Oligonucleotide set and multiplexed amplicon library construction method

Abstract

Provided are an oligonucleotide set and a multiplexed amplicon library construction method, belonging to the technical field of biology. The provided method can, in a system, use a gene-specific multiplex amplification primer and a library amplification primer to, by means of a connection or complementary extension mode, generate a connection product or an extension product, and then, by means of the connection product or the extension product being used as a long primer, complete construction of an amplicon library in one step. Compared with conventional multiplexed amplicon library construction methods, the provided method has the advantage of being able to decrease an uncovering operation step, avoiding the risk of cross contamination.

Description

Oligonucleotide collection and multiplex amplicon library construction method Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an oligonucleotide set and a multiplex amplicon library construction method. Background Art

[0002] Current amplicon library construction methods all use a two-round PCR amplification method to complete library construction. In the first round of amplification, gene-specific primers with a second-round primer recognition sequence (i.e., index sequence) amplify a specific region; in the second round of amplification, primers with a sequence complementary to the index sequence amplify the first-round product to complete the library amplification. This most common method will bring the following problems: 1. After the first round of amplification, purification and recovery are required, and then the second round system is prepared, and the second round of amplification is carried out, and the final product is recovered. In other words, a total of two rounds of amplification and two rounds of recovery are required, which increases the complexity of the operation. 2. Because the first-round product carries the universal sequence recognized by the second-round primers at both ends, when there are many samples being amplified simultaneously, the first-round product recovery and system preparation operations are prone to cross-contamination through aerosols, thereby affecting the authenticity of the identification fragment signal between samples. 3. Because there are many operational steps between the two rounds of amplification and the need to open the lid and add samples, cross-contamination between samples may occur, which in turn hinders the automation of targeted amplicon sequencing.

[0003] Chinese patent CN104093890B and US patent US10876108B2 disclose a method for constructing an amplicon library by first building a library and then using a specific primer at one end and a universal primer at the other end to amplify a specific fragment. This library construction model still requires multiple rounds of amplicon library construction and still suffers from complex operation steps and aerosol cross-contamination. In the application scenario of targeted pathogen sequencing (tNGS), cross-contamination between samples caused by aerosols has seriously affected the automated application and large-scale promotion of targeted pathogen detection technology.

[0004] Therefore, converting two rounds of amplification into one round of amplification is a trend in technological development, and it is also helpful in solving the problem of cross-contamination between samples in targeted pathogen detection applications. Summary of the Invention

[0005] The present invention aims to provide an oligonucleotide pair and a method for constructing a multiplex amplicon library. By ligating or complementary extension, gene-specific multiplex amplification primers and library amplification primers in a multiplex amplification system are combined to generate ligation products or extension products. These ligation products or extension products then serve as long primers to complete the construction of an amplicon library in a single step. Compared to traditional multiplex amplicon library construction methods, the method provided by the present invention reduces the number of uncapping steps and avoids the risk of cross-contamination.

[0006] The first aspect of the present invention includes an oligonucleotide set, which comprises a first oligonucleotide primer and a second oligonucleotide primer, wherein the first oligonucleotide primer comprises a sequencing adapter sequence, a tag sequence and a sequencing primer sequence in sequence from the 5' end to the 3' end, and the second oligonucleotide primer comprises a reverse complementary sequence of a gene-specific primer and a reverse complementary sequence of a sequencing primer in sequence from the 5' end to the 3' end.

[0007] In some embodiments, the first oligonucleotide primer and the second oligonucleotide primer are complementary paired through the sequencing primer sequence and the reverse complement sequence of the sequencing primer.

[0008] In some embodiments, the "reverse complementary sequence" refers to a nucleotide sequence related by the base pairing rules. For example, the reverse complementary sequence of the sequence "5'-ATCG-3'" is "5'-CGAT-3'".

[0009] In some embodiments, the tag sequence is a random sequence used to distinguish different samples to be tested. In some embodiments, the tag sequence is at least 3 nucleotides, such as 4-12 nucleotides, preferably 6-8 nucleotides, such as 8 nucleotides.

[0010] In some embodiments, the gene-specific primer sequence is a specific primer sequence designed according to the target fragment of the DNA sample to be tested. For example, the gene-specific primer sequence comprises at least 3 nucleotides, such as 3-50 nucleotides, such as 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 22, 25, 26, 28, 30, 32, 35, 38, 40, 42, 45, 48 and 50 nucleotides.

[0011] In some embodiments, one or more nucleotides of the reverse complementary sequence of the gene-specific primer contain RNA base modifications, and the modifications are at least one of rA (adenine ribonucleoside), rU (uridine ribonucleoside), rG (guanine ribonucleoside), and rC (cytosine ribonucleoside). In some embodiments, the number of the modifications is at least 1, for example, 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, or 15.

[0012] In some embodiments, when the reverse complementary sequence of the gene-specific primer contains RNA base modifications at multiple nucleotide positions, the multiple nucleotide positions are continuous or discontinuous and are independently one of rA, rU, rG and rC.

[0013] In some embodiments, one or more nucleotide positions in the second oligonucleotide primer contain RNA base modifications, and the modifications are at least one of rA (adenine ribonucleoside), rU (uridine ribonucleoside), rG (guanine ribonucleoside) and rC (cytosine ribonucleoside), and the number of the modifications is at least 1, for example, 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18 or 20.

[0014] In some embodiments, when multiple nucleotide positions in the second oligonucleotide primer contain RNA base modifications, the multiple nucleotide positions are continuous or discontinuous and are independently one of rA, rU, rG and rC.

[0015] In some embodiments, the 3' end of the second oligonucleotide primer contains a blocking modification, which prevents the addition of dNTPs to the 3' end of the second oligonucleotide primer for extension. In some embodiments, the blocking modification is a 3' phosphorylation modification or a spacer modification; preferably, the spacer modification is a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification, or a PC-linker modification.

[0016] In some embodiments, the first oligonucleotide primers include at least a pair of an upstream first oligonucleotide primer and a downstream first oligonucleotide primer.

[0017] In some embodiments, the upstream first oligonucleotide primer comprises an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream first oligonucleotide primer comprises a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5' end to the 3' end.

[0018] In some embodiments, the upstream first oligonucleotide primer does not comprise an (upstream) tag sequence, and / or the downstream first oligonucleotide primer does not comprise a (downstream) tag sequence.

[0019] In some embodiments, the sequencing adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to the Iontorrent platform, the Illumina platform, and the BGI platform).

[0020] In some embodiments, the sequencing adapter sequence includes an upstream sequencing adapter sequence and / or a downstream sequencing adapter sequence.

[0021] In some embodiments, the upstream sequencing adapter sequence is the P5 adapter sequence of the Illumina platform, and the downstream sequencing adapter sequence is the P7 adapter sequence of the Illumina platform; or vice versa.

[0022] In some embodiments, the upstream sequencing adapter sequence is a P1 adapter sequence of an ion torrent platform, and the downstream sequencing adapter sequence is an A adapter sequence of an ion torrent platform, or vice versa.

[0023] In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for single-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for single-ended tag library construction on the MGI platform; or vice versa. In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for double-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for double-ended tag library construction on the MGI platform; or vice versa.

[0024] In some embodiments, the upstream first oligonucleotide primer is an i5 primer of the Illumina platform, and the downstream first oligonucleotide primer is an i7 primer of the Illumina platform; or vice versa.

[0025] In some embodiments, the second oligonucleotide primers include at least a pair of an upstream second oligonucleotide primer and a downstream second oligonucleotide primer.

[0026] In some embodiments, the upstream second oligonucleotide primer comprises, from the 5' end to the 3' end, a reverse complementary sequence of a gene-specific upstream primer and a reverse complementary sequence of an upstream sequencing primer.

[0027] In some embodiments, the downstream second oligonucleotide primer comprises, from the 5' end to the 3' end, a reverse complementary sequence of a gene-specific downstream primer and a reverse complementary sequence of a downstream sequencing primer.

[0028] In some embodiments, the upstream first oligonucleotide primer and the upstream second oligonucleotide primer are complementary paired through the upstream sequencing primer sequence and the reverse complementary sequence of the upstream sequencing primer.

[0029] In some embodiments, the downstream first oligonucleotide primer and the downstream second oligonucleotide primer are complementary paired through the downstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer.

[0030] The second aspect of the present invention provides a composition comprising a DNA polymerase, an endoribonuclease, dNTPs, a buffer component, a metal salt and a primer combination; wherein the primer combination comprises the first oligonucleotide primer described in the first aspect and the second oligonucleotide primer described in the first aspect, in particular, at least one pair of an upstream first oligonucleotide primer and a downstream first oligonucleotide primer and at least one pair of an upstream second oligonucleotide primer and a downstream second oligonucleotide primer.

[0031] In some embodiments, the first oligonucleotide primer and the second oligonucleotide primer can be paired by a sequencing primer sequence and a reverse complementary sequence of the sequencing primer. In some embodiments, the first oligonucleotide primer and the second oligonucleotide primer are each present in the composition in a single-stranded form. In some embodiments, the first oligonucleotide primer and the second oligonucleotide primer are present in the composition in the form of a partial double-stranded structure formed by the sequencing primer sequence and the reverse complementary sequence of the sequencing primer.

[0032] In some embodiments, the first oligonucleotide primer comprises at least one pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer described in the first aspect, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0033] In some embodiments, each pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer differs in that the tag sequence is different.

[0034] In some embodiments, the second oligonucleotide primers comprise at least one pair of the upstream second oligonucleotide primers described in the first aspect and the downstream second oligonucleotide primers described in the first aspect, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0035] In some embodiments, each pair of the upstream second oligonucleotide primer and the downstream second oligonucleotide primer differs in that the gene-specific primer sequences are different.

[0036] In some embodiments, the composition further comprises a DNA sample.

[0037] In some embodiments, the DNA sample is gDNA or cDNA.

[0038] In some embodiments, the DNA polymerase comprises at least one thermostable DNA polymerase.

[0039] In some embodiments, the DNA polymerase comprises at least one thermostable DNA polymerase and one room-temperature DNA polymerase.

[0040] In some embodiments, the thermostable DNA polymerase is Taq DNA polymerase, Pfu DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, or KOD DNA polymerase.

[0041] In some embodiments, the thermostable polymerase is a hot-start polymerase, such as an antibody-blocked polymerase or a ligand-blocked polymerase.

[0042] In some embodiments, the DNA polymerase comprises at least one non-hot-start polymerase and one hot-start polymerase.

[0043] In some embodiments, the room-temperature DNA polymerase is Klenow DNA polymerase, Bst DNA polymerase, or Phi 29 DNA polymerase.

[0044] In some embodiments, the "room-temperature polymerase" described in the present invention refers to a polymerase that can perform polymerization activity at room temperature, such as below 45°C.

[0045] In some embodiments, the endoribonuclease recognizes the RNA / DNA hybrid and cleaves the RNA strand.

[0046] In some embodiments, the endoribonuclease is an endoribonuclease that can recognize ribonucleotide sites and remove phosphodiester bonds; preferably, the endoribonuclease is RNase H2.

[0047] In some embodiments, the endoribonuclease is a homologous protein to RNase H2.

[0048] In some embodiments, the buffer component is one or more of Tris, Tris-HCl, Tris base, or HEPES, preferably Tris.

[0049] In some embodiments, the metal salt is Mg 2+ Salt, preferably MgCl2.

[0050] In some embodiments, the metal salt is Mg 2+ Salt, and K + Salt, Mn 2+ Salt, Cs + Salt, Na + Salt and Ca 2+ One or more salts.

[0051] In some embodiments, the composition further comprises other components known in the art that can help DNA polymerase and endoribonuclease to function, such as glycerol.

[0052] In some embodiments, the composition further comprises a surfactant, such as one or more of anionic surfactants, cationic surfactants, and nonionic surfactants.

[0053] A third aspect of the present invention provides a method for constructing a multiplex amplicon library, comprising the following steps:

[0054] (1) preparing a reaction system comprising at least one pair of first oligonucleotide primers, at least one pair of second oligonucleotide primers, and a sample nucleic acid; wherein the first oligonucleotide primer comprises a sequencing adapter sequence, a tag sequence, and a sequencing primer sequence in sequence from the 5' end to the 3' end, and the second oligonucleotide primer comprises a reverse complementary sequence of a gene-specific primer and a reverse complementary sequence of a sequencing primer in sequence from the 5' end to the 3' end, and the second oligonucleotide primer comprises a cleavable group;

[0055] (2) allowing the 3' end of the first oligonucleotide primer and the 3' end of the second oligonucleotide primer to be reverse complementary and pairing, and extending the first oligonucleotide primer in the 5'-3' direction to obtain an extension product;

[0056] (3) cutting the reverse complementary sequence of the gene-specific primer in the second oligonucleotide primer;

[0057] (4) Performing PCR amplification on the sample nucleic acid using the extension product of step (2) as a primer.

[0058] In some embodiments, the tag sequence is a random sequence used to distinguish different samples. In some embodiments, the tag sequence is at least 3 nucleotides, such as 4-12 nucleotides, preferably 6-8 nucleotides, such as 8 nucleotides.

[0059] In some embodiments, the gene-specific primer sequence is a specific primer sequence designed according to the target fragment of the DNA sample to be tested. For example, the gene-specific primer sequence comprises at least 3 nucleotides, such as 3-50 nucleotides, such as 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 22, 25, 26, 28, 30, 32, 35, 38, 40, 42, 45, 48 and 50 nucleotides.

[0060] In some embodiments, the 3' end of the second oligonucleotide primer contains a blocking modification, which prevents the addition of dNTPs to the 3' end of the second oligonucleotide primer for extension. In some embodiments, the blocking modification is a 3' phosphorylation modification or a spacer modification; preferably, the spacer modification is a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification, or a PC-linker modification.

[0061] In some embodiments, the at least one pair of first oligonucleotide primers includes at least one pair of an upstream first oligonucleotide primer and a downstream first oligonucleotide primer.

[0062] In some embodiments, the upstream first oligonucleotide primer comprises an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream first oligonucleotide primer comprises a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5' end to the 3' end.

[0063] In some embodiments, the upstream first oligonucleotide primer does not comprise an (upstream) tag sequence, and / or the downstream first oligonucleotide primer does not comprise a (downstream) tag sequence.

[0064] In some embodiments, the sequencing adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to the Ion torrent platform, the Illumina platform, and the BGI platform).

[0065] In some embodiments, the sequencing adapter sequence includes an upstream sequencing adapter sequence and a downstream sequencing adapter sequence.

[0066] In some embodiments, the upstream sequencing adapter sequence is the P5 adapter sequence of the Illumina platform, and the downstream sequencing adapter sequence is the P7 adapter sequence of the Illumina platform; or vice versa.

[0067] In some embodiments, the upstream sequencing adapter sequence is the P1 adapter sequence of the Ion torrent platform, and the downstream sequencing adapter sequence is the A adapter sequence of the Ion torrent platform, or vice versa.

[0068] In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for single-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for single-ended tag library construction on the MGI platform; or vice versa. In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for double-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for double-ended tag library construction on the MGI platform; or vice versa.

[0069] In some embodiments, the upstream first oligonucleotide primer is an i5 primer of the Illumina platform, and the downstream first oligonucleotide primer is an i7 primer of the Illumina platform; or vice versa.

[0070] In some embodiments, the at least one pair of second oligonucleotide primers includes at least one pair of an upstream second oligonucleotide primer and a downstream second oligonucleotide primer.

[0071] In some embodiments, the upstream second oligonucleotide primer comprises, from the 5' end to the 3' end, a reverse complementary sequence of a gene-specific upstream primer and a reverse complementary sequence of an upstream sequencing primer.

[0072] In some embodiments, the downstream second oligonucleotide primer comprises, from the 5' end to the 3' end, a reverse complementary sequence of a gene-specific downstream primer and a reverse complementary sequence of a downstream sequencing primer.

[0073] In some embodiments, the upstream first oligonucleotide primer and the upstream second oligonucleotide primer are complementary paired through the upstream sequencing primer sequence and the reverse complementary sequence of the upstream sequencing primer.

[0074] In some embodiments, the downstream first oligonucleotide primer and the downstream second oligonucleotide primer are complementary paired through the downstream sequencing primer sequence and the reverse complementary sequence of the upstream sequencing primer.

[0075] In some embodiments, the first oligonucleotide primer comprises at least one pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer described in the first aspect, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0076] In some embodiments, each pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer differs in that the tag sequence is different.

[0077] In some embodiments, the second oligonucleotide primer comprises at least one pair of the upstream second oligonucleotide primer and the downstream second oligonucleotide primer described in the first aspect, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0078] In some embodiments, each pair of the upstream second oligonucleotide primer and the downstream second oligonucleotide primer differs in that the gene-specific primer sequences are different.

[0079] In some embodiments, the sample nucleic acid is DNA, for example, the DNA is gDNA or cDNA.

[0080] In some embodiments, the cleavable group refers to a nucleotide substrate that can be recognized and cleaved by an endonuclease or an exonuclease.

[0081] In some embodiments, the cleavable group is deoxyuridine triphosphate (dUTP) or deoxyinosine triphosphate (dITP). In some embodiments, when the cleavable group is dUTP, the reaction system further comprises uracil DNA glycosylase (UDG enzyme) or single-strand selective monofunctional uracil-DNA glycosylase (SMUG enzyme); when the cleavable group is dITP, the reaction system further comprises alkyladenine DNA glycosylase (AAG enzyme) or endonuclease V.

[0082] In some embodiments, the cleavable group is an RNA base, and the RNA base is at least one of rA (adenine ribonucleoside), rU (uridine ribonucleoside), rG (guanine ribonucleoside), and rC (cytosine ribonucleoside). In some embodiments, the number of the RNA base is at least 1, such as 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, or 15. In all embodiments, when the cleavable group is an RNA base, the reaction system further comprises an endoribonuclease, which is an endoribonuclease that can recognize ribonucleotide sites and remove phosphodiester bonds; preferably, the endoribonuclease is RNase H2 or a homologous protein of RNase H2.

[0083] In some embodiments, the cleavable group is located at the reverse complement of the gene-specific primer and / or the reverse complement of the sequencing primer in the second oligonucleotide primer.

[0084] In some embodiments, step (3) is to cut the cleavable group on the reverse complementary sequence of the gene-specific primer in the second oligonucleotide primer under the action of a nuclease endonuclease or an exonuclease, for example, to recognize and cut the ribonucleotide site on the reverse complementary sequence of the gene-specific primer under the action of a ribonuclease endonuclease.

[0085] In some embodiments, the first oligonucleotide primers include at least one pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0086] In some embodiments, each pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer differs in that the tag sequence is different.

[0087] In some embodiments, the second oligonucleotide primers comprise at least one pair of the upstream second oligonucleotide primer and the downstream second oligonucleotide primer, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0088] In some embodiments, each pair of the upstream second oligonucleotide primer and the downstream second oligonucleotide primer differs in that the gene-specific primer sequences are different.

[0089] In some embodiments, the reaction system further includes a DNA polymerase.

[0090] In some embodiments, the DNA polymerase comprises at least one thermostable DNA polymerase.

[0091] In some embodiments, the DNA polymerase comprises at least one thermostable DNA polymerase and one room-temperature DNA polymerase.

[0092] In some embodiments, the thermostable polymerase is a hot-start polymerase, such as an antibody-blocked polymerase or a ligand-blocked polymerase.

[0093] In some embodiments, the DNA polymerase comprises at least one non-hot-start polymerase and one hot-start polymerase.

[0094] In some embodiments, the thermostable DNA polymerase is Taq DNA polymerase, Pfu DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, or KOD DNA polymerase.

[0095] In some embodiments, the room-temperature DNA polymerase is Klenow DNA polymerase, Bst DNA polymerase, or Phi 29 DNA polymerase.

[0096] In some embodiments, the reaction of step (2) is performed under the action of a non-hot-start polymerase; and the reaction of step (4) is performed under the action of a hot-start polymerase.

[0097] In some embodiments, the reaction of step (2) is carried out under the action of a room temperature polymerase; and the reaction of step (4) is carried out under the action of a heat-resistant polymerase.

[0098] In some embodiments, the reaction temperature of step (2) is 20-50°C, preferably 25-40°C, for example 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0099] In some embodiments, the reaction time of step (2) is 5-40 minutes, preferably 10-30 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes or 30 minutes.

[0100] In some embodiments, the reaction conditions of step (2) are incubation at 25-40°C for 10-30 minutes, incubation at 25-40°C for 10-25 minutes, incubation at 25-40°C for 10-20 minutes, or incubation at 30°C for 10-20 minutes, including but not limited to any combination of the reaction temperature of the aforementioned step (2) and the reaction time of step (2).

[0101] In some embodiments, the reaction temperature of step (3) is 40-80°C, preferably 60-80°C, for example 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C.

[0102] In some embodiments, the reaction time of step (3) is 5-30 minutes, preferably 5-20 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 5 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes or 20 minutes.

[0103] In some embodiments, the reaction conditions of step (3) are incubation at 60-80°C for 10-20 minutes, incubation at 65-80°C for 10-20 minutes, incubation at 70-80°C for 10-20 minutes, incubation at 70-80°C for 10-15 minutes, incubation at 70-75°C for 10-15 minutes, incubation at 72°C for 10 minutes, or incubation at 72°C for 10 minutes, including but not limited to any combination of the reaction temperature of the aforementioned step (3) and the reaction time of step (3).

[0104] In some embodiments, during the reaction of step (2), the extension product generated by the complementary extension of the first oligonucleotide primer and the second oligonucleotide primer under the action of DNA polymerase is an Index-Panel long primer, and the Index-Panel long primer comprises a sequencing adapter sequence-a tag sequence-a sequencing primer sequence-a gene-specific primer sequence from the 5' end to the 3' end.

[0105] In some embodiments, the reaction system further comprises a buffer component. In some embodiments, the buffer component is one or more of Tris, Tris-HCl, Tris base or HEPES, preferably Tris.

[0106] In some embodiments, the reaction system further comprises a metal salt. In some embodiments, the metal salt is Mg 2+ Salt, preferably MgCl2. In some embodiments, the metal salt is Mg 2+ Salt, and K + Salt, Mn 2+ Salt, Cs + Salt, Na + Salt and Ca 2+ One or more salts.

[0107] In some embodiments, the reaction system further comprises dNTPs.

[0108] In some embodiments, the reaction system further comprises other components known in the art that can help DNA polymerase and endoribonuclease to function, such as glycerol.

[0109] In some embodiments, the reaction system further comprises a surfactant, such as one or more of anionic surfactants, cationic surfactants, and nonionic surfactants.

[0110] In some embodiments, the PCR amplification in step (4) includes one or more cycles (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40) of denaturation, annealing, and extension; preferably, the PCR amplification includes pre-denaturation and multiple cycles (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40) of denaturation, annealing, and extension.

[0111] In some embodiments, the denaturation is high temperature denaturation, and the high temperature denaturation is high temperature denaturation of at least 90°C, such as 90°C, 91°C, 92°C, 93°C, 94°C and 95°C.

[0112] In some embodiments, the denaturation simultaneously achieves: (1) denaturing the sample DNA from double strands to single strands; and (2) inactivating the RNase H2 protein.

[0113] In some embodiments, the PCR amplification is one round of PCR amplification.

[0114] In some embodiments, the PCR amplification conditions can be any conventional PCR amplification conditions known to those skilled in the art.

[0115] In some embodiments, the method further comprises step (5) of purifying the amplified product of the sample nucleic acid.

[0116] In some embodiments, the purification is carried out by a magnetic bead method, a high salt precipitation method, a centrifugal column method or a phenol-chloroform extraction method, preferably a magnetic bead method.

[0117] A fourth aspect of the present invention provides an oligonucleotide primer, wherein the oligonucleotide comprises, from the 5' end to the 3' end, a sequencing adapter sequence-a tag sequence-a sequencing primer sequence-a gene-specific primer sequence.

[0118] The fifth aspect of the present invention provides an oligonucleotide combination, which comprises a first oligonucleotide primer, a third oligonucleotide primer and a bridge primer, wherein the first oligonucleotide primer comprises a sequencing adapter sequence, a tag sequence and a sequencing primer sequence in sequence from the 5' end to the 3' end, the third oligonucleotide primer comprises a universal sequence and a gene-specific primer sequence in sequence from the 5' end to the 3' end, and the bridge primer comprises a reverse complementary sequence of the universal sequence and a reverse complementary sequence of the sequencing primer in sequence from the 5' end to the 3' end.

[0119] In some embodiments, the tag sequence is a random sequence for distinguishing different samples. In some embodiments, the tag sequence is at least 3 nucleotides, such as 4-12 nucleotides, preferably 6-8 oligonucleotides, such as 8 nucleotides.

[0120] In some embodiments, the gene-specific primer sequence is a specific primer sequence designed according to the target fragment of the DNA sample to be tested. For example, the gene-specific primer sequence comprises at least 3 nucleotides, such as 3-50 nucleotides, such as 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 22, 25, 26, 28, 30, 32, 35, 38, 40, 42, 45, 48 and 50 nucleotides.

[0121] In some embodiments, the reverse complementary sequence of the universal sequence refers to a sequence that is partially or completely reverse complementary to the universal sequence, and the reverse complementary sequence of the sequencing primer refers to a sequence that is partially or completely reverse complementary to the sequencing primer.

[0122] In some embodiments, the 5' end of the third oligonucleotide primer contains or does not contain a phosphorylation modification.

[0123] In some embodiments, the 3' end of the bridge primer contains a blocking modification, which prevents dNTP addition to the 3' end of the bridge primer for extension. In some embodiments, the blocking modification is a 3' phosphorylation modification or a spacer modification; preferably, the spacer modification is a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification, or a PC-linker modification.

[0124] In some embodiments, the universal sequence is a random sequence, and the universal sequence is not identical to or complementary to the sequencing adapter sequence, the tag sequence, and / or the sequencing primer sequence.

[0125] In some embodiments, the universal sequence is a fixed sequence that serves to connect the first oligonucleotide primer to the third oligonucleotide primer by being complementary to the bridge primer, and the universal sequence is not identical to or complementary to the sequencing adapter sequence, the tag sequence, and / or the sequencing primer sequence.

[0126] In some embodiments, the universal sequence is not identical to or complementary to the target gene sequence.

[0127] In some embodiments, the universal sequence is balanced in base composition.

[0128] In some embodiments, the length of the universal sequence is 5-200bp, for example, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 15bp, 20bp, 25bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 120bp, 150bp, 160bp, 180bp and 200bp.

[0129] In some embodiments, the first oligonucleotide primers include at least a pair of an upstream first oligonucleotide primer and a downstream first oligonucleotide primer.

[0130] In some embodiments, the upstream first oligonucleotide primer comprises an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream first oligonucleotide primer comprises a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5' end to the 3' end.

[0131] In some embodiments, the upstream first oligonucleotide primer does not comprise an (upstream) tag sequence, and / or the downstream first oligonucleotide primer does not comprise a (downstream) tag sequence.

[0132] In some embodiments, the sequencing adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to ion torrent platform, Illumina platform and BGI platform).

[0133] In some embodiments, the sequencing adapter sequence includes an upstream sequencing adapter sequence and a downstream sequencing adapter sequence.

[0134] In some embodiments, the upstream sequencing adapter sequence is the P5 adapter sequence of the Illumina platform, and the downstream sequencing adapter sequence is the P7 adapter sequence of the Illumina platform; or vice versa.

[0135] In some embodiments, the upstream sequencing adapter sequence is the P1 adapter sequence of the Ion torrent platform, and the downstream sequencing adapter sequence is the A adapter sequence of the Iontorrent platform; or vice versa.

[0136] In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for single-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for single-ended tag library construction on the MGI platform; or vice versa. In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for double-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for double-ended tag library construction on the MGI platform; or vice versa.

[0137] In some embodiments, the upstream first oligonucleotide primer is an i5 primer of the Illumina platform, and the downstream first oligonucleotide primer is an i7 primer of the Illumina platform; or vice versa.

[0138] In some embodiments, the third oligonucleotide primers include at least a pair of an upstream third oligonucleotide primer and a downstream third oligonucleotide primer.

[0139] In some embodiments, the upstream third oligonucleotide primer comprises an upstream universal sequence and a gene-specific upstream primer sequence sequentially from the 5' end to the 3' end.

[0140] In some embodiments, the downstream third oligonucleotide primer comprises a downstream universal sequence and a gene-specific downstream primer sequence sequentially from the 5' end to the 3' end.

[0141] In some embodiments, the bridge primers include at least one pair of an upstream bridge primer and a downstream bridge primer.

[0142] In some embodiments, the upstream bridge primer includes, from the 5' end to the 3' end, a reverse complementary sequence of the upstream universal sequence and a reverse complementary sequence of the upstream sequencing primer.

[0143] In some embodiments, the downstream bridge primer includes, from the 5' end to the 3' end, a reverse complementary sequence of the downstream universal sequence and a reverse complementary sequence of the downstream sequencing primer.

[0144] In some embodiments, the upstream sequencing primer is sequencing primer 1 of the TruSeq library structure, and the downstream sequencing primer is sequencing primer 2 of the TruSeq library structure; or vice versa.

[0145] In some embodiments, the upstream sequencing primer is sequencing primer 1 of the Nextera library structure, and the downstream sequencing primer is sequencing primer 2 of the Nextera library structure; or vice versa.

[0146] In some embodiments, the upstream sequencing primer is sequencing primer 1 of a TruSeq library structure, and the downstream sequencing primer is sequencing primer 2 of a Nextera library structure; or vice versa.

[0147] In some embodiments, the upstream sequencing primer is sequencing primer 1 of the Nextera library structure, and the downstream sequencing primer is sequencing primer 2 of the TruSeq library structure; or vice versa.

[0148] The sixth aspect of the present invention provides a composition comprising DNA polymerase, T4 DNA ligase, ATP, dNTP, a buffer component, a metal salt and a primer combination; the primer combination comprises the first oligonucleotide primer, the third oligonucleotide primer and the bridge primer described in the fifth aspect.

[0149] In some embodiments, the first oligonucleotide primer, the third oligonucleotide primer, and the bridge primer are each present in the composition in a single-stranded form. In some embodiments, the first oligonucleotide primer and the bridge primer are complementary to each other through the sequencing primer sequence, and the third oligonucleotide primer and the bridge primer are complementary to each other through the universal primer sequence to form a double-stranded complementary structure of three primers present in the composition.

[0150] In some embodiments, the first oligonucleotide primer comprises at least one pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer described in the fourth aspect, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0151] In some embodiments, each pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer differs in that the tag sequence is different.

[0152] In some embodiments, the third oligonucleotide primer comprises at least one pair of the upstream third oligonucleotide primer and the downstream third oligonucleotide primer described in the fifth aspect, for example, at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0153] In some embodiments, each pair of the upstream third oligonucleotide primer and the downstream third oligonucleotide primer differs in that the gene-specific primer sequences are different.

[0154] In some embodiments, the bridge primer comprises at least one pair of the upstream bridge primer described in the fifth aspect and the downstream bridge primer described in the fourth aspect, for example, at least one pair or two pairs.

[0155] In some embodiments, each pair of the upstream bridge primer and the downstream bridge primer differs in that the sequencing primers are different.

[0156] In some embodiments, the composition further comprises a DNA sample.

[0157] In some embodiments, the DNA sample is gDNA or cDNA.

[0158] In some embodiments, the 5' end of the third oligonucleotide primer contains or does not contain a phosphorylation modification.

[0159] In some embodiments, when the 5' end of the third oligonucleotide primer does not contain phosphorylation modification, the composition further comprises a substance capable of causing phosphorylation modification of the 5' end of the third oligonucleotide primer, such as T4 polynucleotide kinase (T4 PNK).

[0160] In some embodiments, the mass ratio or molar ratio of the first oligonucleotide primer to the third oligonucleotide primer is N:1, where N is a constant greater than or equal to 1 and less than or equal to 5, such as 1, 2, 3, 4, and 5.

[0161] In some embodiments, the DNA polymerase is any thermostable DNA polymerase known in the art. In some embodiments, the DNA polymerase is one or more of Taq DNA polymerase, Pfu DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, and KOD DNA polymerase.

[0162] In some embodiments, the buffer component is one or more of Tris, Tris-HCl, Tris base, or HEPES, preferably Tris.

[0163] In some embodiments, the metal salt is Mg 2+ Salt, preferably MgCl2.

[0164] In some embodiments, the metal salt is Mg 2+ Salt, and K + Salt, Mn 2+ Salt, Cs + Salt, Na + Salt and Ca 2+ One or more salts.

[0165] In some embodiments, the composition further comprises other substances that can help the components of the composition, such as enzymes, to function, such as glycerol.

[0166] In some embodiments, the composition further comprises a surfactant, such as one or more of anionic surfactants, cationic surfactants, and nonionic surfactants.

[0167] A seventh aspect of the present invention provides a method for constructing a multiplex amplicon library, the method comprising the following steps:

[0168] (1) preparing a reaction system comprising at least one pair of first oligonucleotide primers, at least one pair of third oligonucleotide primers, at least one pair of bridge primers, and a sample nucleic acid; wherein the first oligonucleotide primers sequentially comprise a sequencing adapter sequence, a tag sequence, and a sequencing primer sequence from the 5' end to the 3' end, the third oligonucleotide primers sequentially comprise a universal sequence and a gene-specific primer sequence from the 5' end to the 3' end, and the bridge primers sequentially comprise a reverse complementary sequence of the universal sequence and a reverse complementary sequence of the sequencing primer from the 5' end to the 3' end;

[0169] (2) making the 3' end of the first oligonucleotide primer and the 3' end of the bridge primer reverse complementary pairing, making the 5' end of the third oligonucleotide primer and the 5' end of the bridge primer reverse complementary pairing, and connecting the first oligonucleotide primer and the third oligonucleotide primer to obtain a connection product;

[0170] (3) Performing PCR amplification on the sample nucleic acid using the ligation product described in step (2) as a primer.

[0171] In some embodiments, the tag sequence is a random sequence for distinguishing different samples. In some embodiments, the tag sequence is at least 3 nucleotides, such as 4-12 nucleotides, preferably 6-8 nucleotides, such as 8 nucleotides.

[0172] In some embodiments, the gene-specific primer sequence is a specific primer sequence designed according to the target fragment of the DNA sample to be tested. For example, the gene-specific primer sequence comprises at least 3 nucleotides, such as 3-50 nucleotides, such as 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 22, 25, 26, 28, 30, 32, 35, 38, 40, 42, 45, 48 and 50 nucleotides.

[0173] In some embodiments, the reverse complementary sequence of the universal sequence refers to a sequence that is partially or completely reverse complementary to the universal sequence, and the reverse complementary sequence of the sequencing primer refers to a sequence that is partially or completely reverse complementary to the sequencing primer.

[0174] In some embodiments, the 5' end of the third oligonucleotide primer contains or does not contain a phosphorylation modification.

[0175] In some embodiments, the 3' end of the bridge primer contains a blocking modification, which prevents dNTP addition to the 3' end of the bridge primer for extension. In some embodiments, the blocking modification is a 3' phosphorylation modification or a spacer modification; preferably, the spacer modification is a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification, or a PC-linker modification.

[0176] In some embodiments, the universal sequence is a random sequence, and the universal sequence is not identical to or complementary to the sequencing adapter sequence, the tag sequence, and / or the sequencing primer sequence.

[0177] In some embodiments, the universal sequence is a fixed sequence that serves to connect the first oligonucleotide primer to the third oligonucleotide primer by being complementary to the bridge primer, and the universal sequence is not identical to or complementary to the sequencing adapter sequence, the tag sequence, and / or the sequencing primer sequence.

[0178] In some embodiments, the universal sequence is not identical to or complementary to the target gene sequence.

[0179] In some embodiments, the universal sequence is balanced in base composition.

[0180] In some embodiments, the length of the universal sequence is 10-200bp, for example, 10bp, 15bp, 20bp, 25bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 120bp, 150bp, 160bp, 180bp and 200bp.

[0181] In some embodiments, the at least one pair of first oligonucleotide primers includes at least one pair of an upstream first oligonucleotide primer and a downstream first oligonucleotide primer.

[0182] In some embodiments, the upstream first oligonucleotide primer comprises an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream first oligonucleotide primer comprises a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5' end to the 3' end.

[0183] In some embodiments, the upstream first oligonucleotide primer does not comprise an (upstream) tag sequence, and / or the downstream first oligonucleotide primer does not comprise a (downstream) tag sequence.

[0184] In some embodiments, the sequencing adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to the Iontorrent platform, the Illumina platform, and the BGI platform).

[0185] In some embodiments, the at least one pair of sequencing adapter sequences includes at least one pair of an upstream sequencing adapter sequence and a downstream sequencing adapter sequence.

[0186] In some embodiments, the upstream sequencing adapter sequence is the P5 adapter sequence of the Illumina platform, and the downstream sequencing adapter sequence is the P7 adapter sequence of the Illumina platform; or vice versa.

[0187] In some embodiments, the upstream sequencing adapter sequence is the P1 adapter sequence of the Ion torrent platform, and the downstream sequencing adapter sequence is the A adapter sequence of the Iontorrent platform; or vice versa.

[0188] In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for single-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for single-ended tag library construction on the MGI platform; or vice versa. In some embodiments, the upstream sequencing adapter sequence is the upstream splint sequence for double-ended tag library construction on the MGI platform, and the downstream sequencing adapter sequence is the downstream splint sequence for double-ended tag library construction on the MGI platform; or vice versa.

[0189] In some embodiments, the upstream first oligonucleotide primer is an i5 primer of the Illumina platform, and the downstream first oligonucleotide primer is an i7 primer of the Illumina platform; or vice versa.

[0190] In some embodiments, the third oligonucleotide primers include at least a pair of an upstream third oligonucleotide primer and a downstream third oligonucleotide primer.

[0191] In some embodiments, the upstream third oligonucleotide primer comprises an upstream universal sequence and a gene-specific upstream primer sequence sequentially from the 5' end to the 3' end.

[0192] In some embodiments, the downstream third oligonucleotide primer comprises a downstream universal sequence and a gene-specific downstream primer sequence sequentially from the 5' end to the 3' end.

[0193] In some embodiments, the at least one pair of bridge primers includes at least one pair of an upstream bridge primer and a downstream bridge primer.

[0194] In some embodiments, the upstream bridge primer includes, from the 5' end to the 3' end, a reverse complementary sequence of the upstream universal sequence and a reverse complementary sequence of the upstream sequencing primer.

[0195] In some embodiments, the downstream bridge primer includes, from the 5' end to the 3' end, a reverse complementary sequence of the downstream universal sequence and a reverse complementary sequence of the downstream sequencing primer.

[0196] In some embodiments, the upstream sequencing primer is sequencing primer 1 of the TruSeq library structure, and the downstream sequencing primer is sequencing primer 2 of the TruSeq library structure; or vice versa.

[0197] In some embodiments, the upstream sequencing primer is sequencing primer 1 of the Nextera library structure, and the downstream sequencing primer is sequencing primer 2 of the Nextera library structure; or vice versa.

[0198] In some embodiments, the upstream sequencing primer is sequencing primer 1 of a TruSeq library structure, and the downstream sequencing primer is sequencing primer 2 of a Nextera library structure; or vice versa.

[0199] In some embodiments, the upstream sequencing primer is sequencing primer 1 of the Nextera library structure, and the downstream sequencing primer is sequencing primer 2 of the TruSeq library structure; or vice versa.

[0200] In some embodiments, the at least one pair of first oligonucleotide primers comprises at least one pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer, such as at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0201] In some embodiments, each pair of the upstream first oligonucleotide primer and the downstream first oligonucleotide primer differs in that the tag sequence is different.

[0202] In some embodiments, the at least one pair of third oligonucleotide primers comprises at least one pair of the upstream third oligonucleotide primer and the downstream third oligonucleotide primer, such as at least 1 pair, 5 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs, 200 pairs, 300 pairs and 500 pairs, including but not limited to any natural number between 1-1000.

[0203] In some embodiments, each pair of the upstream third oligonucleotide primer and the downstream third oligonucleotide primer differs in that the gene-specific primer sequences are different.

[0204] In some embodiments, the at least one pair of bridge primers comprises at least one pair of the upstream bridge primer and the downstream bridge primer, such as at least 1 pair or 2 pairs.

[0205] In some embodiments, each pair of the upstream bridge primer and the downstream bridge primer differs in that the sequencing primers are different.

[0206] In some embodiments, the reaction temperature of step (2) is 20-50°C, preferably 25-45°C, for example 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C.

[0207] In some embodiments, the reaction time of step (2) is 1-60 minutes, preferably 5-40 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes.

[0208] In some embodiments, the reaction conditions of step (2) are incubation at 20-50°C for 1-60 minutes, incubation at 20-50°C for 5-40 minutes, incubation at 25-45°C for 5-40 minutes, incubation at 25-40°C for 5-40 minutes, incubation at 25-40°C for 5-35 minutes, incubation at 25-35°C for 5-35 minutes, incubation at 25-35°C for 5-30 minutes or incubation at 37°C for 5-30 minutes, including but not limited to any combination of the foregoing incubation temperatures and incubation times.

[0209] In some embodiments, the reaction system further comprises a DNA ligase, preferably T4 DNA ligase. In some embodiments, during the incubation process of step (2), the ligation product generated by the first oligonucleotide primer and the third oligonucleotide primer under the action of the bridge primer and T4 DNA ligase is an Index-T4-Panel long primer, wherein the Index-T4-Panel long primer comprises, from the 5' end to the 3' end, an adapter sequence-tag sequence-sequencing primer sequence-universal sequence-gene-specific primer sequence.

[0210] In some embodiments, the sample nucleic acid is DNA, for example, the DNA is gDNA or cDNA.

[0211] In some embodiments, the 5' end of the third oligonucleotide primer contains or does not contain a phosphorylation modification.

[0212] In some embodiments, when the 5' end of the third oligonucleotide primer does not contain phosphorylation modification, the composition further includes a substance capable of causing phosphorylation modification of the 5' end of the third oligonucleotide primer, such as T4 polynucleotide kinase (T4 PNK).

[0213] In some embodiments, the mass ratio or molar ratio of the first oligonucleotide primer to the third oligonucleotide primer is N:1, where N is a constant greater than or equal to 1 and less than or equal to 5, such as 1, 2, 3, 4, and 5.

[0214] In some embodiments, the reaction system further comprises an DNA polymerase. In some embodiments, the DNA polymerase is any heat-resistant DNA polymerase known in the art. In some embodiments, the heat-resistant DNA polymerase is one or more of Taq DNA polymerase, Pfu DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, and KOD DNA polymerase.

[0215] In some embodiments, the reaction system further comprises a buffer component. In some embodiments, the buffer component is one or more of Tris, Tris-HCl, Tris base or HEPES, preferably Tris.

[0216] In some embodiments, the reaction system further comprises a metal salt. In some embodiments, the metal salt is Mg 2+ Salt, preferably MgCl2. In some embodiments, the metal salt is Mg 2+ Salt, and K + Salt, Mn 2+ Salt, Cs + Salt, Na + Salt and Ca 2+ One or more salts.

[0217] In some embodiments, the reaction system further comprises dNTPs.

[0218] In some embodiments, the reaction system further comprises other components known in the art that can help DNA polymerase and endoribonuclease to function, such as glycerol.

[0219] In some embodiments, the reaction system further comprises a surfactant, such as one or more of anionic surfactants, cationic surfactants, and nonionic surfactants.

[0220] In some embodiments, the PCR amplification comprises one or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40) cycles of denaturation, annealing, and extension; preferably, the PCR amplification comprises preliminary denaturation and multiple (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40) cycles of denaturation, annealing, and extension.

[0221] In some embodiments, the PCR amplification is one round of PCR amplification.

[0222] In some embodiments, the PCR amplification conditions can be any conventional PCR amplification conditions known to those skilled in the art.

[0223] In some embodiments, the method further comprises step (4) of purifying the amplified product of the sample nucleic acid.

[0224] In some embodiments, the purification is carried out by a magnetic bead method, a high salt precipitation method, a centrifugal column method or a phenol-chloroform extraction method, preferably a magnetic bead method.

[0225] The eighth aspect of the present invention provides an oligonucleotide primer, which comprises, from the 5' end to the 3' end, a linker sequence-a tag sequence-a sequencing primer sequence-a universal sequence-a gene-specific primer sequence.

[0226] The ninth aspect of the present invention provides the use of the method of the third aspect or the seventh aspect in constructing a multiplex amplicon library.

[0227] In the present invention, the sequence and its reverse complementary sequence may be completely complementary or partially complementary, as long as subsequent extension and amplification can be achieved.

[0228] "Perfectly complementary" means that all nucleotide bases of the sequence are capable of pairing with all nucleotide bases of the corresponding reverse complementary sequence.

[0229] "Partially complementary" means that at least 30% (e.g., 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%), but not all, of the bases in the contiguous sequence of the sequence hybridizes to the same number of bases in the entire contiguous sequence of the corresponding reverse complementary sequence.

[0230] The composition of the present invention is used in a multiplex amplification system. BRIEF DESCRIPTION OF THE DRAWINGS

[0231] Figure 1: Schematic diagram of the existing two-round multiplex amplicon library construction;

[0232] Figure 2: Schematic diagram of one-step multiplex amplicon library construction based on reverse panel primers;

[0233] Figure 3: Schematic diagram of one-step multiplex amplicon library construction based on bridge primers and T4 DNA ligase;

[0234] Figure 4: Denaturing polyacrylamide gel electrophoresis of primer extension of test group 1-14 in Example 1;

[0235] Figure 5: Denaturing polyacrylamide gel electrophoresis of primer extension of test group 1-20 in Example 2;

[0236] Figure 6: Denaturing polyacrylamide gel electrophoresis of primer extension of test group 1-6 in Example 3;

[0237] Figure 7: Gel electrophoresis of amplified products of test groups 1-10 in Example 4;

[0238] Figure 8: Denaturing polyacrylamide gel electrophoresis of primers 1-11 of test group 5 after ligation;

[0239] Figure 9: Gel electrophoresis of amplified products of test groups 1-10 in Example 6;

[0240] Figure 10: Final library structures of the 5TS-7NT and 5NT-7TS groups;

[0241] Figure 11: Gel electrophoresis of amplified products of test groups 1-4 in Example 7;

[0242] Figure 12: T4-panel-16 primer sequence for constructing the 5NT-7TS library structure in Example 8;

[0243] Figure 13: T4-panel-16 primer sequences for constructing the 5TS-7NT library structure in Example 8;

[0244] Figure 14: Primer sequence of Panel-16 containing only specific sequences from Example 8;

[0245] Figure 15: Gel electrophoresis of the 16-plex panel one-step amplification products of test groups 1-13 in Example 8;

[0246] Figure 16: Gel electrophoresis of the amplified products of primers P5 and P7 of test groups 1-13 in Example 8;

[0247] Figure 17: Reverse panel primer sequences for library construction in Example 9;

[0248] Figure 18: Panel primer sequences for library construction in Example 9;

[0249] Figure 19: Gel electrophoresis of amplified products of test groups 1-10 in Example 9;

[0250] Figure 20: Peak profiles of target products from the one-tube one-step and two-step libraries of Example 11;

[0251] Figure 21: Number of reads detected by sequencing libraries using the one-tube one-step and two-step methods in Example 11;

[0252] Figure 22: Number of dimers detected by library sequencing using the one-tube one-step and two-step methods in Example 11;

[0253] Figure 23: Number of reads detected by sequencing libraries using the one-tube one-step and two-step methods in Example 10;

[0254] Figure 24: Number of dimers detected by library sequencing using the one-tube one-step and two-step methods in Example 10.

[0255] Specific implementation (embodiment)

[0256] The technical solution of the present invention is further illustrated below with reference to the accompanying drawings and through specific implementation methods. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0257] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0258] Example 1

[0259] The Index primer and the reverse Panel primer are extended by DNA polymerase to generate the Index-panel long primer.

[0260] A conventional Vazyme#NA301 multiplex amplification system was used, with upstream and downstream index primers (Vazyme#TD202) and corresponding upstream and downstream reverse panel primers (Panel-Rp) added to the system. Phanta DNA polymerase (Vazyme#P501) without antibody blocking was further added. The amplification system and component volumes are shown in Table 1. The primer types and lengths in the system and product are shown in Table 2. Vazyme#NA301 contains a hot-start polymerase, while Vazyme#P501 is a non-hot-start polymerase. The primer sequences for Panel-Rp are (direction: 5'-3', the same below):

[0261] Test groups 1-14 with different reaction temperatures and reaction times were set as shown in Table 3, and the reactions were carried out according to the corresponding reaction temperatures and times. The products after each reaction were subjected to denaturing polyacrylamide gel electrophoresis, and the results are shown in FIG4 .

[0262] Table 1

[0263] Table 2

[0264] Table 3

[0265] As shown in Figure 4 , the extension of the Index-panel long primer can be efficiently completed at temperatures between 25°C and 65°C, indicating that the replication of the Index-panel long primer can be efficiently completed by the reverse panel primer.

[0266] This example uses the index primer (Vazyme#TD202) of the Novagen transposon library structure for testing. This approach is also suitable for conventional index primers for traditional Illumina library structures, such as Vazyme#N321 / N322. Simply design the corresponding complementary sequence on the reverse panel primer based on the sequence at the 3' end of the index primer. Furthermore, for sequencing library structures on the BGI platform or other sequencing platforms known in the art, a similar approach can be used, i.e., designing the complementary sequence on the reverse panel primer based on the differences in the primer sequence at the 3' end of the index primer to achieve a one-tube, one-step multiplex amplicon library construction.

[0267] Example 2

[0268] RNase H2 removes RNA base modifications from the reverse panel primer.

[0269] To further test the scheme of inactivating the reverse panel primer after generating the Index-panel long primer through the reverse panel primer, we selected upstream and downstream reverse panel primers (Panel-Rp-R) carrying RNA modifications and combined them with hot-start RNase H2 (IDT#11-03-02-03, which is a ribonuclease that is active at high temperatures and can specifically recognize and excise single-base RNA on double-stranded DNA) to inactivate the reverse panel primer. To test the optimal reaction conditions for RNase H2, the conditions need to ensure that the DNA polymerase preferentially completes the synthesis of the Index-panel long primer before RNase H2 can remove the reverse panel primer. Test groups 1-20 with different extension temperatures and reaction times are set as shown in Table 5, the amplification system is prepared as shown in Table 5, and the reaction is carried out according to the reaction procedure shown in Table 4, where the sequences of Panel-Rp-R are:

[0270] The reaction products were subjected to denaturing polyacrylamide gel electrophoresis, and the results are shown in FIG5 .

[0271] Table 4

[0272] Table 5

[0273] As described above, this example tests the optimal working conditions of RNase H2, which needs to ensure that RNase H2 is inactive during the process of DNA polymerase generating long primers. As can be seen from the results in Figure 5, under the reaction conditions of test groups 1-9, no or only very weak digestion products of the reverse panel primer are present when RNase H2 is added or not added, indicating that RNase H2 has basically no activity under the condition that the reaction temperature of the first step is less than 40°C. Therefore, 25-40°C can be selected as the temperature for the DNA polymerase extension reaction, which can not only allow the DNA polymerase to efficiently generate the Index-Panel long primer, but also ensure that RNase H2 is inactivated. After the reaction temperature is higher than 40°C, as shown in test groups 10-18 in Figure 5, RNase H2 can already exert RNA base cleavage activity, so there is a possibility that the Index primer is not fully extended into a long primer by RNase H2. H2 cleaves the reverse panel primer, causing the long Index-panel primer to terminate at the RNA modified base, ultimately generating an incomplete long Index-panel primer and causing subsequent nonspecific amplification. In summary, the complementary extension reaction of the Panel-Rp-R primer and the Index primer can be performed at 25-40°C, while the RNase H2 cleavage reaction of the Panel-Rp-R primer can be performed at 40-72°C.

[0274] Example 3

[0275] Primer extension using a DNA polymerase and reverse panel primer removal were tested.

[0276] To test the effectiveness of generating long Index-panel primers and removing reverse panel primers when the amplification system contains only a thermostable DNA polymerase, we selected a Vazyme#PM202 amplification system combined with a thermostable DNA polymerase (Vazyme#PM202) and a hot-start RNase H2 (IDT#11-03-02-03) to create a test set as shown in Table 6. The sequences of the Index and Panel-Rp-R primers used in the test were the same as in Example 2. The prepared system is shown in Table 7. Reactions were carried out according to the conditions described in the table, and the reaction products were subjected to denaturing polyacrylamide gel electrophoresis. The results are shown in Figure 6.

[0277] Table 6

[0278] Table 7

[0279] As shown in FIG6 , the extension of the Index-panel long primer can be efficiently completed in an amplification system containing only one thermostable DNA polymerase.

[0280] Example 4

[0281] Primer extension-mediated one-step amplification test.

[0282] Ten amplification experiments were further set up, including GSP primers (GSP-F / R), panel primers, Index primers + panel primers, Index primers + reverse panel primers containing RNA base modifications (Panel-RP-R), and Index primers + reverse panel primers without RNA base modifications (Panel-RP). The specific settings for enzyme and primer addition amounts for each group are shown in Table 8, the components and addition amounts of the amplification system are shown in Table 9, the amplification reaction procedures are shown in Table 10, and the significance and lengths of different primer sets are shown in Table 11. The sequences of some primers are:

[0283] The amplified product was subjected to agarose gel electrophoresis, and the results were shown in FIG7 .

[0284] Table 8

[0285] Table 9

[0286] Table 10

[0287] Table 11

[0288] As shown in Figure 7, test group 1 shows that amplification does not occur if the reverse panel primer is not treated with RNase H2. In the test groups with the addition of RNase H2, increasing the proportion of index primers can increase the output of target products, as shown in test groups 3 and 4. In contrast, test groups 5 and 6, which directly add index and panel primers to the system for amplification, produce a large amount of nonspecific products under the action of phanta DNA polymerase.

[0289] Example 5

[0290] T4 DNA ligase-dependent long primer generation assay using the Index-T4-panel.

[0291] To achieve the generation of Index-T4-panel long primers from Index and T4-panel primers using ligase, we designed a T4 DNA ligase method to connect the Index primer and T4-panel primer using a bridge primer (Splint Oligo, carrying RNA base modifications and a blocking base at the 3' end, such as C3-Spacer) and T4 DNA ligase to generate Index-T4-panel long primers. The T4-Panel primers contain universal sequences and gene-specific primer sequences, while the Panel primers contain sequencing primer sequences and gene-specific primer sequences. To explore the optimal ligation conditions for T4 DNA ligase, test groups 1-11 were set up according to Table 12. The components and contents of the amplification system are shown in Table 13, the amplification program is shown in Table 14, and the meaning and length of each primer are shown in Table 15. Some primer sequences are as follows:

[0292] The amplified product was subjected to denaturing polyacrylamide gel electrophoresis, and the results were shown in FIG8 .

[0293] Table 12

[0294] Table 13

[0295] Table 14

[0296] Table 15

[0297] As shown in FIG8 , this example tested different ligase usage amounts and different ligation conditions. The results of denaturing PAGE gel detection showed that T4 DNA ligase could effectively ligate the Index primer and the T4-Panel primer under the several conditions tested.

[0298] Example 6

[0299] T4 DNA ligase-mediated one-step amplification test

[0300] To test the amplification efficiency of the Index-T4-panel long primer generated by our designed T4 DNA ligase scheme, 10 amplification experiments were further set up, including Index primer + Splint Oligo (bridge primer) + T4-Panel primer, T4-Panel primer, and Panel primer + Index primer. The specific settings such as the enzyme addition amount and primer addition amount of each group are shown in Table 16, the components and addition amounts of the amplification system are shown in Table 17, the amplification reaction procedure is shown in Table 18, and the significance and length of different primer sets are shown in Table 19. The primer sequences are the same as in Example 5.

[0301] The amplified product was subjected to agarose gel electrophoresis, and the results were shown in FIG9 .

[0302] Table 16

[0303] Table 17

[0304] Table 18

[0305] Table 19

[0306] Analysis of results: This embodiment further tests the amplification efficiency of the target fragment by the Index-T4-panel long primer formed by connecting with T4 DNA ligase under different conditions. As shown in Figure 9, it can be seen that the amplification product (456bp) of the Index-T4-Panel long primer after connection is larger than the amplification product (367bp) of the Panel primer that is not connected. The amplification product band brightness level can be used to judge that the amplification efficiency of the Index-T4-Panel long primer reaches a level consistent with the amplification efficiency of the Panel primer. At the same time, the electrophoresis bands of test groups 1-2 and test group 3 show that whether the bridge primer is inactivated by RNase H2 has no effect on the amplification result, that is, the bridge primer will not have other effects on the amplification system. In summary, it is feasible to preferentially connect the Index primer and the T4-Panel primer to form a long primer by T4 DNA ligase and then amplify.

[0307] Example 7

[0308] Testing of different library structures for T4 DNA ligase-mediated one-step amplification.

[0309] To verify that the final library constructed using the one-tube amplicon library construction scheme provided in this invention meets the requirements of the Illumia sequencing platform, we designed a hybrid library structure with a TruSeq library structure on one end and a Nextera library structure on the other end, enabling normal compatible sequencing on the Illumia sequencer. The bridge primers for the 5NT-7TS group (i5 is a Nextera structure, i7 is a TruSeq structure) are Splint-1 and Splint-2, the first oligonucleotide primer is Vazyme#TD202's N5xx primer + Vazyme#N321's DM7xx primer, and the third oligonucleotide primer is T4-Panel-F1 and T4-Panel-F2. The bridge primers for the 5TS-7NT group (i5 is a TruSeq structure, i7 is a Nextera structure) are Splint-3 and Splint-4, the first oligonucleotide primer is Vazyme#N321-DM5xx primer and Vazyme#TD204-N9xx primer, and the third oligonucleotide primer is T4-Panel-F3 and T4-Panel-F4. The corresponding primer sequences are as follows:

[0310] The primer addition settings for each group are shown in Table 20. The components and addition amounts of the amplification system for the 5NT-7TS group are shown in Table 21. The components and addition amounts of the amplification system for the 5TS-7NT group are shown in Table 22. The amplification reaction procedure is shown in Table 23. The final library structures for the 5NT-7TS and 5TS-7NT groups are shown in Figure 10. Agarose gel electrophoresis of the amplified products is shown in Figure 11.

[0311] Table 20

[0312] Table 21

[0313] Table 22

[0314] Table 23

[0315] As shown in Figure 11, the bridge primers designed for the 5NT-7TS and 5NT-7TS library structures can effectively connect the corresponding T4-Panel and Index primers into an Index-T4-panel long primer, and can efficiently complete the amplification of the complete library structure in one step.

[0316] Example 8

[0317] T4 DNA ligase-mediated one-step multiplex panel amplification assay.

[0318] To verify the one-tube one-step amplification scheme for constructing a multiplex amplicon library in the present invention, we further designed 16-plex panel primers for testing. The T4-panel-16 primer sequence for constructing a 5NT-7TS library structure is shown in Figure 12 , the T4-Panel-16 primer sequence for constructing a 5TS-7NT library structure is shown in Figure 13 , and the Panel-16 primer sequence containing only a specific sequence is shown in Figure 14 . The sequence of the bridge primer used in the test is the same as in Example 7 . The test group was set up according to Table 24 , the one-tube one-step amplification system was prepared according to Table 25 , and the reaction was carried out according to the amplification procedure in Table 26 . The amplification results were run on a gel electrophoresis plot to detect the amplification effect, and the results are shown in Figure 15 .

[0319] The product of the one-step amplification of the 16-plex panel was recovered and amplified using the P5 and P7 sequences of the library to verify whether the one-step amplification product contained a valid library of the 16-plex panel. 50 ng of the recovered product was prepared according to Table 27, amplified according to Table 28, and the amplified product was detected on an agarose gel. The results are shown in Figure 16. The sequences of the P5 primer and the P7 primer are:

[0320] Table 24

[0321] Table 25

[0322] Table 26

[0323] Table 27

[0324] Table 28

[0325] As shown in Figure 15 , regardless of the library structure type of 5NT-7TS or 5TS-7NT, the primers of the 16-plex T4-panel can complete the extension of the long primer and amplification of the target fragment, and the electrophoretic bands of the amplified products increase with the increase in the amount of index primer used. Further increase in the amount of index primer will inhibit the target amplification of the multiplex panel.

[0326] As shown in Figure 16, further amplification of the one-step amplification products with P5 and P7 primers revealed that the amplification products of the one-step test groups could be amplified by both P5 and P7 primers (test groups 1-4, 7-10), while the products of the non-one-step amplification system could not be amplified by both P5 and P7 primers (test groups 5, 6, 11, 12, and 13). Furthermore, as the content of the Index primer in the system increased, the number of products amplified by P5 and P7 also increased. However, further addition of the Index primer inhibited amplification of the target product, indicating that the ratio of Index primer to T4-panel primer in the T4 DNA ligase-mediated one-step amplification system can range from 1:1 to 4:1, with a ratio of 2:1 to 3:1 being optimal.

[0327] Example 9

[0328] One-step multiplex panel amplification assay mediated by reverse-complement panel primers containing RNA modifications.

[0329] To verify the RNA-modified reverse complementary primer-mediated one-step amplification scheme for constructing a multiplex amplicon library in the present invention, we further designed a 14-fold RNA-modified reverse panel primer (Panel-Rp-R-14) for testing. The sequences of the 14-fold RNA-modified reverse panel primer (Panel-Rp-R-14) and the normal panel primer are shown in Figures 17 and 18, respectively. The test group was set up according to Table 29, and the one-tube one-step amplification system was prepared according to Table 30. The reaction was carried out according to the amplification program in Table 31. The amplification results were run on gel electrophoresis to detect the amplification effect, and the results are shown in Figure 19.

[0330] Table 29

[0331] Table 30

[0332] Table 31

[0333] As shown in Figure 19, when the molar ratio of index primer to RNA-modified reverse panel primer was 1:1, the one-tube, one-step multiplex amplification method mediated by the RNA-modified reverse panel primer was the most effective. Conventional direct amplification of the panel primer and index mixture yielded fewer target characteristic products, resulting in a low proportion of valid libraries. In summary, the test results of the 14-plex RNA-modified reverse panel demonstrate that the one-tube, one-step method mediated by RNA-modified reverse panel primers is suitable for multiplex amplification.

[0334] Example 10

[0335] One-step multiplex panel amplification assay mediated by reverse-complement panel primers containing RNA modifications.

[0336] To validate the RNA-modified reverse complementary primer-mediated one-step amplification and construction of a multiplex amplicon library, we further designed a 223-fold RNA-modified reverse panel primer (Panel-Rp-R-223) for testing. 100 of these primers target the Escherichia coli genome, 100 target the Staphylococcus aureus genome, and 23 target the Pichia pastoris genome. The sequences of the 223-fold RNA-modified reverse panel primers are:

[0337] There are 223 pairs in total, and two upstream primers are listed here as examples.

[0338] The primer sequences for the 223-plex normal panel are:

[0339] There are 223 pairs in total, all of which have the same sequencing primer sequence at the 5' end, and different gene-specific sequences are designed according to the target site (two upstream sequences are listed here as an example).

[0340] The pathogen template was 293T gDNA, Escherichia coli genomic DNA, Staphylococcus aureus genomic DNA, and Pichia pastoris genomic DNA, mixed at a ratio of 1000 / 1 (1 μg 293T + 1 ng E. coli, 1 ng E. coli, and 1 ng Pichia pastoris) to obtain the simulated pathogen sample DNA. The two-step method system preparation is shown in Tables 38 and 39, and the one-tube preparation system containing RNA-modified reverse panel primers is shown in Table 40. The two-step method amplification program is shown in Tables 42 and 43, and the one-tube amplification program is shown in Table 41. The amplified library was sequenced on a sequencing machine. The number of reads detected by sequencing analysis is shown in Figure 20, and the number of dimers is shown in Figure 21.

[0341] Table 38

[0342] Table 39

[0343] Table 40

[0344] Table 41

[0345] Table 42

[0346] Table 43

[0347] As shown in Figures 23 and 24, the one-tube library construction scheme mediated by 223-plex reverse panel primers containing RNA modifications can also detect target reads. The number of species target reads detected is slightly lower than that of the two-step method, and the number of primer dimers is slightly higher than that of the two-step method. However, the target can still be effectively amplified, indicating that the multiplex amplicon library construction method mediated by reverse complementary panel primers containing RNA modifications can effectively amplify the target fragments and construct the library.

[0348] Example 11

[0349] T4 DNA ligase-mediated one-step 223-plex panel amplification assay.

[0350] To verify the ability of the T4 ligase-mediated one-step amplification method of the present invention to construct multiplex amplicon libraries under super-multiplex conditions, we further designed a T4-panel primer set (T4-Panel-223) with a 223-plex 5TS-7NT library structure for testing, of which 100-plex targets the Escherichia coli genome, 100-plex targets Staphylococcus aureus, and the remaining 23-plex targets the Pichia pastoris genome. The panel primer sequences for the 223-plex 5TS-7NT library structure are:

[0351] There are 223 pairs in total, all of which have the same universal sequence at the 5' end, and different gene-specific sequences are designed according to the target site (two upstream sequences are listed here as an example).

[0352] The primer sequences for the 223-plex normal panel are:

[0353] There are 223 pairs in total, all of which have the same sequencing primer sequence at the 5' end, and different gene-specific sequences are designed according to the target site (two upstream sequences are listed here as an example).

[0354] The sequences of bridge primers Splint 3 and Splint 4 are shown in Example 7. Pathogen templates were 293T gDNA, Escherichia coli genomic DNA, Staphylococcus aureus genomic DNA, and Pichia pastoris genomic DNA, mixed in the following ratios: 10 μg 293T + 1 ng Escherichia coli, 1 ng E. coli, and 1 ng Pichia pastoris to obtain pathogenic simulated sample DNA. The amplification system was prepared according to Table 32, and amplification was performed according to the amplification program in Table 35. The conventional two-step amplification system was prepared according to Tables 33 and 34, and the reaction program was performed according to Tables 36 and 37, respectively. The amplified results were recovered and the concentration was determined using Qubit, and the product peak shape was detected using Qseq, and then sequenced. The Qseq peak profile of the library is shown in Figure 20, the number of reads detected by sequencing analysis is shown in Figure 21, and the number of dimers is shown in Figure 22.

[0355] Table 32

[0356] Table 33

[0357] Table 34

[0358] Table 35

[0359] Table 36

[0360] Table 37

[0361] As shown in Figure 20, the target product peaks of the T4 DNA ligase-mediated one-tube targeted multiplex amplicon library are clear. Furthermore, high-throughput sequencing data from Figures 20 and 21 show that the one-tube and two-step methods detected consistent target reads for these three pathogens, with slightly higher primer dimers in the T4 DNA ligase-mediated one-tube one-step library construction than in the two-step method.

Claims

1. An oligonucleotide set, the oligonucleotide set comprising a first oligonucleotide primer and a second oligonucleotide primer, the first oligonucleotide primer sequentially comprising a sequencing adapter sequence, a tag sequence, and a sequencing primer sequence from the 5'-end to the 3'-end, and the second oligonucleotide primer sequentially comprising the reverse complementary sequence of a gene-specific primer and the reverse complementary sequence of a sequencing primer from the 5'-end to the 3'-end.

2. The oligonucleotide set according to claim 1, wherein, One or more nucleotide sites of the reverse complementary sequence of the gene-specific primer contain RNA base modifications.

3. The oligonucleotide set according to claim 1, wherein, The 3'-end of the second oligonucleotide primer contains a blocking modification, preferably a 3'-phosphorylation modification or an inter-arm modification, more preferably a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification, or a PC-linker modification.

4. The oligonucleotide set according to claim 2, wherein, The first oligonucleotide primer includes an upstream first oligonucleotide primer and a downstream first oligonucleotide primer. The upstream first oligonucleotide primer sequentially comprises an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5'-end to the 3'-end. The downstream first oligonucleotide primer sequentially comprises a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5'-end to the 3'-end. The second oligonucleotide primer includes an upstream second oligonucleotide primer and a downstream second oligonucleotide primer. The upstream second oligonucleotide primer sequentially comprises the reverse complementary sequence of a gene-specific upstream primer and the reverse complementary sequence of an upstream sequencing primer from the 5'-end to the 3'-end. The downstream second oligonucleotide primer sequentially comprises the reverse complementary sequence of a gene-specific downstream primer and the reverse complementary sequence of a downstream sequencing primer from the 5'-end to the 3'-end. Preferably, the upstream sequencing adapter sequence is the P5 adapter sequence of the Illumina platform, and the downstream sequencing adapter sequence is the P7 adapter sequence of the Illumina platform.

5. The oligonucleotide set according to claim 4, wherein, The first oligonucleotide primer includes at least one pair of upstream first oligonucleotide primers and downstream first oligonucleotide primers, and the second oligonucleotide primer includes at least one pair of upstream second oligonucleotide primers and downstream second oligonucleotide primers.

6. A composition comprising a DNA polymerase, a ribonuclease, dNTPs, buffer components, metal salts, and a primer combination; wherein, The primer combination comprises the oligonucleotide set recited in claim 5.

7. The composition according to claim 6, wherein, The composition further includes a DNA sample; preferably, the DNA sample is gDNA or cDNA.

8. The composition according to claim 6, wherein, The DNA polymerase comprises at least one heat-resistant DNA polymerase. Preferably, the DNA polymerase comprises at least one heat-resistant DNA polymerase and one room-temperature DNA polymerase.

9. The composition according to claim 6, wherein, The ribonuclease is an endonuclease capable of recognizing ribonucleotide sites and cleaving phosphodiester bonds; preferably, the ribonuclease is RNase H2 or a homologous protein of RNase H2.

10. A method for constructing a multiplex amplicon library, the method comprising the following steps: (1) Prepare a reaction system comprising at least a pair of first oligonucleotide primers, at least a pair of second oligonucleotide primers, and sample nucleic acid; wherein, The first oligonucleotide primer sequentially includes a sequencing adapter sequence, a tag sequence, and a sequencing primer sequence from the 5'-end to the 3'-end. The second oligonucleotide primer sequentially includes the reverse complementary sequence of a gene-specific primer and the reverse complementary sequence of a sequencing primer from the 5'-end to the 3'-end. A cleavable group is included in the second oligonucleotide primer; (2) Make the 3'-ends of the first oligonucleotide primer and the second oligonucleotide primer complementary in an antiparallel manner, and extend the first oligonucleotide primer in the 5'-3' direction to obtain an extension product; (3) Cleave the reverse complementary sequence of the gene-specific primer in the second oligonucleotide primer; (4) Use the extension product described in step (2) as a primer to perform PCR amplification on the sample nucleic acid.

11. The method according to claim 10, wherein, The 3'-end of the second oligonucleotide primer contains a blocking modification, preferably a 3'-phosphorylation modification or a spacer modification, more preferably a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification, or a PC-linker modification.

12. The method according to claim 10, wherein, The at least one pair of first oligonucleotide primers includes at least one pair of upstream first oligonucleotide primers and downstream first oligonucleotide primers. The upstream first oligonucleotide primer sequentially includes an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5'-end to the 3'-end. The downstream first oligonucleotide primer sequentially includes a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5'-end to the 3'-end. The at least one pair of second oligonucleotide primers includes at least one pair of upstream second oligonucleotide primers and downstream second oligonucleotide primers. The upstream second oligonucleotide primer sequentially includes the reverse complementary sequence of a gene-specific upstream primer and the reverse complementary sequence of an upstream sequencing primer from the 5'-end to the 3'-end. The downstream second oligonucleotide primer sequentially includes the reverse complementary sequence of a gene-specific downstream primer and the reverse complementary sequence of a downstream sequencing primer from the 5'-end to the 3'-end. Preferably, the upstream sequencing adapter sequence is the P5 adapter sequence of the Illumina platform, and the downstream sequencing adapter sequence is the P7 adapter sequence of the Illumina platform.

13. The method according to claim 10, wherein, The sample nucleic acid is DNA; preferably, the DNA is gDNA or cDNA.

14. The method according to claim 10, wherein, The cleavable group is an RNA base, and the RNA base is at least one of rA (adenosine ribonucleoside), rU (uridine ribonucleoside), rG (guanosine ribonucleoside), and rC (cytidine ribonucleoside).

15. The method according to claim 14, wherein, An endoribonuclease is further included in the reaction system, and the endoribonuclease is an endonuclease capable of recognizing ribonucleotide sites and excising phosphodiester bonds; preferably, the endoribonuclease is RNase H2 or a homologous protein of RNase H2.

16. The method according to claim 15, wherein, A DNA polymerase is further included in the reaction system; preferably, the DNA polymerase includes at least one heat-resistant DNA polymerase.

17. The method according to claim 16, wherein, The DNA polymerase includes at least one heat-resistant DNA polymerase and one room-temperature DNA polymerase.

18. The method according to claim 16, wherein, The DNA polymerase comprises at least one non-thermostart polymerase and one thermostart polymerase.

19. The method according to claim 17, wherein, The reaction of step (2) is carried out under the action of a room temperature polymerase; the reaction of step (4) is carried out under the action of a heat-resistant polymerase. The method according to claim 18, wherein The reaction of step (2) is carried out under the action of a non-thermostart polymerase; the reaction of step (4) is carried out under the action of a thermostart polymerase.

21. The method according to any one of claims 19 and 20, wherein The conditions for the reaction of step (2) are incubation at 25-40 °C for 10-30 min, and the PCR amplification of step (4) includes denaturation, annealing and extension.

22. The method according to claim 15, wherein, The reaction conditions for step (3) are incubation at 60-80 °C for 10-20 min.

23. The method according to claim 10, wherein, The reaction system further includes one or more of metal salts, buffer components and dNTPs.

24. An oligonucleotide primer, which sequentially comprises a sequencing adapter sequence - a tag sequence - a sequencing primer sequence - a gene-specific primer sequence from the 5'-end to the 3'-end.

25. An oligonucleotide combination, which comprises a first oligonucleotide primer, a third oligonucleotide primer and a bridge primer. The first oligonucleotide primer sequentially comprises a sequencing adapter sequence, a tag sequence and a sequencing primer sequence from the 5'-end to the 3'-end. The third oligonucleotide primer sequentially comprises a universal sequence and a gene-specific primer sequence from the 5'-end to the 3'-end. The bridge primer sequentially includes the reverse complementary sequence of the universal sequence and the reverse complementary sequence of the sequencing primer from the 5'-end to the 3'-end.

26. The oligonucleotide combination according to claim 25, wherein, The 3'-end of the bridge primer contains a blocking modification, preferably a 3'-phosphorylation modification or an interarm modification, more preferably a Spacer C3 modification, a Spacer C6 modification, a Spacer C9 modification, a dSpacer modification or a PC-linker modification.

27. The oligonucleotide combination according to claim 26, wherein, The first oligonucleotide primer includes an upstream first oligonucleotide primer and a downstream first oligonucleotide primer. The upstream first oligonucleotide primer sequentially comprises an upstream sequencing adapter sequence, an upstream tag sequence and an upstream sequencing primer sequence from the 5'-end to the 3'-end. The downstream first oligonucleotide primer sequentially comprises a downstream sequencing adapter sequence, a downstream tag sequence and a downstream sequencing primer sequence from the 5'-end to the 3'-end. The third oligonucleotide primer includes an upstream third oligonucleotide primer and a downstream third oligonucleotide primer. The upstream third oligonucleotide primer sequentially comprises an upstream universal sequence and a gene-specific upstream primer sequence from the 5'-end to the 3'-end. The downstream third oligonucleotide primer sequentially comprises a downstream universal sequence and a gene-specific downstream primer sequence from the 5'-end to the 3'-end. The bridge primer includes an upstream bridge primer and a downstream bridge primer. The upstream bridge primer sequentially includes the reverse complementary sequence of the upstream universal sequence and the reverse complementary sequence of the upstream sequencing primer from the 5'-end to the 3'-end. The downstream bridge primer sequentially includes the reverse complementary sequence of the downstream universal sequence and the reverse complementary sequence of the downstream sequencing primer from the 5'-end to the 3'-end.

28. The oligonucleotide combination according to claim 27, wherein, The first oligonucleotide primer includes at least a pair of upstream first oligonucleotide primers and downstream first oligonucleotide primers, the third oligonucleotide primer includes at least a pair of upstream third oligonucleotide primers and downstream third oligonucleotide primers, and the bridge primer includes at least a pair of upstream bridge primers and downstream bridge primers.

29. A composition, the composition comprising a DNA polymerase, T4 DNA ligase, ATP, dNTP, buffer components, metal salts, and a primer combination; the primer combination comprises the oligonucleotide combination according to claim 28.

30. The composition according to claim 29, wherein, The composition further comprises a DNA sample; preferably, the DNA sample is gDNA or cDNA.

31. The composition according to claim 29, wherein, The 5'-end of the third oligonucleotide primer contains a phosphorylation modification, or, the 5'-end of the third oligonucleotide primer does not contain a phosphorylation modification, and the composition further comprises T4 polynucleotide kinase.

32. The composition according to claim 29, wherein, The mass ratio or molar ratio of the first oligonucleotide primer to the third oligonucleotide primer is N:1, where N is a constant greater than or equal to 1 and less than or equal to 5.

33. A method for constructing a multiplex amplicon library, the method comprising the following steps: (1) Prepare a reaction system comprising at least a pair of first oligonucleotide primers, at least a pair of third oligonucleotide primers, at least a pair of bridge primers, and sample nucleic acid; wherein, The first oligonucleotide primer sequentially comprises a sequencing adapter sequence, a tag sequence, and a sequencing primer sequence from the 5'-end to the 3'-end, the third oligonucleotide primer sequentially comprises a universal sequence and a gene-specific primer sequence from the 5'-end to the 3'-end, and the bridge primer sequentially comprises the reverse complementary sequence of the universal sequence and the reverse complementary sequence of the sequencing primer from the 5'-end to the 3'-end; (2) Make the 3'-ends of the first oligonucleotide primer and the bridge primer be reverse complementary paired, make the 5'-ends of the third oligonucleotide primer and the bridge primer be reverse complementary paired, and ligate the first oligonucleotide primer and the third oligonucleotide primer to obtain a ligation product; (3) Use the ligation product in step (2) as a primer to perform PCR amplification on the sample nucleic acid.

34. The method according to claim 33, wherein, The 3'-end of the bridge primer contains a blocking modification, preferably 3'-phosphorylation modification or an inter-arm modification, more preferably Spacer C3 modification, Spacer C6 modification, Spacer C9 modification, dSpacer modification, or PC-linker modification. The method according to claim 33, wherein, The sample nucleic acid is DNA, preferably gDNA or cDNA.

36. The method according to claim 33, wherein, The 5'-end of the third oligonucleotide primer contains a phosphorylation modification, or, the 5'-end of the third oligonucleotide primer does not contain a phosphorylation modification, and the reaction system further comprises T4 polynucleotide kinase.

37. The method according to claim 33, wherein, The mass ratio or molar ratio of the first oligonucleotide primer to the third oligonucleotide primer is N:1, where N is a constant greater than or equal to 1 and less than or equal to 5.

38. The method according to claim 36, wherein, The at least one pair of first oligonucleotide primers includes at least one pair of upstream first oligonucleotide primers and downstream first oligonucleotide primers. The upstream first oligonucleotide primer sequentially includes an upstream sequencing adapter sequence, an upstream tag sequence, and an upstream sequencing primer sequence from the 5'-end to the 3'-end. The downstream first oligonucleotide primer sequentially includes a downstream sequencing adapter sequence, a downstream tag sequence, and a downstream sequencing primer sequence from the 5'-end to the 3'-end. The at least one pair of third oligonucleotide primers includes at least one pair of upstream third oligonucleotide primers and downstream third oligonucleotide primers. The upstream third oligonucleotide primer sequentially includes an upstream universal sequence and a gene-specific upstream primer sequence from the 5'-end to the 3'-end. The downstream third oligonucleotide primer sequentially includes a downstream universal sequence and a gene-specific downstream primer sequence from the 5'-end to the 3'-end. The at least one pair of bridge primers includes at least one pair of upstream bridge primers and downstream bridge primers. The upstream bridge primer sequentially includes the reverse complementary sequence of the upstream universal sequence and the reverse complementary sequence of the upstream sequencing primer from the 5'-end to the 3'-end. The downstream bridge primer sequentially includes the reverse complementary sequence of the downstream universal sequence and the reverse complementary sequence of the downstream sequencing primer from the 5'-end to the 3'-end.

39. The method according to claim 33, wherein, The reaction conditions of step (2) are incubation at 20-50°C for 1-60 min. The method according to claim 38, wherein, The reaction system further includes a DNA ligase, preferably T4 DNA ligase.

41. The method according to claim 40, wherein, The reaction system further includes a DNA polymerase, preferably a thermostable DNA polymerase, more preferably Taq DNA polymerase.

42. The method according to claim 33, wherein, The reaction system further includes one or more of metal salts, buffer components, and dNTPs.

43. An oligonucleotide primer, which sequentially includes an adapter sequence - tag sequence - sequencing primer sequence - universal sequence - gene-specific primer sequence from the 5'-end to the 3'-end.

44. Use of the method according to any one of claims 10-23 and claims 33-42 in multiplex amplicon library construction.

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