Novel library preparation method based on oligo DNA ligation reaction and continuous reaction of DNA amplification due to ligated oligo DNA

Abstract

Provided is a technology capable of preparing a library containing a high proportion of a target product through an economical and simple operation when simultaneously performing sequence analysis of a plurality of specimens through one-time analysis using a next-generation sequencer.　This method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence comprises the following steps (1a) and (2a): step (1a) for reacting three types of oligo DNA pairs, that is, the following (ia)-(iiia) in the presence of a ligase and thereby ligating the following (iia) oligo DNA and (iiia) oligo DNA through a nick repair reaction using a DNA ligase to synthesize a ligation primer pair (a ligation primer (F) and a ligation primer (R) in fig. 5), wherein (ia) is an oligo pair (an oligo (F) for ligation and an oligo (R) for ligation in fig. 5) for ligation which is characterized by having a sequence complementary to a sequence (hereinafter, a ligation sequence) necessary for ligating the oligo DNA in the nick repair reaction using the ligase, (iia) is a 3'LG oligo pair (3'LG oligo (F) and 3'LG oligo (R) in fig. 5) which has a portion of the sequence of the 3' end side of the ligation sequence at the 5' end side thereof, has a sequence specific to a gene to be analyzed at the 3' end side thereof, and has a phosphorylated 5' end, and (iiia) is a 5'LG oligo pair (5'LG oligo (F) and 5'LG oligo (R) in fig. 5) characterized by having an artificial nucleic acid sequence, such as a barcode sequence, at the 5' end side thereof and having a portion of the sequence of the 5' end side of the ligation sequence at the 3' end side thereof; and step (2a) for performing gene amplification using the ligation primer pair synthesized in the step (1).

Description

A novel library preparation method based on the sequential reaction of oligo DNA ligation and DNA amplification using the ligated oligo DNA

[0001] The present invention relates primarily to assays for detecting or sequencing specific nucleic acid sequences. In certain embodiments, the present invention provides amplification methods in which one or more artificially designed nucleic acid sequences (hereinafter referred to as artificial nucleic acid sequences), including barcode sequences, which are artificial nucleic acid sequences for identifying analytes, are added to a target nucleic acid sequence.

[0002] Detection of specific nucleic acid sequences is used in many fields, such as genetic diagnosis in medicine, hygiene inspection in food, and environmental monitoring.

[0003] In addition, with the recent rise of next-generation sequencers (hereinafter referred to as NGS), nucleic acid sequencing has become much easier and cheaper than conventional sequencing methods, and is therefore gaining popularity as a new analytical technique. In NGS, in order to provide a mixture of amplification products derived from multiple samples, specific artificial nucleic acid sequences including barcode sequences for identifying the samples must be added to both ends of the nucleic acid fragments to be sequenced, and a nucleic acid amplification reaction step for this purpose is required in addition to the step of amplifying the target nucleic acid sequence.

[0004] It is also possible to obtain nucleic acid fragments for NGS using primers specific to the target nucleic acid sequence, to which a specific artificial nucleic acid sequence containing a barcode sequence has been added in advance at the 5' end (prior art [1]). However, this method requires the preparation of primers with different barcode sequences for each type of nucleic acid sequence, equal to the number of samples, which is extremely uneconomical (Figure 1).

[0005] A two-stage PCR method (prior art [2]) is commonly used as an economical means of obtaining an amplification product with an artificial nucleic acid sequence attached (Non-Patent Document 1). In this method, a first PCR is performed using a primer with a portion of the 3' end of an artificial nucleic acid sequence attached to the 5' end of a specific sequence (hereinafter referred to as the artificially attached specific primer). This produces a product derived from the target nucleic acid (hereinafter referred to as the specific product). After purifying this product, a second PCR is performed using this as a template with a primer with an artificial nucleic acid sequence, such as a barcode sequence, attached to the 3' end (hereinafter referred to as the artificial sequence primer). These two PCR amplifications produce an amplification product with artificial nucleic acid sequences attached to both ends of the target nucleic acid (hereinafter referred to as the target product) (Figure 2).

[0006] However, in the prior art [2], PCR must be performed twice, and the specific product obtained by the first PCR must be purified and its concentration adjusted, making the process complicated and time-consuming.

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

[0008] According to Patent Documents 1 and 2, by lowering the concentration of the artificially added specific primers compared to the concentration of the artificial sequence primers, the artificial sequence primers that can bind to the artificial nucleic acid sequences added in common to all target sequences can dominate the amplification reaction, thereby enabling the target product to be obtained efficiently, and in the case of multiple target nucleic acids, variation in amplification efficiency between genes can be suppressed (Figure 3).

[0009] However, when preparing nucleic acid sequences for NGS using the prior art [3], the artificial primers used at high concentrations are long, approximately 60-70 bases long, increasing the likelihood of primer dimers being produced. When multiple target nucleic acid sequences are used, the number of artificially added specific primers used increases, resulting in the frequent production of primer dimers derived from these primers. This leads to the problem of an increased proportion of sequences other than the target sequence being analyzed during NGS.

[0010] In addition, as the number of target nucleic acid sequences increases, the total amount of artificially added specific primers increases, resulting in the problem that the artificial sequence primers cannot dominate the amplification reaction, and the proportion of nucleic acid sequences to which no artificial nucleic acid sequence is added increases.

[0011] A method (prior art [4]) that can solve the above-mentioned problems has been proposed (Patent Document 3). Prior art [4] discloses a method in which, in a reaction for adding an artificial nucleic acid sequence to a target gene sequence, in addition to an artificially added specific primer, two pairs of artificial sequence primers are used: a long artificial sequence primer (hereinafter referred to as "long artificial sequence primer" in Figure 4) and a short artificial sequence primer (hereinafter referred to as "short artificial sequence primer" in Figure 4) to suppress amplification of nonspecific sequences derived from the primers and efficiently amplify the target product. Furthermore, it is described that the target product can be more efficiently amplified by lowering the concentration of the artificially added specific primer and the artificial sequence primer containing a barcode sequence, etc. (primer pair Y) and increasing the concentration of the short primer (primer pair Z).

[0012] However, in the prior art [4], at least three sets of primers are used: an artificially added specific primer pair, a long artificial sequence primer pair, and a short artificial sequence primer pair. Since the three 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 one PCR reaction solution.

[0013] Due to the fundamental characteristics described above, the prior art [4] has the following problems: (1) it requires strict optimization of reaction conditions for each target nucleic acid, which requires an excessive amount of time and effort; and (2) it is less sensitive than conventional PCR, which uses only one primer pair, and therefore requires a larger number of initial templates (target genes).

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

[0015] In NGS analysis, quantification of the target product is essential to ensure that the amount of target product added falls within the range specified by each NGS instrument manufacturer. Quantification of the target product is commonly achieved using simple and low-cost methods such as electrophoresis, fluorometry, and absorbance measurement, as well as quantitative PCR (qPCR), a relatively complex and expensive method. Prior art techniques have struggled to introduce artificial nucleic acid sequences into all specific products, resulting in the synthesis of not only the target product but also specific products without the artificial nucleic acid sequence (hereinafter referred to as non-target products). While the former simple and low-cost method cannot accurately distinguish between the target product and non-target products, it can be used without problems if the proportion of the target product among the specific products is extremely high, since the difference between the quantification value calculated assuming all specific products are the target product and the true amount of the target product is negligible. On the other hand, when the proportion of the target product among the specific products is low, qPCR, a complex and costly method, is required, as it can distinguish the presence or absence of artificial nucleic acid sequences and quantify only the target product. In the aforementioned prior art [5], not only the target product but also many specific products without the artificial nucleic acid sequence are synthesized, which requires quantification using qPCR, which is both complicated and costly.

[0016] In addition, in the case of a method such as the NGS manufactured by Oxford Nanopore Technologies (hereinafter referred to as ONT), which requires the addition of double-stranded DNA (hereinafter referred to as "adapter") to both ends of a target product using ligase or the like, the adapter sequence is added not only to the target product but also to non-target products, which are then sequenced. Since non-target products do not have a barcode sequence attached, it is not possible to identify the genetic sequence from which the sample originated. Therefore, depending on the proportion of non-target products, unusable genetic sequence information is output from the NGS. Since there is a limit to the genetic sequence information output per NGS analysis, the amount of usable genetic sequence information decreases as the amount of unusable genetic sequence information increases. Therefore, in the case of ONT's NGS, it is extremely important to reduce the amount of unusable genetic sequence information as much as possible and increase the proportion of target products in order to reduce costs. Under normal operating conditions in the prior art [5], the ratio of the target product to the specific products is around 50%, which means that 50% of the output gene sequence information cannot be used, and the cost of 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 among all specific products, it is necessary to use oligo DNA (artificially added specific SL primer and SL oligo with artificial 3' sequence) that has an intramolecular secondary structure in the 5'-terminal region and has a substance (blocker) inserted into the sequence chain that has the function of blocking DNA elongation, or to add a heat-resistant DNA polymerase, SD polymerase (Biolon), in an amount greater than that specified in the manufacturer's protocol. However, the former oligo DNA is lengthened because a sequence needs to be added for the purpose of forming an intramolecular secondary structure, and the need to insert a blocker increases the synthesis cost. In the latter, the amount of SD polymerase added needs to be 10 times the amount specified in the manufacturer's protocol to achieve sufficient effect, leading to high reagent costs.

[0018] As described above, in the prior art [5], there are problems with economy and operability in the process of quantifying the target product and the process aimed at increasing the ratio of the target product.

[0019] Applied and Environmental Microbiology, 2013 September 79(17):5112-5120.

[0020] WO2006 / 023919, Patent Publication No. 2012-522517, Japanese Patent No. 5997407, Japanese Patent Publication No. 2022-170729, Japanese Patent Publication No. 2022-170730

[0021] Therefore, when using NGS to simultaneously perform sequence analysis of multiple samples in a single analysis, there is a need for a technology that can economically and easily prepare a library containing a high proportion of the target product.

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

[0023] As a result, (1a) a step of synthesizing a linking primer pair (linking primer (F) and linking primer (R) in FIG. 5 ) by reacting three types of oligo DNA pairs (ia) to (iiia) below in the presence of a ligase to link the oligo DNA (iia) and the oligo DNA (iiia) below by a nick repair reaction using DNA ligase, (ia) a linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5 ) characterized by having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter referred to as linking sequence) in a nick repair reaction using ligase, (iia) a 3′LG oligo pair (3′LG oligo (F) and 3′LG oligo (R) in FIG. 5 ) having a part of the sequence on the 3′-end side of the linking sequence at the 5′-end, a sequence specific to the gene to be analyzed at the 3′-end, and having a phosphorylated 5′-end, (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 the linking sequence at the 3' end. (2a) A step of performing gene amplification using the linked primer pair synthesized in step (1) above. By directly adding the unpurified reaction solution obtained in step (1a) above to the reaction solution (2a) above and performing the two reactions (1a) and (2a) above, or by allowing the two reactions (1a) and (2a) above to proceed sequentially in the order of (1a) and (2a) in a reaction solution to which all necessary components (DNA polymerase, DNA ligase, dNTPs, etc.) have been added without opening the reaction tube, it was possible to prepare a library containing a high proportion of the target product in an economical and simple operation.In another embodiment, (1b) a step of synthesizing a linked primer pair (linked primer (F) and linked primer (R) in Figure 6) by linking two types of oligo DNA pairs (ib) and (iib) below by click chemistry reaction: (ib) a 3' CC oligo pair having a sequence specific to the gene to be analyzed and whose 5' end has been modified with a click chemistry functional group (3' CC oligo (F) and 3' CC oligo (R) in Figure 6); (iib) a 5' CC oligo pair having an artificial nucleic acid sequence such as a barcode sequence and whose 3' end has been 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 of carrying out gene amplification using the linked primer pair synthesized in the above step (1b). The unpurified reaction solution obtained in (1b) above was directly added to the reaction solution in (2b) above to carry out the two reactions (1b) and (2b) above, or the two reactions (1b) and (2b) above were carried out successively in the order of (1b) and (2b) in a reaction solution to which all necessary components (DNA polymerase, dNTPs, etc.) had been added without opening the reaction tube, thereby enabling the preparation of a library containing a high proportion of the target product in an economical and simple operation.

[0024] In the above reactions (2a) and (2b) (gene amplification reactions), only the ligated primer functions as a primer, and the unligated oligo DNA (hereinafter referred to as "unligated oligo"), which has a lower dissociation temperature than the ligated primer, does not function as a primer. By increasing the reaction temperature after the initial synthesis of the target product within a range that does not significantly reduce the efficiency of gene amplification by the ligated primer, 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] This technology can be applied to analyses for a variety of purposes. For example, it can comprehensively analyze genes that may cause cancer in order to identify the location of genetic mutations. This technology can also be suitably used when using NGS in various SNP analyses, gene mutation analyses, gene expression analyses, and microbiota analyses. However, the scope of application is not limited as long as the test target is a nucleic acid amplification product and multiple target genes are analyzed by adding an artificial nucleic acid sequence to identify the sample.

[0026] The gist of the present 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, the method comprising the following steps (1a) and (2a): (1a) A step of synthesizing a linking primer pair (linking primer (F) and linking primer (R) in FIG. 5 ) by reacting three types of oligo DNA pairs (ia) to (iiia) below in the presence of a ligase to link the oligo DNA (iia) and the oligo DNA (iiia) below by a nick repair reaction using DNA ligase. (ia) A linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5 ) characterized by having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter referred to as a linking sequence) in a nick repair reaction using ligase. (iia) A 3′LG oligo pair (3′LG oligo (F) and 3′LG oligo (R) in FIG. 5 ) having a part of the sequence on the 3′-end side of the linking sequence at the 5′-end, a sequence specific to the gene to be analyzed at the 3′-end, and having a phosphorylated 5′-end. (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 part of the 5' end sequence of the linking sequence at the 3' end. (2a) A step of carrying out gene amplification using the linking primer pair synthesized in the step (1) above. [2] The method of [1], which uses a linking oligo whose 3' end has been modified to prevent DNA elongation. [3] A kit for preparing a target product by the method of [1], which includes at least one pair of each of the three types of oligo DNA pairs (ia) to (iiia) below.(ia) A linking oligo pair having a complementary sequence to the linking sequence (linking oligo (F) and linking oligo (R) in Figure 5); (iia) A 3'LG oligo pair having a part of the 3'-terminal sequence of the linking sequence at its 5'-terminal side, a sequence specific to the gene to be analyzed at its 3'-terminal side, and phosphorylated at its 5'-terminal (3'LG oligo (F) and 3'LG oligo (R) in Figure 5); (iiia) A 5'LG oligo pair having an artificial nucleic acid sequence such as a barcode sequence at its 5'-terminal side, and a part of the 5'-terminal sequence of the linking sequence at its 3'-terminal side (5'LG oligo (F) and 5'LG oligo (R) in Figure 5); [4] The kit according to [3], which contains a linking oligo whose 3'-terminal side has been modified to prevent DNA elongation. [5] The kit according to [3] or [4], further containing a DNA polymerase and a 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, the method comprising the following steps (1b) and (2b): (1b) a step of linking two types of oligo-DNA pairs, (ib) and (iib) below, by click chemistry reaction to synthesize a linked primer pair (linked primer (F) and linked primer (R) in Figure 6); (ib) a 3' CC oligo pair, having a sequence specific to the gene to be analyzed and having the 5' end modified with a click chemistry functional group (3' CC oligo (F) and 3' CC oligo (R) in Figure 6); (iib) a 5' CC oligo pair, having an artificial nucleic acid sequence such as a barcode sequence and having the 3' end 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 of carrying out gene amplification 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 of each of the two types of oligo-DNA pairs, (ib) and (iib) below.(ib) A 3' CC oligo pair having a sequence specific to the gene to be analyzed and having the 5' ends modified with a click chemistry functional group (3' CC oligo (F) and 3' CC oligo (R) in Figure 6). (iib) A 5' CC oligo pair having an artificial nucleic acid sequence such as a barcode sequence and having the 3' ends 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 according to [7], further comprising a DNA polymerase. [9] The method according to any one of claims 1, 2 or 6, wherein the 5' ends of the 5' LG oligo in Figure 5 or the 5' CC oligo in Figure 6 are 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 unligated oligos do not function as primers and the efficiency of gene amplification by the ligated primers is not significantly reduced.

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

[10] , in which two or more types of 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] , in which two or more types of 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 one of [1], [2], [6], and [9] to

[12] , in which all steps are continuously reacted in a single reaction without opening the reaction tube.

[14] The gene amplification method is a PCR (Polymerase Chain Reaction) method, an LCR (Liagase Chain Reaction) method, an RPA (Recombinase Polymerase Amplification) method, a SIBA (Strand-Invasion Based Amplification) method, a LAMP (Loop-mediated isothermal amplification) method, an SDa (Strand displacement amplification) method, or a NEAR (Nicking Endonuclease Amplification) method. The method according to any one of [1], [2], [6], and [9] to

[13] , which is any one of the following methods: the Helicase-Dependent Amplification (HDA) method, the Nucleic Acid Sequence-Based Amplification (NASBA) method, the Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids (ICAN) method, the Rolling Cycle method, and the Smart Amplification Process (SMAP) method.

[15] The method of

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

[16] The method of

[14] or

[15] , wherein the PCR enzyme uses 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 products, quantifying the target product concentrations based on the fluorescence values ​​of the dye, and mixing the gene amplification reaction solutions so that the target products are at an arbitrary concentration ratio based on the quantification results.

[18] The method of

[17] , wherein purification is performed on a mixture of target products.

[19] The method of

[17] or

[18] , wherein fluorescence of the dsDNA-specific dye is obtained during the gene amplification process.

[20] The method of any one of

[17] to

[19] , in which gene amplification and acquisition of dsDNA-specific dye fluorescence are carried out using real-time PCR.

[21] The method of any one of

[17] to

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

[22] The method of any one of

[17] to

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

[23] The method of

[22] , in which the dissociation-corrected fluorescence intensity value is calculated using the following formula: Dissociation-corrected fluorescence intensity value = F. n，ＴＴ / F n，ＤＮ F ｎ，ＴＴ　　　　 F: Fluorescence intensity measured at the temperature at which only the target product exists as dsDNA in any cycle (n) (TT; abbreviation for Temperature at which only the target product exists as dsDNA) ｎ，ＤＮ　　　　 : Actual fluorescence intensity value measured at a temperature at which all DNA dissociates in an arbitrary cycle (n) (DN; abbreviation for DeNature)

[24] The method of any of

[17] to

[22] , in which, in a cycle in which an increase in fluorescence value derived from a dsDNA-specific dye due to gene amplification does not occur in all gene amplification reactions to be analyzed, the dissociation-corrected fluorescence intensity value calculated by the method of claim 22 is corrected to be the same value in all reactions, and the target product is quantified based on the final corrected fluorescence intensity value obtained.

[25] The method of

[22] , in which the final corrected fluorescence intensity value is calculated using the following formula: Final corrected fluorescence intensity value = (F ｆｃ，ＴＴ / F ｆｃ，ＤＮ ) / (F ＣＢＦＩ，ＴＴ / F ＣＢＦＩ，ＤＮ ) F ｆｃ，ＴＴ F: Fluorescence intensity measured at the temperature at which only the target product exists as dsDNA (TT; abbreviation for Temperature at which only the targeted product exists as dsDNA) in the final cycle (fc; abbreviation for final cycle) ｆｃ，ＤＮF: Fluorescence intensity measured at the temperature at which all DNA dissociates in the final cycle (fc) (DN; abbreviation for DeNature) ＣＢＦＩ，ＴＴ F: Measured fluorescence intensity at the temperature at which only the target product exists as dsDNA in the cycle before the increase in fluorescence associated with gene amplification is confirmed (CBFI; abbreviation for Cycle Before Fluorescence Increases) ＣＢＦＩ，DN

[26] The method of any of

[17] to

[25] , in which the target product is quantified based on the dissociation-corrected fluorescence intensity value calculated by the method described in

[22] or

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

[24] or

[25] .

[27] The method of any of

[17] to

[26] , in which the dsDNA-specific dye is a low-inhibitory dsDNA dye.

[28] The method of any of

[17] to

[27] , in which the low-inhibitory dsDNA dye is at least one dye selected from the group consisting of pGreen and LC GREEN PLUS.

[29] A method for preparing a target product mixture using one of the methods described in

[17] to

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

[17] to

[28] .

[30] A kit for performing NGS analysis, comprising a dsDNA-specific dye, the kit preparing a mixture of target products for NGS analysis by the method of

[29] .

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

[32] The method of

[31] , wherein the target product is amplified using a PCR enzyme having proofreading activity, and then adding a single A base to the 3' end of the target product by adding an inactivator of the PCR enzyme and a DNA polymerase having A-adding activity to the 3' end to the reaction solution.

[33] The method of

[31] or

[32] , wherein the target product to which a single A base is added to the 3' end is the target product prepared by the method of claim 9.

[34] A method for modifying a target product, which comprises adding an adapter sequence using a ligase to a mixture of target products prepared by the method of

[29] or the kit of

[30] , using the method of any one of

[31] to

[33] .

[0027] The present invention relates to: (ia) a linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5) characterized by having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter referred to as linking sequence) in a nick repair reaction using ligase; (iia) a 3'LG oligo pair (3'LG oligo (F) and 3'LG oligo (R) in FIG. 5) having a part of the 3'-terminal sequence of the linking sequence at the 5'-terminal side, a sequence specific to a gene to be analyzed at the 3'-terminal side, and the 5'-terminal is phosphorylated; (iiia) a 5'LG oligo pair (5'LG oligo (F) and 5'LG oligo (R) in FIG. 5) having an artificial nucleic acid sequence such as a barcode sequence at the 5'-terminal side, and a part of the 5'-terminal sequence of the linking sequence at the 3'-terminal side. and a reaction reagent such as a DNA polymerase for carrying out an amplification reaction, and a reaction reagent for carrying out a DNA ligase reaction for linking the oligo-DNAs described in (iia) and (iiia) above.

[0028] Alternatively, a method for obtaining a desired product derived from one or more target nucleic acids by using an oligo DNA set composed of: (ib) a 3' CC oligo pair having a sequence specific to the gene to be analyzed and having its 5' end modified with a click chemistry functional group (3' CC oligo (F) and 3' CC oligo (R) in Figure 6); or (iib) a 5' CC oligo pair having an artificial nucleic acid sequence such as a barcode sequence and having its 3' end 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).

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

[0030] The reaction solution used in the embodiment described in FIG. 5 contains the oligo DNA set (ia) to (iiia), amplification reagents including DNA polymerase, and reagents including DNA ligase for carrying out the ligase reaction, and the desired target product is obtained by carrying out the following reaction steps (1a) and (2a) using these. Reaction step (1a): By mixing the three types of oligo DNA pairs (ia) to (iiia) in a reaction solution, the 5'LG oligo (F or R) and the 3'LG oligo (F or R) are bound to the linking oligo (F or R) in a form adjacent to each other, and a ligase recognizes the linking site and links the 5'LG oligo (F or R) and the 3'LG oligo (F or R) by a nick repair reaction. Reaction step (2a): A reaction step in which a target product is obtained by gene amplification using the linking primer pair (F and R) obtained in step (1a). The reaction solution used in the embodiment described in Figure 6 contains the oligo DNA sets (ib) and (iib) and amplification reagents such as DNA polymerase, and the target product is obtained by using these to carry out the reaction steps (1b) and (2b) below. Reaction step (1b): By mixing the oligo DNAs (F or R) described in (ib) and (iib) in a reaction solution, a 5' CC oligo (F or R) and a 3' CC oligo (F or R), each modified with a click chemistry functional group, are linked by click chemistry. Reaction step (2b): A reaction step in which a target product is obtained by gene amplification using the linked primer pair (F or R) obtained in step (1b). Figure 7 shows that if the 3' LG oligo (F or R) that could not be linked to the 5' LG oligo (F or R) in reaction step (2a) functions as a primer and the gene amplification reaction proceeds, the product will lack 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. Therefore, it is necessary to suppress the product (non-target product), and a method for suppressing this is described.Specifically, this suppression method takes advantage of the fact that the ligated primer is longer than the unligated oligo and, as a result, has a higher dissociation temperature for the ligated primer than for the unligated oligo. After the target product derived from the ligated primer is first synthesized (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 that of the initial template), the annealing temperature is raised to a temperature that is sufficient to suppress the binding of the unligated oligo without interfering with the binding of the ligated primer to the target product, thereby suppressing the amplification of non-target products and allowing the amplification of the target product to predominate. Note that while Figure 7 shows an example of reaction step (2a), the method shown in Figure 7 can also be used for reaction step (2b), whereby the amplification of non-target products can be suppressed and the amplification of the target product can predominate.

[0031] The present invention can be embodied in a variety of variations, as follows: (1) Two or more pairs of 3'LG oligos or 3'CC oligos containing target gene-specific sequences are used, and the reaction steps (1a), (2a), or (2a), (2b) described above are performed to obtain two or more target products. (2) By using two linking oligos with different sequences and corresponding 3'LG and 5'LG oligos, different artificial nucleic acid sequences can be introduced at each end of the target product. For example, this variation is effective when different barcode sequences are introduced at each end and sequence separation (demultiplexing) is performed for not only the barcode sequences but also combinations thereof. (3) The reaction steps (1a), (2a), or (2a), (2b) described above can be performed independently, or the reaction steps can be arbitrarily separated and used depending on the purpose. However, an embodiment in which all necessary materials are added to the same reaction solution before the start of the reaction and the reaction steps proceed continuously in a single reaction is advantageous in terms of economy and simplicity. (4) The linking oligo, which serves as a scaffold for linking the 3'LG oligo and the 5'LG oligo, preferably functions only in reaction step (1a) and not in reaction step (2a), which is the gene amplification reaction. For this reason, it is desirable to label the 3' end of the linking oligo so that it cannot be extended by DNA polymerase. Such labeling is generally achieved by modification with phosphorylation, fluorescent dyes, amino linkers, biotin, etc., but any modification method that can prevent extension of the 3' end by DNA polymerase is sufficient. (5) The ligase used in reaction step (1a) can be any type of ligase as long as it has the activity of repairing nicks in double-stranded DNA. However, since ligase does not contribute to the 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 the case of a gene amplification method in which the reaction temperature is high, such as PCR, a non-thermostable ligase will be inactivated.Specific examples of non-thermostable ligases that have the activity of repairing nicks in double-stranded DNA include T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, PBCV-1 DNA ligase, Chlorella virus DNA ligase, E. coli DNA ligase, etc. On the other hand, examples of heat-stable ligases that have the activity of repairing nicks in double-stranded DNA include Taq DNA ligase, 9. o Examples of suitable thermostable ligases include NAD DNA ligase. These thermostable ligases maintain a certain level of activity in reaction step (2a) (gene amplification step) even when a gene amplification reaction is performed at a high reaction temperature, and therefore may have some effect on reaction step (2a). However, 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. Therefore, in such a situation, the use of a thermostable 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 compositions required for each reaction are different. (6) The click chemistry functional group used to modify the oligo-DNA used in reaction step (1b) may be any functional group that can modify the oligo-DNA and enables the linkage of a 5' CC oligo and a 3' CC oligo. However, the bonding between an azide group and an alkyne group, a typical click chemistry reaction, requires copper ions as a catalyst, and these ions cause DNA damage and DNA strand scission, making this not particularly preferable as a click chemistry reaction used in the present invention. On the other hand, dibenzocyclooctyne (DBCO), azadibenzocyclooctyne (ADIBO), bicyclononyne (BCN), 4,8-diazacyclononyne (DACN), etc. are known as functional groups that undergo click chemistry reactions with azide groups 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) The DNA polymerase used in reaction steps (2a) and (2b) may be any polymerase that can be used for gene amplification. However, in the reaction steps (2a) and (2b) where the oligo-DNAs are ligated, the presence of an activated DNA polymerase may inhibit the ligation reaction of the oligo-DNAs. In particular, in reaction step (2a), if the DNA polymerase is activated when the 5'LG oligo is ligated to the ligation oligo, the 5'LG oligo will be extended using the ligation oligo as a template, preventing the 3'LG oligo from ligating to the ligation oligo. As a result, the ligation of the 5'LG oligo and the 3'LG oligo by the ligase may be inhibited. For these reasons, in the present invention, it is desirable to use a hot-start DNA polymerase that is inactive in the temperature range where the oligo-DNAs are ligated in reaction step (1a). (8) The gene amplification reaction used in reaction steps (2a) and (2b) may be a 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 Iso-thermal Amplification), SDA (Strand Displacement Amplification), and NEAR (Nicking Endonuclease Amplification). Examples of such amplification methods include the Helicase-Dependent Amplification (HDA) method, the Nucleic Acid Sequence-Based Amplification (NASBA) method, the Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids (ICAN) method, the Rolling Cycle method, and the Smart Amplification Process (SMAP) method.

[0032] The present invention is expected to have the following advantages: (1) A library containing a high percentage of target products with artificial nucleic acid sequences attached to their termini can be obtained in a single-step process. (2) In the present invention, after the target product is initially synthesized, the annealing temperature can be increased to limit the primers contributing to the gene amplification reaction to ligated primers only. This eliminates the need to use primers for amplification using the target nucleic acid as a template and primers for amplification using an amplification product derived from the target nucleic acid as a template in the same reaction solution, as in the prior art methods [3] and [4], and suppresses the generation of nonspecific amplification products such as primer dimers. This allows the minimum required amount of initial template to be kept lower than in the prior art described above, ensuring sensitivity similar to that of general gene amplification using only primers specific to the target nucleic acid. (3) In the case of a method such as the 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 using primers different from those used in the amplification 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 case of the present invention, even in a one-step closed reaction system, by changing the temperature conditions, it is possible to completely separate the reaction of ligating two oligo-DNAs (reaction steps (1a) and (1b)) and the amplification reaction (reaction steps (2a) and (2b)), allowing each reaction to be carried out under optimal temperature conditions. Furthermore, since the oligo-DNA sequences contributing to the reaction of ligating two oligo-DNAs can react 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. This allows for simple and rapid construction of a reaction system similar to general gene amplification. (4) In the case of a method using a primer specific to the target nucleic acid sequence, to which a specific artificial nucleic acid sequence containing a barcode sequence has been added in advance at the 5' end (prior art [1]), a target product suitable for NGS analysis can be obtained in a simple one-step process. However, it is necessary to prepare primers with different barcode sequences for each target nucleic acid, which is extremely uneconomical. For example, if 100 samples are analyzed simultaneously in one analysis using a method that identifies samples by the combination of barcode sequences added to both ends of the sample and the sequences, 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 per target nucleic acid, for a total of 20 types of primers.As one set of such primers is 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 a 3'LG oligo or a 3'CC oligo, and the addition of the artificial nucleic acid sequence containing the barcode sequence is carried out by a 5'LG oligo or a 5'CC oligo, so the type of oligo DNA required for each depends on the type of target nucleic acid for the former and the number of samples to be analyzed for the latter. For example, assuming the same assumptions as above (100 types of target nucleic acids, 100 samples), there are 200 types of 3'LG oligos or 3'CC oligos, including forward and reverse primers, and 200 types of 5'LG oligos or 5'CC oligos, including forward and reverse primers. In a method of linking oligo DNAs using ligase, if the artificial nucleic acid sequences added to both ends are the same, only one type of linking oligo is required, resulting in a total of 401 types of oligo DNAs, including the oligo. Even if the artificial nucleic acid sequences added to both ends are different, only two types of linking oligos are required, resulting in a total of 402 types of oligo DNAs, including the oligo. Furthermore, in a method of linking oligo DNAs using click chemistry, no linking oligos are required, so only 400 types are required. In addition, since there is no need to change the sequence of 5'LG oligos, 5'CC oligos, and linking oligos depending on the target nucleic acid, they can be used in common regardless of the target nucleic acid, and can be used efficiently. (5) Because the concentration of the target product to be subjected to NGS needs to be constant, it is necessary to adjust the concentration after the target product is created. However, in the prior art [5], unless special labeling substances or oligo-DNAs with structures (3' artificial sequence-introduced SL oligos) or primers (artificially added specific SL primers) are used, or a high concentration of SD polymerase is added, non-target products are synthesized at a high rate. Therefore, it is not possible to use low-cost and simple methods such as electrophoresis, fluorometry, and absorptiometry, which make it difficult to specifically detect and quantify only the target product. Therefore, 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, the present invention makes it possible to prepare a library in which the target product accounts for the majority (95% or more) of the specific products, without using special labeling substances or oligo-DNA structures, using standard manufacturer-recommended enzyme amounts. This allows for accurate quantification of the target product even with the low-cost and simple method described above. As described above, the present invention allows for quantification of the target product more simply and at lower cost than prior art [5]. (6) In analyses that require the addition of adapters required for analysis to both ends of the target product using ligase, such as the NGS system manufactured by ONT, adapters are added not only to the target product but also to non-target products, which are then sequenced. Since non-target products do not have barcode sequences, it is impossible to identify the genetic sequence information from which sample the non-target products originate. Therefore, an increase in non-target products leads to an increase in unusable genetic sequence information. Therefore, reducing these products is extremely important for reducing the analysis costs of the NGS method. While the percentage of the target product under normal operating conditions in the prior art [5] is around 50%, it is possible to increase the percentage of the target product up to approximately 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 high cost of synthesizing these oligo DNAs and adding SD polymerase has led to a need for an improvement. On the other hand, the present invention does not require oligo DNA modified with special substances, and by adding DNA polymerase at the manufacturer's recommended concentration, it is possible to increase the percentage of the target product up to 95%. This makes it possible to increase the percentage of the target product more simply and at lower cost than the prior art [5]. As a result, it can be suitably used as a library preparation method for the ONT NGS and other systems.

[0035] According to the present invention, a library containing a high percentage of a target product can be prepared economically and easily.

[0036] This diagram shows the principle of prior art [1]. This method uses a primer specific to a target nucleic acid sequence, to which a specific artificial nucleic acid sequence containing a barcode sequence has been added at its 5' end, to obtain a target product suitable for NGS. This method requires the preparation of primers with different barcode sequences for each type of nucleic acid sequence, as many as the number of samples. This diagram shows the principle of prior art [2]. This method involves a first amplification using an artificially added specific primer, in which a portion of the 3' end of the artificial nucleic acid sequence has been added at the 5' end of the specific sequence, to obtain a specific product derived from the target nucleic acid. This product is then purified and used as a template for a second amplification using an artificial sequence primer with an artificial nucleic acid sequence, such as a barcode sequence, at its 3' end. These two rounds of amplification yield a target product with artificial nucleic acid sequences added to both ends of the target nucleic acid. This diagram shows the principle of prior art [3]. This method involves setting the concentration of the artificially added specific primer lower than the concentration of the artificial sequence primer, and adding both primers to the same reaction solution for amplification, thereby obtaining the target product in a single amplification. This diagram shows the principle of prior art [4]. In this method, in the reaction of adding an artificial nucleic acid sequence to a target nucleic acid sequence, in addition to the artificial addition-specific primer, two pairs of artificial sequence primers, a long artificial sequence primer and a short artificial sequence primer, are used, and by increasing the concentration of the short artificial sequence primer above the concentrations of the artificial addition-specific primer and the long artificial sequence primer, it is possible to amplify the target product more efficiently. Reaction scheme of a new library preparation method based on ligation. Reaction scheme of a new library preparation method based on click chemistry. Principle diagram of the amplification of non-target products by unlinked oligos and their problems, and the suppression of amplification of non-target products by increasing the reaction temperature during gene amplification. Steps in the existing quantification and purification process. Flow diagram of the library end modification method recommended by Nanopore, Inc. Simplification and speedup of the library quantification and purification process using a dsDNA-specific fluorescent dye. Speedup and cost reduction of the library end modification method using a 5'LG oligo with a phosphorylated 5' end and A-attachment mix. A diagram showing the amount of specific product under each reaction condition performed in Example 1. Electrophoresis (PAGE) photograph after the ligase reaction in Example 2. Comparison of the theoretical composition ratio of DNA-Mock and the measured composition ratio of the target product in Example 3.Comparison of the measured composition ratio of the target product and the measured composition ratio of non-target products in Example 3. Graph showing the number of reads for each target gene in Example 4. Electrophoresis (PAGE) photograph after click chemistry reaction. Comparison of the theoretical composition ratio of DNA-Mock and the measured composition ratio of the target product in Example 6. Comparison of the measured composition ratio of the target product and the measured composition ratio of non-target products in Example 6. Graph showing the number of reads for each target gene in Example 7. (a) Relationship between double-stranded DNA concentration after purification and uncorrected fluorescence intensity at 90°C (b) Relationship between double-stranded DNA concentration after purification and final corrected fluorescence intensity. The flow of the new process and the time-saving effect are shown, applying the details of Examples 8 to 11.

[0037] The embodiments of the present invention will now 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 of synthesizing a linking primer pair (linking primer (F) and linking primer (R) in FIG. 5 ) by reacting three types of oligo DNA pairs (ia) to (iiia) below in the presence of a ligase to link the oligo DNA (iia) and the oligo DNA (iiia) below by a nick repair reaction using DNA ligase. (ia) A linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5 ) characterized by having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter referred to as a linking sequence) in a nick repair reaction using ligase. (iia) A 3′LG oligo pair (3′LG oligo (F) and 3′LG oligo (R) in FIG. 5 ) having a part of the sequence on the 3′-end side of the linking sequence at the 5′-end, a sequence specific to the gene to be analyzed at the 3′-end, and having a phosphorylated 5′-end. (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 the linking sequence at the 3' end. (2a) A step of performing gene amplification using the linking primer pair synthesized in step (1) above. In a method including the reaction steps (1a) and (2a), the linking 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 the former and latter sequences are preferably 4 to 30 bases long, preferably 6 to 20 bases long, and more preferably 8 to 12 bases long.

[0039] The 3'-ends of the ligation oligos are preferably labeled to prevent their extension by DNA polymerase. Such labeling is generally achieved by phosphorylation, fluorescent dyes, amino linkers, biotin, or modification with bases that do not form complementary strands with the target DNA, but any modification method that can prevent the extension of the 3'-end 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 5' end sequence of the former may be 4 to 30 bases long, preferably 6 to 20 bases long, and more preferably 8 to 12 bases long, while the 3' end sequence of the latter may be set to a sequence length generally preferred for gene amplification primers, and no special design dependent on the method of the present invention is required. Furthermore, the 5' end of the 3'LG oligo must be phosphorylated to enable ligation to the 5'LG oligo by ligase.

[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 the oligo DNA present on the NGS flow cell, and a sequence at its 3' end that is complementary to the sequence at the 3' end of the linking oligo. The sequence and length of the former at the 5' end are determined by the NGS used, so they can be set according to the sequence recommended by the NGS manufacturer and do not require special settings dependent on the method of the present invention. The latter sequence at the 3' end is preferably 4 to 30 bases long, preferably 6 to 20 bases long, and more preferably 8 to 12 bases long.

[0042] If it is desired to add different artificial nucleic acid sequences to both ends, this can be achieved by making the sequence of the linking oligo at one end different from the sequence of the linking oligo at the other end, and designing the sequence at the 5' end of the 3'LG oligo that binds to the linking oligo pair and the sequence at the 3' end of the 5'LG oligo so that they are complementary to the sequences.

[0043] Furthermore, it is desirable to set the dissociation temperatures of the 5'-terminal sequence of the 3'LG oligo to be linked to the linking oligo and the 3'-terminal sequence of the 5'LG oligo, using the optimal temperature of each ligase as a guide. Specifically, the dissociation temperature is preferably ±15°C, preferably ±10°C, and more preferably ±5°C, relative to the ligase reaction temperature. It is also desirable to make the dissociation temperatures of the 3'LG oligo and the 5'LG oligo relative to the linking oligo as consistent as possible, so as to ensure that the 3'LG oligo and the 5'LG oligo are reliably linked to the linking oligo at the ligase reaction temperature.

[0044] The DNA polymerase used in the method including the reaction steps (1a) and (2a) may be any type of polymerase that can be used for gene amplification. However, if the DNA polymerase is present in an activated state during the oligo-DNA ligation step, the ligation reaction of the oligo-DNAs may be inhibited. In particular, during the reaction step (1a) (ligase-mediated ligation reaction), if the DNA polymerase is present in an activated state when the 5'LG oligo is ligated 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 ligating to the ligation oligo. As a result, ligation of the 3'LG oligo and the 5'LG oligo by the ligase may be inhibited. For these reasons, in the present invention, it is desirable to use a hot-start DNA polymerase that is inactive in the temperature range in which the oligo-DNA ligation in the reaction step (1a) is carried out.

[0045] Furthermore, when the target product obtained by the present invention is subjected to NGS analysis, it is desirable that the product contain as few base incorporation errors resulting from gene amplification as possible. Therefore, it is desirable to use a polymerase having proofreading activity (3'→5' exonuclease activity) that can suppress base incorporation errors.

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

[0047] In the reaction step (2a), an effective method for suppressing the amplification of non-target products that occur as a result of the 3'LG oligo that was unable to ligate to the 5'LG oligo functioning as a primer is to raise the reaction temperature immediately after the initial synthesis of the target product derived from the ligated primer to a temperature that does not interfere with the annealing of the ligated primer but that suppresses the annealing of the unligated oligo (3'LG oligo).By carrying out this step, it is possible to suppress the amplification of non-target products and allow the amplification of the target product to predominate.

[0048] For example, when PCR, the most common gene amplification method, is used as the gene amplification method, the annealing temperature in any cycle following the second cycle (cycle 3) in which the target product derived from the linked primer is first synthesized, can be raised to a temperature that does not interfere with annealing of the linked primer to the target product but suppresses annealing of the unlinked oligo (3'LG oligo), thereby suppressing amplification of non-target products and allowing amplification of the target product to dominate. The cycle in which the annealing temperature is raised is preferably cycles 3 to 11, preferably cycles 3 to 5, and more preferably cycle 3. Assuming that the amplification efficiency is close to 100%, since the amplification product derived from the linked primer is synthesized in equimolar amounts with the initially added template DNA in cycle 2, gene amplification proceeds well even if the annealing temperature is raised to a temperature at which only the linked primer functions as a primer from cycle 3 onwards and the 3'LG oligo has difficulty functioning as a primer. On the other hand, if the amount of product derived from the linked primer at the second cycle is low due to factors such as a low initial template concentration or low efficiency of binding of the target gene-specific sequence in the primer to the initial template, and raising the annealing temperature from the third cycle onwards does not result in a sufficient amount of target product, 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 can ensure a sufficient amount of product derived from the linked primer in the initial cycle with a low annealing temperature, and subsequently increasing the annealing temperature can ensure the final required amount of target product. The annealing temperature after temperature increase can 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 extension activity of the DNA polymerase does not decrease to the point where gene amplification does not proceed normally, using the proportion of target product in the specific product targeted by each practitioner (e.g., 95% of the target product in the specific product) as an indicator. Specifically, the annealing temperature is preferably 60 to 90°C, more preferably 65 to 85°C, and even more preferably 68 to 80°C.When PCR is used as the gene amplification method, if the annealing temperature after temperature increase set as described above is close to the optimal extension temperature of the DNA polymerase, two-step PCR in which annealing and extension are performed in the same step may be performed. Note that, as with ordinary gene amplification, the annealing temperature in the initial cycle of gene amplification in the present invention is a value that should be determined from the dissociation temperature of the sequence region specific to the target gene, and is generally set to a temperature 2 to 5°C lower than the dissociation temperature, but in the present invention, the annealing temperature may also be set according to a commonly known method.

[0049] On the other hand, when the gene amplification method is an isothermal gene amplification method such as the SIBA method, the SDA method, or the NEAR method, the reaction temperature is constant. Therefore, by raising the reaction temperature after the time required for the target product to be first obtained has elapsed, it is possible to suppress the amplification of non-target products as described above and to make the amplification of the target product dominant.

[0050] Regarding the DNA ligase used in the method including the reaction steps (1a) and (2a), any type of ligase can be used as long as it has the activity of repairing nicks in double-stranded DNA. However, since ligase activity is not necessary during gene amplification reactions (except for the LCR method), it is desirable to use a non-thermostable ligase that is completely inactivated in the step. Specific examples of non-thermostable ligases that have the activity of repairing 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 that have the activity of repairing nicks in double-stranded DNA include Taq DNA ligase and 9 oExamples of suitable ligases include N DNA ligase. Since thermostable ligases maintain their activity even in gene amplification reactions, they may have some effect on the gene amplification reaction. However, if the dissociation temperature between the linked oligo and the unlinked oligo (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 a situation, the use of a thermostable ligase will not have any effect on the gene amplification reaction and can be used without any problems. On the other hand, since the LCR method uses a thermostable DNA ligase for gene amplification, when this amplification method is adopted, it is reasonable that all reactions can be carried out with a single ligase as long as the oligo ligation reaction in the reaction step (1a) proceeds with the same ligase as in the LCR method. Note that Taq DNA ligase and E. coli DNA ligase are NAD + Since some of the enzymes are dependent on ATP, and the others are ATP-dependent enzymes, it is advisable to add the aforementioned substances to the reaction solution.

[0051] The present invention further 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 of linking two types of oligo DNA pairs (ib) and (iib) below by a click chemistry reaction to synthesize a linked primer pair (linked primer (F) and linked primer (R) in Figure 6). (ib) A 3' CC oligo pair having a sequence specific to a gene to be analyzed and having the 5' end modified with a click chemistry functional group (3' CC oligo (F) and 3' CC oligo (R) in Figure 6). (iib) A 5' CC oligo pair having an artificial nucleic acid sequence such as a barcode sequence and having the 3' end 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 of carrying out gene amplification using the linked primer pair synthesized in the above step (1b). In a method comprising the above reaction steps (1b) and (2b), The CC oligo pair has a sequence specific to the gene to be analyzed, but the sequence may be set to a number of sequences generally preferred for gene amplification primers, and no special considerations specific to the method of the present invention are required. Furthermore, to enable linkage between the 3' CC oligo and 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 between the 3' end of the 5' CC oligo and the functional group modified at the 3' end of the 5' CC oligo.

[0052] The 5' CC oligo pair, including the barcode sequence for identifying the sample and the artificial nucleic acid sequence, such as the adapter for binding to the oligo DNA present on the NGS flow cell, is specified for each NGS device, so it can be set according to the sequence recommended by the NGS manufacturer, and no special considerations are required depending on the method of the present invention. Furthermore, as mentioned above, to enable linkage with a 3' CC oligo via a click chemistry reaction, the 3' end of the oligo is modified with a functional group that generates click chemistry between the 5' end of the 3' CC oligo and the functional group modified at the 5' end of the 3' CC oligo. Typical functional groups used in click chemistry include azide groups and alkyne groups, but these reactions require copper ions as catalysts, and these ions can cause DNA damage and scission, making them unsuitable for the click chemistry reactions used in the present invention. Meanwhile, dibenzocyclooctyne (DBCO), azadibenzocyclooctyne (ADIBO), bicyclononyne (BCN), 4,8-diazacyclononyne (DACN), and the like are known as functional groups that undergo click chemistry reactions with azide groups 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).

[0053] In the reaction step (2b), an effective method for suppressing the amplification of non-target products that occur as a result of the 3' CC oligo that was unable to ligate to the 5' CC oligo functioning as a primer is to raise the reaction temperature immediately after the initial synthesis of the target product derived from the ligated primer to a temperature that does not interfere with the annealing of the ligated primer and that can suppress the annealing of the unligated oligo (the 3' CC oligo in the reaction step (2b)), as in the reaction step (2a).By carrying out this step, it is possible to suppress the amplification of non-target products and allow the amplification of the target product to predominate.

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

[0055] Furthermore, in the above-described reaction steps (1a), (2a), and reaction steps (1b), (2b), by using oligo pairs containing target gene-specific sequences in the same number 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 types of target products can be obtained in a single reaction.

[0056] The above-mentioned reaction steps (1a), (2a), and reaction steps (1b3), (2b4) can all be carried out continuously in one reaction without opening the reaction tube.

[0057] The gene amplification methods in the reaction steps (2a) and (2b) include PCR (Polymerase Chain Reaction), LCR (Liagase Chain Reaction), RPA (Recombinase Polymerase Amplification), SIBA (Strand-Invasion Based Amplification), LAMP (Loop-Mediated Iso-thermal Amplification), SDa (Strand Displacement Amplification), and NEAR (Nicking Endonuclease). Amplification can be performed by the Amplification Reaction method, the Helicase-dependent amplification (HDA) method, the Nucleic Acid Sequence-Based Amplification (NASBA) method, the Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids (ICAN) method, the Rolling Cycle method, the Smart Amplification Process (SMAP) method, or the like.

[0058] The present invention also provides a kit for preparing a target product, which contains at least one pair of each of the following three types of oligo DNA pairs (ia) to (iiia). The method for preparing the target product has been described above (FIG. 5). (ia) A linking oligo pair characterized by having a complementary sequence to the linking sequence (linking oligo (F) and linking oligo (R) in Figure 5). (iia) A 3'LG oligo pair having a part of the 3'-end sequence of the linking sequence at its 5'-end, a sequence specific to the gene to be analyzed at its 3'-end, and phosphorylated at its 5'-end (3'LG oligo (F) and 3'LG oligo (R) in Figure 5). (iiia) A 5'LG oligo pair characterized by having an artificial nucleic acid sequence such as a barcode sequence at its 5'-end, and having a part of the 5'-end sequence of the linking sequence at its 3'-end (5'LG oligo (F) and 5'LG oligo (R) in Figure 5). The linking oligo (ia) in the kit of the present invention may be modified at its 3'-end to prevent DNA elongation.

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

[0060] The requirements for the primers, oligo DNA, DNA polymerase, DNA ligase, etc. that constitute the kit of the present invention are as described above.

[0061] The present invention also provides a kit for preparing a target product, which includes at least one pair of each of the following two types of oligo-DNA pairs (ib) and (iib). The method for preparing the target product is described above (FIG. 6). (ib) A 3' CC oligo pair having a sequence specific to a gene to be analyzed and having its 5' end modified with a click chemistry functional group (3' CC oligo (F) and 3' CC oligo (R) in FIG. 6). (iib) A 5' CC oligo pair having an artificial nucleic acid sequence such as a barcode sequence and having its 3' end 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 FIG. 6). The kit of the present invention may further include a DNA polymerase.

[0062] The elements constituting the kit of the present invention, such as oligo DNA and DNA polymerase, are as described above.

[0063] The two types of kits of the present invention described above may also contain other components such as a reaction vessel, a reaction buffer, an instruction manual, and a target nucleic acid for a positive control test.

[0064] An embodiment of the present invention that aims to simplify and speed up the process will be described in detail below.

[0065] In the present invention, shortening the gene amplification reaction time 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 three-step PCR to a two-step PCR during the gene amplification (PCR) reaction, or (3) increasing the annealing ( / extension) temperature in the two-step PCR described in (2).

[0066] Regarding the high-speed PCR enzyme described as (1), the use of this enzyme can shorten the time required for extension, and therefore it is possible to shorten the gene amplification reaction time.

[0067] Examples of high-speed PCR enzymes that can be used in the present invention include repliQa HiFi ToughMix (manufactured by Quantabio) and KOD ONE PCR Master Mix (manufactured by Toyobo Co., Ltd.). However, any high-speed PCR enzyme that can be suitably applied to the present invention will suffice, and the scope of application of the present invention is not limited by the type of enzyme.

[0068] Regarding the change from 3-step PCR to 2-step PCR described as (2), the time required to transition to the omitted step and the temperature holding time in that step are reduced, thereby making it possible to shorten the gene amplification reaction time.

[0069] Regarding the increase in the annealing ( / extension) temperature described as (3), this change narrows the temperature change range (the difference between the dissociation temperature and the annealing temperature), which in turn reduces the time required for the temperature change, thereby shortening the gene amplification reaction time.

[0070] An embodiment of the present invention for simplifying and speeding up the purification of the target product obtained and the quantification of DNA concentration will be described in detail below.

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

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

[0073] The above-mentioned problem can be solved by adding a necessary and sufficient amount of a fluorescent dye (hereinafter referred to as a low-inhibitory dsDNA dye) that emits fluorescence by specifically binding to double-stranded DNA and that is characterized by low PCR inhibition to the gene amplification reaction solution, measuring the fluorescence of the reaction tube directly after the reaction is completed, 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). However, any low-inhibitory dsDNA dye that is suitably applicable to the present invention may be used, and the scope of application of the present invention is not limited by the type of dye.

[0075] Since the above-mentioned low inhibitory dsDNA dye has little PCR inhibition, it is possible to amplify the target product even if the substance is added to the reaction solution at a high concentration. As a result, even if the concentration of the target product increases, it is possible to maintain the amount of dye bound per molecule of the target product at a saturation amount. Therefore, even in the latter half of the amplification reaction when the target product concentration increases, there is a positive correlation between the concentration of the target product and the fluorescence intensity. As described above, by adding a high concentration of 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 completed, the library can be quantified simply and quickly.

[0076] On the other hand, since non-specific products such as primer dimers are frequently amplified during the gene amplification reaction, in order to accurately quantify the concentration of the target product using a low-inhibitory dsDNA dye, it is necessary to clearly distinguish and detect the fluorescence derived from the target product and the fluorescence derived from the non-specific products.

[0077] The length of the target product is often longer than that of the non-specific products, and therefore the dissociation temperature of the target product is generally higher than that of the non-specific products. By utilizing this property and detecting the fluorescence from the low-inhibitory dsDNA dye at a temperature where the non-specific products dissociate but the target product exists as double-stranded DNA, the concentration of the target product can be accurately quantified.

[0078] By using a real-time PCR device to perform gene amplification according to the present invention, it is possible to simultaneously measure fluorescence at any timing and temperature, and therefore, upon completion of amplification according to the present invention, quantification of the target product can be completed from the fluorescence intensity value obtained above. In this way, by using a real-time PCR device, it is possible to quantitate the target product more quickly and easily than by separately measuring fluorescence after gene amplification, and therefore it can be said to be more suitable as a gene amplification device for use in the present invention.

[0079] When using the real-time PCR device described above for gene amplification according to the present invention, it is necessary to use a different reaction tube for each sample, and fluorescence must be measured from the outside of each reaction tube. Therefore, even if reaction solutions with the same composition are measured, differences in the optical properties between the reaction tubes will result in differences in the obtained fluorescence values. Therefore, in order to accurately quantify the target product, it is desirable to correct for differences in the optical properties of the reaction tubes. Below, we describe an embodiment for correcting for differences in the optical properties of the reaction tubes.

[0080] The temperature of the denaturation step in the gene amplification process is set to the temperature at which all double-stranded DNA contained in the reaction tube dissociates, so theoretically, the fluorescence intensity (fluorescence intensity from free low-inhibitory dsDNA dye not bound to dsDNA) is constant regardless of the degree of gene amplification. This characteristic was used to correct 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 denaturation step of the same cycle, it became possible to roughly correct for differences in optical properties between reaction tubes. The specific calculation formula is shown below. Dissociation-corrected fluorescence intensity value = F n，ＴＴ / F n，ＤＮ F ｎ，ＴＴ　　　　 F: Fluorescence intensity measured at the temperature at which only the target product exists as dsDNA (TT; abbreviation for Temperature at which only the targeted product exists as dsDNA) in any cycle (n) ｎ，ＤＮ　　　　 : Fluorescence intensity measured at the temperature at which all DNA dissociates in any cycle (n) (DN; abbreviation for DeNature)

[0081] Theoretically, the fluorescence intensity value corrected by the fluorescence intensity of the dissociation step described above (hereinafter referred to as the dissociation-corrected fluorescence intensity value) should be the same regardless of the reaction tube in the cycle before an increase in fluorescence due to gene amplification is confirmed, but in reality, slight differences in the dissociation-corrected fluorescence intensity value in that cycle were confirmed between reaction tubes. For this reason, the dissociation-corrected fluorescence intensity value for each cycle was divided by the dissociation-corrected fluorescence intensity value for an arbitrary cycle in which no increase in fluorescence intensity due to gene amplification was observed in any reaction tube, thereby making the dissociation-corrected fluorescence intensity values ​​for all reaction tubes uniform at any cycle.

[0082] The specific calculation formula for obtaining the above-mentioned final corrected fluorescence intensity value is shown below: Final corrected fluorescence intensity value = (F ｆｃ，ＴＴ / F ｆｃ，ＤＮ ) / (F ＣＢＦＩ，ＴＴ / F ＣＢＦＩ，DN ) F ｆｃ，ＴＴ　　　　 F: Fluorescence intensity measured at the temperature at which only the target product exists as dsDNA in the final cycle (fc) (TT; Temperature at which only the target product exists as dsDNA) ｆｃ，ＤＮ　　　　 : Actual fluorescence intensity value at the dissociation step (DN) in the final cycle F ＣＢＦＩ，ＴＴ　　　 F: Measured fluorescence intensity at the temperature at which only the target product exists as dsDNA in the cycle before the increase in fluorescence associated with gene amplification is confirmed (CBFI; Cycle Before Fluorescence Increases) ＣＢＦＩ，DN : Fluorescence intensity measured at the dissociation step in the cycle before the increase in fluorescence associated with gene amplification was confirmed

[0083] The quantitative value of dsDNA obtained by this calculation method based on the corrected fluorescence intensity (hereinafter referred to as the final corrected fluorescence intensity value) was more accurate than the quantitative value of dsDNA quantified based on uncorrected fluorescence intensity (see Example 9 below). Therefore, it can be said that the above calculation method is suitable as an embodiment for correcting differences in the optical properties of reaction tubes.

[0084] The above-described embodiments (1) enable rapid and simple library quantification by simply measuring the fluorescence of the reaction tube directly. (2) Library purification, which previously required separate tube-by-tube purification to accurately quantify DNA concentration, can now be performed on the mixture itself, significantly reducing the labor required for library purification and leading to overall process time and cost savings (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 of the above methods. The present invention also provides a kit for performing NGS analysis, including a dsDNA-specific dye, which prepares a target product mixture for NGS analysis using the above method. The present invention also provides a method for preparing a mixture containing two or more target products with different barcode sequences, which includes adding a dsDNA-specific dye to each gene amplification reaction solution containing the target products, quantifying the target product concentration based on the fluorescence value of the dye, and mixing the gene amplification reaction solutions to achieve a desired concentration ratio for each target product based on the quantification results (Figure 9).

[0085] When performing NGS analysis of the target product obtained according to the present invention using a sequencer manufactured by Nanopore, it is necessary to add adapter sequences provided by the company to both ends of the target product to be analyzed. Various kits for adding such adapter sequences are commercially available from Nanopore, but the following describes an embodiment for simplifying the protocol using the Nanopore Ligation Sequencing Kit, which is characterized by adding the adapter sequence using ligase.

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

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

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

[0089] A-attachment to the 3' end can be achieved 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 3'-end A-attachment activity. Simply adding this reagent directly to the unpurified reaction solution after amplification of the target product allows A-attachment to the 3' end of the target product. While the use of KOD is required, if the expected effects are confirmed, using this reagent can significantly simplify the 3'-end A-attachment process (two steps: reagent addition and incubation) and reduce the time required. Furthermore, the reagent cost per use is one-fifth (600 yen) of the manufacturer's recommended method, allowing for significant cost savings. The present invention also provides a method for modifying a target product (FIG. 11), which comprises targeting a target product amplified using a primer phosphorylated at the 5' end in the above-described method for preparing a target product, adding a single A base to the 3' end of the product, and then adding an adapter sequence to the product using a ligase. The present invention also provides a method for modifying a target product, which targets a mixture of target products prepared by the above-described method or kit, and adds an adapter sequence to the product using a ligase.

[0090] The above method (Figure 11) makes it possible to process both ends of the target product obtained by the present invention, and if NGS analysis of the end-processed target product is possible, it will be possible to reduce the time and reagent costs required for the process by approximately 80% (time: approximately 50 minutes, reagent costs: reduced by 2,500 yen per run).

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

[0092] <Effect of Ligase Reaction Composition on Gene Amplification (PCR)> It is anticipated that the ligase required for the ligase reaction and other reaction components may adversely affect PCR, a widely used gene amplification method. In this example, this effect was investigated.

[0093] Specifically, the oligo DNAs shown in Table 2 were used, and a ligase reaction and PCR were carried out continuously in one reaction tube without opening the tube, and the above-mentioned effects were evaluated from the quantitative values ​​of the specific products obtained. All of the oligo DNAs used in the examples described herein were synthesized by Nippon Gene Research Institute, Ltd.

[0094] The target gene in this example was the 16S rRNA gene derived from eubacteria, and the template used was DNA-Mock (manufactured by NBRC (National Institute of Technology and Evaluation, Biotechnology Center)), which contains a mixture of 15 types of eubacterial genomes at known concentrations.

[0095] Table 3 shows the reaction conditions.

[0096] The reaction solution composition for each condition is shown in Table 4. The gray cells indicate the areas where the settings differ between conditions.

[0097] The reaction solution in Table 4 was subjected to a reaction under the temperature conditions in Table 3 using a PCR amplification device (Life Touch (manufactured by Nippon Genetics Co., Ltd.)).

[0098] After the reaction was completed, the reaction solution was diluted 10,000 times with TE buffer. Subsequently, the diluted reaction solution was used as a sample to quantify specific products using real-time qPCR targeting the gene-specific sequence. The gene-specific sequence was the same as the gene-specific sequence used in the practice of the present invention and listed in Table 2. The real-time PCR device used was Roter-Gene Q (Qiagen).

[0099] The reaction solution composition (including the sequences of the primers used) and the qPCR conditions are shown in Tables 5 and 6. The real-time PCR device used was Roter-Gene Q (Qiagen).

[0100]

[0101] The results are shown in Figure 12. Quantitation was carried out twice for each condition, and the values ​​in Figure 12 represent the average values.

[0102] As can be seen from Figure 12, no specific products were detected when using Ampligase Thermostable DNA Ligase, a heat-resistant ligase, or Hi-T4 DNA Ligase, a non-heat-resistant ligase, under the conditions of the manufacturer's recommended buffer concentration (x1) (conditions [1] and [5]).

[0103] Furthermore, for both ligases, when the buffer concentration was one-third of the manufacturer's recommended concentration (conditions [2] and [6]), the quantitative values ​​were significantly lower than those under other conditions in which specific products were detected (conditions [3], [4], [7], [8]).

[0104] These results demonstrate that the ligase reaction buffer contains substances that inhibit PCR, both for thermostable and non-thermostable ligases, and that specific products cannot be obtained when added at the manufacturer's recommended concentration. However, specific products were confirmed when the reaction buffer was added at one-third the manufacturer's recommended concentration (conditions [2] and [6]), and good amplification of specific products was confirmed when the reaction buffer was added at one-quarter or less the manufacturer's recommended concentration (conditions [3], [4], [7], [8]).

[0105] Based on the above results, in the following Examples 3 and onward, the reaction buffer for ligase was added at 1 / 4 the concentration recommended by the manufacturer. + Hi-T4 DNA Ligase is an ATP-dependent enzyme, and Hi-T4 DNA Ligase, a non-thermostable ligase, is an ATP-dependent enzyme. Although the buffer compositions differ, as mentioned above, they showed similar trends in PCR inhibition.

[0106] <Confirmation of Oligo DNA Ligation by Electrophoresis (PAGE) Based on Ligase Reaction> To confirm the ligation of oligo DNAs by ligase, the oligo DNAs shown in Table 2 of Example 1 were used, and the ligase reaction was performed without PCR using the reaction composition shown in Table 4. The resulting reaction mixture was then subjected to electrophoresis (PAGE analysis). Table 7 shows the reaction conditions for Example 2. Because the thermostable ligase was not inactivated under these conditions (conditions [1] to [4]), all eight reactions were immediately frozen after completion and stored frozen until electrophoresis. The reactions were performed using a PCR amplifier (Life Touch, manufactured by Nippon Genetics Co., Ltd.), as in Example 1. <Confirmation of Oligo DNA Ligation by Electrophoresis (PAGE) Based on Ligase Reaction> To confirm the ligation of oligo DNAs by ligase, the oligo DNAs shown in Table 2 of Example 1 were used, and the ligase reaction was performed without PCR using the reaction composition shown in Table 4. The resulting reaction mixture was then subjected to electrophoresis (PAGE analysis). Table 7 shows the reaction conditions for Example 2. Because the thermostable ligase was not inactivated under these conditions (conditions [1] to [4]), all eight samples were immediately frozen after the reaction was completed and stored frozen until electrophoresis. The reaction was carried out using a PCR amplification device (Life Touch (Nippon Genetics)) as in Example 1.

[0107] The results are shown in FIG.

[0108] Under all conditions, only the ligation primer (81 bases long) formed by ligating the 3'LG oligo and the 5'LG oligo, and the ligation oligo (22 bases long) that served as a scaffold for ligation and remained unchanged, were observed. Successful ligation of the oligos by the ligase was confirmed under all conditions. These results confirmed that the ligase reaction proceeded well even under conditions [1] and [5] where no specific product was observed.

[0109] <Confirmation of the Composition Ratio of the Target Product (Ligase Reaction System)> In this example, the composition ratio of the target product to the specific product in the present invention (ligase reaction system) was confirmed using NGS manufactured by Nanopore.

[0110] First, using the oligo DNA shown in Table 2 of Example 1, a ligase reaction and a PCR reaction were carried out consecutively without opening the reaction tube, using the reaction composition shown in Table 8 and the reaction conditions shown in Table 9. The difference between reaction conditions A and B shown in Table 8 is that while reaction condition B does not change the temperature during the reaction, reaction condition A is a three-step PCR, similar to reaction condition B, for only the first two cycles of PCR, and subsequent cycles are two-step PCR, in which annealing and extension are performed in the same step (72°C). Under reaction condition A, the temperature is raised to the annealing temperature during the gene amplification reaction, at which the unlinked 3'LG oligo, which has a low dissociation temperature, does not function as a primer. This suppresses the amplification of non-target products, while preferentially allowing the ligated primer, which has a high dissociation temperature, to function, thereby improving the proportion of the target product. The target gene in this example was the 16S rRNA gene derived from eubacteria, as in Example 1, and DNA-Mock (manufactured by NBRC) was used as the template, as in Example 1.

[0111] The resulting reaction product was purified using magnetic beads for DNA purification (AMPure XP, manufactured by Beckman Coulter) according to the manufacturer's protocol. The purified product was then quantified using Quant-iT PicoGreen dsDNA Assay Kits (manufactured by Invitrogen) according to the kit's protocol, and measurements were performed using a fluorescent plate reader (DTX800, manufactured by Beckman Coulter).

[0112] Next, the purified product was treated using a Nanopore library preparation kit (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 double-stranded DNA (adapters) containing sequences and molecules (motor proteins) required for analysis to both ends of the target product using ligase.

[0113] The final product obtained above was quantified using the same Quant-iT PicoGreen dsDNA Assay Kits as described above, followed by sequencing analysis of the product using a Nanopore NGS (MinION Mk1B). The flow cell used for the NGS analysis was a Nanopore Flongle, with one flow cell used per condition. This is because non-target products do not have barcode sequences, and therefore, when multiple samples are simultaneously analyzed using a single flow cell, it is not possible to identify which sample condition the non-target product originated from, making it impossible to determine the composition ratio of the target product for each condition.

[0114] Next, base calling was performed using MinKNOW, software provided by Nanopore (base calling is the process of converting raw sequence data obtained from a flow cell into a genetic sequence. This process was performed in the most accurate SUP mode), and demultiplexing (the process of separating sequence information for each sample using a barcode sequence as a marker) was performed.The obtained sequence data was then converted into base sequence information, and bacteria were identified based on the genetic sequence information using EPI2ME, a bioinformatics platform provided by Nanopore.

[0115] The composition ratio of each product under each condition is shown in Table 10. Gene sequence information was obtained but was not recognized as a 16S rRNA gene by EPI2ME, which was determined to be genetic information derived from a non-specific product. Next, genetic information recognized as a 16S rRNA gene by EPI2ME was determined to be genetic information derived from a specific product, and among these, sequence information in which a barcode sequence was recognized by MinKNOW and multiplexed was determined to be genetic sequence information derived from the target product, and information to which a barcode sequence was not added was determined to be genetic sequence information derived from a non-target product.

[0116]

[0117] The results are shown in Tables 10 and 11, Figures 14 and 15. Table 10 shows the composition ratios of target products, non-target products, and non-specific products under each condition. The only difference between conditions [1] and [3], and between conditions [2] and [4], is the type of ligase used. However, the composition ratios of each product were similar, confirming that the impact of differences in ligase on the composition ratios 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] is the reaction temperature conditions. Under conditions [1] and [3], only the first two cycles of three-step PCR are performed in the PCR process, followed by two-step PCR (reaction condition A in Table 9). Under conditions [2] and [4], the PCR is completed under the same conditions as the three-step PCR under conditions [1] and [3] without changing the temperature conditions (reaction condition B in Table 9). Due to the difference in reaction temperature conditions, the composition ratio of the target product was significantly higher under conditions [1] and [3] (reaction condition A) than under conditions [2] and [4] (reaction condition B). From these results, it was determined that, as described above, by increasing the annealing temperature during PCR (reaction condition A), the unlinked 3'LG oligo with a low dissociation temperature did not function as a primer, thereby suppressing the amplification of non-target products and non-specific products, while allowing the linked primer with a high dissociation temperature to function preferentially, thereby improving the proportion of the target product.

[0119] Table 11 shows the theoretical composition ratio of the 16S rRNA genes derived from 15 types of bacteria contained in DNA-Mock and the actually measured composition ratio under each condition.

[0120] Next, a graph showing the relationship between the theoretical composition ratio and the actually measured composition ratio based on the numerical data in Table 11 is shown in Figure 14. The linearity in Figure 14 is an approximate function, and the R 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 there was a certain positive correlation between the theoretical composition ratio and the measured composition ratio, and that this correlation was almost equivalent regardless of the conditions. From the above results, it was confirmed that even under conditions in which the annealing temperature was changed during PCR (reaction condition A), NGS analysis results similar to those obtained under conditions in which the annealing temperature was not changed as usual (reaction condition B) were obtained. Therefore, it was assumed that the difference in the above reaction conditions had little impact on the composition ratio of the target gene.

[0121] Figure 15 shows the correlation between the actual measured composition ratio of the target product and the actual measured composition ratio of the non-target product. As in Figure 14, the linear function is an approximation function, and the R 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 that there is an extremely high positive correlation between the measured composition ratios of the target product and non-target products, and that this correlation is nearly constant regardless of the conditions. These results suggest that the presence or absence of a barcode sequence has very little effect on the results of NGS analysis.

[0122] <Simultaneous Analysis of Multiple Gene Items (Ligase Reaction System)> In this example, multiple target nucleic acids were targeted, and oligos were ligated using ligase, and the target products were amplified using the ligated oligos continuously without opening the reaction tube. NGS analysis was then performed on the resulting target products to investigate the feasibility of simultaneous analysis of multiple genes according to the present invention. The oligo DNAs used in this example are shown in Table 12. The target genes in this example were five human genes, and five pairs of 3'LG oligos corresponding to each target gene were prepared.

[0123] The target product was prepared five times, and the target products obtained from the five reactions were analyzed in a single NGS analysis. Therefore, five 5'LG oligos with different barcode sequences were prepared for each reaction in order to separate the gene sequence information, and a different 5'LG oligo was used for each reaction. The linking 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, one 5'LG oligo and one linking oligo were used per reaction.

[0124] In this example, the target gene was a human gene, and therefore, commercially available human genomic DNA (manufactured by Roche) was used as the template to be added, and 100 ng of this was added per reaction tube.

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

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

[0127] Subsequently, as in Example 3, the purified product was treated using a library preparation kit (Ligation Sequencing Kit [SQK-LSK110]) manufactured by Nanopore, Inc., according to the kit's protocol.

[0128] The final product obtained above was quantified by a fluorometric method using the same Quant-iT PicoGreen dsDNA Assay Kits as above, and then sequence analysis was performed using an NGS (MinION Mk1B) manufactured by Nanopore, Inc. The flow cell used for the NGS analysis was a Flongle manufactured by Nanopore, Inc.

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

[0130]

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

[0132] As shown in Figure 16, gene sequence information was obtained for all five items analyzed. Furthermore, the standard deviation between reactions shown in Table 14 indicates that the standard deviation (%) of the number of reads due to differences in reactions was approximately 10% (6.5-14.1%), confirming that the variation in the number of 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] From the above, it was confirmed that by utilizing the present invention (a method for preparing a target product using ligase), it is possible to prepare target products for multiple genes in a closed reaction tube, and by performing NGS analysis of the products, it is possible to perform sequence analysis of multiple types of genes simultaneously.

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

[0136] From the above, it was confirmed that by utilizing the present invention (a method for preparing a target product using ligase), it is possible to simultaneously obtain information on the sequences of multiple genes present in multiple samples.

[0137] <Confirmation of oligo-DNA ligation based on click chemistry by electrophoresis (PAGE)> To confirm the ligation of oligo-DNA using click chemistry, the oligo-DNAs shown in Table 15 were used, and the reaction compositions shown in Table 16 were incubated at room temperature for 10 minutes. The resulting reaction mixture was then subjected to electrophoresis (PAGE analysis). The difference in reaction composition between each condition is the difference in the oligo-DNA added. The difference between conditions [1], [2] and [3], [4] is the presence or absence of the addition of a ligation oligo. The difference between conditions [1] and [2] and between conditions [3] and [4] is whether a forward oligo-DNA or a reverse oligo-DNA was used. The reaction volume was 20 μl.

[0138] The results of the above study are shown in FIG.

[0139]

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

[0141] On the other hand, in conditions [1] and [2], a linking oligo was added to act as a scaffold for bringing the 3'CC oligo and 5'CC oligo into close proximity with each other in the hopes of improving the reactivity of click chemistry. However, no significant difference was observed in the intensity of the bands for the linked primer resulting from the ligation of the 3'CC oligo and 5'CC oligo, the unlinked 5'CC oligo, and the unlinked 3'CC oligo compared to conditions [3] and [4] in which no linking oligo was added. From these results, the effect of adding a linking oligo could not be confirmed in this study.

[0142] <Confirmation of the Composition Ratio of the Target Product (Click Chemistry Reaction System)> In this example, the composition ratio of the target product to the specific product in 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 consecutively without opening the reaction tubes, using the reaction composition shown in Table 18 and the reaction conditions shown in Table 19.

[0144] Note that a linking oligo was not used in this example because the effect of the linking oligo could not be confirmed in Example 5. For this reason, the same 5' CC oligo was added to the 5' end of the 3' CC oligo on both the forward and reverse sides, and only one type of 5' CC oligo was used per reaction.

[0145] There are two conditions, and the composition of the reaction solution is the same regardless of the conditions, with only the reaction conditions differing between the two conditions.

[0146]

[0147]

[0148] The difference between reaction condition A (condition [1]) and reaction condition B (condition [2]) listed in Table 19 is that while reaction condition B does not change the temperature conditions during PCR, reaction condition A uses the same three-step PCR as reaction condition B for only the first two cycles of PCR, and subsequent cycles use two-step PCR in which annealing and extension are performed in the same step (72°C). Reaction condition A raises the annealing temperature midway, thereby suppressing the amplification of non-target products by setting temperature conditions such that unlinked 3'CC oligos with low dissociation temperatures do not function as primers, while preferentially allowing linked primers with high dissociation temperatures to function, thereby improving the proportion of the target product. Note that the target gene in this example, like Examples 1 and 3, is a 16S rRNA gene derived from eubacteria, and like Examples 1 and 3, DNA-Mock (manufactured by NBRC) was used as the template. Subsequent steps are the same as in Example 3 above.

[0149] The results are shown in Tables 20 and 21, and in FIGS.

[0150] First, 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] is the PCR temperature conditions. Under condition [1], only the first two cycles of three-step PCR are performed in the PCR process, followed by two-step PCR (reaction condition A in Table 19). Under condition [2], PCR is completed under the same temperature conditions as the three-step PCR under condition [1] without changing the PCR temperature (reaction condition B in Table 19). Due to the difference in reaction temperature conditions, the target product composition ratio was significantly improved under condition [1] (reaction condition A) compared to condition [2] (reaction condition B). These results confirmed that, as in Example 3, the target product composition ratio can be improved by increasing the annealing temperature during PCR (reaction condition A).

[0152] Table 21 shows the theoretical composition ratio of the 16S rRNA genes derived from 15 types of bacteria contained in DNA-Mock and the measured composition ratio under each condition. Next, based on the numerical data in Table 21, a graph showing the relationship between the theoretical composition ratio and the measured composition ratio under each condition is shown as Figure 18.

[0153] The lines in FIG. 18 represent approximate functions, and the R ２The values ​​were 0.64 under condition [1] and 0.68 under condition [2], and the slope of the function was 1.5 under condition [1] and 1.42 under condition [2], and no significant differences were observed. These results suggest that there is a positive correlation between the theoretical composition ratio and the measured composition ratio, and that this correlation is almost equivalent regardless of the conditions. From the above results, as in Example 3, even under conditions in which the annealing temperature was changed during PCR (reaction condition A), it was shown that NGS analysis results were similar to those obtained under conditions in which the annealing temperature was not changed as usual (reaction condition B), and it was determined that the differences in the above reaction conditions had no or negligible effect on the NGS analysis results.

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

[0155] As in FIG. 18, the linear function is an approximate function, and the R ２ The value was 0.97 under both conditions [1] and [2], and the slope of the function was 1.1 under condition [1] and 1.04 under condition [2], with almost no difference observed between the conditions. These results suggest that there is an extremely high positive correlation between the measured composition ratios of the target product and non-target products, and that this correlation is almost constant regardless of the conditions. From the above results, as in Example 3 above, it was confirmed that the presence or absence of a barcode sequence has an extremely small effect on the NGS analysis results.

[0156] <Simultaneous analysis of multiple gene items (click chemistry reaction system)> In this example, multiple target nucleic acids were targeted, and oligos were linked by click chemistry and the target products were amplified using the linked oligos in succession without opening the reaction tube. NGS analysis was then performed on the resulting target products, thereby examining the possibility of simultaneous analysis of multiple genes according to the present invention.

[0157] The oligo DNAs used in this example are shown in Table 22. The target genes in this example were the same five human genes as in Example 4, and five pairs of 3'CC oligo pairs corresponding to each target gene were prepared.

[0158] The target product preparation reaction was performed five times, and sequence information was obtained for the target products obtained from the five reactions in a single NGS analysis. Therefore, in order to separate the gene sequence information for each reaction, five 5'CC oligos with different barcode sequences were prepared, and a different 5'CC oligo was used for each reaction. Note that, since the effect of the linking oligo could not be confirmed in Example 5 above, no linking oligo was used in this example, as in Example 6. Therefore, the same 5'CC oligo was added to the 5' end of the 3'CC oligo on both the forward and reverse sides, so only one type of 5'CC oligo was used per reaction.

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

[0160] The target product was prepared using the reaction solution composition shown in Table 23 under the reaction temperature conditions shown as reaction condition A in Table 19. The subsequent steps were the same as those in Example 4.

[0161]

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

[0163] 20 shows that gene sequence information was obtained for all five items analyzed. Furthermore, the standard deviations between reactions shown in Table 24 indicate that the standard deviation (%) of the number of reads due to differences in reactions was 5.7 to 18.4%, confirming that the variation in the number of 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] From the above, it was confirmed that the method of the present invention (a method for preparing a target product using click chemistry) can be used to prepare target products for multiple genes by performing only one reaction, and that sequence information for multiple genes can be obtained all at once by analyzing the products by NGS.

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

[0167] From the above, it was confirmed that by utilizing the method of the present invention (a method for preparing a target product using click chemistry), it is possible to simultaneously obtain information on multiple gene sequences present in multiple samples.

[0168] <Study on Reducing Reaction Time in Gene Amplification Process> In this example, we investigated the method for reducing the gene amplification reaction time in the patent. Specifically, we attempted to reduce the reaction time by (1) using a high-speed PCR enzyme, (2) changing from a three-step PCR to a two-step PCR during the gene amplification (PCR) reaction, and (3) increasing the annealing ( / extension) temperature in the two-step PCR described in (2). It is preferable that the high-speed PCR enzyme used has an extension rate in gene amplification faster than 10 seconds / kb.

[0169] The target gene in this example is the 16S rRNA gene derived from eubacteria, as in Example 1, and the template is also DNA-Mock (manufactured by NBRC), as in Example 1, and 10 ５ The oligo DNA used in this example was the oligo DNA shown in Table 2 of Example 1.

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

[0171] Amplification of the target product was carried out using the reaction solution composition shown in Table 26 and under the reaction temperature conditions shown in Table 27.

[0172] Amplification of the target product was confirmed by electrophoresis (apparatus used: 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, KAPA2G Fast HotStart DNA Polymerase (manufactured by Kapa Biosystems) and PrimeSTAR GXL DNA Polymerase did not result in amplification under the high-speed PCR conditions with short extension times set in this example, but the target products were confirmed under all conditions for the other three enzymes.

[0174] The table also lists the actual reaction time under each condition, as well as the reduction in reaction time based on the reaction time (124 minutes) in a three-step PCR (Test No. 1) using TITANIUM Taq DNA polymerase, a PCR enzyme with an average extension rate. These results demonstrate that the reaction time can be reduced by performing only the first two cycles of three-step PCR and then changing to two-step PCR (Test Nos. 6-10). This is because, in two-step PCR, annealing and extension were performed at a high and identical temperature, thereby reducing the time required for temperature transition. Furthermore, by setting the annealing and extension temperatures at higher temperatures (Test Nos. 11-15), the reaction time could be further reduced compared to Test Nos. 6-10, which used lower temperatures.

[0175] The conditions under which amplification of the target product was confirmed and the highest reaction time reduction rate based on Test No. 1 were Test No. 15 using repliQa HiFi ToughMix (Quantabio), with a reaction time of 35 minutes and a reaction time reduction rate of 72%. Next was Test No. 14 using KOD ONE PCR Master Mix (Toyobo), with a reaction time of 36 minutes and a reaction time reduction rate of 71%.

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

[0177] Furthermore, repliQa HiFi ToughMix (Quantabio) and KOD ONE PCR Master Mix (Toyobo), which enabled a reduction in reaction time, are both polymerases with proofreading activity (3'->5' exonuclease activity) that can suppress base incorporation errors. Therefore, they have excellent properties as DNA polymerases when preparing target products for NGS analysis, and can be said to be suitable DNA polymerases for carrying out the method of the present invention.

[0178] <Simplifying and speeding up the target product quantification and purification process using low-inhibitory dsDNA dyes [1]> The following study was conducted to verify the effectiveness of simplifying and speeding up the target product quantification and purification process using the aforementioned inhibitory dsDNA dyes.

[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 the 16S rRNA gene derived from eubacteria, as in Example 1, and DNA-Mock (manufactured by NBRC) was used as the template, as in Example 1, and 10 16S rRNA gene equivalents were used per reaction tube. ５ Added to be 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 2x, which is twice the usual concentration (the product concentration is stated as 20x, and no clear concentration is indicated). The other reaction solution compositions were those using KOD ONE PCR Master Mix, as described under condition [4] in Table 26.

[0181] The reaction temperature conditions, except for the number of cycles, were based on the conditions of Test No. 4 listed in Table 27. The number of cycles was set to 25, 27, 32, and 40 cycles so that the concentration of the target product would change, and one reaction was performed for each cycle number 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 carried out using a Rotor-Gene Q 2plex (Qiagen) real-time PCR device, and fluorescence measurements were taken at 90°C and 98°C for each cycle. At 90°C, nonspecific products such as primer 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. Meanwhile, 98°C is measured during the dissociation step, in the absence of double-stranded DNA, and therefore, theoretically, the fluorescence intensity will be constant regardless of the degree of gene amplification.

[0184] The final corrected fluorescence intensity value was calculated using the above-mentioned formula for calculating the final corrected fluorescence intensity value, using the fluorescence intensity values ​​at 90°C and 98°C obtained as described above. More specifically, the fluorescence measurement value at 90°C in the final cycle was calculated by multiplying it by the actual fluorescence intensity measurement value (F in the formula) at a temperature where only the target product exists as dsDNA. ｆｃ，ＴＴ ), and the fluorescence measurement value at 98°C in the final cycle was used as the actual fluorescence intensity value in the dissociation step (F in the calculation formula). ｆｃ，ＤＮ The number of cycles before the increase in fluorescence due to gene amplification was confirmed was set to 10, and the fluorescence intensity value at 90°C in that cycle was designated as F ＣＢＦＩ，ＴＴ The fluorescence intensity value at 98°C is F ＣＢＦＩ，DN The above four values ​​were substituted into the above-mentioned calculation formula to determine the final corrected fluorescence intensity value.

[0185] Results: Amplification of the target product was confirmed by electrophoresis (apparatus used: Agilent 2100 Bioanalyzer Electrophoresis System (Agilent Technologies)), and no bands derived from non-specific products were observed in any of the reactions. From these results, it was determined that the quantification results of double-stranded DNA using PicoGreen were the result of measuring only the target product.

[0186] To confirm the effect of fluorescence correction, Fig. 21(a) shows the relationship between the quantitative value of double-stranded DNA after purification and the uncorrected fluorescence intensity in the final cycle in each reaction tube, and Fig. 21(b) shows the relationship between the quantitative value of double-stranded DNA after purification and the final corrected fluorescence intensity in the final cycle in each reaction tube. These results demonstrate a positive correlation between the concentration of double-stranded DNA after purification and fluorescence intensity, regardless of whether fluorescence correction was performed or not.

[0187] In addition, the correlation coefficient is higher in Figure 21(b), where fluorescence correction was performed, than in Figure 21(a), where no correction was performed, indicating that the amount of double-stranded DNA can be quantified more accurately by performing fluorescence correction using the above-mentioned method.

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

[0189] Method: Twelve activated sludge samples were prepared in the laboratory after acclimatization with artificial sewage, and DNA was extracted from the samples using the Extract Soil DNA Kit Plus ver. 2 (manufactured by BioDynamics) according to the manufacturer's protocol.

[0190] The target product was obtained by the patented method using the DNA obtained above as a template. The oligo DNAs used in this example were the linking oligo and the 3'LG oligo containing the gene-specific sequence shown in Table 2 of Example 1.

[0191] For the 5'LG oligos containing barcode sequences, those listed in Table 29 were used so that 12 samples could be analyzed simultaneously in a single NGS analysis, and a pair of forward and reverse 5'LG oligos with the same barcode sequence were used so that the barcode sequences of the target products would be the same on the forward and reverse sides.

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

[0193] The target product was quantified by calculating the final corrected fluorescence intensity value in the final cycle (40 cycles) using the method described in Example 9, and the target product was quantified from the calculated value using the relational equation in Figure 21(b) as a calibration curve.

[0194] Based on this calculated value, the unpurified reaction solutions of each sample were mixed in the test system so that the target products were equimolar, and this mixture was purified using Agencourt AMPure XP (manufactured by Beckman Coulter).

[0195] On the other hand, for the control system, equal amounts of the unpurified reaction solution were collected and mixed, and this mixture was used as the control and purified using Agencourt AMPure XP (manufactured by Beckman Coulter).

[0196] The double-stranded DNA concentration of the two mixtures obtained above was determined using Quant-iT PicoGreen dsDNA Assay Kits (manufactured by Invitrogen) according to the kit's protocol.

[0197] The two-type mixture of double-stranded DNAs obtained above was subjected to NGS analysis under the conditions described in Example 3, using an NGS system manufactured by Nanopore.

[0198] The two-type mixture of double-stranded DNAs obtained above was subjected to NGS analysis under the conditions described in Example 3, using Nanopore NGS.

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

[0200] Next, base calling was performed using MinKNOW, software provided by Nanopore (base calling is the process of converting raw sequence data obtained from a flow cell into a gene sequence. This process was performed in the most accurate SUP mode), and demultiplexing (the process of separating sequence information for each sample using a barcode sequence as a marker) was performed.The obtained sequence data was then converted into base sequence information, and 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 in which equal volumes of reaction solution were mixed, the variation in the number of reads per sample was large, with a standard deviation (%) of 26%. On the other hand, in the test system in which the dsDNA concentration in the reaction solution was measured using a low-inhibitory dsDNA dye, the variation in the number of reads per sample was kept low, with a standard deviation (%) of 13%, half that of the control system.

[0202] The results of this Example and Example 9 show that by adding a low-inhibitory dsDNA dye with little 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, and by mixing the reaction solution based on the quantitative value, it is possible to arbitrarily adjust the number of reads (number of gene sequences) per sample.

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

[0204] <Simplifying and reducing the cost of the end processing process using a Nanopore sequencer and the company's Ligation Sequencing Kit> When performing NGS analysis using a Nanopore sequencer, it is necessary to add adapter sequences provided by the company to both ends of the target product to be analyzed. Various kits for adding such adapter sequences are sold by Nanopore, but in this example, we examine how to simplify and reduce the cost of the protocol using the Nanopore Ligation Sequencing Kit, which is characterized by adding the adapter sequences using a ligase.

[0205] Specifically, we investigated whether it would be possible to simplify and reduce the cost of the protocol assuming the use of the Nanopore kit by using the aforementioned (1) 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 a preparation solution of the unpurified target product.

[0206] Method: The target gene in this example is the 16S rRNA gene derived from eubacteria, as in Example 1, and the template is also the same as in Example 1, DNA-Mock (manufactured by NBRC). ５ Added to be a copy.

[0207] The oligo DNAs used in this example were those shown in Table 2 of Example 1 for the ligation oligo and the 3'LG oligo containing a gene-specific sequence.

[0208] The 5'LG oligos used were those listed in Table 31. More specifically, in the test system using the A-attachment mix, 5'LG oligos with phosphorylated 5'-ends (oligos listed in Nos. 1 and 2 in Table 31) were used, and in the control system in which terminal modification was performed according to the manufacturer's recommended protocol, 5'LG oligos with non-phosphorylated 5'-ends (oligos listed in Nos. 3 and 4 in Table 31) were used.

[0209] Amplification of the target product was carried out under the conditions described in Example 9, with the number of cycles being 40 for both the test system and the control system.

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

[0211] Subsequently, the terminally modified target product was purified using Agencourt AMPure XP (manufactured by Beckman Coulter) according to the manufacturer's protocol to obtain a purified terminally modified target product.

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

[0213] The concentration of the double-stranded DNA in 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 above was subjected to end treatment using the NEB Next Ultra II End repair / dA-tailing Module reagents (manufactured by New England BioLabs) recommended by Nanopore, in accordance with the kit's protocol (see Table 1).

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

[0216] The adapter-attached product obtained above was quantified using the same Quant-iT PicoGreen dsDNA Assay Kits as above, using a fluorescent measurement method. The product was then subjected to sequence analysis using a Nanopore NGS (MinION Mk1B). The flow cell used for the NGS analysis was Nanopore's Flongle (version: R10.4.1). Since different barcode sequences were attached to the final products of the test and control systems, a solution containing equimolar amounts of each final product was prepared, and then NGS analysis was performed on this mixture. Gene sequence information for both products was simultaneously obtained in a single NGS analysis.

[0217] Next, base calling was performed using MinKNOW, software provided by Nanopore (base calling is the process of converting raw sequence data obtained from a flow cell into a genetic sequence. This process was performed in the most accurate SUP mode), and demultiplexing (the process of separating sequence information for each sample using a barcode sequence as a marker) was performed.The obtained sequence data was then converted into base sequence information, and bacteria were identified based on the genetic sequence information using EPI2ME, a bioinformatics platform provided by Nanopore.

[0218] Results: The above results are shown in Tables 32 and 33. From the results in Table 32, no significant differences were observed in the analysis results between the test system and the control system in the comparison items such as the number of reads obtained, the barcode identification rate indicating the proportion of products to which a barcode is linked, and the phylogenetic classification name identification rate indicating the proportion of products derived from the gene to be analyzed (16S rRNA gene). Similarly, from the results in Table 33, no significant differences were observed in the microbial composition ratio.

[0219] The above results prove the effectiveness of the terminal modification method for the target product described in this example, and show that the adoption of this method makes it possible to simplify, speed up, and reduce the cost of the protocol using a Nanopore sequencer and its Ligation Sequencing Kit.

[0220] The flow of the new process and the time-saving effect achieved by applying the details of Examples 8 to 11 are shown in Figure 22. Note that the above is based on the premise that 10 samples are processed simultaneously.

[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] Using the low-inhibitory dsDNA dye with low PCR inhibition described in Examples 9 and 10, it was shown that the DNA concentration of the target product can be easily and quickly quantified by directly measuring the fluorescence in the reaction tube after amplification of the target product without going through a DNA purification process. Furthermore, based on this quantitative value, target products from multiple samples can be mixed at the target concentration without going through a DNA purification process, and DNA purification can be performed on this mixture, eliminating the need to purify each product separately. As a result, the method described in Examples 9 and 10 can reduce the time required for purification by approximately 50 minutes (DNA quantification process: 20 minutes, purification process: 30 minutes) and significantly reduce the cost of purification (a 90% reduction when 10 samples are processed simultaneously).

[0223] The application of the terminal modification method described in Example 11 is limited to analysis using a Nanopore sequencer and a Nanopore Ligation Sequencing Kit, which require terminal modification of the target product for analysis. However, by adopting this method, it was possible to reduce the time by approximately 50 minutes (reduction rate: 80%). In addition, reagent costs were reduced by using NEB Next Ultra II End repair / dA-tailing Module reagents (manufactured by New England BioLabs) and A-attachment mix (manufactured by Toyobo Co., Ltd.).

[0224] The methods described in Examples 8 to 10 are applicable to the steps common to amplicon analysis using NGS (amplification, purification, and quantification of the target product), and are therefore recognized as effective methods when performing analysis using NGS other than that of Nanopore.

[0225] As described above, by applying the details of Examples 8 to 11 to the amplification of the target product according to the present invention and to the steps subsequent to the amplification step, it became possible to shorten the time by 190 minutes (approximately 3 hours) (shortening rate: 48%) compared to the existing steps.

[0226] The present invention can be used as a novel method for preparing a gene library.

[0227] <SEQ ID NOs: 1 to 72> Shows the base sequences of the oligo DNAs used in the examples.

Claims

1. 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 of synthesizing a linked primer pair (linked primer (F) and linked primer (R) in FIG. 5 ) by reacting the following three oligo DNA pairs (ia) to (iiia) in the presence of a ligase to link the following oligo DNA (iia) and oligo DNA (iiia) by a nick repair reaction using DNA ligase: (ia) A ligation oligo pair (ligation oligo (F) and ligation oligo (R) in Figure 5) characterized by having a complementary sequence to a sequence required for ligating oligo DNAs (hereinafter referred to as a ligation sequence) in a nick repair reaction using a ligase. (iia) A 3'LG oligo pair having a part of the 3'-terminal sequence of the linked sequence at the 5'-terminal side, a sequence specific to the gene to be analyzed at the 3'-terminal side, and phosphorylated at the 5'-terminal (3'LG oligo (F) and 3'LG oligo (R) in Figure 5). (iiia) A 5'LG oligo pair (5'LG oligo (F) and 5'LG oligo (R) in Figure 5) characterized in that it has an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a part of the 5' end sequence of the linking sequence at the 3' end. (2a) a step of carrying out gene amplification using the linked primer pair synthesized in the step (1) above.

2. A kit for preparing a target product by the method of claim 1, comprising at least one pair of each of the following three types of oligo DNA pairs (ia) to (iiia): (ia) A linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5) having a complementary sequence to the linking sequence. (iia) A 3'LG oligo pair having a part of the 3'-terminal sequence of the linked sequence at the 5'-terminal side, a sequence specific to the gene to be analyzed at the 3'-terminal side, and phosphorylated at the 5'-terminal (3'LG oligo (F) and 3'LG oligo (R) in Figure 5). (iiia) A 5'LG oligo pair (5'LG oligo (F) and 5'LG oligo (R) in Figure 5) characterized in that it has an artificial nucleic acid sequence such as a barcode sequence at the 5' end and a part of the 5' end sequence of the linking sequence at the 3' end.

3. 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 of synthesizing a linked primer pair (linked primer (F) and linked primer (R) in Figure 6) by linking two types of oligo DNA pairs (ib) and (iib) below by a click chemistry reaction: (ib) A pair of 3'CC oligos having a sequence specific to the gene to be analyzed and whose 5' ends are 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 modified at the 3' end 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 of carrying out gene amplification using the linked primer pair synthesized in the step (1b) above.

4. A kit for preparing a target product by the method of claim 3, comprising at least one pair of each of the following two types of oligo DNA pairs (ib) and (iib): (ib) A pair of 3'CC oligos having a sequence specific to the gene to be analyzed and whose 5' ends are 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 modified at the 3' end 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).

5. 4. The method according to claim 1, wherein all steps are carried out continuously in one reaction without opening the reaction tube.

6. The method described in claim 1 or 3, 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, and the PCR enzyme uses at least one DNA polymerase selected from the group consisting of repliQa HiFi ToughMix and KOD ONE PCR Master Mix.

7. A method for preparing a mixture containing two or more target products having different barcode sequences, the method 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 the gene amplification reaction solutions so that the target products are at an arbitrary concentration ratio based on the quantification results.

8. The method according to claim 7, wherein the purification is carried out on a mixture of target products.

9. The method according to claim 7, wherein the fluorescence of the dsDNA-specific dye is obtained during the gene amplification process.

10. The method according to claim 7, wherein gene amplification and acquisition of fluorescence from the dsDNA-specific dye are carried out using real-time PCR.

11. The method according to claim 7, wherein the fluorescence measurement is carried out at a temperature where only the target product exists as double-stranded DNA.

12. A method for modifying a target product, comprising: targeting a target product amplified using a primer whose 5' end is phosphorylated in the method described in claim 1; adding a single base A to the 3' end of the product; and then adding an adapter sequence to the product using a ligase.

13. The method according to claim 12, wherein a target product is amplified using a PCR enzyme having proofreading activity, and then a single base A is added to the 3' end of the target product by adding an inactivator of the PCR enzyme and a DNA polymerase having activity of adding A to the 3' end to the reaction solution.