for 2 minutes (until the liquid is clear), and carefully aspirate 30 μL of supernatant into a new 0.2 mL PCR tube for subsequent reactions or store at -20°C.

[0258] The purified product was quantified using the Qubit dsDNA Assay Kit, following the instructions for use.

[0259] (7) One-step preparation and sequencing of DNA nanospheres (DNB):

[0260] The purified product obtained in step (6) above was used to prepare DNA nanospheres using the DNBSEQ one-step method (any commercial kit in the art or a publicly available one-step DNB construction method can be used), and then the obtained DNA nanospheres were sequenced using DNBSEQ-T7.

[0261] In this embodiment, the purified product obtained in step (6) can also be cyclized into a single-stranded circular (ssCir) library using a cyclization kit (any commercial kit in the art or a publicly available double-stranded DNA cyclization method can be used), and then DNA nanospheres can be prepared and sequenced using DNBSEQ-T7.

[0262] In this embodiment, the one-step DNB construction method is performed according to the operation steps corresponding to DNBSEQ-T7 sequencing (see instruction manual), and the sequencing process is carried out on the machine and data analysis according to the standard operation procedure of DNBSEQ-T7 sequencing.

[0263] Example 2

[0264] In this embodiment, a method for constructing a large-scale whole-genome library is provided, and the construction process is the same as in Embodiment 1.

[0265] In this embodiment, new porcine gDNA was obtained as a test sample, and a whole-genome library was prepared according to the following method (200 ng / sample). The prepared whole-genome library was sequenced on a DNBSEQ-T7 sequencer using PE150 sequencing, with a sequencing depth of 1× per sample. The obtained sequencing data was then analyzed. The data analysis included parameters such as the volume of data per sample (CV), the proportion of clean reads, and the distribution of inserted fragments, to verify the effectiveness of the sequencing method for the library constructed based on the whole-genome library construction method described in this embodiment.

[0266] The specific method is as follows:

[0267] (1) Sample acquisition:

[0268] New porcine gDNA was obtained as a test sample according to the method in Example 1. 200 ng of each test sample was homogenized to ensure a consistent concentration for subsequent steps. In this step, the volume of each test sample after homogenization was 18 μL.

[0269] (2) Construction of the interruption-end repair plus A mixed reaction system:

[0270] Construct a mixed reaction system of interruption-end repair plus A according to the table below.

[0271] Table 5. Components and content of the interruption-end repair plus A mixed reaction system

[0272] (3) Add the prepared break-end repair plus A mixed reaction system from step (2) to 18 μL of homogenized test sample for reaction. The resulting mixed system is 25 μL. The incubation conditions are: 30℃ for 8.5 min, 80℃ for 10 min. After the reaction, the reaction product can be stored at 4℃ for later use, or directly used in subsequent steps. Incubation can be performed using a PCR instrument or other conventional temperature-controlled incubation devices in the field. Before using PCR, a hot cap should be applied at a temperature of 80℃.

[0273] (4) Connector connection:

[0274] In this embodiment, the connector sequence used is as follows:

[0275] Phosphorylated linker B chain:

[0276] 5'- / Phos / -TCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO: 1).

[0277] Phosphorylated linker T-chain:

[0278] 5'- / Phos / -AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCTTGATAAGGTCGCCATGCC-3' (SEQ ID NO: 2).

[0279] In the above linker sequence, " / / " represents a modifying group, such as / X / indicating the presence of a modifying group X; "Phos" indicates phosphorylation modification; and N represents any one of A, T, C, or G.

[0280] It should be understood that the connector sequence in this embodiment is only shown as an example. Those skilled in the art can reasonably adjust the design of the connector sequence according to the actual detection object, so as to use it for library construction of the corresponding object, including but not limited to SEQ ID NO:1 and 2 mentioned above.

[0281] Preparation of the connector solution:

[0282] 20 μL of the phosphorylated B-chain and 20 μL of the phosphorylated T-chain of the above-mentioned adapter were mixed thoroughly to obtain a 25 μM adapter stock solution. The adapter buffer was composed of 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, and 50 mM NaCl, based on the final concentration. The 25 μM adapter stock solution was incubated at room temperature for at least half an hour, and then further diluted for subsequent steps or stored at -20°C for later use. In this embodiment, the 25 μM adapter stock solution was further diluted with TE buffer to a 6 μM adapter solution.

[0283] Add the diluted adapter solution (6 μM) to the reaction product obtained in step (3) above and mix thoroughly. Then add the ligation reaction reagent. Vortex mix and then briefly centrifuge. Then place the centrifuged product in a PCR instrument for reaction under the following conditions: 25℃ for 15 min. After the reaction, the reaction product can be stored at 4℃ for later use or used directly in subsequent steps. Before using PCR, a hot capping is required at a temperature of 30℃. In this step, a total of 42 μL of reaction product was obtained.

[0284] The composition of the connecting reaction reagents is shown in the table below.

[0285] Table 6. Components and content of the reagents used in the ligation reaction

[0286] (5) Add the ligation termination reaction reagent to the 42 μL of reaction product obtained in step (4) above, vortex mix and then perform instantaneous centrifugation to terminate the ligation reaction.

[0287] The composition of the reagent used to terminate the reaction is shown in the table below.

[0288] Table 7. Components and content of the ligation termination reaction reagent

[0289] (6) Pooling:

[0290] The ligation products obtained in the above steps (a total of 384) were randomly divided into 4 tubes of 96 each and mixed in equal volumes (pooling). The amount of each ligation product used was 10 μL.

[0291] Take 120 μL of the mixture from each tube, purify it with magnetic beads, and then transfer it to a new 0.2 mL PCR tube. The specific procedure is as follows: Add 30 μL of TE buffer to each 120 μL mixture, then add 36 μL of magnetic beads. Vortex to mix, incubate at room temperature for 5 minutes, then place on a magnetic rack to incubate for 2 minutes (until the liquid is clear). Carefully aspirate the supernatant into a new 0.2 mL PCR tube (only the supernatant is retained in this step). Add another 15 μL of magnetic beads to the supernatant for further purification, vortex to mix, incubate at room temperature for 5 minutes, then place on a magnetic rack to incubate for 2 minutes (until the liquid is clear), and discard the supernatant. Keep the PCR tube on the magnetic rack, add 200 μL of 80% ethanol to the 0.2 mL PCR tube, let stand for 30 seconds, and discard the supernatant. Wash once more with 200 μL of 80% ethanol, discarding as much residual ethanol as possible using a small-range pipette, and air dry at room temperature. Resuspend the magnetic beads in 32 μL of TE buffer, vortex to mix, bind at room temperature for 10 minutes, place on a magnetic rack to bind for 2 minutes (until the liquid is clear), and carefully aspirate 30 μL of supernatant into a new 0.2 mL PCR tube for subsequent reactions or store at -20°C.

[0292] The purified product was quantified using the Qubit dsDNA Assay Kit, following the instructions for use.

[0293] (7) One-step preparation and sequencing of DNA nanospheres (DNB):

[0294] The purified product (24 ng) obtained after the step was used to prepare DNA nanospheres using a one-step method (any commercial kit in the art or a publicly available one-step DNB construction method can be used), and then the obtained DNA nanospheres were sequenced using a DNBSEQ-T7 PE150.

[0295] In this embodiment, the one-step DNB construction method is performed according to the operation steps corresponding to DNBSEQ-T7 PE150 sequencing (see instruction manual), and the sequencing process is carried out on the machine and data analysis according to the standard operation procedure of DNBSEQ-T7 PE150 sequencing.

[0296] Example 3

[0297] In this embodiment, a method for constructing a large-scale whole-genome library is provided, and the construction process is the same as in Embodiment 1.

[0298] In this embodiment, new porcine gDNA was obtained as a test sample, and a whole-genome library was prepared according to the following method (200 ng / sample). The prepared whole-genome library was sequenced on a DNBSEQ-T7 sequencer using PE150 sequencing, with a sequencing depth of 1× per sample. The obtained sequencing data was then analyzed. The data analysis included parameters such as the volume of data per sample (CV), the proportion of clean reads, and the distribution of inserted fragments, to verify the effectiveness of the sequencing method for the library constructed based on the whole-genome library construction method described in this embodiment.

[0299] The specific method is as follows:

[0300] (1) Sample acquisition:

[0301] New porcine gDNA was obtained as a test sample according to the method in Example 1. 200 ng of each test sample was homogenized to ensure a consistent concentration for subsequent steps. In this step, the volume of each test sample after homogenization was 18 μL.

[0302] (2) Construction of the interruption-end repair plus A mixed reaction system:

[0303] Construct a mixed reaction system of interruption-end repair plus A according to the table below.

[0304] Table 8. Components and content of the interruption-terminal repair plus A mixed reaction system

[0305] (3) Add the prepared break-end repair plus A mixed reaction system from step (2) to 18 μL of homogenized test sample for reaction. The resulting mixed system is 25 μL. The incubation conditions are: 30℃ for 8.5 min, 60℃ for 60 min. After the reaction, the reaction product can be stored at 4℃ for later use, or directly used in subsequent steps. Incubation can be performed using a PCR instrument or other conventional temperature-controlled incubation devices in the field. Before using PCR, a hot cap should be applied at a temperature of 80℃.

[0306] (4) Connector connection:

[0307] In this embodiment, the connector sequence used is as follows:

[0308] Phosphorylated linker B chain:

[0309] 5'- / Phos / -TCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO: 1).

[0310] Phosphorylated linker T-chain:

[0311] 5'- / Phos / -AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCTTGATAAGGTCGCCATGCC-3' (SEQ ID NO: 2).

[0312] In the above linker sequence, " / / " represents a modifying group, such as / X / indicating the presence of a modifying group X; "Phos" indicates phosphorylation modification; and N represents any one of A, T, C, or G.

[0313] It should be understood that the connector sequence in this embodiment is only shown as an example. Those skilled in the art can reasonably adjust the design of the connector sequence according to the actual detection object, so as to use it for library construction of the corresponding object, including but not limited to SEQ ID NO:1 and 2 mentioned above.

[0314] Preparation of the connector solution:

[0315] 20 μL of the phosphorylated B-chain and 20 μL of the phosphorylated T-chain of the above-mentioned adapter were mixed thoroughly to obtain a 25 μM adapter stock solution. The adapter buffer was composed of 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, and 50 mM NaCl, based on the final concentration. The 25 μM adapter stock solution was incubated at room temperature for at least half an hour, and then further diluted for subsequent steps or stored at -20°C for later use. In this embodiment, the 25 μM adapter stock solution was further diluted with TE buffer to a 6 μM adapter solution.

[0316] Add the diluted adapter solution (6 μM) to the reaction product obtained in step (3) above and mix thoroughly. Then add the ligation reaction reagent. Vortex mix and then briefly centrifuge. Then place the centrifuged product in a PCR instrument for reaction under the following conditions: 25℃ for 15 min. After the reaction, the reaction product can be stored at 4℃ for later use or used directly in subsequent steps. Before using PCR, a hot capping is required at a temperature of 30℃. In this step, a total of 42 μL of reaction product was obtained.

[0317] The composition of the connecting reaction reagents is shown in the table below.

[0318] Table 9. Components and content of the reagents used in the linkage reaction

[0319] (5) Add the ligation termination reaction reagent to the 42 μL of reaction product obtained in step (4) above, vortex mix and then perform instantaneous centrifugation to terminate the ligation reaction.

[0320] The composition of the reagent used to terminate the reaction is shown in the table below.

[0321] Table 10 Components and content of ligation termination reaction reagents

[0322] (6) Pooling:

[0323] The ligation products obtained in the above steps (a total of 384) were randomly divided into 4 tubes of 96 each and mixed in equal volumes (pooling). The amount of each ligation product used was 10 μL.

[0324] Take 120 μL of the mixture from each tube, purify it with magnetic beads, and then transfer it to a new 0.2 mL PCR tube. The specific procedure is as follows: Add 30 μL of TE buffer to each 120 μL mixture, then add 36 μL of magnetic beads. Vortex to mix, incubate at room temperature for 5 minutes, then place on a magnetic rack to incubate for 2 minutes (until the liquid is clear). Carefully aspirate the supernatant into a new 0.2 mL PCR tube (only the supernatant is retained in this step). Add another 15 μL of magnetic beads to the supernatant for further purification, vortex to mix, incubate at room temperature for 5 minutes, then place on a magnetic rack to incubate for 2 minutes (until the liquid is clear), and discard the supernatant. Keep the PCR tube on the magnetic rack, add 200 μL of 80% ethanol to the 0.2 mL PCR tube, let stand for 30 seconds, and discard the supernatant. Wash once more with 200 μL of 80% ethanol, discarding as much residual ethanol as possible using a small-range pipette, and air dry at room temperature. Resuspend the magnetic beads in 32 μL of TE buffer, vortex to mix, bind at room temperature for 10 minutes, place on a magnetic rack to bind for 2 minutes (until the liquid is clear), and carefully aspirate 30 μL of supernatant into a new 0.2 mL PCR tube for subsequent reactions or store at -20°C.

[0325] The purified product was quantified using the Qubit dsDNA Assay Kit, following the instructions for use.

[0326] (7) One-step preparation and sequencing of DNA nanospheres (DNB):

[0327] The purified product (24 ng) obtained after the step was used to prepare DNA nanospheres using a one-step method (any commercial kit in the art or a publicly available one-step DNB construction method can be used), and then the obtained DNA nanospheres were sequenced using a DNBSEQ-T7 PE150.

[0328] In this embodiment, the one-step DNB construction method is performed according to the operation steps corresponding to DNBSEQ-T7 PE150 sequencing (see instruction manual), and the sequencing process is carried out on the machine and data analysis according to the standard operation procedure of DNBSEQ-T7 PE150 sequencing.

[0329] Example 4

[0330] The results and data from the above embodiments are summarized to analyze the feasibility of the large-scale whole-genome library construction method described in the above embodiments.

[0331] Figure 2 shows the length detection result of a randomly selected pig ear-derived gDNA sample after fragmentation using the method described in this embodiment of the invention. The detection was performed using an Agilent 2100 bioanalyzer system; please refer to the instruction manual for specific operation details.

[0332] The results showed that the fragments of the gDNA nucleic acid to be tested after being fragmented by the method in the embodiments of the present invention had a size distribution of 100-3000bp, with the peak value of the fragments ranging from 350-800bp. The results of each nucleic acid to be tested randomly selected from each embodiment were similar to these results, indicating that the fragmentation effect of the method in the embodiments of the present invention is stable, and it can reliably obtain DNA fragments with a size distribution of 100-3000bp and a peak value of 350-800bp.

[0333] Taking Example 1 as an example, the difference in sequencing quality between the large-scale PCR-free library preparation and sequencing obtained by the method in the present invention and the sequencing quality of the library obtained by the conventional Fast PCR-free library preparation method was analyzed (all detection parameters were obtained based on the sequencing results using conventional analysis software in the field). The results are shown in the table below and Figures 3-5.

[0334] Table 11 shows the data quality and single-sample analysis results of 384 samples obtained from sequencing using the library construction method described in this embodiment of the invention and the conventional Fast PCR-free library construction method.

[0335] As can be seen from the above results, the sequencing quality of the large-scale PCR-free library preparation and sequencing achieved by the method in the embodiments of the present invention is basically similar to that of the conventional Fast PCR-free library preparation method currently used as the standard method. Both methods exhibit good sequencing quality on the BGI Genomics proprietary high-throughput sequencing platform DNBSEQ-T7, indicating that the large-scale PCR-free library preparation and sequencing achieved by the method in the embodiments of the present invention are accurate and can replace the standard method. However, compared to the conventional Fast PCR-free library preparation method, the method in the embodiments of the present invention allows for single-tube processing and enables the co-detection of multiple samples (up to 96 samples in this embodiment), thereby greatly improving detection efficiency and reducing detection costs, thus providing a superior alternative to the conventional Fast PCR-free library preparation method.

[0336] Example 5

[0337] This embodiment provides a nucleic acid library construction kit or apparatus suitable for use with the methods described in the above embodiments. The nucleic acid library construction kit or apparatus includes a one-tube reaction premix, adapters, ligation reaction reagents, and ligation termination reaction reagents. The one-tube reaction premix includes or consists of the following components: Tris-HCl, dATP, dNTP Mix, magnesium chloride, calcium chloride, T4 PNK, Klenow fragment, rTaq DNA polymerase, T4 DNA polymerase, DNase I (RNase-free), glycerol, and water. The adapter includes or consists of a universal primer sequence and a tag sequence. The universal primer sequence can be obtained through conventional design (according to technical manuals or textbooks in the field), commercial purchase, or any conventional method, depending on the target detection object. The tag sequence can be a barcode sequence or an index sequence. The ligation reaction reagent includes or consists of the following components: PNK buffer, ATP, PEG8000, T4 DNA ligase, and water. The ligation termination reaction reagent is a high-salt reaction reagent with a salt content greater than or equal to 1 mol / L. The salt can include any soluble metal salt or non-metal salt capable of affecting the ligation of nucleic acid molecules.

[0338] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a nucleic acid library, comprising: a. Fragment the nucleic acid to be tested, repair the ends, and add an A to the 3' end of the nucleic acid fragment. b. Terminate or delay the fragmentation reaction, and add a linker and ligation reagent to initiate the ligation reaction. c. Add a ligation termination reagent to terminate or delay the ligation reaction. d. Mix the multiple ligation products from step c, and purify the mixed product to obtain a nucleic acid library; The reactions described in steps a, b, and c are all carried out in the same tube.

2. The construction method according to claim 1, characterized in that, In step d, after purification, the amplification reaction or extension reaction is further included, and the amplification reaction includes isothermal amplification reaction or PCR amplification reaction. The isothermal amplification reaction includes rolling circle amplification.

3. The construction method according to claim 1, characterized in that, Step b further includes a cyclization reaction, which includes: adding an oligonucleotide fragment to step b and performing a single-strand cyclization reaction on the nucleic acid to be tested with adapter sequences at both ends.

4. The construction method according to claim 1, characterized in that, The steps include: Step d is followed by a cyclization reaction, in which oligonucleotide fragments and ligases are added to the purified product of step d to perform a single-strand cyclization reaction on the test nucleic acid with adapter sequences at both ends.

5. The construction method according to claim 1, characterized in that, The termination or delay of the fragmentation reaction in step b includes subjecting the system obtained in step a to high-temperature treatment, wherein the high-temperature treatment is performed at a temperature of 60°C-120°C for a time of 10 min-60 min. Preferably, the high-temperature treatment temperature is 60℃-80℃.

6. The construction method according to claim 5, characterized in that, The linking reagents mentioned in step b include: Nucleotide ligase; Preferably, the nucleic acid ligase includes a DNA ligase; Preferably, the DNA ligase includes, but is not limited to, T4 DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV.

7. The construction method according to claim 6, characterized in that, The ligation reaction reagent in step b further includes a salt ion buffer, wherein the concentration of the salt ion buffer is greater than or equal to 0.05 mol / L, preferably 0.05 to 0.5 mol / L.

8. The construction method according to claim 1, characterized in that, The adapter described in step b includes a universal primer sequence and a tag sequence.

9. The construction method according to claim 1, characterized in that, The ligation termination reagent mentioned in step c is a high-salt reagent; Preferably, the salt content in the connection termination reaction reagent is greater than or equal to 1 mol / L, more preferably 1-20 mol / L; Preferably, the connection termination reaction reagent further includes a buffer solution and a chelating agent; Preferably, the chelating agent includes amino acid compounds, hydroxy acid compounds, phosphates, and carboxylic acid compounds.

10. The construction method according to claim 1, characterized in that, In step d, equal amounts of multiple linker products are mixed; Preferably, the equal quantities include at least one of equal volume, equal mass, and equal concentration.

11. The construction method according to claim 2, characterized in that, The nucleic acid to be tested is derived from at least one of DNA, RNA, or DNA-RNA hybrid chains; Preferably, the DNA includes cDNA, cfDNA, dsDNA, and ssDNA; Preferably, the RNA includes mRNA, lncRNA, and miRNA.

12. The construction method according to claim 1, characterized in that, The total reaction time for steps a to d is less than or equal to 3 hours, preferably 50 to 150 minutes.

13. A nucleic acid library construction kit or apparatus, characterized in that, The nucleic acid library construction kit or package includes a one-tube reaction premix, adapter, ligation reaction reagent, and ligation termination reaction reagent. The one-tube reaction premixed reagent includes: fragmented enzyme and fragmented reaction reagent; The one-tube reaction premixed reagent further includes: 5'-hydroxytransferase and polymerase or their active fragments; Preferably, the 5'-hydroxytransferase comprises T4 polynucleotide kinase (T4 PNK); Preferably, the polymerase or its active fragment comprises DNA polymerase or its active fragment; Preferably, the DNA polymerase or its active fragment includes the Klenow fragment, recombinant Taq DNA polymerase, and T4 DNA polymerase; The connection termination reaction reagent contains a high-salt reaction reagent; Preferably, the salt content in the connection termination reaction reagent is greater than or equal to 1 mol / L, more preferably 1-20 mol / L.

14. The nucleic acid library construction kit or apparatus according to claim 13, characterized in that, The ligation reaction reagent includes a salt ion buffer, wherein the concentration of the salt ion buffer is greater than or equal to 0.05 mol / L, preferably 0.05 to 0.5 mol / L.

15. The nucleic acid library construction kit or apparatus according to claim 13, characterized in that, The connection termination reaction reagent further includes a buffer solution and a chelating agent; Preferably, the chelating agent includes amino acid compounds, hydroxy acid compounds, phosphates, and carboxylic acid compounds.

16. The nucleic acid library construction kit or apparatus according to any one of claims 13-15, characterized in that, The connector includes a universal primer sequence and a tag sequence.

17. The application of the construction method according to any one of claims 1-12 or the nucleic acid library construction kit or apparatus according to any one of claims 13-16 in gene sequencing.

18. A nucleic acid library, characterized in that, The nucleic acid library is constructed using the construction method described in any one of claims 1-12 or the nucleic acid library construction kit or apparatus described in any one of claims 13-16.

19. The nucleic acid library according to claim 18, characterized in that, The nucleic acid library is a genomic library; Preferably, the nucleic acid library is derived from multiple ligation products; Preferably, the number of connection products is greater than or equal to 2; Preferably, the number of the connection products is 2-1000, more preferably 2-500.

20. A sequencing method, comprising the following steps: The library is constructed using the construction method described in any one of claims 1-12 or the nucleic acid library construction kit or apparatus described in any one of claims 13-16, and the library is then sequenced.

21. The sequencing method according to claim 20, characterized in that, The sequencing method also includes analyzing the data obtained from sequencing; Preferably, the analysis includes analyzing the data to obtain at least one of the following: The data included the number of reads, Q30 of the data, barcode splitting rate, GC content, CV of a single sample, average sequencing depth of a single sample, and 1× coverage.

22. A sequencing product, characterized in that, The sequencing products include nucleic acid library construction kits or packages as described in any one of claims 13-16, and sequencing or sequencing aids.

23. The sequencing product according to claim 23, characterized in that, The sequencing or sequencing-aided products include at least one of the following: sequencers, nucleic acid extraction products, reverse transcription products, and nucleic acid purification products.

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