Novel library preparation method based on a sequential reaction of oligo-DNA ligation and DNA amplification by ligated oligo-DNA.

Abstract

To provide a technology that enables the economical and simple preparation of a library containing a high proportion of the target product when performing sequencing analysis of multiple samples simultaneously in a single analysis using a next-generation sequencer. [Solution] A method for preparing a mixture containing two or more target products with different barcode sequences, comprising adding a dsDNA-specific dye to each gene amplification reaction solution containing the target product, quantifying the concentration of each target product from the fluorescence value of the dye, and mixing each gene amplification reaction solution so that each target product is in an arbitrary concentration ratio based on the quantification results.

Description

[Technical Field]

[0001] The present invention relates primarily to assays for detecting or sequencing specific groups of nucleic acid sequences. In certain embodiments, the present invention provides an amplification method in which one or more artificially designed nucleic acid sequences (hereinafter referred to as "artificial nucleic acid sequences"), including a barcode sequence or the like, which is an artificial nucleic acid sequence for identifying a sample, are added to a target nucleic acid sequence. [Background technology]

[0002] The detection of specific nucleic acid sequences is used in many fields, including genetic diagnosis in medicine, hygiene testing in food, and environmental monitoring.

[0003] In addition, with the recent rise of next-generation sequencers (NGS), nucleic acid sequencing has become significantly easier and cheaper compared to conventional sequencing methods, and is gaining popularity as a new analytical technique. In NGS, to provide a mixture of amplification products from multiple samples, a specific artificial nucleic acid sequence containing a barcode sequence for identifying the sample must be attached to both ends of the nucleic acid fragment to be sequenced. Therefore, a nucleic acid amplification reaction step is required in addition to the step of amplifying the target nucleic acid sequence.

[0004] It is possible to obtain nucleic acid fragments for NGS even when using primers that have a specific artificial nucleic acid sequence containing a barcode sequence pre-attached to the 5' end of a primer specific to the target nucleic acid sequence (prior art [1]). However, this method is extremely uneconomical because it requires preparing primers with different barcode sequences for each sample for each type of nucleic acid sequence (Figure 1).

[0005] A two-step PCR method (prior art [2]) is commonly used as an economical means of obtaining an amplified product with an artificial nucleic acid sequence attached (Non-Patent Literature 1). In this method, first, a PCR is performed using a primer (hereinafter referred to as an artificial attachment-specific primer) in which a portion of the 3' end of an artificial nucleic acid sequence is attached to the 5' end of a specific sequence to obtain a product derived from the target nucleic acid (hereinafter referred to as the specific product). Subsequently, after purifying this product, a second PCR is performed using this as a template with a primer (hereinafter referred to as an artificial sequence primer) having an artificial nucleic acid sequence such as a barcode sequence at its 3' end. Through the above two PCR amplifications, an amplified product (hereinafter referred to as the target product) with artificial nucleic acid sequences attached to both ends of the target nucleic acid can be obtained (Figure 2).

[0006] However, the prior art [2] requires two PCR tests, and the specific product obtained from the first PCR test must be purified and its concentration adjusted, making the process complicated and time-consuming.

[0007] As an economical and simple means of obtaining a target product, a method has been disclosed in which both an artificial addition-specific primer pair and an artificial sequence primer pair are simultaneously added to a single PCR reaction solution, and the amplification reaction is carried out, thereby obtaining the target product in a single PCR (prior art [3]) (Patent Documents 1 and 2).

[0008] According to Patent Documents 1 and 2, by lowering the concentration of the artificially added specific primer to that of the artificial sequence primer, the artificial sequence primer capable of binding to the artificial nucleic acid sequence commonly added to all target sequences can predominantly carry out the amplification reaction. This allows for efficient acquisition of the target product, and in the case of multiple target nucleic acids, it is possible to suppress variations in amplification efficiency between genes (Figure 3).

[0009] However, when preparing nucleic acid sequences for NGS using prior art [3], the artificial sequence primers used at high concentrations become long chains of approximately 60-70 base pairs, increasing the likelihood of primer dimer production. When multiple nucleic acid sequences are targeted, the number of artificial addition-specific primers used increases, leading to frequent production of primer dimers derived from these primers. This results in a problem where the proportion of sequences other than the target sequence increases during NGS analysis.

[0010] In addition, as the number of target nucleic acid sequences increases, the total amount of artificial addition-specific primers increases. As a result, artificial sequence primers cannot dominantly perform the amplification reaction, and the proportion of nucleic acid sequences without artificial nucleic acid sequences increases.

[0011] A method (prior art [4]) has been proposed that can solve the aforementioned problems (Patent Document 3). In the prior art [4], in a reaction to add an artificial nucleic acid sequence to a target gene sequence, a method is disclosed in which, in addition to an artificial addition-specific primer, two pairs of artificial sequence primers are used: a long-chain artificial sequence primer (hereinafter, long-chain artificial sequence primer; "primer pair Y" in Figure 4) and a short-chain artificial sequence primer (hereinafter, short-chain artificial sequence primer; "primer pair Z" in Figure 4). This method suppresses the amplification of nonspecific sequences derived from the primers and efficiently amplifies the target product. Furthermore, it is stated that by lowering the concentration of the artificial addition-specific primer and the artificial sequence primer containing a barcode sequence, etc. (primer pair Y), and increasing the concentration of the short-chain primer (primer pair Z), the target product can be amplified more efficiently.

[0012] However, prior art [4] uses at least three sets of primers, including a pair of artificially added specific primers, a pair of long-chain artificial sequence primers, and a pair of short-chain artificial sequence primers. Since three types of PCR reactions proceed simultaneously, it is necessary to strictly optimize the reaction conditions so that the three sets of primers function simultaneously and properly in a single PCR reaction solution.

[0013] Based on the above-described principle characteristics, the prior art [4] has the following problems: (1) strict optimization of reaction conditions is required for each target nucleic acid, which requires excessive time and labor for this operation; (2) compared with ordinary PCR that uses only one set of primer pairs, it has lower sensitivity and requires more initial templates (target genes).

[0014] A method (prior art [5]) that can solve the problems of the above prior art [4] has also been proposed (Patent Documents 4 and 5), but this prior art also has the following problems shown below.

[0015] In NGS analysis, in order to make the added amount of the target product fall within the range specified by each NGS device manufacturer, quantification of the said product is essential. For quantification of the target product, simple and low-cost methods such as electrophoresis, fluorescence photometry, and absorbance photometry, as well as quantitative PCR (hereinafter referred to as qPCR), which is a relatively complicated and high-cost method, are generally used. In the prior art, since it is difficult to introduce artificial nucleic acid sequences into all specific products, not only the target product but also specific products without added artificial nucleic acid sequences (hereinafter referred to as non-target products) are synthesized. Although the former simple and low-cost method cannot accurately distinguish between the target product and the non-target product, when the proportion of the target product in the specific products is extremely high, the difference between the quantification value obtained by assuming that all specific products are target products and the true amount of the target product can be ignored, so it can be used without problems. On the other hand, when the proportion of the target product in the specific products is low, although it is a complicated and high-cost method, there is a need to use the qPCR method that can distinguish the presence or absence of artificial nucleic acid sequences and quantify only the target product. In the above-mentioned prior art [5], since not only the target product but also many specific products without added artificial nucleic acid sequences are synthesized, quantification using complicated and high-cost qPCR is required.

[0016] In addition, in the case of a method that requires adding a double-stranded DNA (hereinafter referred to as an adapter) to which sequences and molecules (motor proteins) necessary for analysis are added, such as NGS manufactured by Oxford Nanopore Technologies (hereinafter referred to as ONT), using Ligase or the like, to both ends of the target product, the adapter sequence is added not only to the target product but also to non-target products, and they will be subjected to sequence analysis. Since the barcode sequence is not added to non-target products, it is impossible to identify the gene sequence derived from which sample. Therefore, depending on the proportion of non-target products, gene sequence information that cannot be used will be output from NGS. Since the upper limit of the gene sequence information output by one NGS analysis is limited, as the gene sequence information that cannot be used increases, the available gene sequence information decreases. Therefore, in the case of ONT's NGS, it is very important to reduce non-target products as much as possible and increase the proportion of target products in order to reduce costs. The proportion of the target product in the specific product under the normal implementation conditions of the prior art [5] is around 50%, which indicates that 50% of the output gene sequence information cannot be used, meaning that the cost for obtaining the target gene sequence information is doubled compared to a sample that does not contain non-target products.

[0017] In the prior art [5], in order to increase the proportion of the target product in all specific products, an oligo DNA (artificially added specific SL primer and 3' artificial sequence-introducing SL oligo) inserted with a substance (blocker) having an intramolecular secondary structure in the 5' end region and a function of blocking DNA elongation in the sequence strand needs to be used, or it is necessary to add SD polymerase (Biolon), a heat-resistant DNA polymerase, in an amount exceeding the specified amount in the manufacturer's protocol. However, for the former oligo DNA, since it is necessary to add sequences for the purpose of forming an intramolecular secondary structure, it becomes longer, and since it is necessary to insert a blocker, the synthesis cost becomes high. For the addition amount of the latter SD polymerase, in order to obtain sufficient effects, it is necessary to add 10 times the specified amount in the manufacturer's protocol, which leads to a high reagent cost.

[0018] Thus, in the prior art [5], there are challenges related to economic efficiency and operability in the process of quantifying the target product and the process aimed at improving the proportion of the target product. [Prior art documents] [Non-patent literature]

[0019] [Non-Patent Document 1] Applied and Environmental Microbiology, 2013 September 79(17):5112-5120. [Patent Documents]

[0020] [Patent Document 1] WO2006 / 023919 [Patent Document 2] Special Publication 2012-522517 [Patent Document 3] Patent No. 5997407 [Patent Document 4] Japanese Patent Publication No. 2022-170729 [Patent Document 5] Japanese Patent Publication No. 2022-170730 [Overview of the Initiative] [Problems that the invention aims to solve]

[0021] Therefore, when performing sequencing analysis of multiple samples simultaneously in a single analysis using NGS, there is a need for a technology that can prepare a library containing a high proportion of the target product economically and with simple operations. [Means for solving the problem]

[0022] In order to solve the above-mentioned problems, the inventors of the present invention have conducted extensive research on a method for obtaining a library containing a high proportion of the target product in an economical and simple manner (see Figures 5-7).

[0023] the result, (1a) A step to synthesize a pair of linking primers (linking primer (F) and linking primer (R) in Figure 5) by reacting the three types of oligoDNA pairs (ia) to (iiia) below in the presence of a ligase, thereby linking the oligoDNA (iia) below and the oligoDNA (iiia) below by a nick repair reaction with DNA ligase. (ia) A ligation oligo pair (ligation oligo (F) and ligation oligo (R) in Figure 5) characterized by having a complementary sequence of the sequence necessary for ligating oligo DNA in a ligase-mediated nick repair reaction (hereinafter referred to as the ligation sequence). (iia) A 3'LG oligo pair (3'LG oligo(F) and 3'LG oligo(R) in Figure 5) that has a portion of the 3' end of the linked sequence at the 5' end, and a sequence specific to the target gene at the 3' end, with the 5' end phosphorylated. (iiia) A 5'LG oligo pair (5'LG oligo(F) and 5'LG oligo(R) in Figure 5) characterized by having an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a portion of the 5' end sequence of a linked sequence at the 3' end. (2a) A step in which gene amplification is carried out using the linked primer pair synthesized in step (1) above. By directly adding the unpurified reaction solution obtained in (1a) above to the reaction solution in (2a) above and carrying out the two reactions in (1a) and (2a), or by proceeding with the two reactions in (1a) and (2a) in the order of (1a) and (2a) without opening the reaction tube in a reaction solution to which all the necessary components (DNA polymerase, DNA ligase, dNTPs, etc.) have been added, it was possible to prepare a library containing a high proportion of the target product economically and with simple operation. In another aspect, (1b) A step to synthesize a pair of linking primers (linking primer (F) and linking primer (R) in Figure 6) by linking two types of oligo DNA pairs, (ib) and (iib) below, by click chemistry. (ib) A pair of 3'CC oligonucleotides that have a sequence specific to the gene being analyzed and whose 5' end is modified with a click chemistry functional group (3'CC oligo(F) and 3'CC oligo(R) in Figure 6). (iib) A pair of 5'CC oligos having an artificial nucleic acid sequence such as a barcode sequence, and whose 3' end is modified with a functional group that undergoes a click chemistry reaction with the functional group modified in (ib) above (5'CC oligo(F) and 5'CC oligo(R) in Figure 6). (2b) A step in which gene amplification is carried out using the linked primer pair synthesized in step (1b) above. By directly adding the unpurified reaction solution obtained in (1b) above to the reaction solution in (2b) above and carrying out the two reactions in (1b) and (2b), or by proceeding with the two reactions in the order of (1b) and (2b) in a reaction solution containing all the necessary components (DNA polymerase, dNTPs, etc.) without opening the reaction tube, it was possible to prepare a library containing a high proportion of the target product economically and with simple operation.

[0024] In the reactions (2a) and (2b) described above (gene amplification reactions), only the ligation primers functioned as primers, and unligated oligo DNA (hereinafter referred to as unligated oligo), which has a lower dissociation temperature compared to the ligation primers, did not function as primers. By increasing the reaction temperature after the initial synthesis of the target product, within a range that did not significantly reduce the efficiency of gene amplification by the ligation primers, it was possible to prepare a library containing a high proportion of the target product (Figure 7). The present invention was completed based on this discovery.

[0025] Such technologies can be applied to analyses for various purposes. For example, they can be used to comprehensively analyze genes that may cause cancer in order to identify the location of gene mutations. In addition, this technology is suitably usable when using NGS in various SNP analyses, gene mutation analyses, gene expression analyses, and microbiome analyses. However, its scope of application is not limited to methods that analyze multiple target genes by adding artificial nucleic acid sequences and identifying the sample, as long as the target of the test is a nucleic acid amplification product.

[0026] The gist of this invention is as follows: [1] A method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, comprising the steps (1a) and (2a) below. (1a) A step to synthesize a pair of linking primers (linking primer (F) and linking primer (R) in Figure 5) by reacting the three types of oligoDNA pairs (ia) to (iiia) below in the presence of a ligase, thereby linking the oligoDNA (iia) below and the oligoDNA (iiia) below by a nick repair reaction with DNA ligase. (ia) A ligation oligo pair (ligation oligo (F) and ligation oligo (R) in Figure 5) characterized by having a complementary sequence of the sequence necessary for ligating oligo DNA in a ligase-mediated nick repair reaction (hereinafter referred to as the ligation sequence). (iia) A 3'LG oligo pair (3'LG oligo(F) and 3'LG oligo(R) in Figure 5) that has a portion of the 3' end of the linked sequence at the 5' end, and a sequence specific to the target gene at the 3' end, with the 5' end phosphorylated. (iiia) A 5'LG oligo pair (5'LG oligo(F) and 5'LG oligo(R) in Figure 5) characterized by having an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a portion of the 5' end sequence of a linked sequence at the 3' end. (2a) A step in which gene amplification is carried out using the linked primer pair synthesized in step (1) above. [2] The method described in [1], using a ligation oligo having a modification at its 3' end to prevent DNA elongation. [3] A kit for preparing the target product by the method described in [1], comprising at least one pair each of the three types of oligoDNA pairs described in (ia) to (iiia) below. (ia) Linking oligo pairs characterized by having complementary sequences of linking sequences (linking oligo(F) and linking oligo(R) in Figure 5) (iia) A 3'LG oligo pair (3'LG oligo(F) and 3'LG oligo(R) in Figure 5) that has a portion of the 3' end of the linked sequence at the 5' end, and a sequence specific to the target gene at the 3' end, with the 5' end phosphorylated. (iiia) A 5'LG oligo pair (5'LG oligo(F) and 5'LG oligo(R) in Figure 5) characterized by having an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a portion of the 5' end sequence of a linked sequence at the 3' end. [4] The kit described in [3], which includes a ligation oligo having a modification at its 3' end to prevent DNA elongation. [5] The kit according to [3] or [4] further comprising DNA polymerase and DNA ligase. [6] A method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, comprising the steps (1b) and (2b) below. (1b) A step to synthesize a pair of linking primers (linking primer (F) and linking primer (R) in Figure 6) by linking two types of oligo DNA pairs, (ib) and (iib) below, by click chemistry. (ib) A pair of 3'CC oligonucleotides that have a sequence specific to the gene being analyzed and whose 5' end is modified with a click chemistry functional group (3'CC oligo(F) and 3'CC oligo(R) in Figure 6). (iib) A pair of 5'CC oligos having an artificial nucleic acid sequence such as a barcode sequence, and whose 3' end is modified with a functional group that undergoes a click chemistry reaction with the functional group modified in (ib) above (5'CC oligo(F) and 5'CC oligo(R) in Figure 6). (2b) A step in which gene amplification is carried out using the linked primer pair synthesized in step (1b) above. [7] A kit for preparing a target product by the method of claim 6, comprising at least one pair each of the following two oligo DNA pairs: (ib) and (iib). (ib) A pair of 3'CC oligonucleotides that have a sequence specific to the gene being analyzed and whose 5' end is modified with a click chemistry functional group (3'CC oligo(F) and 3'CC oligo(R) in Figure 6). (iib) A pair of 5'CC oligos having an artificial nucleic acid sequence such as a barcode sequence, and whose 3' end is modified with a functional group that undergoes a click chemistry reaction with the functional group modified in (1) above (5'CC oligo(F) and 5'CC oligo(R) in Figure 6). [8] The kit described in [7] further includes DNA polymerase. [9] The method according to any one of claims 1, 2, or 6, wherein the 5' end of the 5'LG oligo in Figure 5 or the 5'CC oligo in Figure 6 is phosphorylated.

[10] The method according to any one of [1], [2], [6], or [9], wherein the annealing temperature in the gene amplification reaction is increased within a range in which unbound oligos do not function as primers and the efficiency of gene amplification by bound primers is not significantly reduced.

[11] The method according to any one of [1], [2], [9], or

[10] , wherein two or more target products are obtained by using the same number of 3'LG oligo pairs as the target nucleic acid.

[12] The method according to [6], [9], or

[10] , wherein two or more target products are obtained by using the same number of 3'CC oligo pairs as the target nucleic acid.

[13] The method according to any of [1], [2], [6], [9] to

[12] , wherein the entire process is carried out continuously in a single reaction without opening the reaction tube.

[14] The gene amplification method is one of the following methods: PCR (Polymerase Chain Reaction), LCR (Liagase Chain Reaction), RPA (Recombinase Polymerase Amplification), SIBA (Strand-Invasion Based Amplification), LAMP (Loop-mediated isothermal amplification), SDa (Strand displacement amplification), NEAR (Nicking Endonuclease Amplification Reaction), HDA (Helicase-dependent amplification), NASBA (Nucleic Acid Sequence-Based Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), Rolling Cycle, or SMAP (Smart Amplification Process), as described in [1], [2], [6], [9] to

[13] .

[15] The method described in

[14] , wherein the gene amplification method is a PCR method using at least one DNA polymerase selected from the group consisting of PCR enzymes having an extension rate faster than 10 seconds / kb.

[16] The method according to

[14] or

[15] , wherein the PCR enzyme is at least one DNA polymerase selected from the group consisting of repliQa HiFi ToughMix and KOD ONE PCR Master Mix.

[17] A method for preparing a mixture containing two or more target products with different barcode sequences, comprising adding a dsDNA-specific dye to each gene amplification reaction solution containing the target product, quantifying the concentration of each target product from the fluorescence value of the dye, and mixing each gene amplification reaction solution so that each target product is in an arbitrary concentration ratio based on the quantification results.

[18] The method according to

[17] , wherein the mixture of the target product is purified.

[19] The method according to

[17] or

[18] for obtaining fluorescence of a dsDNA-specific dye during the gene amplification process.

[20] A method according to any of

[17] to

[19] , wherein gene amplification and acquisition of fluorescence of a dsDNA-specific dye are performed using real-time PCR.

[21] A method according to any of

[17] to

[20] , wherein fluorescence measurement is performed at a temperature in which only the target product exists as double-stranded DNA.

[22] The method according to any one of

[17] to

[21] , wherein the target product is quantified based on a dissociation-corrected fluorescence intensity value obtained by correcting the fluorescence measurement value from a dsDNA-specific dye measured at a temperature in which at least the target product can exist in a double-stranded state with the fluorescence measurement value from a dsDNA-specific dye measured at a temperature in which all DNA dissociates.

[23] The method of

[22] for determining the dissociation-corrected fluorescence intensity value using the following formula. Dissociation-corrected fluorescence intensity value = F n,TT / F n,DN F n,TT : In any cycle (n), only the target product exists as dsDNA. Temperature (TT; T emperature at which only the T (Abbreviation for "argeted product exists as dsDNA") Measured fluorescence intensity values F n,DN : Measured at the temperature at which all DNA dissociates in any given cycle (n). Measured fluorescence intensity (DN; D e N (Abbreviation for ature)

[24] In any of the methods of

[17] to

[22] , in a cycle in which the increase in fluorescence value derived from dsDNA-specific dyes associated with gene amplification does not occur in all gene amplification reactions to be analyzed, the target product is quantified based on the final corrected fluorescence intensity value obtained by correcting the dissociation-corrected fluorescence intensity value calculated by the method described in claim 22 to make it the same value in all reactions. The method described in

[22] for obtaining the finally corrected fluorescence intensity value by the following formula. Finally corrected fluorescence intensity value = (F fc,TT / F fc,DN ) / (F CBFI,TT / F CBFI,DN ) F fc,TT : The actually measured fluorescence intensity value measured at the temperature (TT; f inal c ycle's abbreviation) at which only the target product exists as dsDNA in the final cycle (fc; T emperature at which only the T argeted product exists as dsDNA's abbreviation). F fc,DN : The actually measured fluorescence intensity value (DN; D e N ature's abbreviation) measured at the temperature at which all DNA dissociates in the final cycle (fc). F CBFI,TT : The actually measured fluorescence intensity value at the temperature at which only the target product exists as dsDNA in the cycle (CBFI; C ycle B efore F luorescence I ncreases's abbreviation) before the fluorescence increase accompanying gene amplification is confirmed. F CBFI,DN : The actually measured fluorescence intensity value measured at the temperature at which all DNA dissociates in the cycle (CBFI) before the fluorescence increase accompanying gene amplification is confirmed. Quantification of the target product is performed based on the dissociation-corrected fluorescence intensity value calculated by the method described in

[22] or

[23] , or the finally corrected fluorescence intensity value calculated by the method described in

[24] or

[25] , by the method described in any of

[17] to

[25] . The method described in any of

[17] to

[26] , where the dsDNA-specific dye is a low-inhibition dsDNA dye.

[28] The method according to any one of

[17] to

[27] , wherein the low-inhibitory dsDNA dye is at least one dye selected from the group consisting of pGreen and LC GREEN PLUS. A method for preparing a target product mixture using one of the methods described in

[29]

[17] to

[28] , or a method for preparing a target product mixture using any combination of two or more methods.

[30] A kit for performing NGS analysis, comprising a dsDNA-specific dye, wherein a mixture of target products is prepared for NGS analysis by the method described in

[29] .

[31] A method for modifying a target product, comprising adding a single A base to the 3' end of a target product amplified using a primer with a phosphorylated 5' end in the method described in [1] or [2], and then adding an adapter sequence to the target product using a ligase.

[32] The method according to

[31] , wherein the target product is amplified using a PCR enzyme having proofreading activity, and then an inactivator of the PCR enzyme and a DNA polymerase having 3' end A addition activity are added to the reaction mixture to add a single A nucleotide to the 3' end of the target product.

[33] The method according to

[31] or

[32] , wherein the target product to which a single A base is to be added to the 3' end is the target product prepared by the method according to claim 9. A method for modifying a target product, wherein an adapter sequence is added to a mixture of the target product prepared by the method described in

[34] or

[29] , or the kit described in

[30] , using a ligase, by the method described in any of

[31] to

[33] .

[0027] The present invention (ia) A ligation oligo pair (ligation oligo (F) and ligation oligo (R) in Figure 5) characterized by having a complementary sequence of the sequence necessary for ligating oligo DNA in a ligase-mediated nick repair reaction (hereinafter referred to as the ligation sequence). (iia) A 3'LG oligo pair (3'LG oligo(F) and 3'LG oligo(R) in Figure 5) that has a portion of the 3' end of the linked sequence at the 5' end, and a sequence specific to the target gene at the 3' end, with the 5' end phosphorylated. (iiia) A 5'LG oligo pair (5'LG oligo(F) and 5'LG oligo(R) in Figure 5) characterized by having an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a portion of the 5' end sequence of a linked sequence at the 3' end. The present invention provides a method for obtaining a target product derived from one or more target nucleic acids using an oligo DNA set composed of the above, reaction reagents such as DNA polymerase for carrying out amplification reactions, and reaction reagents for carrying out DNA ligase reactions for linking the oligo DNA described in (iia) and (iiia).

[0028] or (ib) A pair of 3'CC oligonucleotides that have a sequence specific to the gene being analyzed and whose 5' end is modified with a click chemistry functional group (3'CC oligo(F) and 3'CC oligo(R) in Figure 6). (iib) A pair of 5'CC oligos having an artificial nucleic acid sequence such as a barcode sequence, and whose 3' end is modified with a functional group that undergoes a click chemistry reaction with the functional group modified in (ib) above (5'CC oligo(F) and 5'CC oligo(R) in Figure 6). This invention provides a method for obtaining a target product derived from one or more target nucleic acids using an oligoDNA set composed of the above, and reaction reagents such as DNA polymerase for carrying out amplification reactions.

[0029] The present invention provides, for example, a method for amplifying a single nucleic acid sequence in a sample and obtaining a target product in which an artificial nucleic acid sequence containing a barcode sequence is attached to both ends of the amplified product. The details are described below (see Figures 5-7).

[0030] The reaction solution used in the embodiment shown in Figure 5 includes the oligo DNA sets described in (ia) to (iiia) above, amplification reagents including DNA polymerase, and reagents including DNA ligase for carrying out the ligase reaction. The target product is obtained by carrying out the reaction steps (1a) and (2a) below using these. Reaction step (1a): The three types of oligoDNA pairs described in (ia) to (iiia) above are mixed in the reaction solution, causing the 5'LG oligo (F or R) and 3'LG oligo (F or R) to bind to the linking oligo (F or R) adjacent to each other. The ligase recognizes this linking site, and a nick repair reaction links the 5'LG oligo (F or R) and 3'LG oligo (F or R). Reaction step (2a): A reaction step in which the target product is obtained by gene amplification using the linked primer pair (F and R) obtained in step (1a). The reaction solution used in the embodiment shown in Figure 6 includes the oligo DNA sets (ib) and (iib) mentioned above, amplification reagents including DNA polymerase, etc., and the target product is obtained by carrying out the reaction steps (1b) and (2b) below using these. Reaction step (1b): The oligo DNA (F or R) described in (ib) and (iib) above is mixed in the reaction solution, thereby linking the 5' CC oligo (F or R) and 3' CC oligo (F or R), each modified with the functional group of click chemistry, via click chemistry. Reaction step (2b): A reaction step in which the target product is obtained by gene amplification using the linked primer pair (F and R) obtained in step (1b). Figure 7 shows that in the reaction step (2a), if the 3'LG oligo (F or R) that could not be linked to the 5'LG oligo (F or R) functions as a primer and the gene amplification reaction proceeds, the product will not contain the barcode sequence contained in the 5'LG oligo (F or R). Therefore, even if gene sequence information can be obtained, it will be impossible to identify which sample the information originated from. Figure 7 also describes the need to suppress this product (non-target product) and how to suppress it. Specifically, this suppression method utilizes the fact that the linked primer is longer than the unlinked oligo, and as a result, the dissociation temperature of the linked primer is higher than that of the unlinked oligo. After the target product derived from the linked primer is synthesized first (for example, in the case of PCR, the target product is first synthesized in the second cycle, and assuming a synthesis efficiency of 100%, the amount of target product in the second cycle will be the same as the initial template), the annealing temperature is raised to a temperature at which there is no impediment to the binding of the linked primer to the target product, and the binding of the unlinked oligo is sufficiently suppressed. This suppresses the amplification of non-target products and prioritizes the amplification of the target product. Although Figure 7 shows the reaction step (2a) as an example, the same method described in Figure 7 can be used for reaction step (2b) to suppress the amplification of non-target products and prioritize the amplification of the target product.

[0031] The embodiments of the present invention have a variety of variations, as shown below. (1) Two or more target products are obtained by using two or more pairs of 3'LG oligo pairs or 3'CC oligo pairs containing target gene-specific sequences and carrying out the reaction steps described in (1a), (2a) or (2a), (2b). (2) By using two types of ligation oligos with different sequences, and by using 3'LG oligos and 5'LG oligos corresponding to those oligos, it is possible to introduce different artificial nucleic acid sequences to each end of the target product. For example, when introducing different barcode sequences to each end and performing sequence separation (demultiplexing) including not only the barcode sequences but also their combinations, the above variations are effective. (3) The reaction steps (1a), (2a), or (2a), (2b) can each be carried out individually, and the reaction steps can be arbitrarily separated and used depending on the purpose. However, the embodiment in which all necessary materials are added to the same reaction solution before the start of the reaction and the reaction steps are carried out continuously in a single reaction is advantageous in terms of economy and simplicity. (4) The ligation oligo, which serves as a scaffold for linking the 3'LG oligo and the 5'LG oligo, should ideally function only in reaction step (1a) and not in reaction step (2a), which is a gene amplification reaction. For this reason, it is desirable to label the 3' end of the ligation oligo so that it is not elongated by DNA polymerase. Such labeling is generally done by modifications such as phosphorylation, fluorescent dyes, aminolinkers, and biotin, but any modification method that can prevent elongation of the 3' end by DNA polymerase is acceptable. (5) Any type of ligase can be used in reaction step (1a) as long as it has the activity to repair Nicks in double-stranded DNA. However, since the ligase does not contribute to reaction step (2a), which is a gene amplification step, it is desirable to use a ligase that is completely inactivated in this step. For example, in gene amplification methods where the reaction temperature is high, such as PCR, non-heat-resistant ligases will be inactivated. Examples of non-heat-resistant ligases with the activity to repair Nicks in double-stranded DNA include T3 DNA Ligase, T4 DNA Ligase, T7 DNA Ligase, PBCV-1 DNA Ligase, Chlorella virus DNA Ligase, and E. coli DNA Ligase. On the other hand, examples of heat-resistant ligases with the activity to repair Nicks in double-stranded DNA include Taq DNA Ligase and 9 oExamples include N DNA Ligase. These heat-resistant ligases maintain a certain level of activity in reaction step (2a) (gene amplification step) even when a gene amplification reaction with a high reaction temperature is employed. Therefore, although they may have some effect on reaction step (2a), if the dissociation temperature between the linked oligo and the unlinked oligo (3'LG oligo and 5'LG oligo) is sufficiently lower than the reaction temperature of reaction step (2a), the ligase will not act. In such situations, using a heat-resistant ligase will not affect reaction step (2a) and can be used without any problems. Note that Taq DNA Ligase and E. coli DNA Ligase are NAD + It is known that some enzymes are ATP-dependent, while others are ATP-dependent, and the composition required for each reaction is different. (6) The click chemistry functional group used to modify the oligo DNA in reaction step (1b) can be any functional group that can modify oligo DNA and enables linking of 5' CC oligo and 3' CC oligo. However, the bonding between an azide group and an alkyne group, which is a typical click chemistry reaction, requires copper ions as a catalyst, and these ions cause DNA damage and DNA strand breaks, so they are not very desirable for the click chemistry reaction used in the present invention. On the other hand, dibenzocyclooctin (DBCO), azadibenzocyclooctin (ADIBO), bicyclononine (BCN), 4,8-diazacyclononine (DACN), etc., are known as functional groups that undergo a click chemistry reaction with an azide group in the absence of copper ions, and these functional groups can be suitably used as functional groups to modify the oligo DNA used in reaction step (1b). (7) Any DNA polymerase that can be used in reaction steps (2a) and (2b) is acceptable. However, in the steps of ligating oligoDNA in reaction steps (2a) and (2b), the presence of activated DNA polymerase may inhibit the ligation reaction of oligoDNA. In particular, in reaction step (2a), when the 5'LG oligo is bound to the ligation oligo, if the DNA polymerase is in an activated state, the 5'LG oligo will extend using the ligation oligo as a template, preventing the 3'LG oligo from binding to the ligation oligo, and consequently inhibiting the ligase binding of the 5'LG oligo and the 3'LG oligo. For these reasons, in the present invention, it is desirable to use a hot-start type DNA polymerase that is inactive in the temperature range in which the ligation of oligoDNA in reaction step (1a) is carried out. (8) The gene amplification reaction used in reaction steps (2a) and (2b) can be any gene amplification reaction using primers. Specific gene amplification methods include PCR (Polymerase Chain Reaction), LCR (Liagase Chain Reaction), RPA (Recombinase Polymerase Amplification), SIBA (Strand-Invasion Based Amplification), LAMP (Loop-mediated isothermal amplification), SDA (Strand displacement amplification), NEAR (Nicking Endonuclease Amplification Reaction), HDA (Helicase-dependent amplification), NASBA (Nucleic Acid Sequence-Based Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), Rolling Cycle, and SMAP (Smart Amplification Process).

[0032] The following effects can be expected from this invention. (1) In a single step, a library containing a high proportion of the target product with an artificial nucleic acid sequence attached to its terminals can be obtained. (2) In the present invention, after the target product is initially synthesized, the annealing temperature is increased to limit the primers contributing to the gene amplification reaction to only ligation primers. Therefore, unlike the methods of the prior art [3] and [4], there is no need to use primers for amplification using the target nucleic acid as a template and primers for amplification using the amplification product derived from the target nucleic acid as a template in the same reaction solution, and the generation of nonspecific amplification products such as primer dimers can be suppressed. For this reason, the minimum required amount of initial template can be kept lower than in the prior art described above, and sensitivity similar to that of general gene amplification using only primers specific to the target nucleic acid can be ensured. (3) In the case of methods such as those described in prior art [3] and [4], in which amplification using a target nucleic acid as a template and amplification using an amplification product derived from the target nucleic acid with a different primer as a template are carried out simultaneously in the same reaction solution, it is necessary to strictly optimize the reaction conditions so that two or more amplification reactions with different properties proceed simultaneously and appropriately.

[0033] On the other hand, in the present invention, by changing the temperature conditions, it is possible to completely separate the reaction of ligating two oligoDNAs (reaction steps (1a) and (1b)) and the amplification reaction (reaction steps (2a) and (2b)) even in a one-step closed reaction system, and for this reason, both reactions can be carried out under optimal temperature conditions. Furthermore, since the oligoDNA sequences contributing to the reaction of ligating two oligoDNAs can be reacted under the same conditions regardless of the target nucleic acid, the optimization of reaction conditions in the method of the present invention is limited to amplification using the amplification product derived from the target nucleic acid as a template, and therefore, the reaction system can be constructed simply and quickly, similar to general gene amplification. (4) In the case of a method using a primer that has a specific artificial nucleic acid sequence containing a barcode sequence pre-attached to the 5' end of a primer specific to the target nucleic acid sequence (prior art [1]), the target product that can be used for NGS analysis can be obtained in a simple one-step process. However, it is necessary to prepare a primer with a different barcode sequence for each target nucleic acid, which is extremely uneconomical. For example, in a method that identifies a sample by the combination of barcode sequences attached to both ends, if 100 samples are analyzed simultaneously in one analysis and 100 types of target nucleic acids are analyzed per sample, then 10 types of forward primers and 10 types of reverse primers are required for each target nucleic acid, for a total of 20 types of primers, and since such sets are required for each type of target nucleic acid, it is necessary to prepare 2,000 types of primers.

[0034] On the other hand, in the method based on the present invention, the amplification of the target nucleic acid is carried out by 3'LG oligo or 3'CC oligo, and the addition of the artificial nucleic acid sequence including the barcode sequence is carried out by 5'LG oligo or 5'CC oligo. Therefore, the types of oligo DNA required depend on the type of target nucleic acid for the former and on the number of samples to be analyzed for the latter. For example, assuming the same premise as above (target nucleic acid: 100 types, number of samples: 100 samples), there are 200 types of 3'LG oligo or 3'CC oligo including forward / reverse primers, and similarly, there are 200 types of 5'LG oligo or 5'CC oligo including forward / reverse primers. In the method of linking oligo DNA with ligase, if the artificial nucleic acid sequences to be added to both ends are the same, only one type of linking oligo is needed, resulting in a total of 401 types including that oligo. Even if the artificial nucleic acid sequences to be added to both ends are different, only two types of linking oligos are needed, resulting in a total of 402 types of oligo DNA including those oligos. Furthermore, in the click chemistry method for linking oligoDNA, linking oligos are not required, so only 400 types are needed. In addition, 5'LG oligos, 5'CC oligos, and linking oligos do not need to have their sequences changed depending on the target nucleic acid, so they can be used commonly regardless of the target nucleic acid, allowing for efficient use without waste. (5) In order to maintain a constant concentration of the target product for NGS, it is necessary to adjust the concentration after preparing the target product. However, in the prior art [5], unless special labeling substances or oligo DNA with a specific structure (3' artificial sequence-introduced SL oligo) or primers (artificial addition-specific SL primers) are used, or a high concentration of SD polymerase is added, non-target products are synthesized in large proportions. This makes it difficult to specifically detect and quantify only the target product. Therefore, it is not possible to use low-cost and simple methods such as electrophoresis, fluorescence measurement, and absorbance measurement, and it is necessary to use the qPCR method, which can determine the presence or absence of artificial nucleic acid sequences and quantify only the target product. On the other hand, in the present invention, it is possible to prepare a library in which the majority of the specific products (more than 95%) are the target product using standard enzyme usage recommended by the manufacturer, without using special labeling substances or oligo DNA with a specific structure. Therefore, it is possible to accurately quantify the target product even with the low-cost and simple methods described above. As described above, in the present invention, the quantification of the target product can be carried out more simply and at a lower cost than in the prior art [5]. (6) In the case of analyses that require the attachment of adapters necessary for analysis to both ends of the target product using Ligase, etc., such as in the NGS manufactured by ONT, the adapters are attached not only to the target product but also to the non-target product, and these are sequenced. Since the non-target product does not have a barcode sequence, it is not possible to identify which sample the gene sequence information comes from, and therefore an increase in the non-target product leads to an increase in unusable gene sequence information. For this reason, reducing the amount of such product is very important in reducing the analysis cost of the above NGS. In the prior art [5], the proportion of the target product under normal operating conditions is around 50%, but it is possible to increase the proportion of the target product to a maximum of about 90% by using the aforementioned 3' artificial sequence-introduced SL oligo or artificially added specific SL primer, or by adding a high concentration of SD polymerase. However, the cost of synthesizing these oligo DNAs and adding SD polymerase is high, so there was a need for improvement measures. On the other hand, in the present invention, oligoDNA modified with special substances is not required, and by adding DNA polymerase at the manufacturer's recommended concentration, the proportion of the target product can be increased to 95%. Therefore, the proportion of the target product can be increased more easily and at lower cost than in the prior art [5], and as a result, it can be suitably used as a library preparation method for ONT's NGS and the like. [Effects of the Invention]

[0035] This invention makes it possible to prepare a library containing a high proportion of the target product economically and with simple operations. [Brief explanation of the drawing]

[0036] [Figure 1] A diagram illustrating the principle of prior art [1]. This method uses primers that are specific to the target nucleic acid sequence and have a particular artificial nucleic acid sequence containing a barcode sequence pre-attached to the 5' end, in order to obtain a target product that can be subjected to NGS. In this method, a different primer with a different barcode sequence must be prepared for each type of nucleic acid sequence, for each sample. [Figure 2]Principle diagram of prior art [2]. In this method, the first amplification is performed using an artificial addition-specific primer in which a portion of the 3' end of an artificial nucleic acid sequence is attached to the 5' end of a specific sequence to obtain a specific product derived from the target nucleic acid. Subsequently, after purifying this product, a second amplification is performed using this as a template with an artificial sequence primer having an artificial nucleic acid sequence such as a barcode sequence at its 3' end. Through the above two amplifications, a target product can be obtained in which the artificial nucleic acid sequence is attached to both ends of the target nucleic acid. [Figure 3] A diagram illustrating the principle of prior art [3]. In this method, the concentration of the artificially added specific primer is set lower than the concentration of the artificial sequence primer, and both primers are added to the same reaction solution and amplified, thereby obtaining the target product in a single amplification step. [Figure 4] A diagram illustrating the principle of prior art [4]. In this method, in the reaction to add an artificial nucleic acid sequence to a target nucleic acid sequence, in addition to an artificial addition-specific primer, two pairs of artificial sequence primers, a long-chain artificial sequence primer and a short-chain artificial sequence primer, are used, and by making the concentration of the short-chain artificial sequence primer higher than the concentration of the artificial addition-specific primer and the long-chain artificial sequence primer, it is possible to amplify the target product more efficiently. [Figure 5] Reaction scheme for a novel library preparation method based on ligation. [Figure 6] Reaction scheme for a novel library preparation method based on click chemistry. [Figure 7] A diagram illustrating the amplification of non-target products by unlinked oligonucleotides, the problems associated with this amplification, and the principle of suppressing the amplification of non-target products by increasing the reaction temperature during gene amplification. [Figure 8] Existing quantitative and purification process [Figure 9] Simplification and acceleration of library quantification and purification processes using dsDNA-specific fluorescent dyes. [Figure 10] Flowchart of Nanopore's recommended library end-modification method [Figure 11] Rapid and low-cost library end modification using 5'LG oligonucleotides with phosphorylated 5' ends and A-attachment mix. [Figure 12] This figure shows the amount of specific products under each reaction condition carried out in Example 1. [Figure 13] Electrophoresis (PAGE) image after the ligase reaction in Example 2. [Figure 14] Comparison of the theoretical composition ratio of DNA-Mock and the measured composition ratio of the target product in Example 3. [Figure 15] Comparison of the measured composition ratio of the target product and the measured composition ratio of the non-target product in Example 3. [Figure 16] A graph showing the number of reads for each target gene in Example 4. [Figure 17] Electrophoresis (PAGE) image after the click chemistry reaction. [Figure 18] Comparison of the theoretical composition ratio of DNA-Mock and the measured composition ratio of the target product in Example 6. [Figure 19] Comparison of the measured composition ratio of the target product and the measured composition ratio of the non-target product in Example 6. [Figure 20] A graph showing the number of reads for each target gene in Example 7. [Figure 21] (a) Relationship between purified double-stranded DNA concentration and uncorrected fluorescence intensity at 90°C (b) Relationship between purified double-stranded DNA concentration and final corrected fluorescence intensity [Figure 22] The flow and time-saving effect of a new process applying the descriptions in Examples 8-11 are shown. [Modes for carrying out the invention]

[0037] The embodiments of the present invention will be described in detail.

[0038] The present invention provides a method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, the method comprising the following steps (1a) and (2a). (1a) A step to synthesize a pair of linking primers (linking primer (F) and linking primer (R) in Figure 5) by reacting the three types of oligoDNA pairs (ia) to (iiia) below in the presence of a ligase, thereby linking the oligoDNA (iia) below and the oligoDNA (iiia) below by a nick repair reaction with DNA ligase. (ia) A ligation oligo pair (ligation oligo (F) and ligation oligo (R) in Figure 5) characterized by having a complementary sequence of the sequence necessary for ligating oligo DNA in a ligase-mediated nick repair reaction (hereinafter referred to as the ligation sequence). (iia) A 3'LG oligo pair (3'LG oligo(F) and 3'LG oligo(R) in Figure 5) that has a portion of the 3' end of the linked sequence at the 5' end, and a sequence specific to the target gene at the 3' end, with the 5' end phosphorylated. (iiia) A 5'LG oligo pair (5'LG oligo(F) and 5'LG oligo(R) in Figure 5) characterized by having an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a portion of the 5' end sequence of a linked sequence at the 3' end. (2a) A step in which gene amplification is carried out using the linked primer pair synthesized in step (1) above. In the method comprising the reaction steps (1a) and (2a) described above, the ligating oligo has a sequence at its 5' end that is complementary to a portion of the 5' end sequence of the 3'LG oligo, and a sequence at its 3' end that is complementary to a portion of the 3' end sequence of the 5'LG oligo, and both sequences are preferably 4 to 30 bases long, more preferably 6 to 20 bases long, and more preferably 8 to 12 bases long.

[0039] It is desirable to label the 3' end of the ligation oligo pair to prevent elongation by DNA polymerase. Common methods of labeling include phosphorylation, fluorescent dyes, aminolinkers, biotin, and bases that do not form a complementary strand with the target DNA; however, any modification method that prevents 3' end elongation by DNA polymerase is acceptable.

[0040] The 3'LG oligo pair has a sequence at its 5' end that is complementary to the 5' end sequence of the ligation oligo, and a sequence at its 3' end that is specific to the gene to be analyzed. The former 5' end sequence is often 4 to 30 nucleotides long, preferably 6 to 20 nucleotides long, and more preferably 8 to 12 nucleotides long, while the latter 3' end sequence can be set to a number of sequences generally preferred for gene amplification primers, and no special settings dependent on the present invention are required. Furthermore, the 5' end of the 3'LG oligo must be phosphorylated in order to enable ligase linkage with the 5'LG oligo.

[0041] The 5'LG oligo pair has an artificial nucleic acid sequence at its 5' end, such as a barcode sequence for identifying the sample or an adapter for binding to oligo DNA present on the NGS flow cell, and a sequence at its 3' end that is complementary to the sequence at its 3' end of the ligation oligo. The former 5' end sequence and sequence length are determined by the NGS used, so they should be set according to the NGS manufacturer's recommended sequence, and no special settings dependent on the present invention are required. The latter 3' end sequence is often 4 to 30 nucleotides long, preferably 6 to 20 nucleotides long, and more preferably 8 to 12 nucleotides long.

[0042] If you want to add different artificial nucleic acid sequences to both ends, you can achieve this by making the sequence of the ligation oligo at one end different from the sequence of the ligation oligo at the other end, and designing the sequence of the 5' end of the 3'LG oligo that binds to the ligation oligo pair and the sequence of the 3' end of the 5'LG oligo to be complementary to those sequences.

[0043] Furthermore, it is desirable to set the dissociation temperatures for the 5'-terminal sequence of the 3'LG oligo bound to the ligating oligo, and the 3'-terminal sequence of the 5'LG oligo, using the optimal temperature of each ligase as a guideline. Specifically, the dissociation temperatures should be ±15°C, preferably ±10°C, and more preferably ±5°C relative to the ligase reaction temperature. In addition, it is desirable to keep the dissociation temperatures of the 3'LG oligo and 5'LG oligo as similar as possible to the ligating oligo, in order to ensure that the 3'LG oligo and 5'LG oligo are reliably bound to the ligating oligo at the ligase reaction temperature.

[0044] Regarding the DNA polymerase used in the method comprising the reaction steps (1a) and (2a) described above, any type of polymerase that can be used for gene amplification may be used. However, if DNA polymerase is present in an activated state during the oligo DNA ligation step, the oligo DNA ligation reaction may be inhibited. In particular, in reaction step (1a) (ligation reaction by ligase), if DNA polymerase is present in an activated state when the 5'LG oligo is bound to the ligation oligo, the 3' end of the 5'LG oligo will be extended using the ligation oligo as a template, preventing the 3'LG oligo from binding to the ligation oligo, and consequently potentially inhibiting the ligase binding of the 3'LG oligo and the 5'LG oligo. For these reasons, in the present invention, it is desirable to use a hot-start type DNA polymerase that is inactive in the temperature range in which the oligo DNA ligation in reaction step (1a) is carried out.

[0045] Furthermore, when the target product obtained in this invention is subjected to NGS analysis, it is desirable that the product has as few base incorporation errors originating from gene amplification as possible. Therefore, it is desirable to use a polymerase that has proofreading activity (3'→5' exonuclease activity) capable of suppressing base incorporation errors.

[0046] Examples of DNA polymerases possessing the above characteristics include PrimeSTAR® HS DNA Polymerase (Takara Bio Inc.), KOD plus (Toyobo Co., Ltd.), Exact Polymerase (Five Prime Inc.), repliQa HiFi ToughMix (Quanta Bio Inc.), and KOD ONE PCR Master Mix (Toyobo Co., Ltd.).

[0047] In the reaction step (2a) described above, a method to suppress the amplification of non-target products resulting from the functioning of 3'LG oligos that could not be linked to 5'LG oligos as primers is to raise the reaction temperature immediately after the target product derived from the linked primers is first synthesized, in a way that does not hinder the annealing of the linked primers but suppresses the annealing of the unlinked oligos (3'LG oligos). By performing this step, it is possible to suppress the amplification of non-target products and prioritize the amplification of the target product.

[0048] For example, if the PCR method, the most representative gene amplification method, is adopted as the gene amplification method described above, the annealing temperature in any cycle from the second cycle (third cycle) onward, after the second cycle in which the target product derived from the linked primer is first synthesized, can be raised to a temperature that does not hinder the annealing of the linked primer to the target product, while suppressing the annealing of the unlinked oligo (3'LG oligo). This allows for the suppression of amplification of non-target products and the dominance of amplification of the target product. The cycle in which the annealing temperature is raised is preferably from the 3rd to the 11th cycle, more preferably from the 3rd to the 5th cycle, and more preferably from the 3rd cycle. Assuming that the amplification efficiency is close to 100%, since the amplification product derived from the linked primer is synthesized equimolarly with the initially added template DNA in the second cycle, gene amplification will proceed well even if the annealing temperature is raised to a temperature in the third cycle onward where only the linked primer functions as a primer and it is difficult for the 3'LG oligo to function as a primer. On the other hand, if the initial template concentration is low, or if the efficiency of binding of the target gene-specific sequence in the primer to the initial template is low, resulting in a small amount of product derived from the linked primer at the second cycle, and increasing the annealing temperature from the third cycle onward does not yield a sufficient amount of the target product, then increasing the initial cycle, in which the annealing temperature is set based on the dissociation temperature of the target gene-specific sequence, by 3 to 10 cycles, it is possible to secure a sufficient amount of product derived from the linked primer in the initial cycle with a low annealing temperature, and then ensure the final amount of the target product even if the annealing temperature is increased thereafter. The annealing temperature after heating should be set within a temperature range where the amplification efficiency of the linked primer is sufficiently higher than that of the unlinked 3'LG oligo, and where the DNA polymerase elongation activity does not decrease to the point where gene amplification does not proceed normally, using the ratio of the target product to the specific product targeted by each implementer (for example, a ratio of the target product to the specific product of 95%) as an indicator. The annealing temperature is preferably 60 to 90°C, more preferably 65 to 85°C, and more preferably 68 to 80°C.When PCR is used as the gene amplification method, if the annealing temperature after heating, as set above, is close to the optimal extension temperature of DNA polymerase, a two-step PCR in which annealing and extension are performed in the same step may be used. The annealing temperature in the initial gene amplification cycle of the present invention should be determined from the dissociation temperature of the sequence region specific to the target gene, as with normal gene amplification. Generally, the annealing temperature is often set to a temperature 2 to 5°C lower than the aforementioned dissociation temperature, but in the present invention as well, it may be set according to a generally known method.

[0049] On the other hand, in the case of isothermal gene amplification methods such as the SIBA method, SDA method, and NEAR method, the reaction temperature is constant. Therefore, after the time has elapsed for the target product to be first obtained, it is possible to raise the reaction temperature to suppress the amplification of non-target products and to prioritize the amplification of the target product, as described above.

[0050] Regarding the DNA ligase used in the method including the reaction steps (1a) and (2a) described above, any type of ligase can be used as long as it has the activity to repair Nicks in double-stranded DNA. However, since ligase activity is not required during the gene amplification reaction (except in the LCR method), it is desirable to use a non-heat-stable ligase that is completely inactivated in this step. Specific examples of non-heat-stable ligases with the activity to repair Nicks in double-stranded DNA include T3 DNA Ligase, T4 DNA Ligase, T7 DNA Ligase, PBCV-1 DNA Ligase, Chlorella virus DNA Ligase, and E. coli DNA Ligase. On the other hand, examples of heat-stable ligases with the activity to repair Nicks in double-stranded DNA include Taq DNA Ligase and 9 oExamples include N DNA ligase. Although heat-resistant ligases maintain their activity even during gene amplification reactions and may therefore have some influence on the reaction, if the dissociation temperature between linked and unlinked oligos (3'LG oligo and 5'LG oligo) is sufficiently low compared to the annealing temperature of gene amplification, the ligase will not act. In such situations, using a heat-resistant ligase will not affect the gene amplification reaction in any way and can be used without problems. On the other hand, since the LCR method uses a heat-resistant DNA ligase for gene amplification, if the oligo linking reaction in reaction step (1a) proceeds with the same ligase as in the LCR method, then it is rational because all reactions can be carried out with a single type of ligase. Note that Taq DNA Ligase and E. coli DNA Ligase are NAD + Since one is an ATP-dependent enzyme and the others are ATP-dependent enzymes, it is advisable to add each of the aforementioned substances to the reaction solution.

[0051] Furthermore, the present invention provides a method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, the method comprising the following steps (1b) and (2b). (1b) A step to synthesize a pair of linking primers (linking primer (F) and linking primer (R) in Figure 6) by linking two types of oligo DNA pairs, (ib) and (iib) below, by click chemistry. (ib) A pair of 3'CC oligonucleotides that have a sequence specific to the gene being analyzed and whose 5' end is modified with a click chemistry functional group (3'CC oligo(F) and 3'CC oligo(R) in Figure 6). (iib) A pair of 5'CC oligos having an artificial nucleic acid sequence such as a barcode sequence, and whose 3' end is modified with a functional group that undergoes a click chemistry reaction with the functional group modified in (ib) above (5'CC oligo(F) and 5'CC oligo(R) in Figure 6). (2b) A step in which gene amplification is carried out using the linked primer pair synthesized in step (1b) above. In the method comprising the reaction steps (1b) and (2b) described above, the 3' CC oligo pair has a sequence specific to the gene to be analyzed, but this sequence can be set to a number of sequences that is generally preferred as a gene amplification primer, and no special considerations unique to the present invention are required. Furthermore, in order to enable ligation of the 3' CC oligo with the 5' CC oligo by a click chemistry reaction, the 5' end of the oligo is modified with a functional group that generates click chemistry with the functional group modified at the 3' end of the 5' CC oligo.

[0052] The 5'CC oligo pairs, including the barcode sequence for identifying the sample and the artificial nucleic acid sequence for the adapter that binds to the oligo DNA present on the NGS flow cell, are specified for each NGS instrument. Therefore, they should be set according to the recommended sequence of the NGS manufacturer used, and no special considerations dependent on the present invention method are necessary. Furthermore, as mentioned above, the 5'CC oligo is modified with a functional group that generates click chemistry with the functional group modified at the 5' end of the 3'CC oligo, in order to enable linkage with the 3' CC oligo via a click chemistry reaction. Typical functional groups for click chemistry include azide groups and alkyne groups, but these reactions require copper ions as a catalyst, and these ions cause DNA damage or DNA strand breaks, making them undesirable for the click chemistry reaction used in the present invention. On the other hand, functional groups known to undergo click chemistry with azide groups in the absence of copper ions include dibenzocyclooctin (DBCO), azadibenzocyclooctin (ADIBO), bicyclononine (BCN), and 4,8-diazacyclononine (DACN). These functional groups can be suitably used as functional groups to modify the oligoDNA used in reaction step (1b).

[0053] In the reaction step (2b), as a method to suppress the amplification of non-target products resulting from the 3'CC oligo that could not be linked to the 5'CC oligo functioning as a primer, it is effective to raise the reaction temperature to a temperature that does not hinder the annealing of the linked primer and suppresses the annealing of the unlinked oligo (3'CC oligo in reaction step (2b)) immediately after the target product derived from the linked primer is first synthesized, similar to the method in reaction step (2a). By performing this step, it is possible to suppress the amplification of non-target products and prioritize the amplification of the target product.

[0054] The timing for raising the reaction temperature in this implementation, and the reaction temperature after the increase, are the same as those described in the method including reaction steps (1a) and (2a) above. Furthermore, the DNA polymerases that can be used in the method including reaction steps (1b3) and (2b4) are the same as those described in the method including reaction steps (1a) and (2a) above.

[0055] Furthermore, by using the same number of oligo pairs containing target gene-specific sequences as the target nucleic acid (3'LG oligo pairs in reaction steps (1a) and (2a), and 3'CC oligo pairs in reaction steps (1b) and (2b)), two or more target products can be obtained in a single reaction.

[0056] Furthermore, the reaction steps (1a), (2a), and (1b3), (2b4) described above can be carried out continuously in a single reaction without opening the reaction tube.

[0057] The gene amplification methods for reaction steps (2a) and (2b) described above can be carried out using methods such as PCR (Polymerase Chain Reaction), LCR (Liagase Chain Reaction), RPA (Recombinase Polymerase Amplification), SIBA (Strand-Invasion Based Amplification), LAMP (Loop-mediated isothermal amplification), SDa (Strand displacement amplification), NEAR (Nicking Endonuclease Amplification Reaction), HDA (Helicase-dependent amplification), NASBA (Nucleic Acid Sequence-Based Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), Rolling Cycle, and SMAP (Smart Amplification Process).

[0058] The present invention also provides a kit for preparing the target product, which includes at least one pair each of the three types of oligoDNA pairs described below (ia) to (iiia). The method for preparing the target product is described above (Figure 5). (ia) Linking oligo pairs characterized by having complementary sequences of linking sequences (linking oligo(F) and linking oligo(R) in Figure 5) (iia) A 3'LG oligo pair (3'LG oligo(F) and 3'LG oligo(R) in Figure 5) that has a portion of the 3' end of the linked sequence at the 5' end, and a sequence specific to the target gene at the 3' end, with the 5' end phosphorylated. (iiia) A 5'LG oligo pair (5'LG oligo(F) and 5'LG oligo(R) in Figure 5) characterized by having an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a portion of the 5' end sequence of a linked sequence at the 3' end. The ligation oligo (ia) of the kit of the present invention may have a modification at its 3' end to prevent DNA elongation.

[0059] The kit of the present invention may further include DNA polymerase and DNA ligase.

[0060] The requirements for the primers, oligoDNA, DNA polymerase, DNA ligase, and other components of the kit of the present invention are as described above.

[0061] Furthermore, the present invention also provides a kit for preparing the target product, which contains at least one pair each of the two types of oligoDNA pairs, (ib) and (iib), described below. The method for preparing the target product is as described above (Figure 6). (ib) A pair of 3'CC oligonucleotides that have a sequence specific to the gene being analyzed and whose 5' end is modified with a click chemistry functional group (3'CC oligo(F) and 3'CC oligo(R) in Figure 6). (iib) A pair of 5'CC oligos having an artificial nucleic acid sequence such as a barcode sequence, and whose 3' end is modified with a functional group that undergoes a click chemistry reaction with the functional group modified in (1) above (5'CC oligo(F) and 5'CC oligo(R) in Figure 6). The kit of the present invention may further include DNA polymerase.

[0062] The components of the kit of this invention, such as oligoDNA and DNA polymerase, are as described above.

[0063] The two types of kits of the present invention described above may also include a reaction vessel, reaction buffer, instructions for use, target nucleic acid for positive control testing, and the like.

[0064] The following describes in detail embodiments of the present invention aimed at simplifying and expediting its implementation.

[0065] In order to shorten the gene amplification reaction time in the present invention, this can be achieved by (1) using a PCR enzyme with a high extension rate (hereinafter referred to as a high-speed PCR enzyme), (2) changing from a 3-step PCR to a 2-step PCR during the gene amplification (PCR) reaction, and (3) raising the annealing ( / extension) temperature in the 2-step PCR described in (2).

[0066] (1) The high-speed PCR enzyme described above can shorten the time required for extension by using this enzyme, thereby shortening the gene amplification reaction time.

[0067] Examples of high-speed PCR enzymes usable in the present invention include repliQa HiFi ToughMix (manufactured by Quantabio Inc.) and KOD ONE PCR Master Mix (manufactured by Toyobo Inc.). However, any high-speed PCR enzyme suitably applicable to the present invention is acceptable, and the scope of application of the present invention is not limited by the type of enzyme.

[0068] (2) The change from 3-step PCR to 2-step PCR, as described above, reduces the time required to transition to the omitted step and the temperature holding time in that step, thereby shortening the gene amplification reaction time.

[0069] Regarding the increase in the annealing temperature ( / extension) temperature described in (3), this change leads to a narrowing of the temperature range (difference between dissociation temperature and annealing temperature), and as a result, the time required for temperature change is reduced, making it possible to shorten the gene amplification reaction time.

[0070] The following describes in detail embodiments for simplifying and accelerating the purification of the target product obtained in this invention and the quantification of DNA concentration.

[0071] When analyzing multiple samples simultaneously in a single NGS analysis, in order to obtain the target number of gene sequences (reads) per sample, it is necessary to quantify the target product from each sample and mix the target products from each sample at the target concentration ratio based on the quantified value. Furthermore, in order to accurately quantify the concentration of the target product, it is necessary to purify the DNA of each product before performing the quantification.

[0072] In this invention, as with general NGS analysis, it is necessary to purify the obtained target product and then measure the DNA concentration of each product, which leads to the following problems: (1) the steps after the amplification reaction of the target product are complicated and time-consuming, and (2) the costs of purifying and quantifying the obtained target product are incurred for each sample, leading to increased costs (see Figure 8).

[0073] The aforementioned problems can be solved by adding a sufficient amount of a fluorescent dye (hereinafter referred to as a low-inhibition dsDNA dye) that specifically binds to double-stranded DNA and emits fluorescence (hereinafter referred to as a dsDNA-specific dye) and exhibits low PCR inhibition to the gene amplification reaction solution, measuring the fluorescence directly in the reaction tube after the reaction is complete, and quantifying the target product based on the measured value.

[0074] Examples of low-inhibitory dsDNA dyes include EvaGreen (manufactured by Biotium) and LC GREEN PLUS (manufactured by BioFire Defense), but any low-inhibitory dsDNA dye suitable for application to the present invention is acceptable, and the scope of application of the present invention is not limited by the type of dye.

[0075] Because the aforementioned low-inhibitory dsDNA dye exhibits minimal PCR inhibition, amplification of the target product is possible even when the substance is added to the reaction solution at high concentrations. As a result, even as the concentration of the target product increases, the amount of dye bound to each molecule of the target product can be maintained at a saturation level. Therefore, a positive correlation exists between the concentration of the target product and the fluorescence intensity, even in the later stages of the amplification reaction when the concentration of the target product increases. Based on the above, by adding a high concentration of the low-inhibitory dsDNA dye to the reaction solution of the present invention and directly measuring the fluorescence intensity without opening the reaction tube after the reaction is complete, the library can be quantified simply and quickly.

[0076] On the other hand, since nonspecific products such as primers and dimers are frequently amplified during the gene amplification reaction, accurately quantifying the concentration of the target product with a low-inhibition dsDNA dye requires distinguishing between fluorescence derived from the target product and fluorescence derived from nonspecific products.

[0077] The length of the target product is often longer than that of the non-specific product, and therefore, the dissociation temperature of the target product is generally higher than that of the non-specific product. By utilizing this characteristic, it is possible to accurately quantify the concentration of the target product by detecting fluorescence derived from a low-inhibition dsDNA dye at a temperature at which the non-specific product dissociates but the target product exists as double-stranded DNA.

[0078] The gene amplification according to the present invention is performed using a real-time PCR instrument, which allows for simultaneous measurement of fluorescence at any desired timing and temperature. Therefore, upon completion of the amplification according to the present invention, the quantification of the target product can be completed using the fluorescence intensity value obtained above. Thus, by using a real-time PCR instrument, the quantification of the target product can be performed more quickly and simply than by separately measuring fluorescence after gene amplification, making it a more suitable gene amplification instrument for use in the present invention.

[0079] In the gene amplification method according to the present invention, when using the aforementioned real-time PCR instrument, it is necessary to use a different reaction tube for each sample, and to measure the fluorescence from the outside of each reaction tube. Therefore, even when measuring reaction solutions of the same composition, differences in the optical properties between reaction tubes will result in differences in the obtained fluorescence values. Accordingly, in order to accurately quantify the target product, it is desirable to correct for the differences in the optical properties of the reaction tubes. The following describes the method for correcting for the differences in the optical properties of the reaction tubes.

[0080] The temperature of the denature step in the gene amplification process is set to the temperature at which all double-stranded DNA in the reaction tube denatures. Theoretically, this results in a constant fluorescence intensity (fluorescence intensity derived from free, low-inhibition dsDNA dyes not bound to dsDNA) regardless of the degree of gene amplification. This characteristic was utilized to compensate for differences in optical properties between reaction tubes. Specifically, by dividing the fluorescence intensity measured at the temperature at which only the target product of each cycle exists as dsDNA by the fluorescence intensity of the denature step in the same cycle, it became possible to roughly compensate for differences in the optical properties of the reaction tubes. The specific calculation formula is shown below. Dissociation-corrected fluorescence intensity value = F n,TT / F n,DN F n,TT : The temperature at which only the target product exists as dsDNA in any cycle (n) (TT; T emperature at which only the T Fluorescence intensity measured using dsDNA (abbreviation for targeted product exists as dsDNA) F n,DN : Measured fluorescence intensity (DN) at the temperature at which all DNA dissociates in any given cycle (n); D e N (Abbreviation for ature)

[0081] The fluorescence intensity values ​​corrected by the fluorescence intensity of the dissociation step described above (hereinafter referred to as dissociation-corrected fluorescence intensity values) should theoretically be the same regardless of the reaction tube in cycles before the fluorescence increase associated with gene amplification is confirmed. However, in reality, slight differences were observed in the dissociation-corrected fluorescence intensity values ​​between reaction tubes in the relevant cycle. Therefore, the dissociation-corrected fluorescence intensity values ​​for each cycle were divided by the dissociation-corrected fluorescence intensity value of an arbitrary cycle in which no increase in fluorescence intensity due to gene amplification was observed in any reaction tube, thereby standardizing the dissociation-corrected fluorescence intensity values ​​for all reaction tubes across an arbitrary cycle.

[0082] The specific calculation formula for obtaining the final corrected fluorescence intensity value mentioned above is shown below. Final corrected fluorescence intensity value = (F fc,TT / F fc,DN ) / (F CBFI,TT / F CBFI,DN ) F fc,TT : The temperature at which only the target product exists as dsDNA in the final cycle (fc) (TT; T emperature at which only the T Measured fluorescence intensity values ​​in targeted product (as dsDNA) F fc,DN : Actual fluorescence intensity at the dissociation step (DN) in the final cycle F CBFI,TT : The cycle before the increase in fluorescence associated with gene amplification is confirmed (CBFI; C ycle B before F luorescence I Measured fluorescence intensity at temperatures where only the target product exists as dsDNA (increases) F CBFI,DN : Measured fluorescence intensity during the dissociation step in the cycle before the increase in fluorescence associated with gene amplification was confirmed.

[0083] The quantified value of dsDNA obtained using the calculation method, based on the corrected fluorescence intensity (hereinafter referred to as the final corrected fluorescence intensity value), was more accurate than the quantified value of dsDNA obtained using the uncorrected fluorescence intensity (see Example 9 below). Therefore, the calculation method described above is suitable as a method for correcting for differences in the optical properties of reaction tubes.

[0084] The aforementioned implementation method allows for (1) rapid and simple quantification of the library simply by measuring the fluorescence of the reaction tube directly. (2) Library purification, which previously had to be performed for each reaction tube in order to accurately quantify DNA concentration, can now be performed on the mixture, significantly reducing the effort required for library purification. This is expected to lead to a reduction in overall process time and cost (Figure 9). The present invention also provides a method for preparing a target product mixture using one of the above methods, or a method for preparing a target product mixture using any combination of two or more methods. Furthermore, the present invention also provides a kit for performing NGS analysis, which includes a dsDNA-specific dye, and the kit is used to prepare a target product mixture for NGS analysis using the above methods. Furthermore, the present invention also provides a method for preparing a mixture containing two or more target products with different barcode sequences, comprising adding a dsDNA-specific dye to each gene amplification reaction solution containing the target product, quantifying the concentration of each target product from the fluorescence value of the dye, and mixing each gene amplification reaction solution so that each target product is in an arbitrary concentration ratio based on the quantification results (Figure 9).

[0085] When performing NGS analysis on the target product obtained in this invention using a sequencer manufactured by Nanopore, it is necessary to attach the adapter sequence provided by Nanopore to both ends of the target product to be analyzed. Various kits for attaching this adapter sequence are sold by Nanopore, but the following describes a simplified method of implementation using Nanopore's Ligation Sequencing Kit, which is characterized by attaching the adapter sequence using ligase.

[0086] When using the aforementioned kit, it is necessary to add a single A base to the 3' end of the target product and phosphorylate its 5' end in order to enable adapter addition by ligase. For the purpose of performing the above-mentioned end modification, the manufacturer (Nanopore) recommends purifying the target product and then using a specified reagent kit (NEB Next Ultra II End repair / dA-tailing Module reagents (manufactured by New England Biolabs)). However, the processing steps for this reagent kit (Table 1, Figure 10) are complicated and time-consuming (18 steps, approximately 1 hour of work), and the reagent cost is also high at 3,000 yen per use.

[0087] The above problems can be solved by (1) using 5'LG oligo or 5'CC oligo with a phosphorylated 5' end, or (2) using a reagent (A-attachment mix (manufactured by Toyobo Co., Ltd.)) that can add a single A base to the 3' end simply by adding it to an unpurified target product preparation solution. The reasons for this are explained below.

[0088] Regarding (1) above, if a 5'LG oligo or 5'CC oligo with a phosphorylated 5' end is used in the present invention, the 5' end of all the resulting products will be phosphorylated, eliminating the need to separately phosphorylate that end.

[0089] A addition to the 3' end can be performed using the aforementioned A-attachment mix. This reagent contains an anti-KOD antibody that inactivates KOD (a PCR enzyme manufactured by Toyobo Co., Ltd.) and a DNA polymerase with A addition activity to the 3' end. By simply adding this reagent directly to the unpurified reaction mixture after amplification of the target product, A addition to the 3' end of the target product is possible. Although the use of KOD is a prerequisite, if the expected effect is confirmed, the use of this reagent will significantly simplify the A addition process to the 3' end (only two steps: reagent addition and incubation) and reduce the time required. Furthermore, the reagent cost per use is 1 / 5 (600 yen) of the manufacturer's recommended method, resulting in significant cost savings. The present invention also provides a method for modifying the target product (Figure 11), which involves adding a single A base to the 3' end of the target product amplified using a primer with a phosphorylated 5' end, and then adding an adapter sequence to the product using a ligase, in addition to the method for preparing the target product described above. The present invention also provides a method for modifying the target product, which involves adding an adapter sequence to a mixture of the target product prepared by the above method or kit using a ligase.

[0090] Using the method described above (Figure 11), it becomes possible to process both ends of the target product obtained by the present invention. If NGS analysis of the end-processed target product is possible, the time and reagent costs required for the process can be reduced by approximately 80% (time: approximately 50 minutes, reagent cost: 2,500 yen / process reduced). [Table 1] [Examples]

[0091] The present invention will now be described in more detail with reference to examples. [Examples]

[0092] <Influence of ligase reaction composition on gene amplification (PCR)> It is suspected that the ligase required for the ligase reaction, as well as other reaction components, may adversely affect PCR, a widely used gene amplification method. This effect was investigated in this example.

[0093] Specifically, using the oligoDNA shown in Table 2, the ligase reaction and PCR were performed continuously in a single reaction tube without opening the tube, and the effects were evaluated from the quantitative values ​​of the resulting specific products. All oligoDNA used in the examples described herein was synthesized by Nippon Gene Research Institute Co., Ltd.

[0094] The target gene in this example was a 16S rRNA gene derived from bacteria, and the template used was a DNA-Mock (manufactured by NBRC (National Institute of Technology and Evaluation Biotechnology Center)) containing 15 types of bacterial genomes mixed at known concentrations. [Table 2]

[0095] The reaction conditions are shown in Table 3. [Table 3]

[0096] Table 4 lists the reaction solution composition for each condition. The cells shown in gray indicate differences in settings between each condition.

[0097] The reaction solutions shown in Table 4 were subjected to a PCR amplification device (Life touch (manufactured by Nippon Genetics Co., Ltd.)) under the temperature conditions shown in Table 3. [Table 4]

[0098] After the reaction was complete, the reaction solution was diluted 10,000 times with TE buffer. Subsequently, the diluted reaction solution was used as a sample, and the specific product was quantified using real-time qPCR targeting a gene-specific sequence. The gene-specific sequence was the same as the gene-specific sequence listed in Table 2 above, which was used in the implementation of the present invention. A Roter-Gene Q (manufactured by Qiagen) was used as the real-time PCR instrument.

[0099] The reaction mixture composition (including the sequence of primers used) and the qPCR conditions are shown in Tables 5 and 6, respectively. A Roter-Gene Q (Qiagen Corporation) real-time PCR instrument was used. [Table 5]

[0100] [Table 6]

[0101] The results are shown in Figure 12. Quantitative analysis was performed twice for each condition, and the values ​​in Figure 12 represent the average values.

[0102] As shown in Figure 12, no specific products were detected for either Ampligase Thermostable DNA Ligase (a heat-stable ligase) or Hi-T4 DNA Ligase (a non-heat-stable ligase) under the manufacturer's recommended buffer concentration (×1) conditions (Conditions [1] and [5]).

[0103] Furthermore, for both ligases, the quantitative values ​​were significantly lower under conditions where the buffer concentration was 1 / 3 of the manufacturer's recommended concentration (conditions [2] and [6]) compared to other conditions where specific products were detected (conditions [3], [4], [7], and [8]).

[0104] The results above clearly show that both heat-stable and non-heat-stable ligases contain substances in their reaction buffers that inhibit PCR, and that specific products cannot be obtained when added at the manufacturer's recommended concentration. However, specific products were observed when the reaction buffer was added at 1 / 3 of the manufacturer's recommended concentration (conditions [2] and [6]), and it was confirmed that good amplification of specific products occurred when the buffer was added at a concentration of 1 / 4 or less (conditions [3], [4], [7], [8]).

[0105] Based on the above results, in Examples 3 and beyond, the reaction buffer for ligase was added at 1 / 4 the manufacturer's recommended concentration. Note that Ampligase Thermostable DNA Ligase, a heat-resistant ligase, is NAD +Hi-T4 DNA Ligase, an ATP-dependent enzyme and a non-heat-stable ligase, is an ATP-dependent enzyme. Although there are differences in the buffer composition of each, as mentioned above, they showed similar trends in PCR inhibition. [Examples]

[0106] <Confirmation of oligo-DNA linkage based on ligase reaction by electrophoresis (PAGE)> To confirm the linkage of oligoDNA by ligase, the oligoDNA shown in Table 2 of Example 1 was used, and the ligase reaction was performed using the reaction composition described in Table 4 without PCR. After that, the resulting reaction product was subjected to electrophoresis (PAGE analysis). Table 7 shows the reaction conditions for Example 2. Under these conditions, the heat-resistant ligase was not deactivated (conditions [1] to [4]), so after the reaction was completed, all eight conditions were immediately frozen and stored frozen until electrophoresis. The reaction was carried out using a PCR amplification device (Life touch (manufactured by Nippon Genetics Co., Ltd.)) as in Example 1. <Confirmation of oligo-DNA linkage based on ligase reaction by electrophoresis (PAGE)> To confirm the linkage of oligoDNA by ligase, the oligoDNA shown in Table 2 of Example 1 was used, and the ligase reaction was performed using the reaction composition described in Table 4 without PCR. After that, the resulting reaction product was subjected to electrophoresis (PAGE analysis). Table 7 shows the reaction conditions for Example 2. Under these conditions, the heat-resistant ligase was not deactivated (conditions [1] to [4]), so after the reaction was completed, all eight conditions were immediately frozen and stored frozen until electrophoresis. The reaction was carried out using a PCR amplification device (Life touch (manufactured by Nippon Genetics Co., Ltd.)) as in Example 1. [Table 7]

[0107] The results are shown in Figure 13.

[0108] Under all conditions, only the ligation primer (base length: 81 bases) resulting from the linkage of the 3'LG oligo and 5'LG oligo, and the ligation oligo (base length: 22 bases) that serves as a scaffold for ligase linkage and does not change itself were observed, and good oligo linkage by ligase was confirmed under all conditions. From these results, it was confirmed that the ligase reaction proceeds well even under conditions where no specific products were observed [1], [5]. [Examples]

[0109] <Confirmation of the composition ratio of the target product (ligase reaction system)> In this example, the proportion of the target product in the specific products of the present invention (ligase reaction system) was confirmed using NGS manufactured by Nanopore Corporation.

[0110] First, using the oligo DNA shown in Table 2 of Example 1, the ligase reaction and PCR reaction were carried out continuously without opening the reaction tube, using the reaction composition described in Table 8 and the reaction conditions described in Table 9. The difference between reaction conditions A and B described in Table 8 is that in reaction condition B, the temperature conditions are not changed during the reaction, while in reaction condition A, only the first two cycles of PCR are the same 3-step PCR as in reaction condition B, and subsequent cycles are 2-step PCR in which annealing and extension are performed in the same step (72°C). In reaction condition A, the temperature is raised to an annealing temperature during the gene amplification reaction in which the unlinked 3'LG oligo with a low dissociation temperature does not function as a primer, thereby suppressing the amplification of non-target products and preferentially allowing linked primers with a high dissociation temperature to function, thereby aiming to improve the proportion of the target product. The target gene in this example is the same as in Example 1, a 16S rRNA gene derived from eubacteria, and the template is also the same as in Example 1, using DNA-Mock (manufactured by NBRC).

[0111] The resulting reaction product was first purified using a magnetic DNA purification bead (AMPure XP, Beckman Coulter) according to the manufacturer's protocol. Subsequently, the purified product was quantified using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol, and the measurement was performed using a fluorescence plate reader (DTX800, Beckman Coulter).

[0112] Next, the purified product was processed using the Nanopore Ligation Sequencing Kit [SQK-LSK110] according to the kit's protocol. This kit repairs and phosphorylates both ends of the purified product, and then adds a double-stranded DNA (adapter) containing the sequences and molecules (motor proteins) necessary for analysis to both ends of the target product using ligase.

[0113] The final product obtained as described above was quantified using the fluorescence measurement method with the same Quant-iT PicoGreen dsDNA Assay Kits as described above. Following this, sequencing analysis was performed on the product using Nanopore's NGS (MinION Mk1B). Nanopore's Flongle flow cell was used for the NGS analysis, with one flow cell used per condition. This is because non-target products do not have barcode sequences; therefore, if multiple samples are analyzed simultaneously using a single flow cell, it becomes impossible to identify which condition the non-target product originated from, thus preventing the determination of the proportion of target products for each condition.

[0114] Next, base calling (the process of converting raw sequence data acquired from a flow cell into gene sequences; this process was performed in the SUP mode, which offers the highest accuracy) and demultiplexing (the process of separating sequence information for each sample using barcode sequences as markers) were performed using MinKNOW software provided by Nanopore. After converting the obtained sequence data into base sequence information, bacterial identification based on the gene sequence information was performed using EPI2ME, a bioinformatics platform provided by Nanopore.

[0115] Table 10 shows the composition ratio of each product under each condition. Gene information for which gene sequence information was obtained but was not recognized as a 16S rRNA gene by EPI2ME was judged to be gene information derived from a non-specific product. Next, gene information for which a 16S rRNA gene was recognized by EPI2ME was judged to be gene information derived from a specific product. Of these, sequence information for which a barcode sequence was recognized by MinKNOW and multiplexed was judged to be gene sequence information derived from the target product, and sequence information for which a barcode sequence was not attached was judged to be gene sequence information derived from a non-target product. [Table 8]

[0116] [Table 9]

[0117] The results are shown in Tables 10 and 11, and Figures 14 and 15. Table 10 shows the composition ratios of the target product, non-target product, and non-specific product under each condition. The only difference in conditions between conditions [1] and [3], and between conditions [2] and [4], was the type of ligase used, but the composition ratios of each product were similar, confirming that the influence of the ligase difference on the composition ratio of each product was relatively small.

[0118] On the other hand, the difference in conditions between conditions [1] and [2], and between conditions [3] and [4], lies in the reaction temperature conditions. In conditions [1] and [3], the PCR process involves performing 3-step PCR only for the first two cycles, followed by 2-step PCR (reaction condition A in Table 9). In contrast, conditions [2] and [4] are the same as the 3-step PCR in conditions [1] and [3], but the temperature conditions are not changed, and the PCR is completed (reaction condition B in Table 9). Due to the differences in reaction temperature conditions, the composition ratio of the target product was significantly higher in conditions [1] and [3] (reaction condition A) than in conditions [2] and [4] (reaction condition B). Based on these results, as described above, it was determined that by increasing the annealing temperature during PCR (reaction condition A), unlinked 3'LG oligos with low dissociation temperatures did not function as primers. As a result, amplification of non-target and nonspecific products was suppressed, while linked primers with high dissociation temperatures were preferentially allowed to function, thereby improving the proportion of the target product. [Table 10]

[0119] Table 11 shows the theoretical and experimental proportions of 16S rRNA genes derived from 15 different bacteria included in DNA-Mock. [Table 11]

[0120] Next, based on the numerical data in Table 11, Figure 14 shows a graph illustrating the relationship between the theoretical and measured composition ratios. The linear function in Figure 14 is an approximation function, and the R of that function is... 2The values ​​ranged from 0.67 to 0.68, and the slope of the function ranged from 1.45 to 1.56. These results confirmed that a certain positive correlation exists between the theoretical and measured composition ratios, and that this correlation is almost equivalent regardless of the conditions. From these results, it was confirmed that the NGS analysis results were similar to those obtained under the condition where the annealing temperature was changed during PCR (reaction condition A) compared to the condition where the annealing temperature was not changed (reaction condition B). Therefore, it was assumed that the difference in reaction conditions had little effect on the composition ratio of the target gene.

[0121] Figure 15 shows the correlation between the measured composition ratio of the target product and the measured composition ratio of the non-target product. Similar to Figure 14, the linear function is an approximation function, and the R of this function is 2 The values ​​ranged from 0.96 to 0.98, and the slope of the function ranged from 0.81 to 0.99. These results suggest a very high positive correlation between the measured composition ratios of the target product and the non-target product, and that this correlation remains almost constant regardless of the conditions. These results suggest that the presence or absence of barcode sequences has a very small impact on the NGS analysis results. [Examples]

[0122] <Simultaneous analysis of multiple gene parameters (ligase reaction system)> In this example, we investigated the possibility of simultaneous analysis of multiple genes according to the present invention by continuously performing oligo linkage using ligase and amplification of the target product by the linked oligo without opening the reaction tube, targeting multiple target nucleic acids, and then performing NGS analysis on the obtained target product. Table 12 shows the oligoDNA used in this example. The target genes in this example were five human genes, and five pairs of 3'LG oligonucleotides were prepared corresponding to each target gene.

[0123] The target product was prepared five times, and the target products from the five reactions were analyzed in a single NGS analysis. Therefore, to separate the gene sequence information for each reaction, five 5'LG oligos with different barcode sequences were prepared, and a different 5'LG oligo was used for each reaction. The ligation oligo used was the same as that used in Example 3. In this example, since the same artificial nucleic acid sequence is added to both ends of the target product, only one 5'LG oligo and one ligation oligo are used per reaction.

[0124] Since the target gene in this example is a human-derived gene, commercially available human genomic DNA (manufactured by Roche) was used as the additive template, and 100 ng of this was added per reaction tube.

[0125] The target product was prepared using the reaction solution composition described in Table 13, and under the reaction temperature conditions described as Reaction Condition A in Table 9.

[0126] The resulting reaction products were first purified using a magnetic DNA purification bead (AMPure XP, Beckman Coulter) according to the manufacturer's protocol. Subsequently, the purified products were quantified using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol, and the measurements were performed using a fluorescence plate reader (DTX800, Beckman Coulter). Based on these quantification values, the concentrations of the target products for each reaction were adjusted to contain equimolar amounts.

[0127] Next, similar to Example 3, the purified product was processed using the Nanopore Ligation Sequencing Kit [SQK-LSK110] according to the kit's protocol.

[0128] The final product obtained as described above was quantified using the fluorescence measurement method with the same Quant-iT PicoGreen dsDNA Assay Kits as described above, and then sequenced using Nanopore's NGS (MinION Mk1B). The flow cell used for the NGS analysis was Nanopore's Flongle.

[0129] After the NGS analysis was completed, base calling (performed in SUP mode, which offers the highest base calling accuracy) and demultiplexing (separating sequence information for each reaction using barcode sequences as markers) were performed using MinKNOW software provided by Nanopore. The resulting sequence data was converted into base sequence information, and the number of gene sequences (hereinafter referred to as reads) was counted for each target gene. [Table 12]

[0130] [Table 13]

[0131] The results are shown in Figure 16 and Table 14. [Table 14]

[0132] As shown in Figure 16, gene sequence information was obtained for all five items analyzed. Furthermore, from the standard deviations between reactions listed in Table 14, the standard deviation (%) of reads due to differences in reactions was approximately 10% (6.5-14.1%), confirming that the variability in reads per reaction was low.

[0133] Furthermore, although the standard deviation (%) of the average number of reads between genes was relatively high at 57.7%, as mentioned above, a considerable number of reads were obtained for all genes, and the results were judged to be sufficiently practical.

[0134] Based on the above, it has been confirmed that by utilizing the present invention (a method for preparing a target product using ligase), it is possible to prepare a target product targeting multiple genes in a closed reaction tube, and that by performing NGS analysis on the product, it is possible to perform sequencing analysis of multiple types of genes simultaneously.

[0135] Furthermore, in this embodiment, the number of reads for each reaction was measured by using 5'LG oligos with different barcode sequences for each different reaction. However, by applying 5'LG oligos with different barcode sequences for each different sample, it is also possible to perform sequencing analysis of different samples simultaneously.

[0136] Based on the above, it has been confirmed that by using the present invention (a method for preparing a target product using ligase), it is possible to obtain information on multiple gene sequences present in multiple samples all at once. [Examples]

[0137] <Confirmation of oligo-DNA linkage by electrophoresis (PAGE) based on click chemistry> To confirm the linking of oligoDNA by click chemistry, the oligoDNAs shown in Table 15 were used, and the reaction was carried out at room temperature for 10 minutes using the reaction compositions described in Table 16. The resulting reaction products were then subjected to electrophoresis (PAGE analysis). The difference in reaction composition under each condition was due to the difference in the added oligoDNA. The difference between conditions [1] and [2] and conditions [3] and [4] was the presence or absence of linking oligoDNA. The difference between conditions [1] and [2], and between conditions [3] and [4], was whether forward-side oligoDNA or reverse-side oligoDNA was used. The reaction volume was 20 μl.

[0138] The results of the above study are shown in Figure 17. [Table 15]

[0139] [Table 16]

[0140] Under all conditions, a ligation primer (base length: 81 bases) resulting from the ligation of 3'CC oligo and 5'CC oligo was confirmed, confirming oligo ligation by click chemistry.

[0141] On the other hand, under conditions [1] and [2], linking oligos were added to serve as scaffolds for the 3'CC oligo and 5'CC oligo to move closer together, in the hope of improving the reactivity of click chemistry. However, no significant difference in the intensity of each band was observed for the linking primers resulting from the linking of the 3'CC oligo and 5'CC oligo, the unlinked 5'CC oligo, or the unlinked 3'CC oligo, compared to conditions [3] and [4] where no linking oligo was added. As a result, the effect of adding linking oligos could not be confirmed in this study. [Examples]

[0142] <Confirmation of the composition ratio of the target product (crick chemistry reaction system)> In this example, the proportion of the target product in the specific products of the present invention (click chemistry reaction system) was confirmed using NGS manufactured by Nanopore.

[0143] The oligo-DNAs shown in Table 17 were used, and the click chemistry reaction and PCR reaction were carried out continuously without opening the reaction tube, using the reaction composition described in Table 18 and the reaction conditions described in Table 19.

[0144] In this example, since the effect of the linking oligo was not confirmed in Example 5, the linking oligo was not used. Therefore, the same 5'CC oligo was added to both the forward and reverse ends of the 3'CC oligo, and only one type of 5'CC oligo was used per reaction.

[0145] There are two conditions; the reaction solution composition is the same regardless of the conditions, and only the reaction conditions differ between the two conditions. [Table 17]

[0146] [Table 18]

[0147] [Table 19]

[0148] The difference between reaction condition A (condition [1]) and reaction condition B (condition [2]) described in Table 19 is that in reaction condition B, there is no change in temperature conditions during PCR, while in reaction condition A, only the first two cycles of PCR are performed as a 3-step PCR, the same as in reaction condition B, and subsequent cycles are performed as a 2-step PCR, with annealing and extension performed in the same step (72°C). In reaction condition A, the annealing temperature is increased midway through the reaction to suppress the amplification of non-target products by setting the temperature conditions so that unbound 3'CC oligos with low dissociation temperatures do not function as primers, while the composition ratio of the target product is improved by preferentially allowing bound primers with high dissociation temperatures to function. The target gene in this example is a 16S rRNA gene derived from eubacteria, the same as in Examples 1 and 3, and the template is also the same as in Examples 1 and 3, using DNA-Mock (manufactured by NBRC). The subsequent steps are the same as in Example 3 described above.

[0149] The results described above are shown in Tables 20 and 21, Figure 18, and Figure 19.

[0150] Table 20 shows the composition ratios of the target product, non-target product, and non-specific product under each condition.

[0151] The difference between conditions [1] and [2] lies in the PCR temperature conditions. In condition [1], the PCR process involves performing a 3-step PCR only for the first two cycles, followed by a 2-step PCR (reaction condition A in Table 19). In contrast, condition [2] uses the same conditions as the 3-step PCR in condition [1], but without changing the temperature conditions, to complete the PCR (reaction condition B in Table 19). Due to the difference in reaction temperature conditions, the composition ratio of the target product was significantly improved in condition [1] (reaction condition A) compared to condition [2] (reaction condition B). This result confirms that, similar to Example 3, the composition ratio of the target product can be improved by raising the annealing temperature during the PCR process (reaction condition A). [Table 20]

[0152] Table 21 shows the theoretical and experimental proportions of 16S rRNA genes derived from 15 different bacteria included in DNA-Mock. Next, based on the numerical data in Table 21, Figure 18 shows a graph illustrating the relationship between the theoretical composition ratio and the measured composition ratio for each condition. [Table 21]

[0153] The linear function in Figure 18 represents the approximation function, and the R of that function is 2The values ​​were 0.64 for condition [1] and 0.68 for condition [2], and the slope of the function was 1.5 for condition [1] and 1.42 for condition [2], with no significant differences observed. This result suggests that there is a positive correlation between the theoretical and measured composition ratios, and that this correlation is almost the same regardless of the condition. Based on these results, it was shown that, similar to Example 3, the same NGS analysis results can be obtained even when the annealing temperature is changed during PCR (reaction condition A) as with the condition where the annealing temperature is not changed as usual (reaction condition B), and it was determined that the difference in reaction conditions has no effect on the NGS analysis results, or that it can be ignored.

[0154] Figure 19 shows the correlation between the measured composition ratio of the target product and the measured composition ratio of the non-target product.

[0155] Similar to Figure 18, the linear function is an approximation function, and the R of that function is 2 The values ​​were 0.97 for both conditions [1] and [2], and the slopes of the functions were 1.1 for condition [1] and 1.04 for condition [2], with almost no difference observed between the conditions. This result suggests that there is an extremely high positive correlation between the measured composition ratio of the target product and the non-target product, and that this correlation is almost constant regardless of the condition. Based on these results, it was confirmed that, similar to Example 3 above, the presence or absence of barcode sequences has an extremely small impact on the NGS analysis results. [Examples]

[0156] <Simultaneous analysis of multiple gene parameters (crick chemistry reaction system)> In this example, we investigated the possibility of simultaneous analysis of multiple genes according to the present invention by continuously performing oligo linkage by click chemistry and amplification of the target product using the linked oligo without opening the reaction tube, targeting multiple target nucleic acids, and then performing NGS analysis on the obtained target product.

[0157] The oligo DNA used in this example is shown in Table 22. The target genes in this example were the same 5 human genes as in Example 4 above, and 5 pairs of 3’CC oligos corresponding to each target gene were prepared.

[0158] The preparation reaction of the target product was carried out 5 times, and the target products obtained from the 5 reactions were subjected to NGS analysis once to obtain sequence information. Therefore, in order to separate the gene sequence information for each reaction, 5 5’CC oligos with different barcode sequences were prepared, and different 5’CC oligos were used for each reaction. In addition, since the effect of the ligation oligo could not be confirmed in Example 5 above, the ligation oligo was not used in this example either, as in Example 6. Therefore, the same 5’CC oligo was added to both the forward side and the reverse side at the 5’ end of the 3’CC oligo, so only 1 type of 5’CC oligo was used per reaction.

[0159] The added template in this example was the same as in Example 4 above, and commercially available human genomic DNA (manufactured by Roche) was used, and 100 ng of this was added per reaction tube.

[0160] The preparation of the target product was carried out based on the reaction temperature conditions described as Reaction Condition A in Table 19 with the reaction solution composition described in Table 23. The subsequent steps were the same as those in Example 4 above.

Table 22

[0161]

Table 23

[0162] The results are shown in Figure 20 and Table 24.

Table 24

[0163] Figure 20 shows that gene sequence information was obtained for all five items analyzed. Furthermore, from the standard deviations between reactions listed in Table 24, the standard deviation (%) of reads due to differences in reactions ranged from 5.7% to 18.4%, confirming that the variability in reads per reaction was low.

[0164] Furthermore, although the standard deviation (%) of the average number of reads between genes was relatively high at 57.6%, as mentioned above, a considerable number of reads were obtained for all genes, and the results were judged to be sufficiently practical.

[0165] Based on the above, it has been confirmed that the present invention (a method for preparing a target product using click chemistry) can be used to prepare a target product targeting multiple genes in just one reaction, and that sequence information of multiple genes can be obtained simultaneously by performing NGS analysis on the product.

[0166] Furthermore, in this embodiment, the number of reads for each reaction was measured by using 5'CC oligos with different barcode sequences for each different reaction. However, by applying 5'CC oligos with different barcode sequences for each different sample, it is also possible to perform sequencing analysis of different samples together.

[0167] Based on the above, it has been confirmed that by utilizing the present invention (a method for preparing a target product using click chemistry), it is possible to obtain information on multiple gene sequences present in multiple samples all at once. [Examples]

[0168] <Investigation into shortening reaction time in gene amplification processes> In this example, we investigated methods for shortening the gene amplification reaction time described in the patent. Specifically, we attempted to shorten the reaction time by (1) shortening the reaction time by using a high-speed PCR enzyme, (2) changing from a 3-step PCR to a 2-step PCR during the gene amplification (PCR) reaction, and (3) raising the annealing ( / extension) temperature in the 2-step PCR described in (2). The high-speed PCR enzyme used should preferably have an extension rate in gene amplification faster than 10 seconds / kb.

[0169] The target gene in this example is a 16S rRNA gene derived from eubacteria, as in Example 1. The template is also the same as in Example 1, using DNA-Mock (manufactured by NBRC), and this is applied in quantities of 10 units per reaction tube in terms of 16S rRNA gene equivalent. 5 I added it so that it would be a copy. Furthermore, the oligoDNA used in this example was the oligoDNA shown in Table 2 of Example 1.

[0170] Table 25 lists the DNA polymerases used in this example. TITANIUM Taq DNA polymerase (Clontech) is not a high-speed PCR enzyme. PrimeSTAR GXL DNA polymerase (Takara Bio) was added at twice the normal amount according to the manufacturer's protocol to enable a high-speed extension reaction. The other three enzymes are all high-speed PCR enzymes. Roter-Gene Q (Qiagen) was used for the gene amplification (PCR) reaction.

[0171] Amplification of the target product was carried out using the reaction solution composition described in Table 26, based on the reaction temperature conditions in Table 27.

[0172] The amplification of the target product was confirmed by electrophoresis (using an Agilent 2100 Bioanalyzer electrophoresis system [manufactured by Agilent Technologies]), and the reaction time was measured from the start to the end of the reaction.

[0173] The results are shown in Table 28. Of the five enzymes used, amplification was not observed with KAPA2G Fast HotStart DNA Polymerase (KAPA Biosystems) and PrimeSTAR GXL DNA Polymerase under the short extension time high-speed PCR conditions set in this example. However, the target product was confirmed under all conditions for the other three enzymes.

[0174] The table shows the measured reaction time for each condition, as well as the rate of reduction in reaction time relative to the reaction time (124 minutes) of a 3-step PCR (Test No. 1) using TITANIUM Taq DNA polymerase, a PCR enzyme with an average extension rate. These results indicate that the reaction time can be shortened by performing 3-step PCR only for the first two cycles, and then switching to 2-step PCR (Tests No. 6-10). This is because, in 2-step PCR, annealing and extension were performed at high and uniform temperatures, thus reducing the time required for temperature changes. Furthermore, by setting the annealing and extension temperatures to even higher levels (Tests No. 11-15), it was possible to further shorten the reaction time compared to Tests No. 6-10, which used lower temperatures.

[0175] The condition that confirmed amplification of the target product and showed the highest rate of reduction in reaction time relative to Test No. 1 was Test No. 15, using repliQa HiFi ToughMix (Quanta Bio), with a reaction time of 35 minutes and a reaction time reduction of 72%. Next was Test No. 14, using KOD ONE PCR Master Mix (Toyobo Co., Ltd.), with a reaction time of 36 minutes and a reaction time reduction of 71%.

[0176] From the above, although it is necessary to select a high-speed PCR enzyme, by (1) shortening the reaction time by using a high-speed PCR enzyme, (2) changing from 3-step PCR to 2-step PCR during the gene amplification (PCR) reaction, and (3) increasing the annealing ( / extension) temperature in the 2-step PCR of (2) above, it was confirmed that the reaction time of the method of the present invention can be significantly reduced.

[0177] In addition, repliQa HiFi ToughMix (manufactured by Quantabio) and KOD ONE PCR Master Mix (manufactured by Toyobo), for which the reaction time could be reduced, are both polymerases having proofreading activity (3'→5' exonuclease activity) capable of suppressing base incorporation errors. Therefore, since they also have excellent characteristics as DNA polymerases when preparing target products for NGS analysis, they can be said to be DNA polymerases suitable for the implementation of the method of the present invention.

Table 25

Table 26

Table 27

Table 28

Example

[0178] <Simplification / Speeding up of the Target Product Quantification and Purification Process Using a Low-Inhibition dsDNA Dye [1]> For the purpose of verifying the effectiveness of the simplification / speeding up of the target product quantification and purification process using the above-mentioned inhibitory dsDNA dye, the following investigations were carried out.

[0179] Method: The oligo DNA used in this example was the oligo DNA shown in Table 2 of Example 1. The target gene in this example was a 16S rRNA gene derived from eubacteria, the same as in Example 1. The template was also the same as in Example 1, using DNA-Mock (manufactured by NBRC), which was added to each reaction tube in terms of 10 units of 16S rRNA gene equivalent. 5 It was added to create a copy.

[0180] In this example, KOD ONE PCR Master Mix (manufactured by Toyobo Co., Ltd.) was used as the DNA polymerase. EvaGreen (manufactured by Biotium) was added to the reaction solution as a low-inhibitory dsDNA dye at a final concentration of 2×, which is twice the usual concentration (note that the product concentration is indicated as 20×, and there is no clear concentration indication). The rest of the reaction solution composition was the same as the reaction solution composition using KOD ONE PCR Master Mix described as conditions [4] in Table 26.

[0181] The reaction temperature conditions, other than the number of cycles, were based on the conditions of Test No. 4 described in Table 27. The number of cycles was set to 25, 27, 32, and 40 cycles to produce a change in the concentration of the target product, and one reaction was performed for each cycle count condition.

[0182] The target product obtained under the above conditions was purified using Agencourt AMPure XP (Beckman Coulter), and the purified product (double-stranded DNA) was quantified using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol. Fluorescence was measured using a fluorescence plate reader (Beckman Coulter DTX800).

[0183] The gene amplification reaction of the present invention was performed using a real-time PCR instrument, Rotor-Gene Q 2plex (manufactured by Qiagen), and fluorescence measurements were performed at 90°C and 98°C for each cycle. At 90°C, nonspecific products such as primers and dimers cannot exist as double-stranded DNA, but the target product (the Tm value of the target product in this example is 90°C) can exist as double-stranded DNA. Therefore, it is assumed that the fluorescence intensity at this temperature reflects the amount of the target product. On the other hand, the measurement at 98°C is performed during the dissociation step, and since fluorescence is measured in a state where double-stranded DNA is absent, theoretically, the fluorescence intensity is constant regardless of the degree of gene amplification.

[0184] The fluorescence intensity values ​​at 90°C and 98°C obtained in the manner described above were used to determine the final corrected fluorescence intensity value using the calculation formula for the final corrected fluorescence intensity value described above. More specifically, the fluorescence measurement value at 90°C in the final cycle was used as the actual fluorescence intensity value at the temperature in which only the target product exists as dsDNA (F in the calculation formula). fc,TT ) and the fluorescence measurement value at 98°C in the final cycle is the actual fluorescence intensity value in the dissociation step (F in the calculation formula). fc,DN ) was set as follows. In addition, the number of cycles before the increase in fluorescence associated with gene amplification was confirmed was set to 10 cycles, and the fluorescence intensity value at 90°C in that cycle was set to F CBFI,TT The fluorescence intensity value at 98°C is F CBFI,DN The above four values ​​were substituted into the calculation formula described above to obtain the final corrected fluorescence intensity value.

[0185] Results: Amplification of the target product was confirmed by electrophoresis (using an Agilent 2100 Bioanalyzer electrophoresis system (Agilent Technologies)). No bands derived from nonspecific products were observed in any of the reactions. Based on these results, it was determined that the quantitative results of double-stranded DNA using PicoGreen measured only the target product.

[0186] To confirm the effect of fluorescence correction, Figure 21(a) shows the relationship between the quantitative value of purified double-stranded DNA and the uncorrected fluorescence intensity of the final cycle in each reaction tube, and Figure 21(b) shows the relationship between the quantitative value of purified double-stranded DNA and the final corrected fluorescence intensity of the final cycle in each reaction tube. These results showed a positive correlation between the concentration of purified double-stranded DNA and the fluorescence intensity, regardless of whether fluorescence correction was applied.

[0187] Furthermore, since Figure 21(b), which includes fluorescence correction, shows a higher correlation coefficient than Figure 21(a), which does not, it was demonstrated that the amount of double-stranded DNA can be more accurately quantified by performing fluorescence correction using the method described above. [Examples]

[0188] <Simplification / rapidification of the quantification and purification process of target products using low-inhibitory dsDNA dyes[2]> Summary: In this example, we investigated whether it is possible to arbitrarily set the number of reads per sample by measuring the concentration of the target product using the double-stranded DNA quantification method described in Example 9.

[0189] Methods: Twelve activated sludge samples were prepared in the laboratory using artificial wastewater. DNA was extracted from these samples using the Extra Soil DNA Kit Plus ver.2 (Biodynamics Inc.) according to the manufacturer's protocol.

[0190] Using the DNA obtained above as a template, the target product was obtained using the said patent method. For the oligo-DNA used in this example, the oligo-DNA shown in Table 2 of Example 1 was used for the ligation oligo and the 3'LG oligo containing the gene-specific sequence.

[0191] For 5'LG oligos containing barcode sequences, those listed in Table 29 were used to enable simultaneous analysis of 12 samples in a single NGS analysis. To ensure that the barcode sequences of the target product were the same on both the forward and reverse sides, a pair of forward and reverse 5'LG oligos with the same barcode sequence were used.

[0192] The template addition amount was 2 μl of the extracted DNA described above per reaction. Other conditions were the same as those described in Example 9, except that the number of cycles was fixed at 40 cycles.

[0193] For the quantitative determination of the target product, the final corrected fluorescence intensity value for the final cycle (40 cycles) was calculated using the method described in Example 9, and the target product was calculated from this value using the relationship shown in Figure 21(b) as a calibration curve.

[0194] Based on the calculated values, the unpurified reaction solutions were mixed in the test system so that the target product of each sample was equimolar. This mixture was then purified using an Agentcourt AMPure XP (Beckman Coulter).

[0195] On the other hand, in the control system, equal volumes of unpurified reaction solution were collected and mixed, and this mixture was purified using an Agentcourt AMPure XP (Beckman Coulter).

[0196] The double-stranded DNA concentrations of the two mixtures obtained above were measured using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol.

[0197] NGS analysis was performed on the two mixtures of double-stranded DNA obtained above, after quantification. The NGS analysis conditions were as described in Example 3, and an NGS system manufactured by Nanopore was used.

[0198] NGS analysis was performed on the two mixtures of double-stranded DNA obtained above, after quantification. The NGS analysis conditions were as described in Example 3, and an NGS system manufactured by Nanopore was used.

[0199] In this embodiment, NGS analysis was performed on a total of 24 samples (12 test samples and 12 control samples). However, by using the 5'LG oligo with different barcode sequences as described above, it is possible to simultaneously analyze up to 12 samples in a single NGS analysis. Therefore, the number of NGS analyses performed in this embodiment is two.

[0200] Next, base calling (the process of converting raw sequence data acquired from flow cells into gene sequences; this process was performed in the SUP mode, which offers the highest accuracy) and demultiplexing (the process of separating sequence information for each sample using barcode sequences as markers) were performed using MinKNOW software provided by Nanopore. After converting the obtained sequence data into base sequence information, the number of reads for each sample was obtained using EPI2ME, a bioinformatics platform provided by Nanopore.

[0201] Results: The results of this example are shown in Table 30. In the control system, where equal volumes of reaction solutions were mixed, there was a large variation in the number of reads per sample, with a standard deviation (%) of 26%. On the other hand, in the test system, where the dsDNA concentration in the reaction solution was measured with a low-inhibition dsDNA dye, the variation in the number of reads per sample was kept low, and the standard deviation (%) was 13%, half that of the control system.

[0202] The results from this example and Example 9 demonstrate that by adding a low-inhibition dsDNA dye with minimal amplification inhibition to the amplification reaction solution before the reaction and performing fluorescence measurement with the reaction tube closed, it is possible to accurately quantify the target product amplified by the method described in this patent. Furthermore, it is shown that the number of reads (number of gene sequences) per sample can be arbitrarily adjusted by mixing the reaction solution based on the quantified value.

[0203] As described above, it was shown that by adding a low-inhibition dsDNA dye with minimal PCR inhibition to the reaction solution for preparing the target product, and then directly measuring the fluorescence of the reaction tube after the reaction is complete, the quantification of the target product can be performed quickly and easily. Furthermore, since the purification of the target product, which previously had to be performed for each reaction tube, can now be performed on the mixture, it is expected that the effort required for target product purification will be significantly reduced, leading to a reduction in overall process time and cost (Figure 9). [Table 29] [Table 30] [Examples]

[0204] <Simplification and cost reduction of end-processing steps based on the use of Nanopore sequencers and their Ligation Sequencing Kits> When performing NGS analysis using a Nanopore sequencer, it is necessary to add adapter sequences provided by Nanopore to both ends of the target product to be analyzed. Various kits for adding these adapter sequences are sold by Nanopore, but in this example, we will investigate a simplified and cost-effective protocol using Nanopore's Ligation Sequencing Kit, which features the addition of the adapter sequence using ligase.

[0205] Specifically, we investigated whether it would be possible to simplify and reduce the cost of the protocol, which is based on the use of the Nanopore kit mentioned above, by using (1) a 5'LG oligo with a phosphorylated 5' end, and (2) a reagent (A-attachment mix (manufactured by Toyobo Co., Ltd.)) that can add a single A base to the 3' end simply by adding it to the unpurified target product preparation solution.

[0206] Method: The target gene in this example is a 16S rRNA gene derived from eubacteria, as in Example 1. The template is also the same as in Example 1, using DNA-Mock (NBRC), and this is applied in quantities of 10 units per reaction tube in terms of 16S rRNA gene equivalent. 5 It was added to create a copy.

[0207] For the oligo-DNA used in this example, the oligo-DNA shown in Table 2 of Example 1 was used for the ligation oligo and the 3'LG oligo containing the gene-specific sequence.

[0208] For the 5'LG oligos, the oligos listed in Table 31 were used. More specifically, in the test system using the A-attachment mix, 5'LG oligos with a phosphorylated 5' terminus (oligos listed in Table 31 No. 1 and 2) were used, while in the control system where terminology modification was performed according to the manufacturer's recommended protocol, 5'LG oligos without phosphorylation at the 5' terminus (oligos listed in Table 31 No. 3 and 4) were used.

[0209] Amplification of the target product was performed using 40 cycles in both the test system and the control system, with all other conditions being those described in Example 9.

[0210] In the test system, a 10x concentration of A-attachment mix was added to the reaction solution containing the target product obtained above, according to the manufacturer's protocol, until the final concentration was 1x. The mixture was then incubated at 60°C for 10 minutes in a PCR instrument (iCycler (Bio-Rad)).

[0211] Next, the end-modified target product described above was purified using an Agentcourt AMPure XP (Beckman Coulter) according to the manufacturer's protocol for the reagent, and the end-modified purified target product was obtained.

[0212] On the other hand, in the control system, the target product was first purified using an Agentcourt AMPure XP (manufactured by Beckman Coulter) according to the manufacturer's protocol for the reagent.

[0213] The double-stranded DNA concentration of the purified product obtained above was determined using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol.

[0214] The purified target product obtained as described above was subjected to end-treatment using NEB Next Ultra II End repair / dA-tailing Module reagents (New England Biolabs), recommended by Nanopore, according to the kit's protocol (see Table 1).

[0215] Adapters were added to both ends of the end-modified purified target product of the test system and the target system obtained above, using the Nanopore Ligation Sequencing Kit [SQK-LSK114], in accordance with the manufacturer's protocol.

[0216] The adapter-attached products obtained above were quantified using the fluorescence measurement method with the same Quant-iT PicoGreen dsDNA Assay Kits as described above. Sequence analysis was then performed on these products using Nanopore's NGS (MinION Mk1B). The flow cell used for the NGS analysis was Nanopore's Flongle (version: R10.4.1). Since the final products of the test system and the control system had different barcode sequences attached, solutions containing equimolar amounts of each final product were prepared. NGS analysis was then performed on this mixture, allowing for the simultaneous acquisition of gene sequence information for both products in a single NGS analysis.

[0217] Next, base calling (the process of converting raw sequence data acquired from a flow cell into gene sequences; this process was performed in the SUP mode, which offers the highest accuracy) and demultiplexing (the process of separating sequence information for each sample using barcode sequences as markers) were performed using MinKNOW software provided by Nanopore. After converting the obtained sequence data into base sequence information, bacterial identification based on the gene sequence information was performed using EPI2ME, a bioinformatics platform provided by Nanopore.

[0218] Results: The results are shown in Tables 32 and 33. Table 32 shows that no significant differences were observed between the test and control systems in comparative items such as the number of reads obtained, the barcode identification rate (indicating the proportion of products with linked barcodes), and the systematic classification name identification rate (indicating the proportion of products derived from the target gene (16S rRNA gene)). Similarly, Table 33 shows no significant differences in microbial composition.

[0219] The results above demonstrate the effectiveness of the end-level modification method for the target product described in this embodiment, and show that adopting this method makes it possible to simplify, accelerate, and reduce the cost of protocols using Nanopore sequencers and Ligation Sequencing Kits. [Table 31] [Table 32] [Table 33]

[0220] Figure 22 shows the flow and time-saving effect of the new process applying the methods described in Examples 8-11. Note that the above assumes simultaneous processing of 10 samples.

[0221] By using the high-speed PCR enzyme described in Example 8 and changing the reaction temperature conditions, it was possible to reduce the reaction time by up to 90 minutes (reduction rate: 56%).

[0222] Examples 9 and 10 demonstrate that by using low-inhibition dsDNA dyes with minimal PCR inhibition, the DNA concentration of the target product can be easily and rapidly quantified by directly measuring the fluorescence of the reaction tube after amplification of the target product, without the need for a DNA purification step. Furthermore, based on this quantified value, it is possible to mix target products from multiple samples at the target concentration without a DNA purification step, and DNA purification can be performed on this mixture, eliminating the need to purify each product individually. As a result, the method described in Examples 9 and 10 can reduce the processing time by approximately 50 minutes (DNA quantification step: 20 minutes, purification step: 30 minutes) and significantly reduce the cost of purification (a 90% reduction assuming simultaneous processing of 10 samples).

[0223] The application of the end-tailing method described in Example 11 is limited to analyses using Nanopore sequencers and Nanopore Ligation Sequencing Kits, where end-tailing of the target product is required for analysis. However, by adopting this method, a time reduction of approximately 50 minutes was achieved (reduction rate: 80%). Furthermore, reagent costs were reduced from NEB Next Ultra II End repair / dA-tailing Module reagents (New England Biolabs) to A-attachment mix (Toyobo Co., Ltd.).

[0224] Furthermore, the methods described in Examples 8-10 are applicable to the common steps (amplification, purification, and quantification of the target product) in amplicon analysis using NGS, and are therefore considered effective methods even when performing analysis with NGS systems other than those from Nanopore.

[0225] By applying the methods described in Examples 8 to 11 to the amplification of the target product and subsequent steps according to the present invention, it was possible to shorten the process by 190 minutes (approximately 3 hours) compared to existing processes (shortening rate: 48%). [Industrial applicability]

[0226] This invention can be used as a novel method for preparing gene libraries. [Sequence Listing Free Text]

[0227] The base sequences of the oligoDNA used in the examples <SEQ ID NOs: 1-72> are shown.

Claims

1. A method for preparing a mixture containing two or more target products with different barcode sequences, comprising adding a dsDNA-specific dye to each gene amplification reaction solution containing the target products, quantifying the concentration of each target product from the fluorescence value of the dye, and mixing each gene amplification reaction solution so that each target product is in a desired concentration ratio based on the quantification results.

2. The method according to claim 1, wherein the purification is carried out with respect to a mixture of target products.

3. The method according to claim 1, wherein fluorescence of a dsDNA-specific dye is obtained during the gene amplification process.

4. The method according to claim 1, wherein gene amplification and acquisition of fluorescence of a dsDNA-specific dye are performed using real-time PCR.

5. The method according to claim 1, wherein fluorescence measurement is performed at a temperature in which only the target product exists as double-stranded DNA.

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