Method for rapidly preparing multiplex PCR sequencing library and application thereof

Abstract

The invention relates to a method for rapidly preparing a multiplex PCR sequencing library and application thereof. The oligonucleotide combination comprises a specific oligonucleotide and a universal primer, the 3'end of the specific oligonucleotide is defined as a universal sequence, the 5 'end of the specific oligonucleotide is defined as a specific sequence, the universal sequence located at the 3 end of the specific oligonucleotide is complementary with the 3' end of the universal primer, and the 3 'end of the universal primer extends under the action of polymerase, so that a product with the universal sequence at the 5' end and the specific sequence at the 3 'end is obtained. The 3'end of the product can be complementary with a to-be-detected target region, and the target region is amplified under the action of polymerase to obtain a product containing a universal sequence and a to-be-detected target sequence; the 3'end is a single fixed sequence, so that the formation of a complementary structure and the generation of a dimer can be effectively avoided.

Description

A method for rapidly preparing multiplex PCR sequencing library and its application Technical Field

[0001] The present invention belongs to the technical field of multiplex PCR amplification, and in particular relates to a method for rapidly preparing a multiplex PCR sequencing library and an application thereof. Background Art

[0002] With the advancement of sequencing technology, the demand for resequencing candidate genomic regions is increasing. Sequencing interest extends beyond a few SNPs, and candidate regions can range from 5 kb to 10 Mb. Traditional Sanger sequencing or whole-genome sequencing is expensive, but target region capture sequencing addresses this challenge. Compared to whole-genome sequencing, target region capture sequencing significantly reduces costs while screening large sample sizes. Target region capture techniques can be broadly categorized into two types: hybridization-based capture sequencing and multiplex PCR-based capture. Both use multiple probes or primers to capture the genomic region of interest at once. Combined with high-throughput sequencing, multiple samples can be sequenced simultaneously to obtain sequence information for the target region. However, the cumbersome experimental workflow and high probe costs of the former limit its clinical application. The latter, on the other hand, offers simpler and more flexible experimental procedures and is suitable for screening and diagnosis of Mendelian diseases, resequencing candidate regions for GWAS, resequencing regions for QTL mapping, and precision medicine research and applications.

[0003] The high-throughput SNP detection service combines multiplex PCR and high-throughput sequencing technologies. Specific primers are designed for the loci to be detected, and multiplex PCR amplification is performed in a single tube. Different samples are distinguished by different labeled (barcoded) primers. After the samples are mixed, the amplicons are sequenced on a sequencing platform. The sequencing results use bioinformatics methods to distinguish different samples, ultimately obtaining SNP information for each locus. This method is suitable for genetic research with different purposes, such as disease genome research, tumor genome research, disease-gene association research, clinical molecular diagnosis, etc. In plant genome research, it can be used for QTL positioning and molecular breeding, and is very suitable for SNP analysis of large-scale samples.

[0004] Although the multiplex PCR experiment is simple to operate and the cost of a single test is very low, it requires repeated testing and optimization of multiple pairs of primers in the early stages of the experiment, which is time-consuming and labor-intensive. Especially in ultra-high-weight PCR, the complexity of the primer sequence makes it easy for the primers to form primer dimers. The formation of primer dimers will rapidly consume the raw materials in the PCR reaction system, causing the PCR to quickly reach a plateau; the primer dimers formed will also be sequenced in subsequent sequencing, forming invalid data, affecting the efficiency of data utilization. The most serious are primers that are prone to forming primer dimers, which will seriously affect the amplification efficiency of the target amplification region corresponding to the primer, resulting in a low sequencing depth of the target, and ultimately affecting the uniformity of the entire amplification system. In addition, the specificity of the primers also greatly affects the performance of multiplex amplification.

[0005] In multiplex amplification, as the number of primer pairs increases, the diversity of the primer 3' ends increases, increasing the likelihood of complementary structures between primers, leading to dimer formation under the action of the polymerase. Many companies eliminate excessive primer dimers through additional enzymatic treatment. For example, Life's Ampliseq technology uses a one-step specific amplification followed by enzymatic digestion to remove primer dimers and specific primer sequences in the amplicon, and finally prepares the product library. The entire process is cumbersome. Later, Paragon invented Clean Plex dimer elimination technology, which uses specific enzymes to eliminate primer dimers only during the multiplex PCR process, similarly solving the problem of primer dimer formation. The resulting products are then amplified using universal primers to complete library preparation, but the entire process still involves too many steps.

[0006] Summary of the Invention

[0007] The first aspect of the present invention aims to provide an oligonucleic acid combination.

[0008] The second aspect of the present invention aims to provide a kit for preparing multiplex PCR sequencing libraries.

[0009] The third aspect of the present invention is to provide a sequencing reagent kit.

[0010] The fourth aspect of the present invention aims to provide a sequencing system.

[0011] The fifth aspect of the present invention aims to provide a multiplex PCR amplification method.

[0012] The sixth aspect of the present invention aims to provide a method for preparing a multiplex PCR sequencing library.

[0013] The seventh aspect of the present invention aims to provide a sequencing method.

[0014] The eighth aspect of the present invention aims to provide a method for detecting gene loci in a target region.

[0015] The ninth aspect of the present invention aims to provide an application of the oligonucleic acid combination of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, or the sequencing system of the fourth aspect.

[0016] In order to achieve the above object, the technical solution adopted by the present invention is:

[0017] In a first aspect, the present invention provides an oligonucleic acid combination.

[0018] 1. An oligonucleotide combination, comprising: a specific oligonucleotide and a universal primer;

[0019] The universal primers include: a first universal primer and a second universal primer;

[0020] The specific oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid;

[0021] The specific oligonucleic acid comprises: a specific sequence and a universal sequence;

[0022] The upstream-specific oligonucleic acid comprises: an upstream-specific sequence and a first universal sequence, wherein the upstream-specific sequence is located at the 5' end of the upstream-specific oligonucleic acid, and the first universal sequence is located at the 3' end of the upstream-specific oligonucleic acid;

[0023] The downstream-specific oligonucleotide comprises: a downstream-specific sequence and a second universal sequence, wherein the downstream-specific sequence is located at the 5' end of the downstream-specific oligonucleotide, and the second universal sequence is located at the 3' end of the downstream-specific oligonucleotide;

[0024] 1) The 3' end sequence of the first universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleotide, and the 3' end sequence of the second universal sequence of the downstream specific oligonucleotide is complementary to a portion or all of the second universal sequence; or

[0025] 2) The 3' end sequence of the second universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleic acid, and the 3' end sequence of the first universal primer is complementary to a portion or all of the second universal sequence of the downstream specific oligonucleic acid.

[0026] 2. an oligonucleotide combination, comprising: a specific oligonucleotide, a second primer, and a universal primer;

[0027] The universal primers include: a first universal primer;

[0028] The specific oligonucleic acid comprises: a specific sequence and a universal sequence, wherein the specific sequence is located at the 5' end of the specific oligonucleic acid, and the universal sequence is located at the 3' end of the specific oligonucleic acid;

[0029] The specific sequence is a first specific sequence, and the universal sequence is a first universal sequence;

[0030] The second primer comprises: a second specific sequence and a second universal sequence, wherein the second specific sequence is located at the 3' end of the second primer, and the second universal sequence is located at the 5' end of the second primer;

[0031] The 3' end sequence of the first universal primer is complementary to a portion or the entire sequence of the first universal sequence of the specific oligonucleic acid.

[0032] Regarding the oligonucleotide combination described in the second point above (i.e., "II. Oligonucleotide combination"), the oligonucleotide combination may or may not include a second universal primer:

[0033] (1) When the oligonucleic acid combination does not include a second universal primer:

[0034] Preferably, the second primer further comprises a functional nucleic acid sequence.

[0035] Preferably, the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0036] Preferably, the second primer further comprises a third tag sequence.

[0037] Preferably, the third tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.

[0038] (2) When the oligonucleotide combination includes a second universal primer:

[0039] Preferably, the universal primer further comprises: a second universal primer; the 3' end of the second universal primer is identical to a partial sequence or the entire sequence of the second universal sequence of the second primer.

[0040] In one embodiment of the present invention, the first specific sequence is an upstream specific sequence, and the second specific sequence is a downstream specific sequence;

[0041] In another embodiment of the present invention, the first specific sequence is a downstream specific sequence, and the second specific sequence is an upstream specific sequence.

[0042] Regarding the oligonucleotide combination described in the first point (i.e., "I. Oligonucleotide combination") and the oligonucleotide combination comprising a second universal primer described in the second point (i.e., "II. Oligonucleotide combination - (II) the oligonucleotide combination comprising a second universal primer"):

[0043] Preferably, the second universal primer comprises a functional nucleic acid sequence.

[0044] Preferably, the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0045] Preferably, the second universal primer further comprises a second tag sequence.

[0046] Preferably, the second tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.

[0047] Regarding the oligonucleotide combinations of the first and second points above (i.e., “Oligonucleotide combination 1” and “Oligonucleotide combination 2”):

[0048] Preferably, the first universal primer comprises a functional nucleic acid sequence.

[0049] Preferably, the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0050] Preferably, the first universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences.

[0051] Preferably, the first universal primer further comprises a first tag sequence.

[0052] Preferably, the first tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.

[0053] Preferably, the first universal sequence is the same as or different from the second universal sequence; further preferably, the first universal sequence is different from the second universal sequence.

[0054] Preferably, the 3' end of the universal sequence of the specific oligonucleic acid is subjected to blocking modification.

[0055] Preferably, the blocking modification comprises at least one of phosphorylation modification, spacer modification, and amino modification; further preferably, the blocking modification comprises phosphorylation modification.

[0056] Preferably, the design of the specific sequence in the specific oligonucleic acid follows conventional primer design principles, but is reverse complementary to the conventional primer sequence, for example: the upstream specific sequence is identical to the upstream negative chain sequence of the target region to be detected or complementary to the positive chain, and the downstream specific sequence is identical to the downstream positive chain sequence of the target region to be detected or complementary to the negative chain sequence.

[0057] Preferably, the design of the second specific sequence of the second primer follows conventional primer design principles and is the same as a conventional primer sequence.

[0058] Preferably, the specific oligonucleic acid comprises multiple groups of specific oligonucleic acids targeting different target areas to be detected.

[0059] Preferably, the second primers comprise multiple sets of second primers targeting different target regions to be detected.

[0060] Preferably, the first tag sequence, the second tag sequence and the third tag sequence may be the same or different.

[0061] Regarding the oligonucleotide combination described in the first point (i.e., "I. Oligonucleotide combination") and the oligonucleotide combination comprising a second universal primer described in the second point (i.e., "II. Oligonucleotide combination - (II) the oligonucleotide combination comprising a second universal primer"):

[0062] Preferably, 1) the 5' end of the first universal primer contains a phosphate group, and the 5' end of the amplified product can be provided with a phosphate group by the first universal primer, thereby avoiding the high cost of special phosphorylation; or

[0063] The 5' end of the second universal primer contains a phosphate group. The second universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.

[0064] Regarding the oligonucleotide combination not containing the second universal primer in the second point above (i.e., “II. Oligonucleotide Combination—(I) The oligonucleotide combination does not contain the second universal primer”):

[0065] Preferably, the 5' end of the first universal primer contains a phosphate group. The first universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.

[0066] The second aspect of the present invention provides a kit for preparing a multiplex PCR sequencing library, comprising: multiple sets of oligonucleotide combinations according to the first aspect of the present invention.

[0067] The plurality of groups are at least two groups, preferably more than two groups.

[0068] Preferably, the kit further comprises: DNA polymerase, Mg 2+ , PCR buffer, and at least one of dNTPs.

[0069] Preferably, the kit comprises: a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is: a lysis reagent.

[0070] Preferably, the kit comprises a nucleic acid extraction reagent combination selected from any one of the following methods: alkaline lysis method, phenol-chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.

[0071] The third aspect of the present invention provides a sequencing reagent kit, comprising: the oligonucleic acid combination of the first aspect of the present invention and / or the reagent kit of the second aspect of the present invention.

[0072] Preferably, the sequencing reagent set further comprises: a sequencing reagent kit.

[0073] A fourth aspect of the present invention provides a sequencing system comprising: at least one of a1) to a3) and a sequencer:

[0074] a1) the oligonucleic acid combination according to the first aspect of the present invention;

[0075] a2) a kit according to the second aspect of the present invention;

[0076] a3) The sequencing reagent set according to the third aspect of the present invention.

[0077] The fifth aspect of the present invention provides a multiplex PCR amplification method, comprising the steps of using multiple sets of oligonucleic acid combinations according to the first aspect of the present invention.

[0078] Preferably, the multiplex PCR amplification method comprises the following steps: obtaining a biological sample, and performing an amplification reaction on the biological sample using multiple sets of oligonucleic acid combinations according to the first aspect of the present invention, wherein the amplification reaction is performed in the same system.

[0079] The plurality of groups are at least two groups, preferably more than two groups.

[0080] Preferably, the same system is specifically not subjected to elution and / or purification treatment.

[0081] Preferably, the biological sample is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, and nucleic acids.

[0082] Preferably, when the biological sample is nucleic acid, the multiplex PCR amplification method does not include a cleavage reaction, and only performs an amplification reaction.

[0083] Preferably, when the biological sample is at least one of cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs, the multiplex PCR amplification method may further include a lysis reaction.

[0084] Preferably, the cleavage reaction and the amplification reaction are performed in the same system.

[0085] Preferably, no extraction or purification step is included after the cleavage reaction and before the amplification reaction.

[0086] Preferably, an extraction and purification step is further included after the cleavage reaction and before the amplification reaction.

[0087] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0088] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0089] Preferably, the lysis method comprises at least one of a physical method, a chemical method, and a biological method.

[0090] The sixth aspect of the present invention provides a method for preparing a multiplex PCR sequencing library, comprising the steps of the multiplex PCR amplification method of the fifth aspect of the present invention to obtain a sequencing library.

[0091] Preferably, a circularization reaction may be further included before obtaining the sequencing library, that is, circularization of the amplified linear library.

[0092] Preferably, the method further comprises the step of purifying the sequencing library.

[0093] Preferably, the purification is performed using magnetic beads.

[0094] A seventh aspect of the present invention provides a sequencing method comprising any one of b1) to b2):

[0095] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0096] b2) The steps of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.

[0097] Preferably, the sequencing method comprises the following steps: preparing a library and sequencing;

[0098] The method for preparing a library is the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.

[0099] Preferably, the sequencing further includes the following steps: library quality inspection.

[0100] An eighth aspect of the present invention provides a method for detecting a target region gene locus, comprising any one of b1) to b3):

[0101] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0102] b2) the step of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention;

[0103] b3) The sequencing method according to the seventh aspect of the present invention.

[0104] Preferably, the method for detecting gene loci in the target region comprises the following steps: obtaining a sequencing library of the target region; obtaining sequencing data; determining the gene loci in the target region;

[0105] The method for obtaining a sequencing library of a target region comprises b1) or b2):

[0106] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0107] b2) The steps of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.

[0108] Preferably, the method for detecting target region gene loci comprises the following steps: obtaining sequencing data; determining target region gene loci;

[0109] The sequencing method is the sequencing method of the seventh aspect of the present invention.

[0110] Preferably, the method for determining the target region gene loci comprises: comparing the obtained sequencing data with a reference genome to determine the bases of the target region gene loci.

[0111] In a ninth aspect, the present invention provides a use of the oligonucleic acid combination of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, or the sequencing system of the fourth aspect.

[0112] Use of the oligonucleic acid combination of the first aspect of the present invention and / or the kit of the second aspect of the present invention in any one of c1) to c8);

[0113] c1) preparing a multiplex PCR sequencing library;

[0114] c2) preparing products for multiplex PCR sequencing library preparation;

[0115] c3) sequencing;

[0116] c4) preparing products for sequencing;

[0117] c5) Detection of gene loci in the target region;

[0118] c6) preparing products for detecting gene loci in target regions;

[0119] c7) multiplex PCR amplification;

[0120] c8) preparing products of multiplex PCR amplification.

[0121] Use of the sequencing reagent set of the third aspect of the present invention and / or the sequencing system of the fourth aspect of the present invention in any one of items c3) to c6);

[0122] c3) sequencing;

[0123] c4) preparing products for sequencing;

[0124] c5) Detection of gene loci in the target region;

[0125] c6) Prepare products for detecting gene loci in target regions.

[0126] The beneficial effects of the present invention are:

[0127] The present invention provides an oligonucleic acid combination, comprising: a specific oligonucleic acid and a universal primer; the 3' end of the specific oligonucleic acid is set as a universal sequence, the 5' end is set as a specific sequence, and the universal sequence at the 3' end of the specific oligonucleic acid is complementary to the 3' end of the universal primer; the 3' end of the universal primer is extended under the action of a polymerase to obtain a product having the universal sequence at the 5' end and the specific sequence at the 3' end; the 3' end of the product can be complementary to a target region to be detected; the target region is amplified under the action of the polymerase to obtain a product containing the universal sequence and the target sequence to be detected; since the 3' end is a single fixed sequence, the formation of complementary structures and the generation of dimers can be effectively avoided, thereby solving the problem that traditional multiplex PCR primers are prone to non-specific amplification and dimers. At the same time, the entire amplification and library construction process can be quickly completed in one tube; the product can be directly used for subsequent sequencing, cloning and other applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] FIG1 is a schematic diagram of target-specific oligonucleotide design.

[0129] FIG2 is a schematic diagram of the reaction of target-specific oligonucleotides and universal primers.

[0130] FIG3 is a schematic diagram of primer amplification in the library preparation method of the present invention.

[0131] FIG4 is a flow chart of the preparation of the target amplification sequencing library in Example 1.

[0132] FIG5 is a flow chart of the preparation of the target amplification sequencing library in Example 2. DETAILED DESCRIPTION

[0133] In the description of the present invention, the terms "first," "second," and "third" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first," "second," or "third" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0134] In a first aspect, the present invention provides an oligonucleic acid combination.

[0135] 1. An oligonucleotide combination, comprising: a specific oligonucleotide and a universal primer;

[0136] The universal primers include: a first universal primer and a second universal primer;

[0137] The specific oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid;

[0138] The specific oligonucleic acid comprises: a specific sequence and a universal sequence;

[0139] The upstream-specific oligonucleic acid comprises: an upstream-specific sequence and a first universal sequence, wherein the upstream-specific sequence is located at the 5' end of the upstream-specific oligonucleic acid, and the first universal sequence is located at the 3' end of the upstream-specific oligonucleic acid;

[0140] The downstream-specific oligonucleotide comprises: a downstream-specific sequence and a second universal sequence, wherein the downstream-specific sequence is located at the 5' end of the downstream-specific oligonucleotide, and the second universal sequence is located at the 3' end of the downstream-specific oligonucleotide;

[0141] 1) The 3' end sequence of the first universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleotide, and the 3' end sequence of the second universal sequence of the downstream specific oligonucleotide is complementary to a portion or all of the second universal sequence; or

[0142] 2) The 3' end sequence of the second universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleic acid, and the 3' end sequence of the first universal primer is complementary to a portion or all of the second universal sequence of the downstream specific oligonucleic acid.

[0143] Preferably, the 3' end sequence of the first universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleic acid, and the 3' end sequence of the second universal sequence of the downstream specific oligonucleic acid is complementary to a portion or all of the second universal sequence of the downstream specific oligonucleic acid.

[0144] 2. an oligonucleotide combination, comprising: a specific oligonucleotide, a second primer, and a universal primer;

[0145] The universal primers include: a first universal primer;

[0146] The specific oligonucleic acid comprises: a specific sequence and a universal sequence, wherein the specific sequence is located at the 5' end of the specific oligonucleic acid, and the universal sequence is located at the 3' end of the specific oligonucleic acid;

[0147] The specific sequence is a first specific sequence, and the universal sequence is a first universal sequence;

[0148] The second primer comprises: a second specific sequence and a second universal sequence, wherein the second specific sequence is located at the 3' end of the second primer, and the second universal sequence is located at the 5' end of the second primer;

[0149] The 3' end sequence of the first universal primer is complementary to a portion or the entire sequence of the first universal sequence of the specific oligonucleic acid.

[0150] In the process of preparing a multiplex PCR sequencing library, the 5' end of a traditional primer (for example, the second primer) is a universal sequence and the 3' end is a specific sequence. The specific sequence at the 3' end is complementary to the target template and extended, which easily produces non-specific amplification and dimers. However, the specific oligonucleotide of the present application has a universal sequence at the 3' end and a specific sequence at the 5' end, and the universal sequence at the 3' end of the specific oligonucleotide is complementary to the 3' end of the universal primer. The 3' end of the universal primer is extended under the action of a polymerase to obtain a product having a universal sequence at the 5' end and a specific sequence at the 3' end. The 3' end of the product can be complementary to the target region to be tested. Under the action of the polymerase, the target region is amplified to obtain a product containing the universal sequence and the target sequence to be tested. In addition, since the 3' end of the specific oligonucleotide with a universal sequence at the 3' end and a specific sequence at the 5' end in the present application is a single fixed sequence, dimers are not easily formed during the PCR amplification process, thereby achieving the purpose of one-step multiplex amplification.

[0151] Regarding the oligonucleotide combination described in the second point above (i.e., "II. Oligonucleotide combination"), the oligonucleotide combination may or may not include a second universal primer:

[0152] (1) When the oligonucleic acid combination does not include a second universal primer:

[0153] Preferably, the second primer further comprises a functional nucleic acid sequence.

[0154] Preferably, the 5' end of the second primer further comprises a functional nucleic acid sequence, that is, the 5' end of the second universal sequence of the second primer further comprises a functional nucleic acid sequence.

[0155] Preferably, the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0156] Preferably, the 5' end of the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0157] Preferably, the second primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the second primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.

[0158] Preferably, the 5' end of the second primer contains one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the 5' end of the second primer contains two or more sequencing primer sequences or sequencing primer complementary sequences.

[0159] Preferably, the second primer further comprises a third tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.

[0160] Preferably, the 5' end of the second primer further comprises a third tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.

[0161] Preferably, the third tag sequence may be a unique molecular identifier (UMI), which is used to count the number of copies of nucleic acid molecules in a sample.

[0162] Preferably, the length of the third tag sequence is 5 to 20 bp.

[0163] Preferably, the third tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the third tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.

[0164] (2) When the oligonucleotide combination includes a second universal primer:

[0165] Preferably, the universal primer further comprises: a second universal primer; the 3' end of the second universal primer is identical to a partial sequence or the entire sequence of the second universal sequence of the second primer.

[0166] Preferably, the 3' end of the second universal primer is identical to a portion or all of the second universal sequence of the second primer.

[0167] In one embodiment of the present invention, the first specific sequence is an upstream specific sequence, and the second specific sequence is a downstream specific sequence;

[0168] In another embodiment of the present invention, the first specific sequence is a downstream specific sequence, and the second specific sequence is an upstream specific sequence.

[0169] Regarding the oligonucleotide combination described in the first point (i.e., "I. Oligonucleotide combination") and the oligonucleotide combination comprising a second universal primer described in the second point (i.e., "II. Oligonucleotide combination - (II) the oligonucleotide combination comprising a second universal primer"):

[0170] Preferably, the second universal primer comprises a functional nucleic acid sequence.

[0171] Preferably, the 5' end of the second universal primer comprises a functional nucleic acid sequence.

[0172] Preferably, the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0173] Preferably, the 5' end of the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0174] Preferably, the second universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the second universal primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.

[0175] Preferably, the 5' end of the second universal primer contains one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the 5' end of the second universal primer contains two or more sequencing primer sequences or sequencing primer complementary sequences.

[0176] Preferably, the second universal primer further comprises a second tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.

[0177] Preferably, the 5' end of the second universal primer further comprises a second tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, for example, a barcode sequence or an index sequence.

[0178] Preferably, the second tag sequence can be a unique molecular identifier (UMI), which is used to count the copy number of nucleic acid molecules in a sample.

[0179] Preferably, the length of the second tag sequence is 5 to 20 bp.

[0180] Preferably, the second tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the second tag sequence is located in the middle of two sections of the sequencing primer sequence or the sequencing primer complementary sequence.

[0181] Regarding the oligonucleotide combinations of the first and second points above (i.e., “Oligonucleotide combination 1” and “Oligonucleotide combination 2”):

[0182] Preferably, the first universal primer comprises a functional nucleic acid sequence.

[0183] Preferably, the 5' end of the first universal primer comprises a functional nucleic acid sequence.

[0184] Preferably, the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0185] Preferably, the 5' end of the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.

[0186] Preferably, the first universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the first universal primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.

[0187] Preferably, the 5' end of the first universal primer contains one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the 5' end of the first universal primer contains two or more sequencing primer sequences or sequencing primer complementary sequences.

[0188] Preferably, the first universal primer further comprises a first tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.

[0189] Preferably, the 5' end of the first universal primer further comprises a first tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, for example, a barcode sequence or an index sequence.

[0190] Preferably, the first tag sequence may be a unique molecular identifier (UMI), which is used to count the copy number of nucleic acid molecules in a sample.

[0191] Preferably, the length of the first tag sequence is 5 to 20 bp.

[0192] Preferably, the first tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the first tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.

[0193] Preferably, the universal sequence is selected from a partial sequencing adapter sequence, a complete sequencing adapter sequence, a sequencing primer binding sequence or any fixed sequence (such as comprising an enzyme cleavage site).

[0194] Preferably, the first universal sequence and the second universal sequence are each independently selected from a partial sequencing adapter sequence, a complete sequencing adapter sequence, a sequencing primer binding sequence or any fixed sequence (such as comprising a restriction enzyme cleavage site).

[0195] Preferably, the sequencing adapter sequence can be any sequencing adapter of any sequencing platform.

[0196] Preferably, the first universal sequence is the same as or different from the second universal sequence; further preferably, the first universal sequence is different from the second universal sequence.

[0197] Preferably, the 3' end of the universal sequence of the specific oligonucleotide is blocked with a modification to prevent extension at the 3' end.

[0198] Preferably, the blocking modification comprises at least one of phosphorylation modification, spacer modification, and amino modification; further preferably, the blocking modification comprises phosphorylation modification.

[0199] Preferably, the design of the specific sequence in the specific oligonucleic acid follows conventional primer design principles, but is reverse complementary to the conventional primer sequence, for example: the upstream specific sequence is identical to the upstream negative chain sequence of the target region to be detected or is reverse complementary to the positive chain, and the downstream specific sequence is identical to the downstream positive chain sequence of the target region to be detected or is reverse complementary to the negative chain sequence.

[0200] Preferably, the design of the second specific sequence of the second primer follows conventional primer design principles and is the same as a conventional primer sequence.

[0201] Preferably, the upstream specific sequence of the upstream specific oligonucleic acid is identical to the upstream negative strand sequence of the target region to be detected or is complementary to the positive strand sequence.

[0202] Preferably, the downstream specific sequence of the downstream specific oligonucleic acid is identical to the downstream positive strand sequence of the target region to be detected or is complementary to the negative strand sequence.

[0203] Preferably, when the first specific sequence of the specific oligonucleic acid is a downstream specific sequence, the first specific sequence of the specific oligonucleic acid is identical to the downstream positive chain sequence of the target region to be detected or is complementary to the negative chain sequence.

[0204] Preferably, when the first specific sequence of the specific oligonucleic acid is an upstream specific sequence, the first specific sequence of the specific oligonucleic acid is identical to the upstream negative chain sequence or complementary to the positive chain sequence of the target region to be detected.

[0205] Preferably, when the second specific sequence of the second primer is an upstream specific sequence, the second specific sequence of the second primer is complementary to the upstream negative chain sequence or identical to the positive chain sequence of the target region to be detected.

[0206] Preferably, when the second specific sequence of the second primer is a downstream specific sequence, the second specific sequence of the second primer is complementary to the positive strand sequence downstream of the target region to be detected or is identical to the negative strand sequence.

[0207] Preferably, the specific oligonucleic acid comprises multiple groups of specific oligonucleic acids targeting different target areas to be detected.

[0208] Preferably, the second primers comprise multiple sets of second primers targeting different target regions to be detected.

[0209] Preferably, the first tag sequence, the second tag sequence and the third tag sequence may be the same or different.

[0210] Preferably, the third tag sequence is different from the first tag sequence.

[0211] Preferably, the second tag sequence is different from the first tag sequence.

[0212] Preferably, the multiple tags can be set as a tag combination in the same primer.

[0213] Regarding the oligonucleotide combination described in the first point (i.e., "I. Oligonucleotide combination") and the oligonucleotide combination comprising a second universal primer described in the second point (i.e., "II. Oligonucleotide combination - (II) the oligonucleotide combination comprising a second universal primer"):

[0214] Preferably, 1) the 5' end of the first universal primer contains a phosphate group, and the 5' end of the amplified product can be provided with a phosphate group by the first universal primer, thereby avoiding the high cost of special phosphorylation; or

[0215] The 5' end of the second universal primer contains a phosphate group. The second universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.

[0216] Regarding the oligonucleotide combination not including the second universal primer in the second point above:

[0217] Preferably, the 5' end of the first universal primer contains a phosphate group. The first universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.

[0218] Preferably, the oligonucleic acid combination for detecting the EGFR gene comprises: a specific oligonucleic acid and a universal primer;

[0219] The specific oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid;

[0220] The universal primers include: a first universal primer and a second universal primer;

[0221] The sequences of the upstream specific oligonucleotides are shown in SEQ ID NO.19 to SEQ ID NO.26, and the sequences of the downstream specific oligonucleotides are shown in SEQ ID NO.27 to SEQ ID NO.34;

[0222] The sequences of the first universal primers are shown in SEQ ID NOs. 43 to 50, and the sequence of the second universal primer is shown in SEQ ID NO. 18.

[0223] Preferably, the oligonucleic acid combination for detecting the EGFR gene comprises: a specific oligonucleic acid, a second primer and a universal primer;

[0224] The universal primers include: a first universal primer;

[0225] The sequences of the second primers are shown in SEQ ID NO.1 to SEQ ID NO.8, and the sequences of the specific oligonucleotides are shown in SEQ ID NO.9 to SEQ ID NO.16;

[0226] The sequences of the first universal primers are shown in SEQ ID NOs. 43 to 50.

[0227] Preferably, the 3' end of the specific oligonucleotides having sequences as shown in SEQ ID NOs. 9 to 16, 19 to 34 is subjected to blocking modification.

[0228] Preferably, the blocking modification comprises at least one of phosphorylation modification, spacer modification, and amino modification; further preferably, the blocking modification comprises phosphorylation modification.

[0229] Preferably, the 5' end of the second universal primer contains a phosphate group. The second universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.

[0230] The second aspect of the present invention provides a kit for preparing a multiplex PCR sequencing library, comprising: multiple sets of oligonucleotide combinations according to the first aspect of the present invention.

[0231] The plurality of groups are at least two groups, preferably more than two groups.

[0232] Preferably, the kit further comprises: DNA polymerase, Mg 2+ , PCR buffer, at least one of dNTPs; further preferably, the kit further comprises: DNA polymerase, Mg 2+ , PCR buffer and dNTPs;.

[0233] Preferably, the kit comprises: a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is: a lysis reagent.

[0234] Preferably, the kit further comprises a nucleic acid extraction reagent combination, specifically a nucleic acid extraction reagent combination for any one of the following methods: alkaline lysis method, phenol-chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.

[0235] Preferably, the nucleic acid extraction reagent combination comprises: at least one of: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent; further preferably, the nucleic acid extraction reagent combination comprises: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent.

[0236] Preferably, the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.

[0237] The third aspect of the present invention provides a sequencing reagent kit, comprising: the oligonucleic acid combination of the first aspect of the present invention and / or the reagent kit of the second aspect of the present invention.

[0238] Preferably, the sequencing reagent set further comprises: a sequencing reagent kit.

[0239] A fourth aspect of the present invention provides a sequencing system comprising: at least one of a1) to a3) and a sequencer:

[0240] a1) the oligonucleic acid combination according to the first aspect of the present invention;

[0241] a2) a kit according to the second aspect of the present invention;

[0242] a3) The sequencing reagent set according to the third aspect of the present invention.

[0243] The fifth aspect of the present invention provides a multiplex PCR amplification method, comprising the steps of using multiple sets of oligonucleic acid combinations according to the first aspect of the present invention.

[0244] Preferably, the multiplex PCR amplification method comprises the following steps: obtaining a biological sample, and performing an amplification reaction on the biological sample using multiple sets of oligonucleic acid combinations according to the first aspect of the present invention, wherein the amplification reaction is performed in the same system.

[0245] The plurality of groups are at least two groups, preferably more than two groups.

[0246] Preferably, the same system is specifically not subjected to elution and / or purification treatment.

[0247] Preferably, the biological sample is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, and nucleic acids; further comprises: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, or nucleic acids.

[0248] Preferably, the nucleic acid can be a nucleic acid obtained by lysing the biological sample A, and the biological sample A is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs; further: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs or nasal swabs.

[0249] Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood, and cerebrospinal fluid.

[0250] Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.

[0251] Preferably, when the biological sample is nucleic acid, the multiplex PCR amplification method does not include a cleavage reaction, and only performs an amplification reaction.

[0252] Preferably, when the biological sample is at least one of cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs, the multiplex PCR amplification method may further include a lysis reaction.

[0253] Preferably, the cleavage reaction precedes the amplification reaction.

[0254] Preferably, the cleavage reaction and the amplification reaction are performed in the same system.

[0255] Preferably, the same system is specifically the same reaction container.

[0256] Preferably, no extraction or purification step is included after the cleavage reaction and before the amplification reaction.

[0257] Preferably, an extraction and purification step is further included after the cleavage reaction and before the amplification reaction to remove impurities such as salts and organic agents.

[0258] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0259] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0260] Preferably, the cleavage reaction and the amplification reaction are performed in different systems.

[0261] Preferably, the cleavage reaction further includes an extraction and purification step to remove impurities such as salts and organic agents.

[0262] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0263] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0264] Preferably, the lysis method comprises at least one of a physical method, a chemical method, and a biological method.

[0265] Preferably, the physical method comprises at least one of a boiling method, a glass bead method, an ultrasonic method, a grinding method, a freeze-thaw method, and a homogenization method.

[0266] Preferably, the chemical method comprises at least one of a surfactant method (SDS method) and an alkaline lysis method.

[0267] Preferably, the biological method comprises an enzymatic method, such as cleavage by enzymes such as lysozyme and proteinase K.

[0268] Preferably, the number of cycles of the amplification is 20 to 30; further 23 to 27.

[0269] Preferably, the amplification system further comprises: DNA polymerase, Mg 2+ , PCR buffer, at least one of dNTPs; further preferably, the amplification system further comprises: DNA polymerase, Mg 2+ , PCR buffer and dNTPs;

[0270] The sixth aspect of the present invention provides a method for preparing a multiplex PCR sequencing library, comprising the steps of the multiplex PCR amplification method of the fifth aspect of the present invention to obtain a sequencing library.

[0271] Preferably, the method further comprises the step of purifying the amplified product.

[0272] Preferably, the purification is performed using magnetic beads.

[0273] Preferably, a circularization reaction may be further included before obtaining the sequencing library, that is, circularization of the amplified linear library.

[0274] A seventh aspect of the present invention provides a sequencing method comprising any one of b1) to b2):

[0275] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0276] b2) The steps of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.

[0277] Preferably, the sequencing method comprises the following steps: preparing a library; sequencing;

[0278] The method for preparing a library is the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.

[0279] Preferably, the sequencing further includes the following steps: library quality inspection.

[0280] In an eighth aspect, the present invention provides a method for detecting a target region gene locus, comprising any one of b1) to b3):

[0281] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0282] b2) the step of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention;

[0283] b3) The sequencing method according to the seventh aspect of the present invention.

[0284] Preferably, the method for detecting gene loci in the target region comprises the following steps: obtaining a sequencing library of the target region; obtaining sequencing data; determining the gene loci in the target region;

[0285] The method for obtaining a sequencing library of a target region comprises b1) or b2):

[0286] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0287] b2) The steps of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.

[0288] Preferably, the method for detecting target region gene loci comprises the following steps: obtaining sequencing data; determining target region gene loci;

[0289] The sequencing method is the sequencing method of the seventh aspect of the present invention.

[0290] Preferably, the method for determining the target region gene loci comprises: comparing the obtained sequencing data with a reference genome to determine the bases of the target region gene loci.

[0291] A method for detecting EGFR gene loci comprises the following steps: obtaining a sequencing library of a target region; obtaining sequencing data; determining the gene loci of the target region;

[0292] The method for obtaining a sequencing library of a target region comprises b1) or b2):

[0293] b1) the steps of the multiplex PCR amplification method according to the fifth aspect of the present invention;

[0294] b2) the step of the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention;

[0295] The oligonucleic acid combination is the oligonucleic acid combination for detecting the EGFR gene according to the first aspect of the present invention.

[0296] Preferably, the method for detecting target region gene loci comprises the following steps: obtaining sequencing data; determining target region gene loci;

[0297] The sequencing method is the sequencing method of the seventh aspect of the present invention;

[0298] The oligonucleic acid combination is the oligonucleic acid combination for detecting the EGFR gene according to the first aspect of the present invention.

[0299] Preferably, the method for determining the target region gene loci comprises: comparing the obtained sequencing data with a reference genome to determine the bases of the target region gene loci.

[0300] In a ninth aspect, the present invention provides a use of the oligonucleic acid combination of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, or the sequencing system of the fourth aspect.

[0301] Use of the oligonucleic acid combination of the first aspect of the present invention and / or the kit of the second aspect of the present invention in any one of c1) to c8);

[0302] c1) preparing a multiplex PCR sequencing library;

[0303] c2) preparing products for multiplex PCR sequencing library preparation;

[0304] c3) sequencing;

[0305] c4) preparing products for sequencing;

[0306] c5) Detection of gene loci in the target region;

[0307] c6) preparing products for detecting gene loci in target regions;

[0308] c7) multiplex PCR amplification;

[0309] c8) preparing products of multiplex PCR amplification.

[0310] Use of the sequencing reagent set of the third aspect of the present invention and / or the sequencing system of the fourth aspect of the present invention in any one of items c3) to c6);

[0311] c3) sequencing;

[0312] c4) preparing products for sequencing;

[0313] c5) Detection of gene loci in the target region;

[0314] c6) Prepare products for detecting gene loci in target regions.

[0315] The present invention is further described in detail below through specific examples.

[0316] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0317] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0318] Example 1 Method for Preparing a Library of EGFR Tumor Hotspot Genes

[0319] The method for preparing a library of EGFR tumor hotspot genes is shown in FIG4 , and includes the following steps:

[0320] (1) Design an EGFR gene tumor hotspot gene capture panel and universal primers. The panel contains 8 pairs of specific oligonucleotides (EX1-EGFR_1R to EX1-EGFR_8R) / primers (EX1-EGFR_1F to EX1-EGFR_8F). The amplicon size is 100 to 200 bp. The specific oligonucleotide / primer sequences are shown in Table 1. The universal primer includes universal primer R. The nucleotide sequence of universal primer R is: (The double-underlined portion is SEQ ID NO.17, the bold portion is SEQ ID NO.51, NNNNNNNNNNN is the tag sequence, which can be random or pre-set, and N is independently selected from A, T, C, and G); (The specific universal primer R is shown in Table 13), the underlined portion is complementary to the 3' end sequence of the specific oligonucleotide (universal sequence).

[0321] (2) PCR reaction

[0322] The PCR system was configured in a PCR tube according to the reaction system shown in Table 2, and the PCR reaction was performed. The reaction program was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 25 cycles; 72°C for 5 min; 12°C ∞. After the reaction, the product was purified using 1.5X AMPure (Beckman Co.) magnetic beads, and finally the purified product was dissolved in 22 μL of elution buffer to obtain the library.

[0323] Table 1 Specific oligonucleotide / primer sequences

[0324]

[0325] Note: The above-mentioned specific oligonucleotides / primers were mixed at a concentration of 2 μM each to obtain a specific oligonucleotide / primer pool with a total concentration of 2 μM; the primer sequence was conventionally designed, i.e., the 5' to 3' end was sequentially: universal sequence and specific sequence; the specific oligonucleotide sequence was designed according to the present invention, i.e., the 5' to 3' end was sequentially: specific sequence and universal sequence, the underlined portion and the sequence complementary to the 3' end of universal primer R.

[0326] Table 2 PCR reaction system

[0327]

[0328] Example 2 Method for Preparing a Library of EGFR Tumor Hotspot Genes

[0329] The method for preparing a library of EGFR tumor hotspot genes is shown in FIG5 , and includes the following steps:

[0330] (1) Design an EGFR gene tumor hotspot gene capture panel and universal primers. The panel contains 8 pairs of specific oligonucleotide sequences, and the amplicon size is 100 to 200 bp. The design sequences of the specific oligonucleotide sequences are shown in Table 3. The universal primers include universal primer F and universal primer R. The nucleotide sequence of universal primer R is: (The double underlined portion is SEQ ID NO.17, the bold portion is SEQ ID NO.51, NNNNNNNNNNN is the tag sequence, which can be random or set in advance, and N is independently selected from A, T, C, and G); (The universal primer R is specifically shown in Table 13), the underlined portion is complementary to the 3' end sequence (universal sequence) of the upstream specific oligonucleotide; the nucleotide sequence of the universal primer F is: P-GAACGACATGGCTACGATCCGACTT (SEQ ID NO.18), P is phosphorylation modification, and the underlined portion is complementary to the 3' end sequence of the downstream specific oligonucleotide.

[0331] (2) PCR reaction

[0332] A PCR system was configured in a PCR tube according to the reaction system shown in Table 4 (the specific oligonucleotide / primer pool (2 μM) shown in Table 1 was replaced with the specific oligonucleotide pool shown in Table 3), and the PCR reaction was performed. The reaction procedure was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 25 cycles; 72°C for 5 min; 12°C ∞. After the reaction, the product was purified using 1.5X AMPure (Beckman Co.) magnetic beads, and finally the purified product was dissolved in 22 μL of elution buffer to obtain a library.

[0333] Table 3 Specific oligonucleotide sequences

[0334]

[0335]

[0336] Note: The above-mentioned specific oligonucleotides were mixed at a concentration of 2 μM each to obtain a specific oligonucleotide pool with a total concentration of 2 μM; the upstream specific oligonucleotide sequence was designed by the present invention, i.e., the 5' to 3' end was sequentially: a specific sequence and a universal sequence, wherein the underlined portion was complementary to the 3' end of the universal primer R; the downstream specific primer sequence was designed by the present invention, i.e., the 5' to 3' end was sequentially: a specific sequence and a universal sequence, wherein the underlined portion was complementary to the 3' end of the universal primer F.

[0337] Table 4 PCR reaction system

[0338]

[0339] Example 3: Method for Preparing a Library of EGFR Tumor Hotspot Genes without Extracting Blood Samples

[0340] The method for preparing a library of EGFR tumor hotspot genes is shown in FIG5 , and includes the following steps:

[0341] (1) Design an EGFR gene tumor hotspot gene capture panel and universal primers. The panel contains 8 pairs of specific oligonucleotide sequences, and the amplicon size is 100 to 200 bp. The design sequences of the specific oligonucleotide sequences are shown in Table 3. The universal primers include universal primer F and universal primer R. The nucleotide sequence of universal primer R is: (The double underlined portion is SEQ ID NO.17, the bold portion is SEQ ID NO.51, NNNNNNNNNNN is the tag sequence, which can be random or set in advance, and N is independently selected from A, T, C, and G); (The universal primer R is specifically shown in Table 13), the underlined portion is complementary to the 3' end sequence (universal sequence) of the upstream specific oligonucleotide; the nucleotide sequence of the universal primer F is: P-GAACGACATGGCTACGATCCGACTT (SEQ ID NO.18), P is phosphorylation modification, and the underlined portion is complementary to the 3' end sequence of the downstream specific oligonucleotide.

[0342] (2) Blood sample lysis

[0343] 1 μL of blood sample was added to 25 μL of TE solution, lysed at 95°C for 10 minutes, centrifuged at 10,000 g for 5 minutes, and 20 μL of supernatant was used for subsequent PCR reaction.

[0344] A PCR system was configured in a PCR tube according to the reaction system shown in Table 4 (the specific oligonucleotide / primer pool (2 μM) shown in Table 1 was replaced with the specific oligonucleotide pool shown in Table 3), and the PCR reaction was performed. The reaction procedure was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 25 cycles; 72°C for 5 min; 12°C ∞. After the reaction, the product was purified using 1.5X AMPure (Beckman Co.) magnetic beads, and finally the purified product was dissolved in 22 μL of elution buffer to obtain a library.

[0345] Comparative Example 1: Preparation Method of EGFR Gene Tumor Hotspot Gene Library

[0346] The method for preparing a library of EGFR tumor hotspot genes comprises the following steps:

[0347] (1) Design an EGFR gene tumor hotspot gene capture panel and universal primers. The panel contains 8 pairs of primers with amplicon sizes ranging from 100 to 200 bp. The primer design sequences are shown in Table 5. The universal primers include universal primer F and universal primer R. The nucleotide sequence of universal primer R is: (The double underlined portion is SEQ ID NO.17, the bold portion is SEQ ID NO.51, NNNNNNNNNNN is the tag sequence, which can be random or set in advance, and N is independently selected from A, T, C, and G); (The universal primer R is specifically shown in Table 13), the underlined portion is the same as the 5' end sequence (universal sequence) of the conventional downstream specific primer; the nucleotide sequence of the universal primer F is: P-GAACGACATGGCTACGATCCGACTT (SEQ ID NO.18), P is phosphorylation modification, and the underlined portion is the same as the 5' end sequence of the conventional upstream specific amplification primer.

[0348] (2) First round of PCR

[0349] A PCR system was prepared in a PCR tube according to the reaction system shown in Table 6, and a PCR reaction was performed. The reaction procedure was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 10 cycles; 72°C for 5 min; 12°C ∞. After the reaction, the product was purified using 1.5X AMPure (Beckman Co.) magnetic beads, and finally the purified product was dissolved in 22 μL of elution buffer to obtain the product.

[0350] (3) Second round of PCR

[0351] The PCR system was configured in a PCR tube according to the reaction system shown in Table 7, and the PCR reaction was performed. The reaction program was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 15 cycles; 72°C for 5 min; 12°C ∞. After the reaction, the product was purified using 1.5X AMPure (Beckman Co.) magnetic beads, and finally the purified product was dissolved in 22 μL of elution buffer to obtain the library.

[0352] Table 5 Conventionally designed specific amplification primers

[0353]

[0354] Note: The above primers were mixed at a concentration of 10 μM each to obtain a primer pool with each primer at a concentration of 10 μM; the upstream specific primer sequence was conventionally designed, i.e., the 5' to 3' end was sequentially: universal sequence and specific sequence, of which the underlined portion was the same as the 3' end of universal primer F; the downstream specific primer sequence was conventionally designed, i.e., the 5' to 3' end was sequentially: universal sequence and specific sequence, of which the underlined portion was the same as the 3' end of universal primer R.

[0355] Table 6 First round PCR reaction system

[0356]

[0357] Table 7 Second round PCR reaction system

[0358]

[0359] Effect Example High-throughput Sequencing Method for EGFR Gene Tumor Hotspot Genes

[0360] The following effect examples are based on the MGISEQ-2000 platform of BGI's sequencer; the reagents used are all derived from the library construction kit and paired-end sequencing kit (hereinafter referred to as PE100 kit) used with the sequencer. The sequencing read length of PE100 is used in the following effect examples; the sequencer and reagent operations used in the process refer to the instructions for use of the platform, and finally data analysis is performed, including data utilization, dimer ratio, alignment ratio, target region data ratio, uniformity and other performance (analysis method reference: Campbell, Nathan R., Stephanie A. Harmon, and Shawn R. Narum. "Genotyping-in-Thousands by sequencing (GT-seq): A cost-effective SNP genotyping method based on custom amplicon sequencing." Molecular ecology resources 15.4 (2015): 855-867.), as follows:

[0361] 1) Library preparation: the library preparation method of Examples 1, 2, 3 or Comparative Example 1;

[0362] 2) Library quality control: The obtained products are quantified and the band size is checked;

[0363] 3) Sequencing: The resulting library was sequenced on the MGISEQ-2000 platform (PE100).

[0364] 4) Data Analysis: The obtained offline data were aligned to the human reference genome (hg19) using BWA software. The alignment rate, specificity, and uniformity were statistically analyzed using samtools. Finally, mutation analysis was performed using GATK software.

[0365] The sequencing data statistics of the libraries obtained in Example 1 and Comparative Example 1 are shown in Table 8, and the mutation detection statistics of Example 1 are shown in Table 9: the unique alignment ratio and the target region data ratio in the sequencing data of the libraries obtained in Example 1 and Comparative Example 1 are comparable, and the data utilization rate in Example 1 is higher than that in Comparative Example 1, the dimer ratio is lower than that in Comparative Example 1, and the method of Example 1 has only one operation step; the detected value of the mutation frequency in Example 1 is comparable to the theoretical value.

[0366] The sequencing data statistics of the libraries obtained in Example 2 and Comparative Example 1 are shown in Table 10, and the mutation detection statistics are shown in Table 11: the unique alignment ratio and the target region data ratio in the sequencing data of the libraries obtained in Example 2 and Comparative Example 1 are comparable, and the data utilization rate in Example 2 is higher than that in Comparative Example 1, and the dimer ratio is lower than that in Comparative Example 1. In addition, the method of Example 2 has only one operation step; the detection value of the mutation frequency in Example 2 is comparable to the theoretical value.

[0367] The sequencing data statistics of the libraries obtained in Example 3 and Comparative Example 1 are shown in Table 12: the unique alignment ratio and target region data ratio in the sequencing data of the libraries obtained in Example 3 and Comparative Example 1 are comparable, the data utilization rate in Example 3 is higher than that in Comparative Example 1, and the dimer ratio is lower than that in Comparative Example 1, and the method of Example 3 has only one operation step; at the same time, the unique alignment ratio, target region data ratio, data utilization rate, and dimer ratio in the sequencing data of the library obtained in Example 3 are comparable to those in Example 2. It can be seen that performing the cleavage reaction and the amplification reaction in the same system does not affect its performance.

[0368] Table 8 Sequencing data statistics of the libraries obtained in Example 1 and Comparative Example 1

[0369]

[0370] Table 9 Mutation detection statistics

[0371]

[0372] Table 10 Sequencing data statistics of the libraries obtained in Example 1 and Comparative Example 1

[0373]

[0374] Table 11 Mutation detection statistics

[0375]

[0376] Table 12 Sequencing data statistics of the libraries obtained in Example 3 and Comparative Example 1

[0377]

[0378] Table 13 Universal Primer R Sequence

[0379]

[0380]

[0381] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Oligonucleotide combination, including: specific oligonucleotide and universal primer; The universal primers include: a first universal primer and a second universal primer; The specific oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid; The specific oligonucleic acid comprises: a specific sequence and a universal sequence; The upstream specific oligonucleotide comprises: an upstream specific sequence and a first universal sequence, wherein, The upstream specific sequence is located at the 5' end of the upstream specific oligonucleic acid, and the first universal sequence is located at the 3' end of the upstream specific oligonucleic acid; The downstream-specific oligonucleotide comprises: a downstream-specific sequence and a second universal sequence, wherein the downstream-specific sequence is located at the 5' end of the downstream-specific oligonucleotide, and the second universal sequence is located at the 3' end of the downstream-specific oligonucleotide; 1) The 3' end sequence of the first universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleotide, and the 3' end sequence of the second universal sequence of the downstream specific oligonucleotide is complementary to a portion or all of the second universal sequence; or 2) The 3' end sequence of the second universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleic acid, and the 3' end sequence of the first universal primer is complementary to a portion or all of the second universal sequence of the downstream specific oligonucleic acid.

2. An oligonucleotide combination, comprising: a specific oligonucleotide, a second primer, and a universal primer; The universal primers include: a first universal primer; The specific oligonucleotide comprises: a specific sequence and a universal sequence, wherein, The specific sequence is located at the 5' end of the specific oligonucleotide, and the universal sequence is located at the 3' end of the specific oligonucleotide; The specific sequence is a first specific sequence, and the universal sequence is a first universal sequence; The second primer comprises: a second specific sequence and a second universal sequence, wherein the second specific sequence is located at the 3' end of the second primer, and the second universal sequence is located at the 5' end of the second primer; The 3' end sequence of the first universal primer is complementary to a portion or the entire sequence of the first universal sequence of the specific oligonucleic acid.

3. The oligonucleic acid combination according to claim 2, characterized in that: The universal primer further comprises: a second universal primer; the 3' end of the second universal primer is identical to a partial sequence or the entire sequence of the second universal sequence of the second primer.

4. The oligonucleic acid combination according to claim 1 or 3, characterized in that: The second universal primer comprises a functional nucleic acid sequence; Preferably, the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence; Preferably, the second universal primer further comprises a second tag sequence; Preferably, the second tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.

5. The oligonucleic acid combination according to claim 2, characterized in that: The second primer further comprises a functional nucleic acid sequence; Preferably, the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence; Preferably, the second primer further comprises a third tag sequence; Preferably, the third tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.

6. The oligonucleic acid combination according to claim 1 or 2, characterized in that: The first universal primer comprises a functional nucleic acid sequence; Preferably, the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence; Preferably, the first universal primer further comprises a first tag sequence; Preferably, the first tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.

7. The oligonucleic acid combination according to claim 1 or 2, characterized in that: The 3' end of the universal sequence of the specific oligonucleic acid is subjected to blocking modification; Preferably, the blocking modification comprises at least one of phosphorylation modification, spacer modification, and amino modification.

8. The oligonucleic acid combination according to claim 1 or 2, characterized in that: The first universal sequence and the second universal sequence are the same or different.

9. The oligonucleic acid combination according to claim 2, characterized in that: 1) The first specific sequence is an upstream specific sequence, and the second specific sequence is a downstream specific sequence; or 2) The first specific sequence is a downstream specific sequence, and the second specific sequence is an upstream specific sequence.

10. The oligonucleic acid combination according to any one of claims 4 to 6, characterized in that: The first tag sequence, the second tag sequence and the third tag sequence may be the same or different. A kit comprising the oligonucleic acid combination according to claim 1 or 2.

12. The kit according to claim 11, characterized in that: The kit further comprises: at least one of DNA polymerase, Mg2+, PCR buffer, and dNTPs; Preferably, the kit comprises: a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is: a lysis reagent; Preferably, the kit comprises: a nucleic acid extraction reagent combination.

13. A sequencing reagent kit comprising: a sequencing reagent kit and the reagent kit according to claim 11.

14. A multiplex PCR amplification method comprising the following steps: obtaining a biological sample, and performing an amplification reaction on the biological sample using the multiple oligonucleotide combinations of claim 1 or 2, wherein the multiple amplification reactions are performed in the same system, and wherein the multiple groups are at least two or more.

15. The multiplex PCR amplification method according to claim 14, wherein: The same system specifically does not undergo elution and / or purification.

16. The multiplex PCR amplification method according to claim 14, wherein: The biological sample is at least one of cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, and nucleic acids; Preferably, when the biological sample is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs, the multiplex PCR amplification method further comprises a lysis reaction; Preferably, the cleavage reaction and the amplification reaction are performed in the same system.

17. The multiplex PCR amplification method according to claim 16, wherein: After the cleavage reaction and before the amplification reaction, an extraction and purification step is also included; Preferably, the lysis method comprises: at least one of a physical method, a chemical method, and a biological method; Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid; Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

18. A method for preparing a multiplex PCR sequencing library, comprising the steps of the multiplex PCR amplification method according to claim 14, to obtain a sequencing library.

19. The method according to claim 18, wherein: The method further comprises the steps of: purifying the amplified product; Preferably, the purification is performed using magnetic beads.

20. A method for detecting gene loci in a target region, comprising the following steps: obtaining a sequencing library of the target region; obtaining sequencing data; determining the gene loci in the target region; The method for obtaining a sequencing library of a target region comprises b1) or b2): b1) the multiplex PCR amplification method according to claim 14; b2) The step of the method for preparing a multiplex PCR sequencing library according to claim 18.

21. The method according to claim 20, characterized in that: The method for determining the target region gene site comprises: comparing the obtained sequencing data with the reference genome to determine the bases of the target region gene site.

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