Method of ligation-coupled PCR

Abstract

The present disclosure provides a method and kit for ligation-coupled PCR. A method is provided in which ligation and PCR, and optionally enzymatic digestion, are performed in a single closed tube. Methods and kits for splint-mediated primer assembly and ligation-coupled PCR are also provided. An important requirement for analysis by next-generation sequencing (NGS) is the preparation of an NGS library. During this process, the nucleic acid of interest can first be fragmented, or if starting from RNA, cDNA can be generated, and multiplex PCR specific to the locus of interest can be performed for many targeted sequencing workflows.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 025,738, filed on May 15, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application was electronically filed in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety. The ASCII copy created on 6 January 1 4 , 2021 is named 85563 - 337864_S L. txt and is 33,9 82 bytes in size.

Background Art

[0003] Background A key requirement for next-generation sequencing (NGS) analysis is the preparation of the NGS library. During this process, the nucleic acid of interest can be first fragmented, cDNA can be generated if starting with RNA, and multiplex PCR specific to the target locus can be performed for many targeted sequencing workflows. Next, the substrate ends are repaired and ligated to the NGS adapter to complete library synthesis. The NGS adapter enables library amplification, clonal amplification of library molecules on a flow cell, and annealing of sequencing primers. The NGS adapter also incorporates a sample-specific index to enable multiplex sequencing of the library, while allowing unique molecular barcoding for each library molecule by using a tag containing random nucleotides (six or more N bases) as needed. During the ligation step, it is possible to construct an NGS library using a full-length indexed adapter, or, in Illumina sequencing, to ligate a truncated adapter containing the nearest neighbor adapter to the substrate, and then incorporate a sample-specific index and further terminal adapter sequences during library amplification using a 5'-tailed indexing PCR primer that anneals to the truncated adapter sequence.

[0004] Traditional methods for preparing Illumina libraries from fragmented DNA, cDNA, or multiplexed PCR amplicons require three steps: 1) DNA polishing, end repair, and A-tailing of the substrate's 3' end; 2) ligation of a Y-shaped or stem-loop adapter containing a double-stranded region with a T nucleotide overhang at the 3' end and two different single-stranded adapter sequences to both substrate DNA strands; and 3) PCR amplification with a primer pair complementary to the two adapter sequences. This method is currently used in many commercially available kits such as the TruSeq Nano DNA Library Kit (Illumina), NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), and KAPA HyperPrep Kits (Roche). The use of DNA substrate A-tailing and T-overhang adapter ligation promotes a reduction in unwanted products such as chimeric library molecules containing two inserts and adapter dimers, and the use of an asymmetric Y-shaped or stem-loop adapter enables the connection of the adapter to both strands of the A-tailed substrate in a single-step reaction without loss of library complexity.

[0005] Certain other methods use blunt-end adapters and avoid adapter dimerization by using adapters that ligate only one DNA strand of the substrate, but they have no means to prevent chimeric library molecules and therefore limit the DNA input to 50 ng. For example, in the method used in kits used for AmpliSeq-targeted multiplex PCR library construction, such as SMARTer ThruPLEX DNA-Seq (Takara) and Ion Torrent DNA Library (ThermoFisher), two flat-end adapters ligate the 5' ends of the substrate DNA molecule, while the 3' end of the DNA substrate remains unligated and is then extended by DNA polymerase in the next step before PCR amplification. Because these kits use a mixture of two different double-stranded adapters, only 50% of a fully ligated DNA fragment contains the two different adapters required for sequencing, and 50% of the fully ligated DNA fragment is lost by ligating two similar adapters that cannot be sequenced, resulting in a DNA, cDNA, or targeted amplicon library with half the yield and library complexity.

[0006] Other targeted sequencing library preparation methods, such as CleanPlex (Paragon Genomics), utilize 5'-tailed target-specific primers, where each forward target-specific primer has a 5' tail containing a first truncated adapter sequence, and each reverse target-specific primer has a 5' tail containing a second truncated adapter, with the indexing primer pair containing a sample-specific index and terminal adapter sequence in the 5' tail, used to complete the first and second NGS adapter sequences. While this is a simple workflow, it can generate a high percentage of primer dimers during the multiplex PCR step, increasing sequencing costs if a further background cleansing step is not incorporated to remove primer dimers. The method also generates directional libraries where each library strand is simply sequenced from one direction. Due to systematic sequencing errors, it is desirable to perform paired end sequencing of each strand from both directions for the highest accuracy sequence data quality for variant calling. Similar to this method, the AmpliSeq method described above also requires the incorporation of several multiplexed PCR enzymatic steps, a further step to remove primer dimers before completion of the NGS library by terminal policing, adapter ligation, and a PCR amplification step as needed.

[0007] The ideal method for targeted sequencing is to generate a non-directional, high-complexity library from a minimum number of enzymatic steps, preventing primer dimer inclusion in the final library. In the previously disclosed targeted multiplexed PCR library preparations (described in U.S. Patent No. 10,316,359, which are incorporated herein by reference in their entirety) commercialized as Accel-Amplicon and Swift Amplicon panels, this workflow overcomes the library complexity limitations of double-stranded adapter ligation, generating a non-directional library and further preventing primer dimer accumulation without incorporating additional steps into the workflow. Unlike AmpliSeq, CleanPlex, and other multiplexed PCR targeted sequencing workflows that require separating primer pairs generating overlapping amplicons into two tubes, this method also enables continuous coverage using primer pairs that generate overlapping amplicons in a single-tube multiplexed PCR reaction. The Swift Amplicon method not only reduces sample tracking errors when processing a large number of samples by utilizing a single-tube workflow, but more importantly, it is suitable for samples such as liquid biopsies / cell-free DNA from blood and other fluids where the input material is limited and sensitivity is required to detect low-frequency variants. Single-tube assays are more sensitive and allow for a greater number of copies of input DNA molecules from limited samples compared to assays requiring individual tubes.

[0008] Following Swift multiplexing PCR, a purification step can be performed, and fully ligated molecules have 100% functionality and non-directionality. Adapter ligation can be performed on a purified multiplexing PCR amplicon similar to a Y-shaped adapter, but this method uses separate 5' and 3' adapters that independently ligate the 5' and 3' ends of each strand of the target-specific amplicon. While this is an efficient and simple workflow, the absence of further PCR amplification steps means that fluorescence analysis methods such as Qubit cannot distinguish between fully ligated and partially ligated functional molecules, resulting in high-precision quantification of the library only by qPCR. Furthermore, similar to the Y-shaped adapter, the 5' and 3' single-stranded adapter tails introduce electrophoresis artifacts on electrophoresis chips such as those used in bioanalyzers (Agilent), leading to inaccurate quantification even with these methods. Therefore, a modified version of the Swift Amplicon method is desired to enable higher library yields. Additionally, for high-throughput applications, a modified version of the method that does not require qPCR for high-precision quantification before sequencing is also desirable. However, these modifications preferably do not introduce further enzymatic or purification steps into the workflow in order to maintain the two-incubation method, which is a preferred standard in the art for targeted library preparation using multiplexed PCR. The novel methods disclosed herein provide solutions for these proposed workflow improvements.

[0009] An ideal method for DNA NGS library preparation would require a minimum number of enzymatic steps (three or fewer), a minimum number of purification steps (two or fewer, including post-PCR purification), near 100% DNA utilization, a wide range of DNA input (preferably a low threshold in the picogram to femtogram range and a high threshold in the microgram input range), the absence of adapter-adapter dimers and DNA chimeras, and the elimination of adapter concentration adjustments, which are present in many currently available kits.

[0010] The previously disclosed NGS library preparation method, commercialized as the Accel-NGS 2S DNA Library Kit described in U.S. Patent No. 10,316,357 (which is incorporated herein by reference in its entirety), overcomes the library complexity limitations of double-stranded adapter ligation libraries inherent in SMARTer, ThruPLEX, and Ion Torrent DNA Library Kits. Furthermore, it eliminates the need for adapter concentration adjustments present in TruSeq Nano, NEBNext Ultra II, and KAPA Hyper Prep kits, preventing the formation of adapter dimers and DNA chimeras across a wide range of DNA concentrations while using similar adapter concentrations for low and high input DNA samples. It is also demonstrated that the AT / GC bias is substantially lower compared to other kits. Therefore, Accel-NGS 2S is an ideal method for small and valuable samples such as DNA and cell-free DNA from ChIPSeq experiments, although it involves numerous enzymatic and purification steps. To be competitive with other methods and attractive for many applications, a modified version of the Accel-NGS 2S method is desirable to substantially reduce the number of enzymatic reaction and purification steps. The novel method disclosed herein provides a solution for these proposed workflow improvements. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Strength Patent No. 10,316,359 [Patent Document 2] U.S. Patent No. 10,316,357 [Overview of the project] [Means for solving the problem]

[0012] overview This disclosure provides a method and kit for ligation-coupled polymerase chain reaction (PCR), as well as a method and kit for sprint-mediated primer generation.

[0013] This specification discloses novel methods designed to increase library yield and overcome the need for qPCR quantification when using the Swift Amplicon multiplexed PCR targeted sequencing method and other methods referenced above. These methods utilize indexing primers in a ligation-coupled PCR reaction. There are two methods, each utilizing one of two different indexing primer designs widely used in the art: an indexing primer containing all sequences unique to each adapter but excluding a sequence common to both adapters at its 3' end, and an indexing primer containing the complete full-length sequence of each adapter, including a sequence common to both adapters at its 3' end. Shorter designs reduce oligonucleotide synthesis costs, while longer designs increase PCR efficiency because they can disrupt the secondary structure formed by the common adapter sequence and its reverse complement when priming denatured library molecules.

[0014] In either method, the Swift multiplexed PCR step can be carried out without modification as previously disclosed and, for reference, outlined below, using multiple target-specific primer pairs, each containing a universal adapter sequence and a 5' tail sequence containing a universal primer complementary to the universal adapter sequence, which includes modifications that facilitate cleavage by the endonuclease required for the subsequent adapter ligation step. PCR is performed using a high-performance proofreading polymerase tolerant of modified bases, with a long annealing time in the first PCR cycle to allow for high complexity of target-specific primer pairs, each at low concentrations, to create universal adapter-labeled amplicons from their target sequences. Following multiple cycles (two or more), PCR is continued with shorter annealing times for a second phase of amplification, using universal primers that anneal to the universal adapter adjacent to each target-specific amplicon. Because the universal primers are used at relatively high concentrations compared to the target-specific primers, the universal primers support the amplification reaction without causing further primer dimerization. Primer dimers that accumulate during limited multiplexing cycles are significantly shorter than the desired amplicon, and their complementary universal adapter sequences cause the primer dimers to form a stable secondary structure at the primer annealing temperature, reducing the efficiency of amplification by a single universal primer. Similarly, when target-specific primer pairs that generate duplicate amplicons are used, undesirable short amplicons resulting from the reverse primer of the first target-specific amplicon and the forward primer of the second target-specific amplicon also reduce the efficiency of amplification by a single universal primer similar to the primer dimers. This is due to the similar stable secondary structure formed by their complementary universal adapter sequences at the primer annealing temperature, enabling a single-tube assay with uniform target coverage.Without this special feature, short amplicons would dominate the PCR reaction, as they could prime the intended first and second amplicons, as well as the short amplicons, from the initial template, leading to a loss of coverage uniformity due to amplicon product imbalance and a significant increase in sequencing costs. For this reason, competing technologies circumvent this drawback in achieving uniform target coverage by separating primer pairs that generate duplicate amplicons into two multiplex PCR tubes.

[0015] Following Swift multiplexing PCR, a bead-based purification step can be performed to remove unused primers and replace the buffer with multiple multiplexing PCR amplicons, each containing a cleavable universal primer at its 5' end. As shown in Figure 1A, instead of continuing with the already disclosed indexed adapter ligation, in one embodiment, the purified multiplexing PCR product is combined with a PCR master mix containing an endonuclease, ligase, DNA polymerase, and deoxynucleotide triphosphate (dNTP), as well as a pair of full-length library indexing primers containing a 3' end sequence common to both adapters. By using full-length indexing primers, the primer corresponding to the 5' adapter can be used in the same ligation-coupled PCR reaction as both an adapter for ligation and a primer for PCR amplification. In addition, a complementary blocker oligonucleotide to the 3' end of the indexing primer corresponding to the 3' adapter can be pre-annealed to this primer as needed. Blocker oligonucleotides prevent this primer from participating in the initial 5' adapter ligation step at the first incubation temperature, but at temperatures below the PCR annealing temperature T mBecause it has or is inactivated, it does not inhibit priming activity during PCR (see Figures 2 and 4). In this method, the reaction mixture is incubated under conditions sufficient to allow the 3' end of a full-length indexing primer containing a 5' adapter sequence to anneal to the 5' end of the reverse complement of the universal adapter and ligate to the 5' end of the amplicon substrate by enabling endonuclease cleavage of the incorporated universal primer on the 5' end of the amplicon substrate, and both the endonuclease and ligation reactions are carried out in the PCR reaction mixture at a first temperature suitable for the activity of both enzymes. The reaction mixture is then heated to a high temperature to inactivate the endonuclease and ligase, denature the DNA substrate for PCR, and activate the polymerase if a hot-start polymerase is used. PCR thermal cycling is then performed for the required number of cycles using the indexing primer that anneals to the reverse complement of the universal adapter containing the truncated 3' adapter sequence and to the reverse complement of the full-length 5' adapter to achieve the desired library yield.

[0016] Following ligation-coupled PCR, a bead-based purification step can be performed, followed by library quantification, pooling, and sequencing. This novel method increases library yield and is suitable for low-copy-number samples with low kilobase target regions. In addition to qPCR, this method also yields a fully ligated, functional molecule-enriched library with double-stranded adapters that can be easily quantified by fluorescence analysis methods such as Qubit or electrophoresis chips such as bioanalyzers. By combining adapter ligation with PCR amplification in a single sealed tube, the additional purification steps usually required between adapter ligation and PCR are avoided, and the addition of PCR reagents following the ligation reaction is avoided if a purification step is not necessary. In this respect, the novel workflow maintains the initial workflow of two enzymatic incubations and two purification steps.

[0017] In an alternative embodiment shown in Figure 1B, purified multiplexed PCR products are combined with endonucleases, truncated 5' adapters, ligases, PCR master mixes, and library indexing primer pairs, each lacking a common 3' terminal sequence in both adapters. In this embodiment, separate 5' adapters are required for ligation because the indexing primers lack the common adapter sequence required for annealing of the sequencing primers. The reaction mixture is incubated under conditions sufficient to allow annealing of the 3' position of the 5' truncated adapter to the 5' portion of the reverse complement of the universal adapter and ligation to the 5' end of the amplicon substrate by enabling endonuclease cleavage of the universal primer incorporated on the 5' end of the amplicon during multiplexed PCR, and both the endonuclease and ligation reactions are carried out in the PCR reaction mixture at a first temperature suitable for the activity of both enzymes. Next, the reaction mixture is heated to a high temperature to inactivate the endonuclease and ligase, denature the DNA substrate for PCR, and activate the polymerase if a hot-start polymerase is used. Then, PCR thermal cycling is performed for the required number of cycles using indexing primers that anneal to the reverse complement of the universal adapter containing the truncated 3' adapter sequence and the reverse complement of the truncated 5' adapter to achieve the desired library yield. The truncated 5' adapter also contains a secondary structure that allows it to participate in ligation but prevents it from being activated as a primer during PCR so that the full-length 5' adapter sequence cannot be truncated from the completed library molecule (see Figures 3A, 3D-3L, 5A-5C).

[0018] Following ligation-coupled PCR, a purification step can be performed, followed by library quantification, pooling, and sequencing. This novel method increases library yield and yields a library enriched with fully ligated functional molecules having double-stranded adapters that can be easily quantified by fluorescence analysis methods such as Qubit or electrophoresis chips such as bioanalyzers, in addition to qPCR. By combining adapter ligation with PCR amplification in a single sealed tube, the additional purification step usually required between adapter ligation and PCR is avoided, and the addition of PCR reagents following the ligation reaction is avoided if a purification step is not necessary. In this respect, this workflow maintains the initial workflow of two enzymatic reactions and two purification steps.

[0019] Furthermore, the novel ligation-coupled PCR methods disclosed above can all be combined with a simple enzymatic library normalization method, which is commercially available as Normalase (and is described in its entirety in U.S. Patent No. 10,961,562, which is incorporated herein by reference). The addition of Normalase simplifies the library pooling step for multiplex sequencing by avoiding the quantification of individual library concentrations and the modification of sample pooling volumes based on individual library concentrations. In contrast, this method yields equimolar library yields, enabling equivolume pooling of each library for a simple, high-throughput post-library processing step prior to sequencing. The only requirements for incorporating this method into the two ligation-coupled PCR methods described above are: 1) each indexing primer must further contain a 5' tail sequence containing three or more consecutive ribonucleotide bases, and two or more deoxynucleotides on the 5' end of the three or more consecutive ribonucleotide bases, which may be rU as an example, without limitation; and 2) the DNA polymerase used in the amplification step must have 3'-to-5' exonuclease proofreading activity to generate a 5' overhang during PCR amplification (see Figures 1C and 1D). PCR efficiency can also be increased by including primer pairs that also contain three or more consecutive ribonucleotide bases on the 5' tail, and two or more deoxynucleotides on the 5' end of the three or more consecutive ribonucleotide bases, each corresponding to terminal 5'P5 and 3'P7 adapter sequences (not shown in Table 1, 18-221, 18-222, and Figures 1C and 1D). As a result, the treated library molecules obtained after incubation of the PCR mixture each contain a 5' overhang with two or more deoxynucleotides and at least one of three or more consecutive ribonucleotide bases for each primer.To obtain the desired target molar amount of each NGS library from the starting volume, the starting volume must be greater than the target volume; therefore, the number of PCR cycles applied must be such that a library yield greater than the target volume is achieved. Following PCR, a bead-based purification step is performed.

[0020] Next, Normalase can be carried out without modifying the previously disclosed enzymatic method, which is outlined below for reference. The purified PCR product is combined with a ligase and a probe complementary to the 5' overhang to produce a first enzymatic reaction mixture, in which the probe is added to each library in an amount equal to the desired target molar amount, and the first reaction mixture is incubated under conditions sufficient to allow ligation of the probe to the 3' end by annealing to the 5' overhang portion of the amplified library molecule, so that the portion of the amplified library molecule ligated to the probe becomes the target molar amount of the processed library molecule. Next, since each library has the same target amount of the processed library molecule, equivolute pooling of each library to be co-sequencing is performed. Since the probe includes a modified version to give resistance to digestion by an enzyme having exonuclease activity, the library pool is combined with an exonuclease in a second enzymatic reaction mixture under conditions sufficient to allow isolation of a selected target amount of the processed library molecule by digesting the processed library molecule that is not ligated to the probe. Next, the second enzyme reaction mixture is thermally inactivated, and the pool becomes available for flow cell loading without further purification steps. If necessary, qPCR quantification of the pool can be performed to confirm the desired final molar concentration to achieve a specific cluster density on the optimal sequencing flow cell.

[0021] In individual embodiments, ligation-coupled PCR reactions can be performed in which long PCR primers, used to introduce additional sequences during the amplification of the target substrate, can be assembled by sprint-mediated ligation before substrate amplification (see Figures 6A-6D). For example, the cost of oligo synthesis can be reduced by assembling NGS library indexing primers compatible with the subsequent enzymatic library normalization by sprint ligation. Instead of preparing full-length primers, each up to 90 nucleotides in length or longer, where only the short sample-specific index sequence differs for each pair of primers, universal 5' and 3' primer subunits are synthesized, and then only short oligonucleotide subunits containing their respective unique sample-specific indices are synthesized, and the two universal sprint oligonucleotides conjugate the 5' index and 3' primer subunits for ligation. The sprint contains complementary sequences in the portions of the two primer subunits to ligate the 3' end of the 5' subunit to the 5' end of the index subunit and the 3' end of the index subunit to the 5' end of the 3' subunit. In this embodiment, any NGS library containing both truncated adapters introduced by any method including adapter ligation or incorporation by PCR using 5'-tailed primers is combined with three subunits and two sprint oligonucleotides for each indexing primer, ligase, and PCR master mix. In this method, the reaction mixture is incubated at a first temperature under conditions sufficient to allow annealing and ligation of the primer subunits in the PCR reaction mixture, and the reaction mixture is then heated to a high temperature to inactivate the ligase, denature the NGS library substrates for PCR, and activate the polymerase if a hot-start polymerase is used.Next, PCR thermal cycling is performed for the required number of cycles using an indexing primer ligation product that anneals to two adapter sequences in the truncated NGS library, incorporating the adapter sequence index and the remainder to achieve the desired library yield. The two sprint oligonucleotides contain a 3' blocking group to prevent priming activity during PCR, and the indexing and 3' primer subunits require a 5' phosphate for sprint ligation. Without limitation, this method can be used to assemble any primer from any number of subunits and sprints for amplification of any desired DNA substrate in a single sealed tube.

[0022] In yet another embodiment, a ligation-coupled PCR reaction can be carried out in which any product assembled by oligonucleotide sprint ligation is also a substrate for PCR amplification in a single sealed tube, such as the synthesis of long DNA products that are longer than the length limit of individual oligonucleotide synthesis. In this method, individual oligonucleotide subunits are tandem synthesized against a single strand of the desired product, and then a sprint oligonucleotide containing sequences complementary to the 3' and 5' portions at the junction of the tandem subunits is synthesized to link the tandem single-strand design. In this method, the subunits and sprint oligonucleotides are combined with a ligase, a PCR master mix, and a forward primer containing the same sequence as the 5' portion of the furthest 5' subunit, and a reverse primer complementary to the 3' end of the furthest 3' subunit. The reaction mixture is incubated under conditions sufficient to allow annealing and ligation of the oligonucleotide subunits in the PCR reaction mixture at a first temperature. The reaction mixture is then heated to a high temperature to inactivate the ligase and, if a hot-start polymerase is used, to activate the polymerase. PCR thermal cycling is performed for the required number of cycles using forward and reverse primers to achieve the desired yield of the double-stranded product. The sprint oligonucleotide contains a 3' blocking group to prevent priming activity during PCR, and the oligonucleotide subunit requires a 5' phosphate for sprint ligation. The subunit can be added to the reactant at a concentration so low that it cannot support PCR, so that only the forward and reverse primers amplify the product, in order to prevent unused subunit oligonucleotides from truncating the fully assembled product by priming during PCR.

[0023] Novel NGS library methods are disclosed herein, but should not be construed as being limited to them, that can reduce the number of steps and decrease the amount of DNA input in the Swift Accel-NGS 2S referenced above. There are at least four such methods, each utilizing an indexing primer in a ligation-coupled PCR reaction, each utilizing one of two different indexing primer designs widely used in the art: an indexing primer containing all sequences unique to each adapter but excluding a sequence common to both adapters at the 3' end; and an indexing primer containing a full-length adapter with a sequence common to both adapters at the 3' end and two different sequences for each adapter; and two different adapter ligation chemistrys used in the art: ligation of a blunt-end adapter to a blunt substrate used to improve adapter ligation efficiency and library yield; and ligation of an adapter having a single T-base overhang to a substrate with a single A-base overhang used to prevent the formation of DNA chimeras and adapter dimers. The design of indexing primers is such that shorter primers reduce the cost of oligonucleotide synthesis, while longer primers can disrupt the secondary structure formed by common 3' adapter sequences and their reverse complements when priming denatured library molecules, thus increasing PCR efficiency. In a sequential two-step adapter ligation workflow where a second 5' adapter ligation is performed in a ligation-coupled PCR reaction, using T / A adapter ligation chemistry during the first 3' adapter ligation step almost completely eliminates adapter dimer formation, reducing the DNA input threshold to femtogram levels, and using blunt-end adapter ligation chemistry allows for a reduction in AT / GC bias.

[0024] In one exemplary method, DNA fragmentation and end repair are performed using standard ultrasonic DNA fragmentation methods, such as an optimized end policing protocol using Covaris and proofreading T4 DNA polymerase, followed by thermal inactivation at 65°C. After thermal inactivation of the T4 DNA polymerase, the DNA is combined with a blunt-end 3' adapter formed by annealing oligonucleotide 1 containing a uracil base and oligonucleotide 2 with a phosphorylated 5' end, T4 DNA ligase, and ligation buffer to carry out the first ligation reaction. To prevent the formation of an adapter dimer, the 3' end base of oligonucleotide 1 is modified to prevent its ligation to the 5' end of the DNA, as previously disclosed. Examples of such modified bases include, but are not limited to, dideoxynucleotides lacking two hydroxyl groups at the 2' and 3' positions in the ribose, such as ddT, and 3'- derivatives lacking one hydroxyl group at the 3' position, such as 3'-dT. A 3' adapter ligated to the 3' end of DNA forms a break with the unligated 5' end of the DNA. The second end of the 3' adapter is completely protected from ligation by the adapter design, i.e., by excluding the 5' phosphate group and placing a blocking group or a ligation-non-ligable base modification on the protruding 3' end.

[0025] In another exemplary method, DNA fragmentation and end repair are performed using standard ultrasonic DNA fragmentation methods such as Covaris and an optimized end policing protocol using proofreading T4 DNA polymerase, followed by incubation with Taq DNA polymerase at 65°C to add a single A base 3' overhang to both DNA ends and thermal inactivate the T4 DNA polymerase. The A-tailed DNA is combined with a 3' adapter formed by annealing oligonucleotide 1, which contains a uracil base and oligonucleotide 2 with a phosphorylated 5' end and a single T (or U) base overhang at the 3' end, along with T4 DNA ligase and ligation buffer, to carry out the first ligation reaction. As a result, a 3' adapter with a single base overhang is joined to both DNA strands. The second end of the 3' adapter is completely protected from ligation by a 3' adapter design, i.e., the exclusion of the 5' phosphate group and the placement of a blocking group or ligation-non-ligable base modification at the protruding 3' end.

[0026] Following ligation of the 3' adapter, which may have a blunt or single-base overhang, a bead-based purification step can be performed to remove unused adapters, enzymes, and exchange buffers. As shown in Figures 1E and 1F, instead of continuing with the already disclosed 5' adapter ligation, SPRI bead purification, and indexing PCR, in one embodiment, the purified DNA product is combined with an endonuclease, ligase, PCR master mix, and a full-length library indexing primer pair containing a 3' terminal sequence common to both 3' adapters. By using full-length indexing primers, the primer corresponding to the 5' adapter can be used in the same ligation-coupled PCR reaction as both the adapter for ligation and the primer for PCR amplification. In addition, a blocker oligonucleotide, as needed and complementary to the 3' end of the indexing primer corresponding to the 3' adapter, may be pre-annealed to this primer. The blocker oligonucleotide prevents this primer from participating in the initial 5' adapter ligation step at the first incubation temperature, but at temperatures below the PCR annealing temperature T mBecause they are either present or inactivated, they do not inhibit priming activity during PCR (see Figures 2B-2G and 4A-4E). In this method, the reaction mixture is incubated under conditions sufficient to allow the 3' end of a full-length indexing primer containing a 5' adapter sequence to anneal to the 5' end of the reverse complement of the universal adapter (first common nucleotide sequence) and ligate to the 5' end of the DNA fragment by enabling endonuclease cleavage of 3' adapter oligonucleotide 8 or 9 containing a uracil base, and both the endonuclease reaction and the ligation reaction are carried out in the PCR reaction mixture at a first temperature suitable for the activity of both enzymes. The reaction mixture is then heated to a high temperature to inactivate the endonuclease and ligase, denature the DNA substrate for PCR, and activate the polymerase if a hot-start polymerase is used. Next, PCR thermal cycling is performed for the required number of cycles using indexing primers that anneal to the reverse complement of the universal adapter containing the truncated 3' adapter sequence and the reverse complement of the full-length 5' adapter to achieve the desired library yield.

[0027] Following ligation-coupled PCR, a bead-based purification step can be performed, followed by library quantification, pooling, and sequencing. This novel method reduces the number of steps while maintaining the key advantages of the Accel-NGS 2S DNA workflow, such as high DNA conversion rates, fewer adapter dimers, a wide range of DNA input volumes, and the elimination of the need for adapter concentration adjustments for samples with variable input volumes. In addition, this method, utilizing 3' adapters with U or T base overhangs, allows for library preparation with femtogram-level DNA input volumes not offered by any commercially available kits. By combining adapter ligation with PCR amplification in a single sealed tube, the additional purification steps typically required between adapter ligation and PCR are avoided, and the addition of PCR reagents following the ligation reaction is avoided if purification is not required. In this respect, the novel workflow has a number of enzymatic incubation and purification steps similar to the most common DNA library kits.

[0028] In other embodiments shown in Figures 1G and 1H, the purified 3' adapter ligation product is combined with an endonuclease, a truncated 5' adapter, a ligase, a PCR master mix, and a library indexing primer pair, each lacking a common 3' terminal sequence in both adapters. In these embodiments, a separate 5' adapter is required for ligation because the indexing primer lacks the common adapter sequence required for sequencing primer annealing. The reaction mixture is incubated under conditions sufficient to allow annealing of the 3' portion of the 5' truncated adapter to the 5' portion of the 3' adapter oligonucleotide 7, which is ligated to the 5' end of the DNA, by enabling endonuclease cleavage of the uracil-containing 3' adapter oligonucleotide 8 or 9, and both the endonuclease and ligation reactions are carried out in the PCR reaction mixture at a first temperature suitable for the activity of both enzymes. Next, the reaction mixture is heated to a high temperature to inactivate the endonuclease and ligase, denature the DNA substrate for PCR, and activate the polymerase if a hot-start polymerase is used. Then, PCR thermal cycling is performed for the required number of cycles using an indexing primer that anneals to sequence 7 containing the truncated 3' adapter sequence to achieve the desired library yield. The truncated 5' adapter also contains a secondary structure to prevent its activity as a primer during PCR, allowing it to participate in ligation but preventing the full-length 5' adapter sequence from being truncated from the finished library molecule (see Figures 3 and 5).

[0029] Following ligation-coupled PCR, a purification step can be performed, followed by library quantification, pooling, and sequencing. This novel method reduces the number of steps while maintaining the key advantages of the Accel-NGS 2S DNA workflow, such as high DNA conversion rates, fewer adapter dimers, a wide range of DNA input volumes, and the elimination of the need for adapter concentration adjustments for samples with variable input volumes. In addition, this method, utilizing 3' adapters with U or T base overhangs, allows for library preparation with femtogram DNA input volumes not provided by any commercially available kits. By combining adapter ligation with PCR amplification in a single sealed tube, the additional purification steps typically required between adapter ligation and PCR are avoided, and the addition of PCR reagents following the ligation reaction is avoided if a purification step is not necessary. In this respect, the novel workflow has a number of enzymatic incubation and purification steps similar to the most common DNA library kits.

[0030] The ligation-coupled PCR method for preparing DNA NGS libraries disclosed above can be combined with a simple enzymatic library normalization method commercially available as Normalase (described in U.S. Patent No. 10,961,562, which is incorporated herein by reference in its entirety). The addition of Normalase simplifies the library pooling step for multiplex sequencing by avoiding the quantification of individual library concentrations and the modification of sample pooling volumes based on individual library concentrations. This method, in turn, yields equimolar library yields, thus enabling equivolume pooling of each library for a simple, high-throughput post-library processing step prior to sequencing. The only requirements for incorporating this method into the two ligation-coupled PCR methods described above are: 1) each indexing primer must contain three or more consecutive ribonucleotide bases and two or more deoxynucleotides on the 5' side of the three or more consecutive ribonucleotides; and 2) the DNA polymerase used in the amplification step must have 3'-to-5' exonuclease proofreading activity to generate a 5' overhang during PCR amplification (similar to the amplicon library shown in Figures 1C and 1D).

[0031] Next, Normalase can be carried out without modifying the previously disclosed enzymatic method, which is outlined below for reference. The purified PCR product is combined with a ligase and a probe complementary to the 5' overhang to produce a first enzymatic reaction mixture, in which the probe is added to each library in an amount equal to the desired target molar amount, and the first reaction mixture is incubated under conditions sufficient to allow ligation of the probe to the 3' end by annealing to the 5' overhang portion of the amplified library molecule, so that the portion of the amplified library molecule ligated to the probe becomes the target molar amount of the processed library molecule. Next, since each library has the same target amount of the processed library molecule, equivolute pooling of each library to be co-sequencing is performed. Since the probe includes a modified version to give resistance to digestion by an enzyme having exonuclease activity, the library pool is combined with an exonuclease in a second enzymatic reaction mixture under conditions sufficient to allow isolation of a selected target amount of the processed library molecule by digesting the processed library molecule that is not ligated to the probe. Next, the second enzyme reaction mixture is thermally inactivated, and the pool becomes available for flow cell loading without further purification steps. If necessary, qPCR quantification of the pool can be performed to confirm the desired final molar concentration to achieve a specific cluster density on the optimal sequencing flow cell. In certain embodiments, for example, the following items are provided: (Item 1) (i) To provide a partially double-stranded DNA substrate comprising a first strand and a second strand, comprising a first 3' overhang, a double-stranded portion and a second 3' overhang, The first chain includes a first 5' end, a first portion, and a second portion in the 5' to 3' direction, The second strand includes, in the 5' to 3' direction, a second 5' end, a third portion, and a fourth portion of the partially double-stranded DNA substrate. The first portion of the first chain and the third portion of the second chain are complementary, forming the double-stranded portion. The second portion of the first chain forms the first 3' overhang, The fourth portion of the second chain forms the second 3' overhang. The second portion of the first strand and the fourth portion of the second strand each include a first common nucleotide sequence located at the 5' end of the first 3' overhang and the 5' end of the second 3' overhang, The second portion of the first strand and the fourth portion of the second strand each contain a second common nucleotide sequence located at 3' with respect to the first common nucleotide sequence. (ii) Adding a plurality of first indexing primers, a plurality of second indexing primers, a ligase, a DNA polymerase, and a deoxynucleotide triphosphate (dNTP) to the partially double-stranded DNA substrate to produce a first reaction mixture, wherein each of the plurality of first indexing primers includes a first 3' terminal portion complementary to the first common nucleotide sequence, and each of the plurality of second indexing primers includes a second 3' terminal portion complementary to the first common nucleotide sequence, and a first 5' portion located 5' relative to the second 3' terminal portion and complementary to the second common nucleotide sequence. (iii) Incubating the first reaction mixture under a first set of conditions including a ligation temperature over the duration of the ligation, the first set of conditions being a) The 3' end portion of the first nucleotide anneals to the first common nucleotide sequence, b) The ligase ligates one of the plurality of first indexing primers to the first 5' end of the first chain and one of the plurality of first indexing primers to the second 5' end of the second chain, In the 5' to 3' direction, a third chain comprising one of the plurality of first indexing primers, the first portion and the second portion, In the 5' to 3' direction, a fourth chain including one of the plurality of first indexing primers, the third portion and the fourth portion It is sufficient to generate a second reaction reaction containing, (iv) Incubating the second reaction mixture under a second set of conditions comprising a first denaturation temperature over a first denaturation duration, a first annealing temperature over a first annealing duration, and a first extension temperature over a first extension duration, wherein the second set of conditions is a) Inactivate the ligase, denature the double-stranded DNA, and activate the DNA polymerase as necessary. b) The second 3' terminal portion and the first 5' portion of one of the plurality of second indexing primers anneal to at least the first common nucleotide sequence and the second common nucleotide sequence of the second portion of the third chain, and the second 3' terminal portion and the first 5' portion of one of the plurality of second indexing primers anneal to at least the first common nucleotide sequence and the second common nucleotide sequence of the fourth portion of the fourth chain, c) The DNA polymerase extends one of the plurality of second indexing primers annealed to the first common nucleotide sequence and the second common nucleotide sequence of the second portion of the third strand, and the DNA polymerase extends one of the plurality of second indexing primers annealed to the first common nucleotide sequence and the second common nucleotide sequence of the fourth portion of the fourth strand, thereby extending the third strand, the fourth strand, the fifth strand A third reaction mixture comprising a sixth chain, wherein the fifth chain comprises, in the 5' to 3' direction, one of the plurality of second indexing primers, the third portion, and the reverse complement of one of the plurality of first indexing primers, and the sixth chain is sufficient to produce a third reaction mixture comprising, in the 5' to 3' direction, one of the plurality of second indexing primers, the first portion, and the reverse complement of one of the plurality of first indexing primers, (v) Incubating the third reaction mixture under a third set of conditions comprising a second denaturation temperature over a second denaturation duration, a second annealing temperature over a second annealing duration, and a second extension temperature over a second extension duration, wherein the third set of conditions is a) Denaturing double-stranded DNA, b) One of the plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers of the fifth chain, and one of the plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers of the sixth chain, c) The DNA polymerase extends one of the plurality of first indexing primers annealed to the reverse complement of one of the plurality of first indexing primers of the fifth strand, and one of the plurality of first indexing primers annealed to the reverse complement of one of the plurality of first indexing primers of the sixth strand, thereby forming a fourth reaction mixture comprising the fifth strand, the sixth strand, the seventh strand, and the eighth strand, wherein the seventh strand is 5' to 3 Sufficient to produce a fourth reaction mixture comprising, in the ' direction, one of the plurality of first indexing primers, the first portion, and the reverse complement of one of the plurality of second indexing primers, and the eighth chain comprising, in the 5' to 3' direction, one of the plurality of first indexing primers, the third portion, and the reverse complement of one of the plurality of second indexing primers, with the seventh chain being complementary to the fifth chain and the eighth chain being complementary to the sixth chain, and (vi) The fourth reaction mixture is incubated under a fourth set of conditions comprising a third denaturation temperature over a third denaturation duration, a third annealing temperature over a third annealing duration, and a third extension temperature over a third extension duration, wherein the fourth set of conditions is sufficient to amplify at least a portion of the plurality of first indexing primers and at least a portion of the plurality of second indexing primers, the fifth and seventh chains and the sixth and eighth chains. A method of ligation-coupled polymerase chain reaction (PCR) including [specific details omitted]. (Item 2) The method according to item 1, wherein steps (i) to (vi) are carried out in a single sealed tube. (Item 3) The method according to item 1, wherein the partially double-stranded DNA substrate has a length of approximately 24 to approximately 6000 bases. (Item 4) The method according to item 1, wherein the first strand and the first portion and the third portion of the second strand, respectively, each have a length of about 20 bases to about 6,000 bases. (Item 5) The method according to item 1, wherein the second portion of the first strand and the fourth portion of the second strand each have a length of about 4 to about 100 bases. (Item 6) The method according to item 1, wherein the first common nucleotide sequence has a length of approximately 1 to 50 bases. (Item 7) The method according to item 1, wherein the first common nucleotide sequence has a length of 13 bases. (Item 8) The method according to item 1, wherein the first common nucleotide sequence includes the sequence of sequence number 127(5'-AGATCGGAAGAGC-3'). (Item 9) The method according to item 8, wherein the first 3' terminal portion and the second 3' terminal portion each contain the sequence of sequence number 126(5'-GCTCTTCCGATCT-3'). (Item 10) The method according to item 1, wherein each of the plurality of first indexing primers has a length of about 20 to about 100 bases, and each of the plurality of second indexing primers has a length of about 20 to about 100 bases. (Item 11) The first common nucleotide sequence and the first 3' terminal portion have a melting temperature (T) higher than the ligation temperature. m ) has the first common nucleotide sequence and the first 3' terminal portion, and the annealing temperature is lower than the first annealing temperature T m The method described in item 1, having the characteristics of item 1. (Item 12) The first common nucleotide sequence and the first 3' terminal portion of the T m However, the temperature is at least 1°C higher than the ligation temperature, and the first common nucleotide sequence and the first 3' terminal portion are T m However, the method according to item 11, wherein the annealing temperature is at least 1°C lower than the first annealing temperature. (Item 13) The first common nucleotide sequence and the first 3' terminal portion have a melting temperature (T) lower than the first annealing temperature. m ) has the first common nucleotide sequence and the second 3' terminal portion, and the annealing temperature is lower than the first T m The method described in item 1, having the characteristics of item 1. (Item 14) The method according to item 1, wherein the second common nucleotide sequence has a length of approximately 1 to approximately 100 bases. (Item 15) The method according to item 1, wherein the second common nucleotide sequence has a length of approximately 1 to approximately 20 bases. (Item 16) The method according to item 1, wherein the second common nucleotide sequence has a length of 20 bases. (Item 17) The ligation temperature is the melting temperature (T) of the partially double-stranded DNA substrate. m The method described in item 1, which is at least 1°C lower than ) (Item 18) The ligation temperature is the melting temperature (T) of the partially double-stranded DNA substrate. m ) A method described in item 1, which is lower than the method described in item 1. (Item 19) The melting temperatures (T) of the third and fourth chains. m The method according to item 1, wherein the temperature is lower than the first denaturation temperature. (Item 20) The method according to item 1, wherein the ligation temperature is approximately 25°C to approximately 40°C. (Item 21) The method according to item 1, wherein the duration of ligation is approximately 5 minutes to approximately 60 minutes. (Item 22) The method according to item 1, wherein the ligation duration is approximately 20 minutes. (Item 23) The method according to item 1, wherein the ligase is a thermally unstable ligase that can be ligated in a low-magnesium buffer. (Item 24) The method according to item 1, wherein the ligase is a T3 DNA ligase. (Item 25) The method according to item 24, wherein the ligase is added in an enzyme unit of about 30 to about 300 units per 50 μL of the first reaction mixture. (Item 26) The method according to item 1, wherein the ligase is temperature-sensitive, and the first denaturation temperature over the first denaturation duration in step (iv)(a) is sufficient to inactivate the ligase. (Item 27) The method according to item 1, wherein the DNA polymerase is a thermostable DNA polymerase having 3'-5' exonuclease proofreading activity, selected from the group consisting of Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), and High Fidelity DNA Polymerase (Qiagen). (Item 28) The method according to item 1, wherein the DNA polymerase is inactive at the ligation temperature. (Item 29) The method according to item 28, wherein the DNA polymerase further comprises a hot-start antibody or aptamer, the hot-start antibody or aptamer raising the activation temperature of the DNA polymerase. (Item 30) The method according to item 29, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 Hot Start DNA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara), and High Fidelity Hot Start DNA Polymerase (Qiagen). (Item 31) The method according to item 1, wherein the DNA polymerase is a hot-start polymerase, and the first denaturation temperature over the first denaturation duration in step (iv)(a) is sufficient to activate the hot-start polymerase. (Item 32) The melting temperature (T) of each of the plurality of second indexing primers, and of the first chain and the second or fourth portion of the second chain. m ) each of the plurality of first indexing primers, and the T of the first chain and the second or fourth portion of the second chain m A higher method, as described in item 1. (Item 33) The method according to item 1, wherein the first denaturation temperature, the second denaturation temperature, and the third denaturation temperature are each independently between approximately 95°C and approximately 98°C. (Item 34) The method according to item 1, wherein the first denaturation duration, the second denaturation duration, and the third denaturation duration are each independently about 30 seconds to about 2 minutes. (Item 35) The method according to item 1, wherein the first annealing temperature, the second annealing temperature, and the third annealing temperature are each independently between approximately 55°C and approximately 65°C. (Item 36) The method according to item 1, wherein the first annealing duration, the second annealing duration, and the third annealing duration are each independently between approximately 10 seconds and approximately 60 seconds. (Item 37) The method according to item 1, wherein the first extension temperature, the second extension temperature, and the third extension temperature are each independently between approximately 62°C and approximately 72°C. (Item 38) The method according to item 1, wherein the first elongation duration, the second elongation duration, and the third elongation duration are each independently approximately 30 seconds to approximately 5 minutes. (Item 39) The method according to item 1, wherein the plurality of first indexing primers and the plurality of second indexing primers are each independently added to the first reaction mixture in a concentration of about 100 nM to about 1 μM. (Item 40) The method according to item 1, wherein the plurality of first indexing primers and the plurality of second indexing primers are each independently added to the first reaction mixture at a concentration of about 100 nM to about 200 nM. (Item 41) Step (ii) further comprises adding a blocker oligonucleotide to the first reaction mixture, wherein the blocker oligonucleotide comprises a 5' portion complementary to at least a portion of the second 3' terminal portion of each of the plurality of second indexing primers, and the melting temperature (T) of each of the blocker oligonucleotide and the plurality of second indexing primers m The method according to item 1, wherein the temperature is higher than the ligation temperature and lower than the first annealing temperature and the third annealing temperature. (Item 42) The T of each of the blocker oligonucleotide and the plurality of second indexing primers m However, the T of each of the blocker oligonucleotides and the plurality of first indexing primers m A higher method, as described in item 41. (Item 43) The method according to item 41, wherein a sufficient amount of the blocker oligonucleotide is added to inhibit the ligation of each of the plurality of second indexing primers to the first 5' end of the first chain and to the second 5' end of the second chain. (Item 44) The method according to item 41, wherein the blocker oligonucleotide is added in an amount equal to 1, 1.5, 2, 4, or 6 times the amount of the plurality of second indexing primers. (Item 45) The method according to item 41, wherein the blocker oligonucleotide is pre-annealed to each of the plurality of second indexing primers. (Item 46) The method according to item 41, wherein the blocker oligonucleotide includes a first further portion located at 3' relative to the 5' portion, which is complementary to each of the plurality of second indexing primers and not complementary to each of the plurality of first indexing primers. (Item 47) The melting temperature (T) between the first further portion and the 5' portion and each of the plurality of second indexing primers m The method according to item 46, wherein the melting temperature of the 5' portion is approximately the same as or higher than the melting temperature between each of the plurality of first indexing primers and each of the plurality of second indexing primers. (Item 48) The T between the first further portion and the 5' portion and each of the plurality of second indexing primers m The method according to item 47, wherein the melting temperature between the 5' portion and each of the plurality of first indexing primers and each of the plurality of second indexing primers is at least 1°C higher. (Item 49) The method according to any one of items 46 to 48, wherein the blocker oligonucleotide further comprises a second further portion located between the 5' portion and the first further portion, the second further portion being not complementary to each of the plurality of first indexing primers and not complementary to each of the plurality of second indexing primers. (Item 50) The method according to item 49, wherein the second further portion has a length of about 1 to about 30 bases. (Item 51) The method according to item 41, wherein the blocker oligonucleotide comprises a sequence that is not complementary to each of the second indexing primers. (Item 52) The method according to item 51, wherein the sequence, which is not complementary to each of the plurality of second indexing primers, has a length of about 1 to about 30 bases. (Item 53) The method according to item 41, wherein the blocker oligonucleotide further comprises a 3' modification for inhibiting polymerase elongation. (Item 54) The method according to item 53, wherein the 3' modification for inhibiting polymerase elongation is selected from the group consisting of a C3 carbon spacer, a hexanediol, spacer 9, spacer 18, a phosphate, 2',3'-dideoxynucleosides ddA, ddT, ddC and ddG, 3'-deoxynucleosides 3'-A, 3'-T, 3'-C and 3'-G, RNA nucleotides such as rU, 3-O-methylnucleotides, or DNA sequences that are not complementary to the adjacent primer sequences, such as polyT, polyA, polyC and polyG, and further comprises nuclease-resistant binding to prevent proofreading polymerase 3'-5' exonuclease activity from removing the DNA sequences that are not complementary to the adjacent primer sequences. (Item 55) The melting temperature (T) between the blocker oligonucleotide and each of the plurality of first indexing primers. m ) is the T between the blocker oligonucleotide and each of the plurality of second indexing primers. m A lower method, as described in item 41. (Item 56) The T between the blocker oligonucleotide and each of the plurality of first indexing primers m However, the T between the blocker oligonucleotide and each of the plurality of second indexing primers m At least 5°C lower, as described in item 55. (Item 57) The method according to item 41, wherein the 5' portion of the blocker oligonucleotide contains the sequence of sequence number 127. (Item 58) The method according to any one of items 46 to 50, wherein the blocker oligonucleotide further comprises a 3' hydroxyl group and a hairpin portion located 3' relative to the first further portion, the hairpin portion comprising a first hairpin arrangement and a second hairpin arrangement, the first hairpin arrangement being located 5' relative to the second hairpin arrangement, the first hairpin arrangement and the second hairpin arrangement being complementary, and the hairpin portion having a melting temperature higher than the first annealing temperature, the second annealing temperature and the third annealing temperature. (Item 59) The method according to item 58, wherein the hairpin portion further includes a third hairpin arrangement located between the first hairpin arrangement and the second hairpin arrangement. (Item 60) The method according to item 59, wherein the third hairpin arrangement forms a loop sufficient to allow the hairpin portion and the further first portion to form a stable stem loop structure. (Item 61) The method according to item 60, wherein the third hairpin sequence has a length of about 4 to about 20 bases. (Item 62) The method according to item 58, wherein the melting temperature of the hairpin portion is higher than the melting temperature between the 5' portion and each of the plurality of second indexing primers, the further portion and each of the plurality of second indexing primers, or both. (Item 63) The method according to any of items 58-60, wherein the second set of conditions is further sufficient for the DNA polymerase to extend the 3' end of the blocker oligonucleotide to produce an extended hairpin blocker. (Item 64) The extension hairpin blocker has a melting temperature (T) higher than the second annealing temperature and the third annealing temperature. m The method described in item 63, which has the following characteristics. (Item 65) The method according to item 63, wherein the extension hairpin blocker has a stable second structure. (Item 66) The melting temperature (T) of the aforementioned extension hairpin blocker m ) from the 5' portion and the further first portion to each of the plurality of second indexing primers m A higher method, as described in item 63. (Item 67) The method according to item 1, wherein each of the plurality of first indexing primers further comprises the sequence of SEQ ID NO: 87. (Item 68) The method according to item 1, wherein each of the plurality of second indexing primers further comprises the sequence of SEQ ID NO: 78. (Item 69) The method of any of the above items, further comprising sequencing the fifth and seventh chains or the sixth and eighth chains. (Item 70) Each of the plurality of first indexing primers comprises a first 5' terminal portion, each of the plurality of second indexing primers further comprises a second 5' terminal portion, each of the first 5' terminal portion and the second 5' terminal portion comprises, in the 5' to 3' direction, a first sequence containing two or more deoxynucleotides and a second sequence containing three or more ribonucleotides, wherein the DNA polymerase has 3' to 5' exonuclease activity, and the fifth and sixth strands However, the fifth and sixth chains further include the second 5' end portion at their 5' ends, and the seventh and eighth chains further include the first 5' end portion at their 5' ends, and the fifth and seventh chains can form a first double-stranded product having a first 5' overhang and a second 5' overhang, and the sixth and eighth chains can form a second double-stranded product having a third 5' overhang and a fourth 5' overhang, and the method, The method involves adding a sufficient amount of probes complementary to each of the first, second, third, and fourth 5' overhangs, as well as a second ligase, to generate a target molar amount of the fifth, sixth, seventh, and eighth chains, wherein a greater amount of the fifth, sixth, seventh, and eighth chains is present than the target molar amount, and the probes include modifications to obtain resistance to digestion by enzymes having 3' exonuclease activity. The fifth chain, sixth chain, seventh chain, eighth chain, second ligase, and probe are incubated under conditions sufficient to generate a pre-normalized reaction mixture, by ligating the probe to the target molar amounts of the fifth chain, sixth chain, seventh chain, and eighth chain. Adding an enzyme having 3'-exonuclease activity to the pre-normalization reaction mixture, and The pre-normalization reaction mixture and the enzyme having exonuclease activity are incubated under conditions sufficient to allow the enzyme having 3'-exonuclease activity to digest the fifth, sixth, seventh, and eighth chains that have not ligated to the probe, thereby generating a normalized next-generation sequencing (NGS) library. A method of any of the above items, further including: (Item 71) The method according to item 70, further comprising sequencing the normalized NGS library. (Item 72) (i) To provide a partially double-stranded DNA substrate comprising a first strand and a second strand, comprising a first 3' overhang, a double-stranded portion and a second 3' overhang, The first chain includes a first 5' end, a first portion, and a second portion in the 5' to 3' direction, The second chain includes a second 5' end, a third portion and a fourth portion in the 5' to 3' direction, The first portion of the first chain and the third portion of the second chain are complementary and form the double-stranded portion. The second portion of the first chain forms the first 3' overhang. The fourth portion of the second chain forms the second 3' overhang. The second portion of the first strand and the fourth portion of the second strand each contain a first common nucleotide sequence located at the first 3' overhang and the 5' end of the second 3' overhang, respectively. The second portion of the first strand and the fourth portion of the second strand each contain a second common nucleotide sequence located at 3' with respect to the first common nucleotide sequence. (ii) Adding a plurality of first indexing primers, a plurality of second indexing primers, a plurality of 5' adapters, a ligase, a DNA polymerase, and a deoxynucleotide triphosphate (dNTP) to the partially double-stranded DNA substrate to produce a first reaction mixture, wherein each of the plurality of first indexing primers includes a first 3' terminal portion that is not complementary to the first common nucleotide sequence, and each of the plurality of second indexing primers includes a second 3' terminal portion that is not complementary to the first common nucleotide sequence, and the second 3' terminal sequence is complementary to the second common nucleotide sequence, and Each of the plurality of 5' adapters includes a third 3' terminal sequence complementary to the first common nucleotide sequence, a first 5' portion located 5' relative to the third 3' terminal sequence, which includes at least a portion of the first 3' terminal portion of each of the plurality of first indexing primers, a second 5' portion located 5' relative to the first 5' portion and complementary to the first 5' portion, and a replication blocker located at the 5' end of the first 5' portion that is capable of inhibiting the DNA polymerase, wherein the 5' adapter can form a hairpin by annealing the first 5' portion to the second 5' portion. (iii) The first reaction reaction, 1) the third 3' terminal portion anneals to the first common nucleotide sequence, and 2) the ligase ligates one of the plurality of 5' adapters to the first 5' end of the first strand and the second 5' end of the second strand, A third chain, comprising one of the plurality of 5' adapters, the first part and the second part, in the direction from 5' to 3', From 5' to 3', a fourth chain including one of the plurality of 5' adapters, the third portion and the fourth portion Incubating under a first set of conditions, including a ligation temperature over a ligation duration, sufficient to produce a second reaction mixture containing, (iv) The second reaction mixture, a) Inactivate the ligase, denature the double-stranded DNA, and activate the DNA polymerase as necessary. b) The second 3' terminal portion of one of the plurality of second indexing primers anneals to the second common nucleotide sequence of the second portion of the third chain, and one of the plurality of second indexing primers anneals to the second common nucleotide sequence of the fourth portion of the fourth chain, c) The DNA polymerase extends one of the plurality of second indexing primers annealed to the second common nucleotide sequence of the second portion of the third strand, and extends one of the plurality of second indexing primers annealed to the second common nucleotide sequence of the fourth portion of the fourth strand, to a third reaction mixture comprising the third strand, the fourth strand, the fifth strand, and the sixth strand, wherein the fifth strand has, in the 5' to 3' direction, one of the plurality of second indexing primers, the third portion, the reverse complement of the first common nucleotide sequence, and the reverse complement of the first 5' portion of one of the plurality of 5' adapters, wherein the sixth strand is incubated under a second set of conditions including a first denaturation temperature over a first denaturation duration, a first annealing temperature over a first extension duration, and a first extension temperature over a first extension duration, which is sufficient to produce a third reaction mixture including, in the 5'-to-3' direction, one of the plurality of second indexing primers, the first portion, the reverse complement of the first common nucleotide sequence, and the reverse complement of the first 5' portion of one of the plurality of 5' adapters. (v) The third reaction reaction, a) Denaturate any double-stranded DNA, b) The first 3' end portion of one of the plurality of first indexing primers anneals to the reverse complement of the first 5' portion of one of the plurality of 5' adapters of the fifth chain, and one of the plurality of first indexing primers anneals to the reverse complement of the first 5' portion of one of the plurality of 5' adapters of the sixth chain, c) The DNA polymerase extends one of the plurality of first indexing primers annealed to the reverse complement of the first 5' portion of one of the plurality of 5' adapters of the fifth strand, and extends one of the plurality of first indexing primers annealed to the reverse complement of the first 5' portion of one of the plurality of 5' adapters of the sixth strand, to a fourth reaction mixture comprising the fifth strand, the sixth strand, the seventh strand and the eighth strand, wherein the seventh strand extends in the 5' to 3' direction to one of the plurality of first indexing primers, the first common nucleo The eighth strand comprises a nucleotide sequence, the first portion, and a reverse complement of one of the plurality of second indexing primers, and is incubated under a third set of conditions including a second denaturation temperature over a second denaturation duration, a second annealing temperature over a second extension duration, and a second extension temperature over a second extension duration, which is sufficient to produce a fourth reaction mixture comprising one of the plurality of first indexing primers, the first common nucleotide sequence, the third portion, and the reverse complement of one of the plurality of second indexing primers, in the 5' to 3' direction. (vi) The fourth reaction reaction described above, a) Denaturate any double-stranded DNA, b) One of the plurality of second indexing primers anneals to the reverse complement of one of the plurality of second indexing primers of the seventh chain, and one of the plurality of second indexing primers anneals to the reverse complement of one of the plurality of second indexing primers of the eighth chain, c) The DNA polymerase extends one of the plurality of second indexing primers annealed to each of the seventh and eighth strands, to form a fifth reaction mixture comprising the seventh, eighth, ninth and tenth strands, wherein the ninth strand comprises, in the 5' to 3' direction, one of the plurality of second indexing primers, the third portion, the reverse complement of the first common nucleotide sequence, and the reverse complement of one of the plurality of first indexing primers, and the tenth strand comprises, in the 5' to 3' direction, the plurality Incubating under a fourth set of conditions including a third denaturation temperature over a third denaturation duration, a third annealing temperature over a third extension duration, and a third extension temperature over a third extension duration, sufficient to produce a fifth reaction mixture comprising one of the second indexing primers, the first portion, the reverse complement of the first common nucleotide sequence, and the reverse complement of one of the plurality of first indexing primers, wherein the seventh and ninth chains are complementary and the eighth and tenth chains are complementary, and (vii) The fifth reaction mixture, at least a portion of the plurality of first indexing primers, at least a portion of the plurality of second indexing primers, and the DNA polymerase are incubated under a fifth set of conditions including a fourth denaturation temperature over a fourth denaturation duration, a fourth annealing temperature over a fourth annealing duration, and a fourth extension temperature over a fourth extension duration, which is sufficient to amplify the seventh, ninth, eighth, and tenth strands. A method for ligation-coupled PCR, including the following: (Item 73) The method according to item 72, wherein steps (i) to (vii) are carried out in a single sealed tube. (Item 74) The method according to item 72, wherein the first 5' portion and the second 5' portion each have a length of about 12 to about 20 bases. (Item 75) The method according to item 72, wherein each of the plurality of 5' adapters further includes an intervening array between the first 5' portion and the second 5' portion. (Item 76) The method according to item 75, wherein the intervening sequence has a length of approximately 4 to 20 bases. (Item 77) The method according to item 72, wherein the replication blocker is selected from the group consisting of a stable debase site, a C3 spacer, a hexanediol, a spacer 9, a spacer 18, three or more rU bases, and a 2'-O-methylRNA base. (Item 78) The method according to item 72, wherein each of the plurality of 5' adapters has a length of about 25 to about 100 bases. (Item 79) The method according to item 72, wherein the partially double-stranded DNA substrate has a length of approximately 24 to approximately 6000 bases. (Item 80) The method according to item 72, wherein the first strand and the first portion and the third portion of the second strand, respectively, each have a length of about 20 bases to about 6,000 bases. (Item 81) The method according to item 72, wherein the second portion of the first strand and the fourth portion of the second strand each have a length of about 4 to about 100 bases. (Item 82) The method according to item 72, wherein the first common nucleotide sequence has a length of about 1 to about 50 bases. (Item 83) The method according to item 72, wherein the first common nucleotide sequence has a length of 13 bases. (Item 84) The method according to item 72, wherein the first common nucleotide sequence includes the sequence of sequence number 127. (Item 85) The method according to item 84, wherein the third 3' end portion of each of the plurality of 5' adapters contains the sequence of sequence number 126. (Item 86) The method according to item 72, wherein each of the plurality of first indexing primers has a length of about 20 to about 100 bases, and each of the plurality of second indexing primers has a length of about 20 to about 100 bases. (Item 87) The method according to item 72, wherein the first common nucleotide sequence and the third 3' terminal portion have a melting temperature higher than the ligation temperature. (Item 88) The method according to item 72, wherein the second 3' terminal portion and the second common nucleotide sequence of each of the plurality of second indexing primers have a melting temperature higher than the first annealing temperature. (Item 89) The method according to item 72, wherein the melting temperature of the first common nucleotide sequence and the third 3' terminal portion is at least 1°C higher than the ligation temperature, and the melting temperature of the first common nucleotide sequence and the third 3' terminal portion is at least 1°C lower than the first annealing temperature. (Item 90) The method according to item 72, wherein the second common nucleotide sequence has a length of approximately 1 to 100 bases. (Item 91) The method according to item 72, wherein the second common nucleotide sequence has a length of about 1 to about 20 bases. (Item 92) The method according to item 72, wherein the second common nucleotide sequence includes the sequence of sequence number 95. (Item 93) The method according to item 72, wherein the ligation temperature is lower than the melting temperature of the partially double-stranded DNA substrate. (Item 94) The method according to item 72, wherein the melting temperatures of the third and fourth chains are lower than the denaturation temperature of the first chain. (Item 95) The method according to item 72, wherein the ligation temperature is approximately 25°C to approximately 40°C. (Item 96) The method according to item 72, wherein the duration of ligation is approximately 5 minutes to approximately 60 minutes. (Item 97) The method according to item 72, wherein the ligation duration is approximately 20 minutes. (Item 98) The method according to item 72, wherein the ligase is a thermally unstable ligase that can be ligated in a low-magnesium buffer. (Item 99) The method according to item 72, wherein the ligase is a T3 DNA ligase. (Item 100) The method according to item 99, wherein the ligase is added in units of about 30 to about 300 enzymes per 50 μL of the first reaction mixture. (Item 101) The method according to item 72, wherein the ligase is temperature-sensitive, and the first denaturation temperature over the first denaturation duration is sufficient to inactivate the ligase. (Item 102) The method according to item 72, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), and High Fidelity DNA Polymerase (Qiagen). (Item 103) The method according to item 72, wherein the DNA polymerase is inactive at the ligation temperature. (Item 104) The method according to item 103, wherein the DNA polymerase further comprises a hot-start antibody or aptamer, the hot-start antibody or aptamer raising the activation temperature of the DNA polymerase. (Item 105) The method according to item 104, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 D Hot Start NA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara), and High Fidelity Hot Start DNA Polymerase (Qiagen). (Item 106) The method according to item 72, wherein the DNA polymerase is a hot-start polymerase, and the first denaturation temperature over the first denaturation duration is sufficient to activate the hot-start polymerase. (Item 107) The method according to item 72, wherein the first denaturation temperature, the second denaturation temperature, the third denaturation temperature, and the fourth denaturation temperature are each independently between approximately 95°C and approximately 98°C. (Item 108) The method according to item 72, wherein the first denaturation duration, the second denaturation duration, the third denaturation duration, and the fourth denaturation duration are each independently between approximately 30 seconds and approximately 2 minutes. (Item 109) The method according to item 72, wherein the plurality of first indexing primers and the plurality of second indexing primers are each independently added to the first reaction mixture in a concentration of about 100 nM to about 1 μM. (Item 110) The method according to item 72, wherein the plurality of first indexing primers and the plurality of second indexing primers are each independently added to the first reaction mixture at a concentration of about 100 nM to about 200 nM. (Item 111) The method according to item 72, wherein the first annealing temperature, the second annealing temperature, the third annealing temperature, and the fourth annealing temperature are each independently between approximately 55°C and approximately 65°C. (Item 112) The method according to item 72, wherein the first annealing duration, the second annealing duration, the third annealing duration, and the fourth annealing duration are each independently between approximately 10 seconds and approximately 60 seconds. (Item 113) The method according to item 72, wherein the first extension temperature, the second extension temperature, the third extension temperature, and the fourth extension temperature are each independently between approximately 60°C and approximately 72°C. (Item 114) The method according to item 72, wherein the first elongation duration, the second elongation duration, the third elongation duration, and the fourth elongation duration are each independently between approximately 30 seconds and approximately 5 minutes. (Item 115) The method according to item 72, wherein each of the plurality of first indexing primers further comprises the sequence of SEQ ID NO: 87. (Item 116) The method according to item 72, wherein each of the plurality of second indexing primers further comprises the sequence of SEQ ID NO: 78. (Item 117) The method according to any one of items 72 to 116, further comprising sequencing the seventh and ninth chains or the eighth and tenth chains. (Item 118) Each of the plurality of first indexing primers comprises a first 5' terminal portion, each of the plurality of second indexing primers further comprises a second 5' terminal portion, each of the first 5' terminal portion and the second 5' terminal portion comprises, in the 5' to 3' direction, a first sequence comprising two or more deoxynucleotides and a second sequence comprising three or more ribonucleotides, wherein the DNA polymerase has 3' to 5' exonuclease activity, and the seventh and eighth strands are The seventh and eighth chains further include the first 5' end portion at their 5' ends, the ninth and tenth chains further include the second 5' end portion at their 5' ends, the seventh and ninth chains can form a first double-stranded product having a first 5' overhang and a second 5' overhang, the eighth and tenth chains can form a second double-stranded product having a third 5' overhang and a fourth 5' overhang, and the method, The method involves adding a second ligase and a probe complementary to each of the first, second, third, and fourth 5' overhangs, in an amount sufficient to generate a target molar amount of the seventh, eighth, ninth, and tenth chains, wherein a greater amount of the seventh, eighth, ninth, and tenth chains is present, and the probes include modifications to obtain resistance to digestion by enzymes having 3' exonuclease activity. The seventh, eighth, ninth, and tenth chains, ligase, and probe are incubated under conditions sufficient to generate a pre-normalized reaction mixture, by ligating the probe to the target molar amounts of the seventh, eighth, ninth, and tenth chains. Adding an enzyme having 3'-exonuclease activity to the pre-normalization reaction mixture, and The pre-normalization reaction mixture and the enzyme having exonuclease activity are incubated under conditions sufficient to allow the enzyme having 3'-exonuclease activity to digest the seventh, eighth, ninth, and tenth chains that have not ligated to the probe, thereby generating a normalized next-generation sequencing (NGS) library. The method described in any of items 72-117, further including the method described in any of items 72-117. (Item 119) To provide an initiating double-stranded DNA substrate molecule comprising a first initiating strand and a second initiating strand, wherein the first initiating strand of the initiating double-stranded DNA substrate molecule comprises a universal primer sequence at its 5' end, the universal primer sequence comprises a dU base inside or at its 3' end, and the second initiating strand of the initiating double-stranded DNA substrate molecule comprises the universal primer sequence at its 5' end. Adding an enzyme capable of cleaving the dU base of the initial double-stranded DNA substrate molecule, and The enzyme capable of cleaving the dU base and the initiating double-stranded DNA substrate molecule is incubated under conditions sufficient to generate the partially double-stranded DNA substrate of step (i). The method of any of the above items, further comprising before step (i). (Item 120) The method according to item 119, wherein the enzyme capable of cleaving the dU base is a combination of uracil DNA glycosylase and endonuclease VIII. (Item 121) To provide an initiating double-stranded DNA substrate molecule comprising a first initiating strand and a second initiating strand, wherein the first initiating strand of the initiating double-stranded DNA substrate molecule comprises a first 5' cleavable sequence, the first 5' cleavable sequence comprises a ribonucleotide inside or at its 3' end, and the second initiating strand of the double-stranded DNA substrate molecule comprises a second 5' cleavable sequence, the second 5' cleavable sequence comprises a ribonucleotide inside or at its 3' end. Adding an enzyme capable of cleaving the ribonucleotides of the first 5' cleavable sequence and the second 5' cleavable sequence, The method according to any one of items 1 to 118, further comprising incubating an enzyme capable of cleaving the ribonucleotides of the first 5' cleavable sequence and the second 5' cleavable sequence, as well as the start double-stranded DNA substrate molecule, under conditions sufficient to produce the partially double-stranded DNA substrate of step (i) prior to step (i). (Item 122) The method according to item 121, wherein the enzyme capable of cleaving the ribonucleotides of the first 5' cleavable sequence and the second 5' cleavable sequence is RNase H. (Item 123) To provide an initiating double-stranded DNA substrate molecule comprising a first initiating strand and a second initiating strand, wherein the first initiating strand of the initiating double-stranded DNA substrate molecule comprises a first 5' cleavable sequence, the first 5' cleavable sequence comprises an inosine base inside or at its 3' end, and the second initiating strand of the double-stranded DNA substrate molecule comprises a second 5' cleavable sequence, the second 5' cleavable sequence comprises an inosine base inside or at its 3' end. Adding an enzyme capable of cleaving the inosine bases of the first 5' cleavable sequence and the second 5' cleavable sequence, The enzyme capable of cleaving the inosine bases of the first 5' cleavable sequence and the second 5' cleavable sequence, and the initiation double-stranded DNA substrate molecule, are incubated under conditions sufficient to generate the partially double-stranded DNA substrate of step (i). The method described in any of items 1 through 118, further including before step (i). (Item 124) The method according to item 123, wherein the enzyme capable of cleaving the inosine base is endonuclease V. (Item 125) To provide fragmented DNA substrate molecules, The process involves performing end repair to generate a blunt-ended double-stranded initiation DNA substrate molecule containing a first initiation strand and a second initiation strand, wherein each of the first and second initiation strands contains a 5' end and a 3' end. Each 3' adapter strand is annealed to a complementary strand containing a dU base and a 3' blocking group, and a plurality of 3' adapter strands containing a 5' phosphate, along with a ligase, are added to the blunt-ended double-stranded initiation DNA substrate molecule. The plurality of 3' adapter strands annealed to a complementary strand containing a dU base, the ligase, and the blunt-ended double-stranded initiation DNA substrate molecule are incubated under conditions sufficient to generate a ligated double-stranded substrate molecule, by ligating one of the plurality of 3' adapter strands to the 3' ends of the first and second strands of the blunt-ended initiation DNA substrate molecule, leaving a gap between each complementary strand and the 5' ends of the first and second initiation strands. If necessary, purify the ligated double-stranded substrate molecule. Adding an enzyme capable of cleaving the dU base of the ligated double-stranded substrate molecule to the ligated double-stranded substrate molecule, and The enzyme capable of cleaving the dU base and the ligated double-stranded substrate molecule are incubated under conditions sufficient to cleave the complementary strand and generate the partially double-stranded DNA substrate of step (i). The method described in any of items 1 through 118, further including the method described in any of items 1 through 118. (Item 126) The method according to item 125, wherein the enzyme capable of cleaving the dU base is a combination of uracil DNA glycosylate and endonuclease VIII. (Item 127) To provide fragmented DNA substrate molecules, The process involves performing end repair to generate a blunt-ended double-stranded initiation DNA substrate molecule containing a first initiation strand and a second initiation strand, wherein each of the first and second initiation strands contains a 5' end and a 3' end. Each 3' adapter strand is annealed to a complementary strand containing a ribonucleotide and a 3' blocking group, and a plurality of 3' adapter strands containing a 5' phosphate, along with a ligase, are added to the blunt-ended double-stranded initiation DNA substrate molecule. The plurality of 3' adapter strands annealed to a complementary strand containing ribonucleotides, the ligase, and the blunt-ended double-stranded initiation DNA substrate molecule are incubated under conditions sufficient to generate a ligated double-stranded substrate molecule, by ligating one of the plurality of 3' adapter strands to the 3' ends of the first and second initiation strands of the blunt-ended initiation DNA substrate molecule, leaving a gap between each complementary strand and the 5' ends of the first and second initiation strands. If necessary, purify the ligated double-stranded substrate molecule. Adding an enzyme capable of cleaving the ribonucleotides of the ligated double-stranded substrate molecule to the ligated double-stranded substrate molecule, and The enzyme capable of cleaving the ribonucleotide and the ligated double-stranded substrate molecule are incubated under conditions sufficient to cleave the complementary strand and generate the partially double-stranded DNA substrate of step (i). The method described in any of items 1 through 118, further including the method described in any of items 1 through 118. (Item 128) The method according to item 127, wherein the enzyme capable of cleaving the ribonucleotide is RNase H. (Item 129) To provide fragmented DNA substrate molecules, The process involves performing end repair to generate a blunt-ended double-stranded initiation DNA substrate molecule comprising a first strand and a second strand, wherein each of the first and second strands includes a 5' end and a 3' end. Each 3' adapter strand is annealed to a complementary strand containing an inosine base and a 3' blocking group, and a plurality of 3' adapter strands containing a 5' phosphate, along with a ligase, are added to the blunt-ended double-stranded initiation DNA substrate molecule. The plurality of 3' adapter strands annealed to a complementary strand containing an inosine base, the ligase, and the blunt-ended double-stranded initiation DNA substrate molecule are incubated under conditions sufficient to generate a ligated double-stranded substrate molecule, by ligating one of the plurality of 3' adapter strands to the 3' ends of the first and second strands of the blunt-ended initiation DNA substrate molecule, leaving a gap between each complementary strand and the 5' ends of the first and second strands. If necessary, purify the ligated double-stranded substrate molecule. Adding an enzyme capable of cleaving the inosine base of the ligated double-stranded substrate molecule to the ligation PCR reaction mixture, and The enzyme capable of cleaving the inosine base and the ligated double-stranded substrate molecule are incubated under conditions sufficient to cleave the complementary strand and generate the partially double-stranded DNA substrate of step (i). The method described in any of items 1 through 118, further including the method described in any of items 1 through 118. (Item 130) The method according to item 129, wherein the enzyme capable of cleaving the inosine base is endonuclease V. (Item 131) To provide fragmented DNA substrate molecules, A single A-tailed double-stranded initiation DNA substrate molecule is generated by performing end repair and A-tailing of a fragmented DNA substrate molecule, wherein each of the first and second initiation strands includes a 5' end and a 3' end, and the single A-tailed double-stranded initiation DNA substrate molecule includes a single A base overhang at each 3' end. Each 3' adapter strand is annealed to a complementary strand containing a 3' terminal dU base and a 3' hydroxyl group, and multiple 3' adapter strands containing a 5' phosphate, as well as a ligase, are added to the single A-tailed double-stranded initiation DNA substrate molecule. The plurality of 3' adapter strands annealed to a complementary strand containing a 3' terminal dU base, the ligase, and the single A-tailed double-stranded initiation DNA substrate molecule are incubated under conditions sufficient to generate a ligated double-stranded substrate molecule by ligating one of the plurality of 3' adapter strands to each single A base overhang and ligating one of the complementary strands to each 5' end of the single A-tailed double-stranded initiation DNA substrate molecule. If necessary, purify the ligated double-stranded substrate molecule. Adding an enzyme capable of cleaving dU bases to the ligated double-stranded substrate molecule, and The enzyme capable of cleaving dU bases and the ligated double-stranded substrate molecule are incubated under conditions sufficient to cleave the complementary strand and generate the partially double-stranded DNA substrate of step (i). The method described in any of items 1 through 118, further including the method described in any of items 1 through 118. (Item 132) The method according to item 131, wherein the enzyme capable of cleaving the dU base is a combination of uracil DNA glycosylate and endonuclease VIII. (Item 133) The method according to item 131, wherein the complementary strand contains an additional dU base within the complementary strand. (Item 134) To provide fragmented DNA substrate molecules, A single A-tailed double-stranded initiation DNA substrate molecule is generated by performing end repair and A-tailing of a fragmented DNA substrate molecule, wherein each of the first and second initiation strands includes a 5' end and a 3' end, and the single A-tailed double-stranded initiation DNA substrate molecule includes a single A base overhang at each 3' end. Multiple 3' adapter strands containing a 5' phosphate, each annealed to a complementary strand containing a 3' terminal T base, a 3' hydroxyl group, and a dU base, and a ligase are added to the single A-tailed double-stranded initiation DNA substrate molecule. The plurality of 3' adapter strands annealed to a complementary strand containing a 3' terminal T base, the ligase, and the single A-tailed double-stranded initiation DNA substrate molecule are incubated under conditions sufficient to generate a ligated double-stranded substrate molecule by ligating one of the plurality of 3' adapter strands to each single A base overhang and ligating one of the complementary strands to each 5' end of the single A-tailed double-stranded initiation DNA substrate molecule. If necessary, purify the ligated double-stranded substrate molecule. Adding an enzyme capable of cleaving dU bases to the ligated double-stranded substrate molecule, and The enzyme capable of cleaving the dU base and the ligated double-stranded substrate molecule are incubated under conditions sufficient to cleave the complementary strand and generate the partially double-stranded DNA substrate of step (i). The method described in any of items 1 through 118, further including the method described in any of items 1 through 118. (Item 135) The method according to item 134, wherein the enzyme capable of cleaving the dU base is a combination of uracil DNA glycosylate and endonuclease VIII. (Item 136) The method according to any one of items 125 to 135, further comprising fragmenting target DNA to produce the fragmented DNA substrate molecule. (Item 137) The method according to any of the above items, wherein purification is not performed between steps (i) to (vi) or (i) to (vii). (Item 138) (i) To provide a double-stranded DNA substrate comprising a first strand and a second strand, wherein the first strand comprises a first 3' end portion and a first 5' end portion, and the second strand comprises a second 3' end portion and a second 5' end portion. (ii) Adding a ligase, a first oligonucleotide, a second oligonucleotide, a third oligonucleotide, a first primer, a DNA polymerase, and deoxynucleotide triphosphate (dNTP) to the double-stranded DNA substrate to produce a first reaction mixture, wherein the first oligonucleotide comprises a third 3' terminal portion complementary to at least a portion of the first 3' terminal portion of the first strand, and a 5' phosphate; the third oligonucleotide comprises a first 3' blocking group; the first primer comprises a fourth 3' terminal portion complementary to at least a portion of the second 3' terminal portion of the second strand, the first 3' blocking group is incapable of polymerase chain extension; the second oligonucleotide has a 3' portion complementary to the 3' portion of the third oligonucleotide; and the first oligonucleotide has a 5' portion complementary to the 5' portion of the third oligonucleotide. (iii) Incubating the first reaction mixture, the first oligonucleotide and the second oligonucleotide under a first set of conditions including a ligation temperature for a ligation duration sufficient to produce a second reaction mixture comprising the first primer, the double-stranded DNA substrate, and a second primer containing the first oligonucleotide ligated to the second oligonucleotide, and (iv) Each cycle independently undergoes at least three PCR cycles on the second reaction mixture, each cycle including a denaturation temperature over a denaturation duration, an annealing temperature over an annealing duration, and an extension temperature over an extension duration, to generate a third reaction mixture containing the double-stranded library molecule with the second primer at the first 5' end of the double-stranded library molecule and the first primer at the second 5' end of the double-stranded library molecule. A method of ligation-coupled polymerase chain reaction (PCR) including [specific details omitted]. (Item 139) (v) The method of item 138, further comprising subjecting the third reaction mixture to a further PCR cycle to amplify the double-stranded library molecules. (Item 140) The method according to item 138, wherein the ligation temperature is lower than the melting temperature of the double-stranded DNA substrate. (Item 141) The method according to item 138, wherein the ligation temperature is lower than the melting temperatures of the first oligonucleotide and the third oligonucleotide, and the ligation temperature is lower than the melting temperatures of the second oligonucleotide and the third oligonucleotide. (Item 142) The method according to item 138, wherein the ligase is a thermally unstable ligase capable of ligation in a low-magnesium PCR buffer. (Item 143) The method according to item 138, wherein the ligase is a T3 DNA ligase. (Item 144) The method according to item 143, wherein the ligase is added in units of about 30 to about 300 enzymes per 50 μL of the first reaction mixture. (Item 145) The method according to item 138, wherein the ligase is temperature-sensitive and the denaturation temperature at the first cycle of the at least three PCR cycles in step (iv) is sufficient to inactivate the ligase. (Item 146) The method according to item 138, wherein step (ii) further comprises adding a fourth oligonucleotide and a fifth oligonucleotide, the second oligonucleotide further comprising a 5' phosphate, the fifth oligonucleotide comprising a second 3' blocking group that is incapable of polymerase chain extension, the fifth oligonucleotide further comprising a 5' portion complementary to the 5' portion of the second oligonucleotide and a 3' portion complementary to the 3' portion of the fourth oligonucleotide, the first set of conditions being further sufficient to generate the second primer comprising the fourth oligonucleotide, the second oligonucleotide and the first oligonucleotide in the 5'-to-3' direction by the ligase ligating the fourth oligonucleotide to the second oligonucleotide. (Item 147) The method according to item 146, wherein the ligation temperature is lower than the melting temperatures of the second oligonucleotide and the fifth oligonucleotide, and the ligation temperature is lower than the melting temperatures of the fourth oligonucleotide and the fifth oligonucleotide. (Item 148) The method according to item 146, wherein the second 3' blocking group is selected from a C3 spacer, a hexanediol, spacer 9, spacer 18, three or more rU bases, a phosphate, and a 2'-O-methyl base. (Item 149) The method according to item 146, wherein the fourth oligonucleotide has a length of about 5 to about 100 bases. (Item 150) The method according to item 146, wherein the fifth oligonucleotide has a length of about 10 to about 50 bases. (Item 151) The method according to item 146, wherein the melting temperatures of the fifth oligonucleotide, the fourth oligonucleotide, and the second oligonucleotide are lower than the annealing temperature and the extension temperature. (Item 152) The method described in any of items 138-151, wherein steps (i) through (iv) are performed in the same tube. (Item 153) The method described in any of items 138-151, wherein purification is not performed between step (iii) and step (iv). (Item 154) The method according to any one of items 138 to 153, wherein the first oligonucleotide has a length of about 5 to about 100 bases. (Item 155) The method according to any one of items 138 to 154, wherein the second oligonucleotide has a length of about 5 to about 100 bases. (Item 156) The method according to any one of items 138 to 155, wherein the third oligonucleotide has a length of about 10 to about 50 bases. (Item 157) The method according to any one of items 138 to 156, wherein the first 3' blocking group is selected from a C3 spacer, a hexanediol, a spacer 9, a spacer 18, three or more rU bases, a phosphate, and a 2'-O-methyl base. (Item 158) The method according to any one of items 138 to 157, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), and High Fidelity DNA Polymerase (Qiagen). (Item 159) The method according to any one of items 138 to 157, wherein the DNA polymerase is inactive at the ligation temperature. (Item 160) The method according to item 159, wherein the DNA polymerase further comprises a hot-start antibody or aptamer, the hot-start antibody or aptamer increasing the activity temperature of the DNA polymerase. (Item 161) The method according to item 160, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 Hot Start DNA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara), and High Fidelity Hot Start DNA Polymerase (Qiagen). (Item 162) The method according to any one of items 138-157, wherein the DNA polymerase is a hot-start polymerase, and the denaturation temperature at the first cycle of the at least three PCR cycles in step (iv) is sufficient to activate the DNA polymerase. (Item 163) The method according to any one of items 138 to 162, wherein the ligation temperature is approximately 25°C to approximately 40°C. (Item 164) The method according to any one of items 138 to 163, wherein the denaturation temperature during the at least three PCR cycles is independently between approximately 95°C and approximately 98°C. (Item 165) The method according to any one of items 138 to 164, wherein the annealing temperature during the at least three PCR cycles is independently between approximately 55°C and approximately 65°C. (Item 166) The method according to any one of items 138 to 165, wherein the extension temperature during the at least three PCR cycles is independently between approximately 62°C and approximately 72°C. (Item 167) The method according to any one of items 138 to 166, wherein the denaturation duration during the at least three PCR cycles is independently between about 30 seconds and about 2 minutes. (Item 168) (i) To provide a double-stranded DNA substrate comprising a first strand and a second strand, wherein the first strand comprises a first 3' end portion and a first 5' end portion, and the second strand comprises a second 3' end portion and a second 5' end portion. (ii) Adding ligase, a first oligonucleotide, a second oligonucleotide, a third oligonucleotide, a fourth oligonucleotide, a fifth oligonucleotide, a sixth oligonucleotide, DNA polymerase and deoxynucleotide triphosphate (dNTP) to the double-stranded DNA substrate to produce a first reaction mixture, wherein the first oligonucleotide comprises a third 3' terminal portion and a 5' phosphate complementary to at least a portion of the first 3' terminal portion of the first strand, the third oligonucleotide comprises a first 3' blocking group, and the second oligonucleotide comprises the third oligonucleotide The first oligonucleotide has a 3' portion complementary to the 3' portion of the rheotide, the first oligonucleotide has a 5' portion complementary to the 5' portion of the third oligonucleotide, the fourth oligonucleotide comprises a fourth 3' terminal portion complementary to at least a portion of the second 3' terminal portion of the second chain, and a 5' phosphate, the sixth oligonucleotide comprises a 5' portion complementary to the 5' portion of the fourth oligonucleotide, a 3' portion complementary to the 3' portion of the fifth oligonucleotide, and a second 3' blocking group, the first 3' blocking group and the second 3' blocking group are incapable of polymerase chain elongation, (iii) Incubating the first reaction mixture under a first set of conditions including a ligation temperature for a ligation duration sufficient to produce a second reaction mixture comprising a first primer containing the fourth oligonucleotide ligated to the fifth oligonucleotide, the double-stranded DNA substrate, and a second primer containing the first oligonucleotide ligated to the second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide anneal to the third oligonucleotide in the 3'-5' direction, and the fourth oligonucleotide and the fifth oligonucleotide anneal to the sixth oligonucleotide in the 3'-5' direction, (iv) Each cycle independently undergoes at least three PCR cycles on the second reaction mixture, each cycle including a denaturation temperature over a denaturation duration, an annealing temperature over an annealing duration, and an extension temperature over an extension duration, to generate a third reaction mixture containing the double-stranded library molecule with the second primer at the first 5' end of the double-stranded library molecule and the first primer at the second 5' end of the double-stranded library molecule. A method of ligation-coupled polymerase chain reaction (PCR) including [specific details omitted]. (Item 169) (v) The method of item 168, further comprising subjecting the third reaction mixture to a further PCR cycle to amplify the double-stranded library molecules. (Item 170) The method according to item 168, wherein the ligation temperature is lower than the melting temperature of the double-stranded DNA substrate. (Item 171) The method according to item 168, wherein the ligation temperature is lower than the melting temperatures of the first oligonucleotide and the third oligonucleotide, the ligation temperature is lower than the melting temperatures of the second oligonucleotide and the third oligonucleotide, the ligation temperature is lower than the melting temperatures of the fourth oligonucleotide and the sixth oligonucleotide, and the ligation temperature is lower than the melting temperatures of the fifth oligonucleotide and the sixth oligonucleotide. (Item 172) The method according to item 168, wherein the ligase is a thermally unstable ligase that can be ligated in a low-magnesium PCR buffer. (Item 173) The method according to item 168, wherein the ligase is a T3 DNA ligase. (Item 174) The method according to item 173, wherein the ligase is added in units of about 30 to about 300 enzymes per 50 μL of the first reaction mixture. (Item 175) The method according to item 168, wherein the ligase is temperature-sensitive and the denaturation temperature at the first cycle of the at least three PCR cycles in step (iv) is sufficient to inactivate the ligase. (Item 176) The method according to any one of items 168 to 175, wherein the first 3' blocking group and the second 3' blocking group are each independently selected from a C3 spacer, a hexanediol, spacer 9, spacer 18, three or more rU bases, a phosphate, and a 2'-O-methyl base. (Item 177) The method according to any one of items 168 to 176, wherein the melting temperatures of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide are lower than the annealing temperature and the extension temperature, and the melting temperatures of the fourth oligonucleotide, the fifth oligonucleotide, and the sixth oligonucleotide are lower than the annealing temperature and the extension temperature. (Item 178) The method described in any of items 168-177, wherein steps (i) through (iv) are performed in the same tube. (Item 179) The method described in any of items 168-177, wherein purification is not performed between step (iii) and step (iv). (Item 180) The method according to any one of items 138 to 179, wherein the first oligonucleotide has a length of about 5 to about 100 bases. (Item 181) The method according to any one of items 138 to 180, wherein the second oligonucleotide has a length of about 5 to about 100 bases. (Item 182) The method according to any one of items 138 to 181, wherein the third oligonucleotide has a length of about 10 to about 50 bases. (Item 183) The method according to any one of items 138 to 182, wherein the fourth oligonucleotide has a length of about 5 to about 100 bases. (Item 184) The method according to any one of items 138 to 183, wherein the fifth oligonucleotide has a length of about 5 to about 100 bases. (Item 185) The method according to any one of items 138 to 141, wherein the sixth oligonucleotide has a length of about 10 to about 50 bases. (Item 186) The method according to any one of items 138 to 185, wherein the first 3'-blocking group and the second 3'-blocking group are each independently selected from the group consisting of a C3 spacer, a hexanediol, spacer 9, spacer 18, three or more rU bases, a phosphate, and a 2'-O-methyl base. (Item 187) The method according to any one of items 138 to 186, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), and High Fidelity DNA Polymerase (Qiagen). (Item 188) The method according to any one of items 138 to 186, wherein the DNA polymerase is inactive at the ligation temperature. (Item 189) The method according to item 188, wherein the DNA polymerase further comprises a hot-start antibody or aptamer, the hot-start antibody or aptamer raising the activation temperature of the DNA polymerase. (Item 190) The method according to item 189, wherein the DNA polymerase is selected from the group consisting of Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 Hot Start DNA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara), and High Fidelity Hot Start DNA Polymerase (Qiagen). (Item 191) The method according to any one of items 138-186, wherein the DNA polymerase is a hot-start polymerase and the denaturation temperature at the first cycle of the at least three PCR cycles in step (iv) is sufficient to activate the DNA polymerase. (Item 192) The method according to any one of items 138 to 191, wherein the ligation temperature is approximately 25°C to approximately 40°C. (Item 193) The method according to any one of items 138 to 192, wherein the denaturation temperature during the at least three PCR cycles is independently between approximately 95°C and approximately 98°C. (Item 194) The method according to any one of items 138 to 193, wherein the annealing temperature during the at least three PCR cycles is independently between approximately 55°C and approximately 65°C. (Item 195) The method according to any one of items 138-194, wherein the extension temperature during the at least three PCR cycles is independently between approximately 62°C and approximately 72°C. (Item 196) The method according to any one of items 138-195, wherein the denaturation duration during at least three PCR cycles is independently about 30 seconds to about 2 minutes. (Item 197) The method according to any one of items 138 to 196, wherein the denaturation temperature during the first cycle of the at least three PCR cycles is higher than the melting temperature of the double-stranded DNA substrate. (Item 198) The method according to any one of items 138-197, wherein step (ii) further comprises adding a seventh oligonucleotide and an eighth oligonucleotide, the second oligonucleotide further comprising a 5' phosphate, the eighth oligonucleotide comprising a third 3' blocking group that is not capable of polymerase chain extension, the eighth oligonucleotide further comprising a 3' portion complementary to the 3' portion of the seventh oligonucleotide and a 5' portion complementary to the second oligonucleotide, the first set of conditions being further sufficient to generate the second primer comprising the seventh oligonucleotide, the second oligonucleotide and the first oligonucleotide in the 5'-3' direction by the ligase ligating the seventh oligonucleotide to the second oligonucleotide. (Item 199) The method according to item 198, wherein the ligation temperature is lower than the melting temperatures of the seventh oligonucleotide, the second oligonucleotide, and the eighth oligonucleotide. (Item 200) The method according to any one of items 198 to 199, wherein the annealing temperature and the extension temperature during at least the first cycle of the at least three PCR cycles are higher than the melting temperatures of the seventh oligonucleotide, the second oligonucleotide, and the eighth oligonucleotide. (Item 201) A first indexing primer including the 3' end portion, A second indexing primer including the 3' end portion, The first ligament and, The first DNA polymerase and A kit that includes this. (Item 202) The kit according to item 201, further comprising a blocker oligonucleotide having a 5' portion that is at least partially complementary to the 3' terminal portion. (Item 203) The kit according to item 202, wherein the blocker oligonucleotide is pre-annealed to the second indexing primer. (Item 204) The kit according to any one of items 202 to 203, wherein the blocker oligonucleotide comprises a first further portion 3' of the 5' portion that is complementary to the second indexing primer and not complementary to the first indexing primer. (Item 205) The kit according to item 204, wherein the blocker oligonucleotide further comprises a 3' hydroxyl group and a hairpin portion located 3' relative to the first further portion, the hairpin portion comprising a first hairpin sequence located 5' relative to a second hairpin sequence, the first hairpin sequence and the second hairpin sequence are complementary and can form a hairpin. (Item 206) The kit according to item 205, wherein the hairpin portion further includes a third hairpin arrangement between the first hairpin arrangement and the second hairpin arrangement. (Item 207) The kit according to item 206, wherein the third hairpin arrangement forms enough loops to enable the first and second hairpin arrangements to form a stable stem loop structure. (Item 208) The kit described in item 206, wherein the third hairpin sequence has a length of approximately 4 to 20 bases. (Item 209) The kit according to any one of items 202 to 204, wherein the blocker oligonucleotide further comprises a second further portion located between the 5' portion and the first further portion, the second further portion being not complementary to the first indexing primer, and the second further portion being not complementary to the second indexing primer. (Item 210) The kit described in item 208, wherein the second further part has a length of approximately 1 to 30 bases. (Item 211) The kit according to any of items 202-204 and 209-211, wherein the blocker oligonucleotide further comprises a 3' modification for inhibiting polymerase elongation. (Item 212) The kit described in item 211, wherein the 3' modification for inhibiting polymerase elongation is selected from the group consisting of C3 carbon spacers, hexanediol, spacer 9, spacer 18, phosphate, 2',3'-dideoxynucleosides ddA, ddT, ddC and ddG, 3'-deoxynucleosides 3'-A, 3'-T, 3'-C and 3'-G, RNA nucleosides such as rU, 3-O-methylnucleotides, and DNA sequences that are not complementary to the second indexing primer. (Item 213) A kit according to any one of items 201 to 212, wherein the first indexing primer and the second indexing primer each independently have a length of about 20 to about 100 bases. (Item 214) A first indexing primer including the first 3' end portion, A second indexing primer including the second 3' end portion, A 5' adapter including a third 3' end portion, The first ligament and, The first DNA polymerase and A kit that includes this. (Item 215) The kit according to item 214, wherein the 5' adapter further comprises a first 5' portion located 5' relative to the third 3' portion, which includes at least a portion of the first 3' portion of the first indexing primer; a second 5' portion located 5' relative to the first 5' portion and complementary to the first 5' portion; and a replication blocker located at the 5' end of the first 5' portion that is capable of inhibiting the DNA polymerase. (Item 216) The kit according to item 215, wherein the first 5' portion and the second 5' portion each have a length of about 12 to about 20 base pairs. (Item 217) The kit according to any one of items 214 to 216, wherein the 5' adapter further includes an intervening array between the first 5' portion and the second 5' portion. (Item 218) The kit described in item 217, wherein the intervening sequence has a length of approximately 4 to 20 bases. (Item 219) The kit according to any of items 214-218, wherein the replication blocker is selected from the group consisting of a stable debase site, a C3 spacer, a hexanediol, a spacer 9, a spacer 18, three or more rU bases, and a 2'-O-methylRNA base. (Item 220) The kit described in any of items 214-219, wherein the 5' adapter has a length of approximately 25 to 100 bases. (Item 221) The kit according to any of items 214-220, wherein the first 5' portion and the second 5' portion can form a hairpin. (Item 222) The kit according to any one of items 201 to 221, wherein the first ligase is a thermally unstable ligase that can be ligated in a low-magnesium buffer. (Item 223) The kit described in item 222, wherein the first ligase described above is T3 DNA ligase. (Item 224) The kit according to any one of items 201 to 223, wherein the first indexing primer further comprises a 5' tail sequence containing the sequence of SEQ ID NO: 1, and the DNA polymerase has 3'-5' exonuclease activity. (Item 225) The kit according to any one of items 201 to 224, wherein the second indexing primer further comprises the 5' tail sequence, and the DNA polymerase has 3'-5' exonuclease activity. (Item 226) The kit according to any one of items 201 to 225, wherein the first indexing primer further comprises a 5' tail sequence containing two or more deoxynucleotides at the 5' end of three or more ribonucleotide bases, and the DNA polymerase has 3'-5' exonuclease activity. (Item 227) The kit according to item 226, wherein the second indexing primer further comprises the 5' tail sequence, and the DNA polymerase has 3'-5' exonuclease activity. (Item 228) A kit according to any one of items 224 to 227, further comprising a probe oligonucleotide having modifications complementary to the 5' tail sequence to obtain resistance to digestion by an enzyme having 3' exonuclease activity, the enzyme having 3' exonuclease activity, and a second ligase. (Item 229) The kit described in item 228, wherein the enzyme having exonuclease activity is exonuclease III. (Item 230) A kit as described in any of items 201-229, further comprising a target-specific primer pair. (Item 231) A kit as described in any of items 201-229, further comprising multiple target-specific primer pairs. (Item 232) A kit as described in any of items 201-231, further comprising a universal primer containing a dU base, ribonucleotide, or inosine base. (Item 233) A kit as described in any of items 230-232, further comprising a second DNA polymerase. (Item 234) A kit as described in any of items 201-233, further comprising an enzyme capable of cleaving dU bases. (Item 235) The kit described in item 234, wherein the enzyme capable of cleaving dU bases is a combination of uracil DNA glycosylase and endonuclease VIII. (Item 236) A kit as described in any of items 201-233, further comprising an enzyme capable of cleaving ribonucleotides. (Item 237) The kit described in item 236, wherein the enzyme capable of cleaving ribonucleotides is RNase H. (Item 238) A kit as described in any of items 201-233, further comprising an enzyme capable of cleaving inosine bases. (Item 239) The kit described in item 238 is one in which the enzyme capable of cleaving inosine bases is endonuclease V. (Item 240) The kit described in any of items 201 to 239, wherein the first DNA polymerase and the second DNA polymerase are each independently selected from the group consisting of Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), and High Fidelity DNA Polymerase (Qiagen). (Item 241) The kit according to any one of items 201 to 240, wherein the first DNA polymerase further comprises a hot-start antibody or aptamer, the hot-start antibody or aptamer raising the activation temperature of the DNA polymerase. (Item 242) The kit described in item 241, wherein the first DNA polymerase is selected from the group consisting of Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 Hot Start DNA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara), and High Fidelity Hot Start DNA Polymerase (Qiagen). (Item 243) A kit according to any one of items 215 to 242, wherein the first indexing primer and the second indexing primer each independently have a length of about 20 to about 100 bases. (Item 244) (i) To provide a partially double-stranded DNA substrate comprising a first strand and a second strand, wherein the partially double-stranded DNA substrate comprises a first 3' overhang, a double-stranded portion and a second 3' overhang, The first chain includes a first 5' end, a first portion, and a second portion in the 5' to 3' direction, The second strand includes, in the 5' to 3' direction, a second 5' end, a third portion, and a fourth portion of the partially double-stranded DNA substrate. The first portion of the first chain and the third portion of the second chain are complementary, forming the double-stranded portion. The second portion of the first chain forms the first 3' overhang. The fourth portion of the second chain forms the second 3' overhang. The second portion of the first strand and the fourth portion of the second strand each contain a first common nucleotide sequence located at the 5' end of the first 3' overhang and the 5' end of the second 3' overhang, (ii) Adding a plurality of first indexing primers, a plurality of second indexing primers, a ligase, a DNA polymerase, and a deoxynucleotide triphosphate (dNTP) to the partially double-stranded DNA substrate to produce a first reaction mixture, wherein each of the plurality of first indexing primers contains a first 3' terminal portion complementary to the first common nucleotide sequence, and each of the plurality of second indexing primers contains a second 3' terminal portion complementary to the first common nucleotide sequence. (iii) Incubating the first reaction mixture under a first set of conditions including a ligation temperature over the duration of the ligation, the first set of conditions being a) The 3' end portion of the first nucleotide anneals to the first common nucleotide sequence, b) The ligase ligates one of the plurality of first indexing primers to the first 5' end of the first chain and one of the plurality of first indexing primers to the second 5' end of the second chain, In the 5' to 3' direction, a third chain comprising one of the plurality of first indexing primers, the first portion and the second portion, In the 5' to 3' direction, a fourth chain including one of the plurality of first indexing primers, the third portion and the fourth portion It is sufficient to generate a second reaction reaction containing, (iv) Incubating the second reaction mixture under a second set of conditions comprising a first denaturation temperature over a first denaturation duration, a first annealing temperature over a first annealing duration, and a first extension temperature over a first extension duration, wherein the second set of conditions is a) Inactivate the ligase, denature the double-stranded DNA, and activate the DNA polymerase as necessary. b) The second 3' end portion of one of the plurality of second indexing primers anneals to the first common nucleotide sequence of the second portion of the third chain, and the second 3' end portion of one of the plurality of second indexing primers anneals to the first common nucleotide sequence of the fourth portion of the fourth chain, c) The DNA polymerase extends one of the plurality of second indexing primers annealed to the first common nucleotide sequence of the second portion of the third strand, and the DNA polymerase extends one of the plurality of second indexing primers annealed to the first common nucleotide sequence of the fourth portion of the fourth strand, to produce a third reaction mixture comprising the third strand, the fourth strand, the fifth strand, and the sixth strand, wherein the fifth strand comprises, in the 5' to 3' direction, one of the plurality of second indexing primers, the third portion, and the reverse complement of one of the plurality of first indexing primers, and the sixth strand comprises, in the 5' to 3' direction, one of the plurality of second indexing primers, the first portion, and the reverse complement of one of the plurality of first indexing primers, (v) Incubating the third reaction mixture under a third set of conditions comprising a second denaturation temperature over a second denaturation duration, a second annealing temperature over a second annealing duration, and a second extension temperature over a second extension duration, wherein the third set of conditions is a) Denaturing double-stranded DNA, b) One of the plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers of the fifth chain, and one of the plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers of the sixth chain, c) The DNA polymerase extends one of the plurality of first indexing primers annealed to the reverse complement of one of the plurality of first indexing primers of the fifth strand, and one of the plurality of first indexing primers annealed to the reverse complement of one of the plurality of first indexing primers of the sixth strand, thereby forming a fourth reaction mixture comprising the fifth strand, the sixth strand, the seventh strand, and the eighth strand, wherein the seventh strand is 5' to 3 Sufficient to produce a fourth reaction mixture comprising, in the ' direction, one of the plurality of first indexing primers, the first portion, and the reverse complement of one of the plurality of second indexing primers, and the eighth chain comprising, in the 5' to 3' direction, one of the plurality of first indexing primers, the third portion, and the reverse complement of one of the plurality of second indexing primers, with the seventh chain being complementary to the fifth chain and the eighth chain being complementary to the sixth chain, and (vi) The fourth reaction mixture is incubated under a fourth set of conditions comprising a third denaturation temperature over a third denaturation duration, a third annealing temperature over a third annealing duration, and a third extension temperature over a third extension duration, wherein the fourth set of conditions is sufficient to amplify at least a portion of the plurality of first indexing primers and at least a portion of the plurality of second indexing primers, the fifth and seventh chains and the sixth and eighth chains. A method of ligation-coupled polymerase chain reaction (PCR) including [specific details omitted]. (Item 245) The method according to any one of items 1 to 118, further comprising step (i) being carried out at step (iii) to provide an initiating double-stranded DNA substrate molecule comprising a first start strand and a second start strand, wherein the first start strand and the second start strand each contain at least one dU base inside the 5' ends of the first start strand and the second start strand, and step (ii) further comprises adding an enzyme capable of cleaving the at least one dU base of the double-stranded DNA substrate molecule, wherein the second set of conditions at step (iv) is further sufficient to inactivate the enzyme capable of cleaving the at least one dU base, and the intermediate of step (iii) is a partially double-stranded DNA substrate that can subsequently be ligated according to step (iii). (Item 246) The method according to item 245, wherein the enzyme capable of cleaving at least one dU base is a combination of UDG and endonuclease VIII. (Item 247) The method according to any one of items 1 to 118, further comprising step (i) being carried out at step (iii) to provide an initial double-stranded DNA substrate molecule comprising a first start strand and a second start strand, wherein the first start strand and the second start strand each contain at least one ribonucleotide inside the 5' ends of the first start strand and the second start strand, respectively, and step (ii) further comprises adding an enzyme capable of cleaving the at least one ribonucleotide of the double-stranded DNA substrate molecule, wherein the second set of conditions at step (iv) is further sufficient to inactivate the enzyme capable of cleaving the at least one ribonucleotide, and the intermediate of step (iii) is a partially double-stranded DNA substrate that can subsequently be ligated according to step (iii). (Item 248) The method according to item 247, wherein the enzyme capable of cleaving at least one ribonucleotide is RNase H. (Item 249) The method according to any one of items 1 to 118, further comprising step (i) being carried out at step (iii) to provide an initial double-stranded DNA substrate molecule comprising a first start strand and a second start strand, wherein the first start strand and the second start strand each contain at least one inosine base inside the 5' ends of the first start strand and the second start strand, respectively, and step (ii) further comprises adding an enzyme capable of cleaving the at least one inosine base of the double-stranded DNA substrate molecule, wherein the second set of conditions at step (iv) is further sufficient to inactivate the enzyme capable of cleaving the at least one inosine base, and the intermediate of step (iii) is a partially double-stranded DNA substrate that can subsequently be ligated according to step (iii). (Item 250) The method according to item 249, wherein the enzyme capable of cleaving at least one inosine base is endonuclease V. (Item 251) The method according to any one of items 245 to 250, wherein the initial double-stranded DNA substrate molecule comprises a complementary strand annealed to the 3' overhang of the initial double-stranded DNA substrate molecule. (Item 252) The method according to item 252, wherein the complementary chain comprises a dU base, a ribonucleotide, or an inosine base. [Brief explanation of the drawing]

[0032] [Figure 1A-1] Figure 1A illustrates an exemplary workflow in which a partially double-stranded DNA substrate is generated by multiplex PCR using target-specific and universal primers with dU bases, followed by endonuclease cleavage to generate a 3' overhang, then 5' adapter ligation, and indexing PCR using two indexing primers, each having a 3' terminal portion complementary to the 5' portion of the 3' overhang (first common nucleotide sequence), with one indexing primer being able to function as a 5' adapter and the other being unable to as it anneals to a blocker oligonucleotide. [Figure 1A-2] Same as above. [Figure 1A-3] Same as above.

[0033] [Figure 1B-1]Figure 1B illustrates an exemplary workflow in which a partially double-stranded DNA substrate is generated by multiplex PCR using target-specific and universal primers with dU bases, followed by endonuclease cleavage to generate a 3' overhang, then 5' adapter ligation using a hairpin adapter having a 3' terminal sequence complementary to the 5' portion of the 3' overhang (first common nucleotide sequence), and indexing PCR using two indexing primers that do not contain a 3' terminal portion complementary to the 5' portion of the 3' overhang (first common nucleotide sequence), but the second indexing primer has a 3' terminal portion complementary to the second common nucleotide sequence on the 3' side of the first common nucleotide sequence. The hairpin contains a portion of the first indexing primer that enables subsequent PCR using the first indexing primer after the first PCR cycle. [Figure 1B-2] Same as above. [Figure 1B-3] Same as above.

[0034] [Figure 1C] Figure 1C illustrates an exemplary workflow in which the indexing primer includes a 5' normalization tail that enables library normalization after ligation-coupled PCR. Figure 1C discloses (T)12(rU)4 as Sequence ID No. 1.

[0035] [Figure 1D] Figure 1D illustrates an exemplary workflow in which the indexing primer includes a 5' normalization tail that enables library normalization after ligation-coupled PCR. Figure 1D discloses (T)12(rU)4 as Sequence ID No. 1.

[0036] [Figure 1E] Figure 1E illustrates an NGS library preparation method that includes DNA repair, ligation of a 3' adapter with blunt ends, and ligation-coupled PCR with full-size indexing primers and primer blockers.

[0037] [Figure 1F] Figure 1F illustrates an NGS library preparation method that includes DNA repair, A-tailing, ligation of a 3' adapter with a U-overhang, and ligation-coupled PCR with full-size indexing primers and primer blockers.

[0038] [Figure 1G] Figure 1G illustrates an NGS library preparation method that includes DNA repair, ligation of a blunt-ended 3' adapter, and ligation-coupled PCR with truncated indexing primers and a hairpin 5' adapter.

[0039] [Figure 1H] Figure 1H illustrates an NGS library preparation method that includes DNA repair, ligation of a 3' adapter with A-tailing and U-overhang, and ligation-coupled PCR with truncated indexing primers and a hairpin 5' adapter.

[0040] [Figure 1I] Figure 1I shows a comparison of adapter-dimer formation in different NGS library protocols.

[0041] [Figure 2A-1] Figure 2A illustrates an exemplary workflow in which a partially double-stranded DNA substrate is generated by endonuclease cleavage, which produces a 3' overhang, followed by 5' adapter ligation, and indexing PCR using two indexing primers, each having a 3' terminal portion complementary to the 5' portion of the 3' overhang (first common nucleotide sequence), with either indexing primer functioning as the 5' adapter. [Figure 2A-2] Same as above. [Figure 2A-3] Same as above.

[0042] [Figure 2B] Figure 2B illustrates an exemplary workflow in which a partially double-stranded DNA substrate is generated by indexing PCR using two indexing primers, each having a 3' terminal portion complementary to the 5' portion of the 3' overhang (first common nucleotide sequence), followed by endonuclease cleavage to produce a 3' overhang, followed by 5' adapter ligation, and then indexing PCR using two indexing primers. One of the indexing primers can function as a 5' adapter, while the other cannot, as it anneals to a blocker oligonucleotide.

[0043] [Figure 2C] Figure 2C shows further details of (a) linear blockers that are inactive during PCR due to having a low Tm, or (b) hairpin blockers that are inactivated during PCR when using the Illumina TruSeq adapter. Figure 2C discloses sequence numbers 78-80, 78-80, 78-79, 81-82, 78-79, and 83-84 in order of appearance.

[0044] [Figure 2D] Figure 2D shows the Illumina TruSeq adapter sequences in terms of multiplex PCR, endonuclease cleavage of the incorporated universal primer, linear blocker 5 with three mismatches pre-annealed to the 3' adapter indexing primer (i7), and ligation of the 3' end of the 5' adapter indexing primer (i5) to the 5' portion of the reverse complement of the universal adapter sequence of the substrate amplicon. Figure 2D discloses sequence numbers 85, 85-87, 42, 78-79, 89, 86, 78-79, 89, 87, 42, and 86 in order of appearance.

[0045] [Figure 2E]Figure 2E shows the Illumina TruSeq adapter sequences in terms of multiplex PCR, endonuclease cleavage of the incorporated universal primer, linear blocker 5 having a pre-annealed 6T insertion into the 3' adapter indexing primer (i7), and ligation of the 3' end of the 5' adapter indexing primer (i5) to the 5' end of the substrate amplicon. Figure 2E discloses sequence numbers 85, 85-87, 42, 78-79, 89, 86, 78-79, 89, 87, 42, and 86 in order of appearance.

[0046] [Figure 2F] Figure 2F represents a linear blocker having three degradable U bases in addition to a Tm-reducing T*G mismatch. Figure 2F discloses sequence numbers 78-79, 49, 78-79, 90, 78-79, and 91 in order of appearance.

[0047] [Figure 2G] Figure 2G represents NGS library preparation, including DNA repair, blunt-end 3' adapter ligation, and ligation-coupled PCR with a full-size indexing primer and an i7 primer blocker containing one mismatch and several degradable dU bases. Figure 2G discloses sequence numbers 92, 92-93, 93, 87, 42, 78-79, 90, 93, 78-79, 90, 87, 42, and 93 in order of appearance.

[0048] [Figure 3A] Figure 3A shows a ligation-coupled PCR reaction using truncated indexing primers i5 and i7 lacking 13 bases at their 3' ends, along with a linear 5' adapter.

[0049] [Figure 3B] Ligation-coupled PCR reaction using truncated indexing primers i5 and i7 lacking 13 bases at the 3' end, and a truncated hairpin 5' adapter.

[0050] [Figure 3C] Figure 3C further illustrates that the stem-loop truncated adapter 5 further includes non-replicable modifications represented by black circles within the loop arrangement, thus preventing the formation of a completely replicated hairpin product.

[0051] [Figure 3D] Figure 3D illustrates the undesirable consequences when a stem loop truncated adapter lacks the non-replicable modifier within the loop array.

[0052] [Figure 3E] Figure 3E illustrates a specific embodiment using the TruSeq Illumina adapter workflow when a hairpin truncated adapter, including an internal non-replicable C3 spacer, is used for ligation-coupled PCR. Figure 3E discloses sequence numbers 85, 85-86, 94, 42, 87, 95, 78, 96-97, 86-87, 95, 78, 96-97, 94, 42, 78, 96, and 93 in order of appearance.

[0053] [Figure 3F] Figure 3F illustrates an alternative embodiment using the TruSeq Illumina adapter workflow, in which a hairpin truncated adapter with an internal non-replicable C3 spacer is used for ligation-coupled PCR. Figure 3F discloses sequence numbers 98, 98, 86, 94, 53, 87, 95, 78, 96-97, 86-87, 95, 78, 96-97, 94, 42, 78, 96, and 93 in order of appearance.

[0054] [Figure 3G]Figure 3G illustrates the Nextera Illumina adapter workflow used for ligation-coupled PCR with a hairpin truncated adapter, full-length indexing primers, and a linear blocker. Figure 3G discloses sequence numbers 99, 99-102, 87, 103, 78, 104-105, 100, 87, 103, 78, 104-105, 101, 103, and 100 in order of appearance.

[0055] [Figure 3H] Figure 3H illustrates another Nextera Illumina adapter workflow in which a hairpin truncated adapter is used with an indexing primer lacking a common adapter sequence for ligation-coupled PCR. In this example, a universal amplicon primer containing dU bases replaces all five T deoxynucleotides, but the nine base sequences at the 3' end are retained due to the absence of T bases when USER cleavage is performed. Figure 3H discloses SEQ ID NOs. 99, 99-102, 87, 106, 78, 107-108, 100, 87, 106, 78, 107-108, 101, 103, 78, 107, and 109 in order of appearance, respectively.

[0056] [Figure 3I] Figure 3I represents NGS library preparation, including DNA repair, A tailing, ligation of a 3' adapter with a U base overhang, and ligation-coupled PCR with a truncated indexing primer and a hairpin 5' adapter with a 13b 3' overhang. Figure 3I discloses SEQ ID NOs. 51, 51, 24, 93-94, 42, 87, 95, 78, 96, 93, 87, 95, 94, 42, 78, 96, and 93 in order of appearance.

[0057] [Figure 3J]Figure 3J illustrates NGS library preparation, including DNA repair, ligation of a blunt-end 3' adapter, and ligation-coupled PCR with a truncated indexing primer and a hairpin 5' adapter with a 13b 3' overhang. Figure 3J discloses SEQ ID NOs. 92, 92–93, 93–94, 42, 87, 95, 78, 96, 93, 87, 95, 94, 42, 78, 96, and 93 in order of appearance.

[0058] [Figure 3K] Figure 3K represents NGS library preparation, including DNA repair, A tailing, ligation of a 3' adapter with a T base overhang, and ligation-coupled PCR with a truncated indexing primer and a hairpin 5' adapter with an 11b 3' overhang. Figure 3K discloses SEQ ID NOs. 52, 52, 24, 93-94, 53, 87, 95, 78, 96, 93, 87, 95, 94, 42, 78, 96, and 93 in order of appearance.

[0059] [Figure 3L] Figure 3L represents NGS library preparation including DNA repair, A tailing, ligation of a 3' adapter with a T base overhang, and ligation-coupled PCR with a full-size indexing primer, a blocker containing one mismatch and several degradable U bases, and a hairpin 5' adapter with a 10b overhang. Figure 3L discloses sequence numbers 110, 110-111, 111, 101-102, 87, 103, 78, 104-105, 112, 87, 103, 78, 104-105, 101, 103, and 100 in order of appearance.

[0060] [Figure 4A] Figure 4A shows exemplary structures of linear and hairpin blockers.

[0061] [Figure 4B]Figure 4B illustrates the workflow using blocker oligonucleotides.

[0062] [Figure 4C] Figure 4C shows one of the blocker and indexing primers.

[0063] [Figure 4D] Figure 4D illustrates a workflow using a hairpin blocker.

[0064] [Figure 4E] Figure 4E illustrates an exemplary workflow of a hairpin blocker, demonstrating that an elongating stem can increase the melting temperature of an elongating hairpin.

[0065] [Figure 5A] Figure 5A shows the hairpin adapter and the first indexing primer.

[0066] [Figure 5B] Figure 5B shows the workflow using a hairpin adapter.

[0067] [Figure 5C] Figure 5C illustrates the workflow using a hairpin adapter.

[0068] [Figure 6A] Figure 6A shows an exemplary workflow for splint-mediated primer assembly.

[0069] [Figure 6B] Figure 6B illustrates in detail how normalase-compatible primers containing (T)12(rU)4 (SEQ ID NO: 1) at their 5' ends are assembled. Figure 6B discloses (T)12(rU)4 as SEQ ID NO: 1.

[0070] [Figure 6C]Figure 6C shows an assembly of TruSeq Illumina indexing primers having the (T)12(rU)4 (SEQ ID NO: 1) sequence, which is compatible with downstream enzyme normalization. Figure 6C discloses SEQ ID NOs. 113-118 and 116-117 in order of appearance.

[0071] [Figure 6D] Figure 6D illustrates the ligation-coupled PCR workflow when the primer assembly of the i5 TruSeq Illumina indexing primer shown above is combined with the assembly of the corresponding i7 indexing primer containing the 3' adapter sequence, annealing of the hairpin blocker to the i7 primer, and ligation of the i5 primer to an amplicon substrate containing the truncated 3' adapter (followed by endonuclease cleavage, not shown). Figure 6D discloses sequence numbers 113–117, 119–122, 83, 82, 93, 118, 116–118, 116–117, 93, 123, 122, 83, and 82 in order of appearance.

[0072] [Figure 7] Figure 7 shows the results of Experiment Example 1. ITS1 rRNA amplicon coverage for Candida albicans observed with IGV, with reads starting from the forward primer plotted at the top and reads from the reverse primer plotted at the bottom of the IGV plot.

[0073] [Figure 8] Figure 8 shows the results of Experimental Example 3. Using T3 DNA ligase for sprint ligation under conditions optimized for PCR, assembly of indexing primers by ligation was observed for over 90%, while T4 DNA ligase was inefficient.

[0074] [Figure 9] Figure 9 discloses sequences 44, 49, 44, 50, 44, 124, 44, 125, 44, and 46 in the order of their appearance.

[0075] Figure 9 shows the double-stranded DNA structure formed by indexing primer i7 having different primer blockers as described in Example 5.

[0076] [Figure 10A] Figure 10A shows the structure of the 3' and 5' adapters of the NGS library described in Example 6. Figure 10A discloses sequence numbers 52, 51, 24, 24, 45, 53, 45, and 42 in the order in which they appear.

[0077] [Figure 10B] Figure 10B shows a Picard plot for the NGS library described in Example 6 (NGS library prep A 50ng).

[0078] [Figure 10C] Figure 10C shows a Picard plot for the NGS library described in Example 6 (NGS library prep A 250ng).

[0079] [Figure 10D] Figure 10D shows a Picard plot for the NGS library described in Example 6 (NGS library prep B 50ng).

[0080] [Figure 10E] Figure 10E shows a Picard plot for the NGS library described in Example 6 (NGS library prep B 250ng).

[0081] [Figure 11A] Figure 11A shows a Bio Analyzer trace for a library prepared from DNA pictogram amounts by the method described in Example 6.

[0082] [Figure 11B]Figure 11B shows a Bio Analyzer trace for a library prepared from DNA pictogram amounts by the method described in Example 6.

[0083] [Figure 11C] Figure 11C shows a Bio Analyzer trace for a library prepared from DNA pictogram amounts by the method described in Example 6. [Modes for carrying out the invention]

[0084] The diagram shows that the reaction takes place in a single sealed tube, and it should be understood that if the brackets are on consecutive pages of the diagram, all steps of all brackets take place in the same sealed tube. For example, Figure 1A spans three pages, but it should be understood that all steps after the multiplex PCR take place in the same "single sealed tube," even though separate brackets appear on each page.

[0085] Detailed explanation This disclosure describes certain embodiments with reference to certain drawings, but the scope is not limited to those. The drawings are schematic and not limiting. In the drawings, the size of some elements may be exaggerated or altered for illustrative purposes and may not be depicted to actual size. Elements of this disclosure are assigned the definite article “a” or “an” on their first appearance, and the definite article “the” or “said” on their second or subsequent appearances, unless otherwise specifically indicated.

[0086] This disclosure provides a description of the accompanying drawings, which illustrate some, but not all, embodiments of the subject matter of this disclosure. In fact, the subject matter may be embodied in many different forms and should not be construed as being limited to the embodiments described herein, rather these embodiments are provided so as to satisfy all legal requirements of this disclosure.

[0087] definition Certain terms are used for convenience in the following explanation and are not limiting. Certain terms used herein indicate direction in the referenced drawings. Unless specifically stated herein, the terms “a,” “an,” and “the” should be understood to mean “at least one,” not limited to a single element. Where used herein, “another” means at least the second or subsequent. The terms include those mentioned above, their derivatives, and similar terms of importance.

[0088] The use of the term "or" in the claims shall mean "and / or" unless it is explicitly stated that it refers only to the alternatives, or that the alternatives are mutually exclusive.

[0089] When used with a number, the term "approximately" is intended to include a + / - 10% range. For example, if the number of amino acids is specified as approximately 200, this includes 180 to 220 (plus or minus 10%).

[0090] As used herein, “low magnesium buffer” can be any buffer having a sufficiently low magnesium level to be suitable for PCR. For example, without limitation, a low magnesium buffer may have 1–2 mM or less magnesium.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those widely understood by those skilled in the art.

[0092] This disclosure provides a method for ligation-coupled PCR. Using this method, adapters can be added to a DNA substrate for next-generation sequencing (NGS) and other applications. Methods for assembling and using sprint-mediated primers by ligation-coupled PCR are also provided. Kits for carrying out the methods of this disclosure are also provided.

[0093] It should be understood that the methods disclosed herein can be used with any start DNA substrate containing, or processable to contain, two 3' overhangs, or any partially double-stranded DNA substrate having two 3' overhangs. This then allows for subsequent ligation of a 5' adapter and PCR amplification of the ligated double-stranded DNA substrate with primers. While the embodiments disclosed herein are disclosed in some cases with respect to specific upstream processing methods, they should not be construed as being limited to those methods only.

[0094] Disclosed is a method of ligation-coupled PCR in which i) a PCR substrate is assembled from DNA subunits by ligation and amplified by PCR, ii) a PCR primer is assembled from DNA subunits by ligation and used for amplification by PCR, or a combination thereof in which the ligation and amplification reactions are performed in a single sealed tube. Regarding i), the PCR substrate is generated by ligation of tandem oligonucleotides coupled with complementary sprint oligonucleotides. Alternatively, the PCR substrate is a truncated NGS library containing a first adapter having cleavable bases, and an endonuclease cleaves one strand of the first adapter to enable annealing and ligation of the second adapter. Regarding ii), the PCR primer is also assembled by ligation of tandem oligonucleotides coupled with complementary sprint oligonucleotides.

[0095] In one embodiment, a multiplexed amplicon workflow for ligation-coupled PCR is shown utilizing full-length indexing primers 3 (first indexing primer) and 4 (second indexing primer) containing a common adapter sequence at their 3' ends (Figure 1A). Universal-tailed target-specific primers P1 and P2 (representing multiple primer pairs) are shown together with universal primer 1, which contains cleavable dU bases in multiplexed PCR and is complementary to the universal tails of P1 and P2. The second reaction involves simultaneously performing the following steps in a single sealed tube: endonuclease cleavage of the incorporated universal primer 1 with the USER enzyme to generate a partially double-stranded substrate DNA molecule 10 containing a first strand 11 and a second strand 12; ligation of the 5' end of the substrate DNA 10 with the 5' adapter indexing primer 3 (first indexing primer) after annealing to at least a portion of the reverse complement of the universal primer sequence 2, i.e., the first common nucleotide sequence located at the 5' end of the overhangs in both 3' overhangs; and, if necessary, preventing the 3' adapter indexing primer 4 (second indexing primer) from participating in the ligation reaction with a pre-annealed linear blocker 5 (because universal primer 1 contains the same sequence as the 3' portion of 3' adapter 4 (second indexing primer)); followed by PCR indexing to amplify the library and complete the adapter sequence. The 5' adapter 3 (first indexing primer), when ligated, can generate the third chain 13 and the fourth chain 14. The blocker prevents the ligation of the second indexing primer 4, for example, by having a melting temperature (T) higher than the temperature at which the ligation reaction occurred. mLigation of the second indexing primer 4 to the substrate DNA molecule 10 does not occur because it can have ). In the first PCR cycle, either the first indexing primer 3 or the second indexing primer 4 can anneal to at least a portion of the reverse complement of the universal primer sequence (containing the first common nucleotide sequence) 2, but when the first indexing primer 3 anneals and extends, it produces a product in which the first indexing primer and its complement are at opposite ends, which can be suppressed in further PCR cycles. Thus, when the second indexing primer 4 anneals to at least a portion of the reverse complement of the universal primer sequence 2, it produces the fifth strand 15 from the third strand 13 as a template and the sixth strand 16 from the fourth strand 14 as a template. Since both the fifth strand 15 and the sixth strand 16 each have a reverse complement of the first indexing primer 3, the first indexing primer 3 can anneal to and extend each in the next PCR cycle to produce the seventh strand 17 and the eighth strand 18, which are complementary to the fifth strand 15 and the sixth strand 16, respectively. In further PCR cycles, these double-stranded products can be further amplified by the first indexing primer 3 and the second indexing primer 4. Figure 1A shows all the steps from endonuclease cleavage via indexing PCR performed in a single sealed tube, but it should be understood that the steps from 5' adapter ligation via indexing PCR can also be performed in a single sealed tube, whether or not endonuclease cleavage (or other enzymatic treatment) is also performed. In all figures and embodiments, it should be understood that the cleavage step for generating partially double-stranded DNA substrates can be performed in a single sealed tube together with ligation and PCR, or it can be performed separately before ligation-coupled PCR. In some embodiments, the enzymatic treatment for obtaining partially double-stranded DNA substrates can be performed in a prior step, or it can be performed as part of the workflow in a single sealed tube, as shown in Figure 1A.

[0096] In another embodiment, a targeted amplicon workflow for ligation-coupled PCR utilizing indexing primers 3 and 4 lacking a common adapter sequence at their 3' ends is shown (Figure 1B). Universal-tailed target-specific primers P1 and P2 (representing multiple primer pairs) are shown together with universal primer 1 containing a cleavable dU base in multiplexed PCR. The second reaction involves simultaneously endonuclease cleavage of the incorporated universal primer 1 with the USER enzyme in a single sealed tube, ligating the truncated 5' hairpin adapter 6, which has been annealed to at least a portion of the reverse complement of universal primer sequence 2 (e.g., the first common nucleotide sequence), to the end of the amplicon DNA, followed by PCR indexing using indexing primers 3 and 4 to amplify the library and complete the adapter sequence. The secondary structure of the hairpin 5' adapter prevents its activity as a primer during PCR from truncating the 5' adapter from the completed library molecule.

[0097] The method in Figure 1A can be modified for downstream enzymatic library normalization by utilizing indexing primers 3 and 4 which further contain a 5' tail sequence, the 5' tail sequence can contain four consecutive U ribonucleotide (rU) bases and can contain a 5' adjacent to a 12T deoxynucleotide (SEQ ID NO: 2) (Figure 1C). To increase PCR efficiency, the 5'(T) 12 Further normalization primer pairs containing (rU)4 tails (SEQ ID NO: 1) as well as terminal P5 and P7 adapter sequences can also be included in the reaction (oligonucleotides 18-221, 18-222 in Table 1, primers not shown in Figure 1C).

[0098] The method in Figure 1B can be modified for downstream enzymatic library normalization by utilizing indexing primers 3 and 4 which further contain a 5' tail sequence, the 5' tail sequence can contain four consecutive U ribonucleotide (rU) bases and can contain a 5' adjacent to a 12T deoxynucleotide (SEQ ID NO: 2) (Figure 1D). To increase PCR efficiency, the 5'(T) 12 Further pairs of 5' normalization primers containing (rU)4 tails (SEQ ID NO: 1), as well as terminal P5 and P7 adapter sequences, can also be included in the reaction (oligonucleotides 18-221, 18-222 in Table 1, primers not shown in Figure 1D).

[0099] In one embodiment, a truncated NGS library workflow for ligation-coupled PCR utilizes full-length indexing primers 3 and 4 (Figure 1E). In this workflow, fragmented DNA undergoes end repair with a policing DNA polymerase such as T4 or T7 DNA polymerase, and ligation of a blunt-ended 3' adapter formed by blunt-ended oligonucleotides 7 and 8, wherein oligonucleotide 8 has one or more cleavable bases such as a U base and a modified nucleotide at the blunt-ended 3' end that does not participate in the ligation reaction (e.g., a modification in which ribose is deleted from either the 3' hydroxyl or both the 3' and 2' hydroxyl groups), and oligonucleotide 7 has a phosphate at the blunt-ended 5' end that participates in the ligation reaction. After bead purification, the final reaction involves simultaneously performing endonuclease cleavage of oligonucleotide 8 with USER enzyme in a single sealed tube, ligating the 5' adapter (indexing primer 3), which has been annealed to the reverse complement of 3' adapter oligonucleotide 7, to the 5' end of the DNA, and, if necessary, preventing indexing primer 4 from participating in the ligation reaction with pre-annealed linear blocker 5 (because indexing primer 4 contains the same sequence as the 3' portion of indexing primer 3). Then, PCR is indexed to amplify the library and complete the adapter sequence.

[0100] The method in Figure 1E can be modified for use with A-tailed DNA. 3'-terminated A-tailing can be achieved by ligating a 3' adapter having a U-base overhang at the 3' end formed by oligonucleotides 7 and 8, after incubation with a DNA polymerase lacking 3' proofreading activity, such as a (3'-exo) Klenow fragment of DNA polymerase I or Taq DNA polymerase, wherein oligonucleotide 9 has at least two cleavable U-bases, including a U-base at its 3' end, which are involved in the ligation reaction, and oligonucleotide 7 has a phosphate at its blunt 5' end, which is also involved in the ligation reaction. After bead purification, the final reaction, as shown in Figure 1F, involves simultaneously performing endonuclease cleavage of oligonucleotide 9 with USER enzyme in a single sealed tube, ligating the 5' end of the DNA with 5' adapter indexing primer 3 after annealing to the reverse complement of 3' adapter oligonucleotide 7, and, if necessary, preventing indexing primer 4 from participating in the ligation reaction with pre-annealed linear blocker 5 (because indexing primer 4 contains the same sequence as the 3' portion of indexing primer 3). Then, PCR is indexed to amplify the library and complete the adapter sequence.

[0101] In another embodiment, a truncated NGS library workflow for ligation-coupled PCR utilizes indexing primers 3 and 4, respectively, for common adapter sequences at their 3' ends (Figure 1G). In this workflow, fragmented DNA undergoes end repair with a policing DNA polymerase such as T4 or T7 DNA polymerase, and ligation of a blunt-ended 3' adapter formed by oligonucleotides 7 and 8, wherein oligonucleotide 8 has one or more cleavable bases, such as a U base, and a modified nucleotide at the blunt-ended 3' end that does not participate in the ligation reaction (e.g., a modification in which ribose is deleted from either the 3' hydroxyl or both the 3' and 2' hydroxyl groups), and oligonucleotide 7 has a phosphate at the blunt-ended 5' end that participates in the ligation reaction. After bead purification, the final reaction involves simultaneously cleaving oligonucleotide 8 with USER enzyme in a single sealed tube, annealing the truncated hairpin 5' adapter 6 to the 5' end of the DNA after annealing it to the reverse complement of 3' adapter oligonucleotide 7, and then amplifying the library by PCR using indexing primers 3 and 4 to complete the adapter sequence. The secondary structure of the hairpin 5' adapter prevents its activity as a primer during PCR, thus preventing truncation of the 5' adapter from the completed library molecule.

[0102] The method in Figure 1G can be modified for use with A-tailed DNA. 3'-terminated A-tailing can be achieved by ligating a 3' adapter formed by oligonucleotides 7 and 8, which have a U-base overhang at the 3' end, after incubation with a DNA polymerase lacking 3' proofreading activity, such as a (3'-exo) Klenow fragment of DNA polymerase I or Taq DNA polymerase. Oligonucleotide 8 has at least two cleavable U-bases, including a U-base at its 3' end, which are involved in the ligation reaction, and oligonucleotide 7 has a phosphate at its blunt 5' end, also involved in the ligation reaction. After bead purification, the final reaction, as shown in Figure 1H, involves simultaneously endonuclease cleavage of oligonucleotide 8 with USER enzyme and ligation of the truncated 5' hairpin adapter 6, which has been annealed to the reverse complement of 3' adapter oligonucleotide 7, to the 5' end of the DNA in a single sealed tube, followed by PCR indexing to amplify the library and complete the adapter sequence. The secondary structure of the hairpin 5' adapter prevents its activity as a primer during PCR, thus preventing truncation of the 5' adapter from the completed library molecule.

[0103] The NGS library preparation method utilizing a blunt-end 3' adapter and a ligation-coupled PCR step (Figures 1E and 1G) offers a substantial improvement to the Accel-NGS 2S DNA library workflow by reducing the number of enzyme steps from five to three and the number of purification steps from five to two, while maintaining the main advantages of Accel-NGS 2S DNA library preparation compared to other available kits, such as high library yield, no need to adjust adapter concentration when changing DNA input, and very low AT / GC bias. The only limitation of the described method is the formation of chimeric DNA at DNA input concentrations exceeding 50–100 ng.

[0104] The NGS library preparation method utilizing a 3' adapter with a U or T overhang and a ligation-coupled PCR step (Figures 1F and 1H) represents a substantial improvement over the Accel-NGS 2S DNA library workflow by reducing the number of enzyme steps from five to three and the number of purification steps from five to two, while retaining all the advantages of the Accel-NGS 2S DNA library, such as high library yield, absence of DNA chimeras, a very wide DNA input range, and the elimination of the need to adjust adapter concentrations when changing DNA input. The absence of adapter dimers and the resulting ability to function with extremely low hemtogram amounts of DNA is a unique characteristic of the workflow shown in Figures 1F and 1H that greatly differentiates it from all previously described NGS library methods. As shown in Figure 1I(a), the strong suppression of adapter dimer formation in the protocol shown in Figure 1G (and 1H) stems from the fact that any oligonucleotide 7 remaining after annealing to primer oligonucleotide 3 and subsequent 3' adapter ligation and purification forms an adapter with a T base overhang that cannot self-ligate when present at low concentrations. As shown in Figure 1I(b, c), adapter dimer formation is also possible in the Accel-NGS 2S (b) and NGS library protocols using the Y adapter (c). In the case of the Accel-NGS 2S library, self-ligation of the blunt adapter (formed by annealing of carry-over 3' adapter 2 and adapter primer 3) can occur even at relatively low adapter concentrations, so adapter dimer formation occurs during the second ligation reaction, and in the case of (c), the T base overhang between the adapter ends * Regardless of T mismatch, adapter dimer formation occurs due to high concentrations of T4 DNA ligase and Y adapter.

[0105] In some embodiments, such as amplicons and NGS libraries with T or U base overhangs, the endonuclease used in ligation-coupled PCR reactions is New England Biolabs' USER enzyme, Qiagen's Uracil Cleavage System, or any other mix of uracil DNA glycosylase (UDG) and endonuclease VIII. In embodiments of NGS libraries utilizing blunt-ended 3' adapters having ddT, 3'-dT, ddG, or 3'-dG bases at the 3' end, where there is no covalent bond between the 3' end of the 3' adapter oligonucleotide and the 5' end of the DNA, destabilization and release of the adapter oligonucleotide can be achieved by incubation with UDG enzyme alone. The UDG enzyme does not cause a break within oligonucleotide 1 but lowers the annealing temperature to below 37°C, dissociating oligonucleotide 8 from complementary 3' adapter oligonucleotide 7 and generating enough debase sites to allow annealing and ligation of 5' adapter indexing primer 3 to the DNA end (Figure 1G).

[0106] How to use blocker oligonucleotides In one embodiment shown in Figure 2A, the workflow presented in Figure 1A can be carried out in the absence of any blockers as needed. As shown, the partially double-stranded DNA substrate molecule 10 includes a first strand 11 that is partially complementary to the second strand 12, and each of the first strand 11 and the second strand 12 forms a 3' overhang 2 at each end of the partially double-stranded DNA substrate molecule 10. With respect to the complementary portion, and for consistency of terminology, this complementary portion may be referred to as the first portion of the first strand and the third portion of the second strand. Throughout this disclosure, it should be understood that the first portion of the first strand and the third portion of the second strand refer to the portion and its sequence. Thus, the PCR product does not contain the first portion but may contain the sequence of the first portion, and is therefore considered to contain the first portion as such. Each overhang also contains a first common nucleotide sequence at the 5' end of each 3' overhang 2. While not intended to be limiting, in Figure 2A, the double-stranded DNA product can be generated by multiplex PCR amplification with a dU base-containing universal primer to include the dU base from universal primer 1, which can be cleaved by an endonuclease to produce a partially double-stranded DNA substrate. In such a case, the 3' overhang 2 can be the reverse complement of universal primer 1. Each of the pair of indexing primers 3 (first indexing primer) and 4 (second indexing primer) is at a temperature higher than the ligation reaction temperature T mBecause the 3' overhang 2 on the substrate amplicon has an identical 3' terminal sequence complementary to the first common nucleotide sequence at its 5' end, both primers can be used in the adapter ligation step. This lack of annealing specificity reduces the formation of functional library molecules that require both 5' and 3' adapters. A functional library is generated by ligation of only the 5' adapter primer 3 (first indexing primer). If the 3' adapter primer 4 (second indexing primer) is ligated, a non-functional library is generated with similar adapters at both ends, as the universal adapter includes a truncated 3' adapter.

[0107] As further shown in Figure 2A, after the ligation step, a third strand 13 and a second oligonucleotide 14 can be obtained, each containing a 3' overhang 2 with one of the first indexing primers 3, either the first portion (in the third strand 13) or the third portion (in the fourth strand 14), and the first common nucleotide sequence, in the 5' to 3' direction. In a further cycle of PCR, the second indexing primer 4 can anneal to the first common nucleotide sequence of the third strand 13 or the fourth strand 14 and be extended by DNA polymerase to produce the fifth strand 15 and the sixth strand 16, respectively, each containing one of the second indexing primers, either the first portion (in the sixth strand 16) or the third portion (in the fifth strand 15), and the reverse complement of the first indexing primer 3', in the 5' to 3' direction. In further PCR cycles, the first indexing primer 3 anneals to the reverse complement of the first indexing primer 3' on the fifth strand 15 or the sixth strand 16, and is then extended by DNA polymerase to generate the seventh strand 17 and the eighth strand 18, which contain the first portion (in the seventh strand 17) or the third portion (in the eighth strand 18) and the reverse complement of the second indexing primer 4' in the 5' to 3' direction. The seventh strand 17 and the eighth strand 18 are complementary to the fifth strand 15 and the sixth strand 16, respectively. Further PCR cycles can allow the first and second indexing primers to amplify the double-stranded seventh strand 17 and the fifth strand 15, as well as the double-stranded eighth strand 18 and the sixth strand 16, to generate the final library. As further shown in Figure 2A, when an oligonucleotide is generated with a first indexing primer on one end and its complement on the other end, for example, when the first indexing primer ligates and the ligated chain is primed, the complementarity of these ends during PCR suppresses the amplification of these undesirable products.The same applies when an oligonucleotide is generated having a second indexing primer at one end and its complement at the other end.

[0108] In an alternative embodiment, a linear blocker 5 is used that is pre-annealed to the full-length 3' adapter indexing primer 4 before being added to the ligation reaction (Figure 2B). This prevents the 3' end of this primer from becoming ligable to the amplicon substrate after it has annealed to sequence 2 at the ligation incubation temperature. However, blocker T m Since the temperature is lower than the PCR annealing temperature, or the blocker is inactivated, the 3' adapter indexing primer 4 can anneal efficiently during PCR. Figure 2C shows (a) low T m (b) A linear blocker that becomes inactive during PCR because it contains i7 indexing primer 4, and a lower T that is inactivated and therefore can anneal to the 3' adapter indexing primer during the ligation reaction but does not anneal during PCR. m Linear blocker 5a or hairpin blocker 5b having: (a) Linear blocker 5a has one or more mismatches (three mismatches are shown) with respect to the universal adapter sequence on the indexing primer 4, or not limited to any one of them, such that the blocker does not inhibit the priming of the indexing primer. mThe insertion of a non-complementary base containing one or more T deoxynucleotides (six shown) reduces the temperature below the PCR annealing temperature. The blocker mismatch or insertion is located at 3' of a sequence complementary to the common adapter sequence, allowing the blocker to anneal to the 3' end of the primer during the ligation step, and the linear blocker also contains a 3'C3 spacer blocking group to prevent elongation. With respect to (b), the hairpin blocker 5b contains a stem-loop hairpin whose 5' overhang allows the blocker to hybridize to the 3' end of the primer during the ligation step, but upon hot-start activation of polymerase, the hairpin blocker elongates from its 3' end, producing a fully double-stranded hairpin that cannot anneal during PCR due to its stable secondary structure at the PCR annealing temperature. Figure 2D shows the lower T due to the mismatch to the adapter sequence. m To further illustrate the method using a linear blocker having the following characteristics, the Illumina TruSeq adapter sequence is shown in multiplex PCR, endonuclease cleavage of the incorporated universal primer 1, pre-annealing to the 3' adapter indexing primer (i7) 4, the linear blocker 5 having three mismatches, and the ligation of the 3' end of the 5' adapter indexing primer (i5) 3 to the 5' portion of the reverse complement of the universal adapter sequence 2 on the substrate amplicon. In this embodiment, the linear blocker has a non-complementary 3' tail sequence to prevent extension by polymerase. Figure 2E shows the lower T by insertion of a 6T deoxynucleotide loop. m To further illustrate the method using a linear blocker having the following characteristics, we show the Illumina TruSeq adapter sequences for multiplex PCR, endonuclease cleavage of the incorporated universal primer 1, pre-annealing to the 3' adapter indexing primer (i7) 4, linear blocker 5 having a 6T insert, and the ligation of the 3' end of the 5' adapter indexing primer (i5) 3 to the 5' end of the substrate amplicon.

[0109] Figure 2F shows T m T that reduces * In addition to G mismatches, it exhibits a linear blocker with three degradable U bases. Incubation with uracil glycosylase further destabilizes its interaction with indexing primer 4 by generating three debase sites within the blocker, which is insufficient for its dissociation from indexing primer 4 during the 5' adapter indexing primer 3 ligation step, but sufficient for its degradation by heat during PCR. Figure 2G shows blunt-end adapter ligation, annealing to DNA 3' adapter oligonucleotide 8 but not covalently fused, UDG cleavage and destabilization, and pre-annealing to 3' adapter indexing primer (i7) 4, T * The Illumina TruSeq adapter sequences are shown for UDG cleavage and destabilization of linear blocker 5 having a G mismatch and three uracil nucleotides, as well as for the ligation of the 3' end of 5' adapter indexing primer (i5) 3 to the 5' end of substrate DNA.

[0110] How to use a hairpin adapter Some alternative embodiments utilize truncated adapters for ligation-coupled PCR. Truncated adapters are required when using indexing primers that lack a common adapter sequence at their 3' end. As shown in Figure 3A, when using a linear truncated adapter 100, the endonuclease cleavage and adapter ligation steps proceed efficiently, but to confer specificity for 5' adapter indexing primer annealing, a linear truncated 5' adapter is required for sufficient annealing and extension during the PCR cycle. mThe presence of temperature leads to truncation of some completed 5' adapters and a decrease in library yield. Figure 3B shows a solution to this problem when using indexing primers 3 and 4, which lack a common adapter sequence at the 3' end, and a hairpin truncated adapter 5 is used for ligation-coupled PCR. Following endonuclease cleavage, annealing and ligation of the 3' single-stranded overhang of the hairpin adapter containing at least a portion of the common adapter sequence follows. To confer specificity for subsequent 5' adapter indexing primer annealing, the hairpin adapter contains its own sequence of the 5' adapter and a reverse complement of this sequence at the 5' end of the blocker to generate a hairpin that also contains a T-loop sequence. This secondary structure reduces its activity as a primer during PCR due to annealing competition with its self-complementarity, and increases the amplified library yield because the hairpin adapter cannot truncate the completed adapter during PCR. Figure 3C further illustrates that the stem-loop truncated adapter 6 also contains non-replicable modifications, represented by black circles, within the loop sequence, thus preventing the formation of a fully replicated hairpin product. When indexing PCR is initiated by indexing primer 4, hairpin replication is truncated by the replication block. This, due to its secondary structure, allows for efficient annealing of indexing primer 3 without competition for annealing from the hairpin adapter, and efficient indexing PCR. Figure 3D shows the undesirable result when the stem-loop truncated adapter 6 lacks non-replicable modifications within the loop sequence. In this case, when indexing PCR is initiated by indexing primer 4, full hairpin replication occurs. As a result, the 3' end of the replicated hairpin anneals and extends further, producing a non-functional, fully double-stranded hairpin library molecule. Therefore, the use of non-replicable modifications is necessary when using truncated hairpin adapters.

[0111] Figure 3E presents a specific embodiment of using the TruSeq Illumina adapter workflow when a hairpin truncated adapter containing an internally non-replicable C3 spacer is used for ligation-coupled PCR. In this example, the universal amplicon containing the dU base is cleaved and removed by USER so that all 6T deoxynucleotides are replaced. Thus, the hairpin truncated adapter 6 contains all 13 bases of the TruSeq common adapter sequence as a 3' overhang for ligation to the substrate. In addition, since indexing primers 3 and 4, which lack the common adapter sequence at their 3' ends, are used, oligo 6, which is part of the 3' adapter, is ligated to the 3' end of sequence 2 on each amplicon at the same time as it anneals to indexing primer 4, in order to provide a sufficient annealing sequence for indexing primer 4 during PCR. Figure 3F shows an alternative embodiment of using the TruSeq Illumina adapter workflow when a hairpin truncated adapter with an internally non-replicable C3 spacer is used for ligation-coupled PCR. In this example, universal amplicon primer 1, containing a dU base, replaces all T deoxynucleotides except for the 3' terminal T base, so only a portion of the common adapter sequence is cleaved and removed by USER cleavage. Therefore, the hairpin truncated adapter contains only 11 of the 13 bases of the common adapter sequence as a 3' overhang for ligation to the substrate. This can reduce bias in endonuclease cleavage by not cleaving the U base adjacent to the amplicon end containing the altered base composition.In addition, since indexing primers 3 and 4, which lack a common adapter sequence at their 3' ends, are used, if the universal primer contains a truncated 3' adapter sequence, oligo 22, which is part of the 3' adapter, is annealed to indexing primer 4 at the same time as ligating to the 3' end of sequence 2 on each amplicon, in order to provide a sufficient annealing sequence for indexing primer 4 during PCR.

[0112] In another alternative embodiment, a Nextera Illumina adapter workflow is used for ligation-coupled PCR with a hairpin truncated adapter, full-length indexing primers, and a linear blocker (Figure 3G). The Nextera adapter contains a 19-nucleotide sequence common to both adapters. In this example, the universal amplicon primer containing the dU base replaces all five T deoxynucleotides, but the 3' terminal 9-nucleotide sequence is retained due to the absence of the T base when USER cleavage is performed. Thus, the hairpin truncated adapter 5 contains the remaining 10 bases of the common adapter sequence as a 3' overhang, and the 5' overhang of the hairpin adapter similarly contains a T-loop with its own additional sequence, its reverse complement, and a non-replicable spacer in the 5' adapter. In this example, full-length Nextera indexing primers 3 and 4 are used but are not suitable for ligation due to the portion of universal sequence 1 remaining after USER cleavage. In addition, a linear blocking oligonucleotide 22 is used to prevent the indexing primer 4 from competing for annealing and ligation by the hairpin adapter 6. In another embodiment, Figure 3H shows another Nextera Illumina adapter workflow in which a hairpin truncated adapter is used with an indexing primer lacking a common adapter sequence for ligation-coupled PCR. In this example, the universal amplicon primer 1 containing dU bases replaces all five T deoxynucleotides, but the 3' terminal 9-base sequence is retained by the absence of the T bases if USER cleavage is performed. Thus, the hairpin truncated adapter 6 contains the remaining 10 bases of the common adapter sequence as a 3' overhang, and the 5' overhang of the hairpin adapter similarly contains a T-loop with its own further sequence, its reverse complement, and a non-replicable spacer in the 5' adapter.In this example, Nextera indexing primers 3 and 4, which lack a 19-base common adapter sequence at their 3' ends, are used. Furthermore, to provide a sufficient annealing sequence for indexing primer 4 during PCR, oligo 22, which contains a portion of the 3' adapter, is ligated to the 3' end of sequence 2 on each amplicon while simultaneously being annealed to indexing primer 4. To generate a universal truncated adapter that anneals to adapters with different index sequences, the truncated adapter 6 needs to span the sample-specific index sequence to achieve an effective annealing temperature, so the sample-specific index sequence is replaced with a universal T-loop.

[0113] The following four embodiments illustrate the use of a hairpin 5' adapter in the ligation-coupled PCR step of an NGS library workflow. Two specific embodiments of using the TruSeq Illumina adapter workflow when a truncated adapter containing an internally non-replicable C3 spacer is used for ligation-coupled PCR are presented in Figures 3I and 3J. In the example shown in Figure 3I, the 3' adapter oligonucleotide 9 containing a dU base is cleaved and removed by USER because all six T deoxynucleotides are replaced. In the example shown in Figure 3J, the absence of a covalent bond between the 3' adapter oligonucleotide 1 and DNA results in the T of oligonucleotide 9 being replaced. m This can be substantially reduced sufficiently for dissociation of oligonucleotide 9 from 3' adapter oligonucleotide 7 and annealing to hairpin adapter 6 by incubation with UDG and generation of six debasement sites. In both embodiments, the hairpin truncated adapter 6 contains all 13 bases of the TruSeq common adapter sequence as a 3' overhang for ligation to the substrate.

[0114] Figure 3K shows an alternative embodiment using the TruSeq Illumina adapter workflow in which a hairpin truncated adapter 5 with an internally non-replicable C3 spacer is used for ligation-coupled PCR. In this example, the 3' adapter oligonucleotide 9 containing the dU base replaces all T deoxynucleotides except the 3' terminal T base, so only a portion of the common adapter sequence is cleaved and removed by USER cleavage. Thus, the hairpin truncated adapter 6 contains only 11 of the 13 bases of the common adapter sequence as a 3' overhang for ligation to the substrate. This can reduce bias in endonuclease cleavage by not cleaving the U bases adjacent to the DNA ends containing the altered base composition.

[0115] In another alternative embodiment, a Nextera Illumina adapter workflow is used for ligation-coupled PCR with a hairpin truncated adapter, full-length indexing primers, and a linear blocker (Figure 3L). The Nextera adapter contains a 19-nucleotide sequence common to both adapters. In this example, the 3' adapter oligonucleotide 9 containing the dU base replaces all five T deoxynucleotides, but the 3' terminal 9-nucleotide sequence is retained due to the absence of the T base when USER cleavage is performed. Thus, the hairpin truncated adapter 6 contains the remaining 10 bases of the common adapter sequence as a 3' overhang, and the 5' overhang of the hairpin adapter similarly contains a T-loop with its own additional sequence, its reverse complement, and a non-replicable spacer in the 5' adapter. In this example, full-length Nextera indexing primers 3 and 4 are used, but are not suitable for ligation due to the portion of universal sequence 1 remaining after USER cleavage. In addition, linear blocking oligonucleotides 22 are used to prevent the indexing primer 4 from competing for annealing and ligation by the hairpin adapter 6.

[0116] Blocker explanation By using full-length indexing primers, each containing the same common adapter sequence at its 3' end, the disclosed blocker oligonucleotide can form a double-stranded structure with the 3' portion of the 3' adapter indexing primer, preventing it from annealing and ligating to the amplicon substrate during 5' adapter indexing primer ligation. This is because the TruSeq Illumina adapter has a 13-base common adapter sequence (complementary to the first common nucleotide sequence), and the Nextera Illumina adapter has a 19-base common adapter sequence, with each of these common adapter sequences having a T mHowever, because the incubation temperature is significantly higher than that of the thermally unstable ligase, the blocker achieves specificity for annealing and ligating one primer in the presence of both primers. The blocker includes the following three criteria for design and function: i) Annealing of the blocker to the 3' adapter indexing primer enables specific ligation of the 5' adapter indexing primer and formation of a functional NGS library, thus preventing a mixture of ligation products containing both the 5' and 3' adapter indexing primers, as well as a reduction in the construction of the functional NGS library; ii) Annealing of the blocker oligonucleotide to the common adapter sequence of the 5' adapter indexing primer is reduced compared to annealing to the 3' adapter indexing primer; and iii) The overall T of the blocker m The temperature is below the PCR annealing temperature, so it cannot inhibit the annealing of the 3' adapter indexing primer during PCR, or the blocker is inactivated by polymerase during PCR so that it cannot inhibit the annealing of the 3' adapter indexing primer. This disclosure describes two blocker designs that satisfy these criteria, namely a linear blocker and a hairpin blocker.

[0117] The linear blocker comprises a binding domain 31, a binding domain 32 located at the 5' end of the blocker, a linker domain 33, and a 3' terminal domain 34 (see Figure 4A). The binding domain 32 is complementary to at least a portion of the common adapter sequence of both indexing primers (13 bases in TruSeq and 19 bases in Nextera). The binding domain 31 is complementary to at least a portion of its own adapter sequence located at 5' of the common adapter sequence of the 3' adapter indexing primer, but does not contain a sample-specific index sequence. The binding domain 31 provides higher complementarity, promoting selective annealing to the 3' adapter indexing primer than the 5' adapter indexing primer. The melting temperature of the binding domain 31 is at least equal to, or preferably higher than, the melting temperature of the binding domain 32, with a difference of at least 1°C or greater. The linker domain 33 is not complementary to any portion of the indexing primer, thus affecting the overall T mThe linker domain is a mismatch domain used to reduce the efficiency of blocker binding to the 3' adapter indexing primer during PCR. The linker domain contains an insertion of a polyT, polyA, or polyC sequence, or any combination of A, T, C, and G bases. Its length can vary from 1 to 50 nucleotides. The linker domain may also contain mismatches or stretches of two or more consecutive mismatch nucleotides that are not insertions of further nucleotides. The 3' terminal domain 34 is used to inhibit polymerase elongation during the PCR phase of ligation-coupled PCR. It may include, without limitation, a C3 carbon spacer, hexanediol, spacer 9, spacer 18, phosphate, RNA nucleotides such as 2',3'-dideoxynucleosides ddA, ddT, ddC and ddG, 3'-deoxynucleosides 3'-A, 3'-T, 3'-C and 3'-G, rU, 3-O-methylnucleotides, or DNA sequences that are not complementary to the adjacent primer sequence, such as poly-T, poly-A, poly-C and poly-G, and may include 3' modifications that further include nuclease-resistant binding to prevent proofreading polymerase 3'-5' exonuclease activity from removing the DNA sequence that is not complementary to the adjacent primer sequence. As shown in Figure 4B, the binding domain 31 confers specificity for annealing the blocker to the 3' adapter indexing primer relative to the 5' adapter indexing primer. While not essential, the best results are achieved by pre-annealing the blocker to the 3' adapter indexing primer before adding it to the ligation reaction. However, the T of domain 32 m This is a temperature higher than the incubation temperature of 37°C (T mBecause the temperature is approximately 48°C, any excess blocker, or blocker dissociated products from the 3' adapter indexing primer, can anneal to the 5' adapter indexing primer via domain 32. However, due to domain 31, annealing to the 3' adapter indexing primer (Figure 4B(b)) is thermodynamically preferred over annealing to the 5' adapter indexing primer (Figure 4B(a)), so that after endonuclease cleavage, the 5' adapter indexing primer is efficiently ligated to the amplicon substrate, and ligation of the 3' adapter indexing primer is absent or minimized. As shown in Figure 4C, during PCR, the non-complementary domain 33 interferes with the base stacking interaction between domains 31 and 32, resulting in the overall stability and T of the blocker. m However, because the temperature is lower than the PCR annealing temperature and significantly lower than the competitive amplicon substrate that is fully complementary to the 3' adapter indexing primer, annealing to the 3' adapter indexing primer does not occur.

[0118] The alternative hairpin blocker contains binding domains 31 and 32 similar to the linear blocker, but lacks a non-complementary linker domain 33 and a sealing 3' end to prevent extension by polymerase. Instead, the hairpin blocker has a stem-loop structure at its 3' end formed by stem domains 35 and 36 and a loop domain 34 (Figure 4D). During the ligation reaction, the hairpin blocker thermodynamically preferentially anneals to the 3' adapter indexing primer due to the complementarity between domains 31 and 32 of both the blocker and the primer (T m At approximately 65-70°C (Figure 4D(b)), annealing to the 5' adapter indexing primer is reduced due to decreased complementarity only with domain 32 between the blocker and primer (T m: Approximately 48°C, Figure 4D(a)). Therefore, ligation specificity for the 5' adapter indexing primer is achieved in the presence of both indexing primers, even though the indexing primers have identical common adapter sequences at their 3' ends. During the PCR phase of ligation-coupled PCR, the 3' end of the stem domain 36 of the hairpin blocker is not blocked, so it can be extended by the DNA polymerase that initiates the self-replication of the hairpin blocker oligonucleotide. Self-replication maintains a high T of approximately 90°C, which is maintained at the PCR annealing temperature. m Because secondary structures are generated, the overall T of domains 31 and 32 m Even at approximately 73°C, which is above the PCR annealing temperature, the ability of the blocker to anneal to the 3' adapter indexing primer and inhibit it is inactivated (see Figure 4E). As a result, the 3' adapter indexing primer can efficiently amplify the template during PCR and is inhibited only in the ligation reaction, conferring specificity to ligation to the amplicon substrate by the 5' adapter indexing primer. The lengths of stem domains 35 and 36 should be sufficient to provide 3' hairpin stability and priming at the PCR annealing temperature, and preferably their melting temperature should be higher than the annealing temperatures of both the PCR reaction and blocker domains 31 and 32. In this case, self-replication of the hairpin blocker oligonucleotide can be carried out at a higher temperature before indexing primer annealing, which occurs at a lower temperature if the blocker has already been inactivated. The length and base composition of loop domain 34 are adaptable, containing 1 to 6 or more T, A, C, or G deoxyribonucleotides, or combinations thereof, enabling the formation of a stable stem-loop structure by blocker domains 35 and 36 (Figure 4D).

[0119] Further destabilization of the interaction between the blocker and the 3' adapter indexing primer can be achieved by replacing one or more T nucleotides in the blocker with dU bases. Incubation with UDG enzyme during the 5' adapter ligation reaction removes the dU bases, generating debase sites that promote fragmentation of the blocker oligonucleotide during the first PCR cycle.

[0120] Hairpin adapter description There are two examples of cases in which indexing primers cannot also be used as adapters for ligation. The first example is when using indexing primers that contain only their own adapter sequences and lack a common adapter sequence at their 3' ends, as this results in an insufficient adapter sequence. In the other example, if the endonuclease cleavage of a universal primer on the amplicon does not produce a cleavage pattern that removes the entire universal primer sequence, leaving an undigested 3' portion of the primer at the amplicon junction, the 3' end of the indexing primer is unsuitable for use as a 5' adapter because it has an insufficient or excessive adapter sequence content at its 3' end (depending on whether an indexing primer lacking a common adapter sequence or a full-length indexing primer containing a common adapter sequence is used). In these two embodiments of the method, a truncated 5' adapter is supplemented to the ligation-coupled PCR. As shown in Figure 5A, compared to a full-length adapter (a), the truncated 5' adapter can be linear (b) or contain a hairpin having a stem-loop structure (c), where the hairpin prevents annealing and extension of the adapter during PCR due to competition with its stable self-complementarity. This makes the hairpin adapter a preferred embodiment for the truncated 5' adapter, as it prevents truncation of the completed 5' adapter during PCR. In contrast, the linear truncated adapter can efficiently anneal, extend, and truncate the completed 5' adapter molecule during PCR, reducing the amplification library yield. Both truncated 5' adapters, although in reverse complement, contain domains 41 and 42 similar to blocker oligonucleotides, so that domain 42 is identical to at least a portion of the common adapter sequence and domain 41 is identical to at least a portion of the unique 5' adapter sequence adjacent to the common adapter sequence. Accordingly, domain 42 is located at the 3' end of the truncated adapter.The truncated hairpin adapter shown in Figure 5A(c) has five domains: a single-stranded domain 42, a double-stranded domain 41 at least partially complementary to domain 45, a replication-blocking domain 43, and a loop domain 44 that forms a stem-loop structure. The length and base composition of domain 42 are indicated by the position of the longest 3' cleavable base in the universal primer used in the multiplex PCR step. If endonuclease cleavage of the universal primer occurs at the 3' end of the primer or at the junction with the amplicon insert (Figure 5B(a)), domain 42 contains the entire common adapter sequence (13 bases for the TruSeq adapter and 19 bases for the Nextera adapter). If endonuclease cleavage of the universal primer occurs internally (Figure 5B(b)), the length of domain 42 is reduced to correspond to the remaining 3' portion of the cleavable primer to restore the continuous common adapter sequence without introducing duplicate bases or gaps into the sequence, and then the ligase seals the break. The length and base composition of the loop domain 44 are adaptable, containing 1 to 6 or more T, A, C, or G deoxynucleotides, or combinations thereof, enabling the formation of a stable stem-loop structure by the adapter domains 41 and 45. During both the ligation and PCR phases of ligation-coupled PCR, the truncated hairpin adapter maintains its stem-loop hairpin conformation due to its high stem Tm, which is higher than the PCR annealing temperature, and as a result does not participate in library amplification as a primer, thus not generating the truncated library product that is produced with linear truncated adapters. As previously mentioned in this application (Figures 3C and D), the replication-blocking domain 43 prevents replication of the ligated stem-loop structure and the formation of non-amplified long hairpin structures during PCR.As shown in Figure 5C, the indexing PCR is initiated by the 3' adapter indexing primer i7, replication is stopped at the replication blocking group, and annealing by the 5' adapter indexing primer i5 allows annealing and extension to complete the indexing PCR cycle.

[0121] Primer assembly method In a different embodiment of ligation-coupled PCR, a method is disclosed in which primer subunits and sprints are used to assemble long indexing primers and combined with NGS library amplification. This is used to reduce primer synthesis costs by combining only short index-specific oligonucleotide subunits with universal primer subunits instead of synthesizing individual full-length indexing primers. As shown in Figure 6A, universal primer subunits 51, 53, 56, and 58, as well as universal sprints 54, 55, 59, and 60, are combined with unique indexing subunits 52 and 57. Primer subunits 51, 52, 56, and 57 contain a 5' phosphate for ligation, and the sprint oligonucleotides contain a 3' blocking group to prevent priming activity during PCR. After annealing and ligating the primer subunits with sprint oligonucleotides at a first temperature suitable for ligation, thermal cycling is performed to amplify the NGS library containing the truncated NGS adapter to complete the adapter sequence and incorporate the sample-specific index sequence. Figure 6B shows (T) at their 5' ends. 12Details of a method for assembling a Normalase-adapted primer containing (rU)4 (SEQ ID NO: 1) are provided. The 5' sequences 52 and 57 required for downstream enzymatic normalization are ligated sprints to indexing primers 51 and 56 having sprints 53 and 58. Primers 51 and 56 require a 5' phosphate for ligation, and sprints 53 and 58 have a 3' blocking group to prevent priming activity during PCR. After annealing and ligating the primer subunits and sprints at a first temperature suitable for ligation, thermal cycling is performed to amplify the NGS library containing the truncated NGS adapter to complete the adapter sequence. In a specific embodiment of the method, Figure 6C shows the (T) adapted for downstream enzymatic normalization. 12 The assembly of the TruSeqIllumina indexing primer having the (rU)4 (SEQ ID NO: 1) sequence is shown. Indexing primer i5, which contains the 5' adapter sequence, has a 3' subunit 51 (22 nucleotides), an intermediate subunit 52 (30 nucleotides) containing the index sequence, and a terminal sequence (T). 12 The primer assembly is assembled from three oligonucleotide subunits, including the 5' subunit 53 (34 bases) containing (rU)4 (SEQ ID NO: 1), and two 3' sealed sprint oligonucleotides 54 and 55. To further illustrate this embodiment, Figure 6D shows a ligation-coupled PCR workflow when the primer assembly of the 4C i5TruSeq Illumina indexing primer described above is combined with the assembly of the corresponding i7 indexing primer containing the 3' adapter sequence, annealing of the hairpin blocker to the i7 primer, and ligation of the i5 primer to an amplicon substrate containing the truncated 3' adapter (followed by endonuclease cleavage, not shown).

[0122] Description of enzymes and reaction conditions Ligation-coupled PCR may include a DNA substrate for ligation, a ligase, a polymerase, a primer pair and substrate for PCR amplification which may be DNA subunit products, and optionally an endonuclease. The polymerase may be a heat-stable hot-start DNA polymerase such as Taq DNA Polymerase, or, preferably for NGS library amplification, a high-performance polymerase having 3'-5' exonuclease proofreading activity. A proofreading polymerase having 3'-5' exonuclease activity is (T) 12 When combined with primers for downstream enzymatic normalization, including the (rU)45' tail sequence (SEQ ID NO: 1), it is necessary to generate a 5' overhang during PCR. Commercially available enzymes include, but are not limited to, Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), and High Fidelity DNA Polymerase (Qiagen). PCR reaction conditions are carried out as recommended by the polymerase supplier without modification, in a reaction volume of 50 μL to allow for 20 μL of library eluate by bead-based purification and 5 μL of primer and adapter addition when using a 2x master mix PCR formulation. The ligase can be any thermally unstable ligase capable of high-efficiency ligation in low-magnesium PCR reaction buffer, including T3 DNA ligase, and a minimum of 30-300 or more enzyme units can be used in a 50 μL ligation-coupled PCR reaction. The endonuclease for removing the strand of the first NGS adapter containing cleavable dU bases is a USER enzyme containing a blend of UDG (Uracil DNA Glycosylase) and endonuclease VIII (NEB), with one enzyme unit used for a 50 μL ligation-coupled PCR reaction.

[0123] The single-tube ligation-coupled PCR includes all the reagents necessary for ligation and PCR, and comprises two separate incubations. The first incubation is carried out at a temperature that allows endonuclease and ligase activity but not polymerase activity, and the reactants are then heated to a high temperature to inactivate the endonuclease and ligase, activate the hot-start polymerase, and denature the substrate for PCR thermal cycling. The first reaction is carried out with heat-unstable enzymes at a temperature of 25–37°C, with 25°C being optimal for T3 ligase and 37°C for USER enzyme, although both can be carried out within this temperature range. This incubation is ideally carried out for 20 minutes to ensure the reaction is complete before PCR, but can be carried out in the range of 5–60 minutes. The thermal inactivation temperature and incubation time are determined by the hot-start requirements of the polymerase used in the method, and are typically 95-98°C for 30 seconds to 2 minutes or longer, followed by the annealing and extension temperature and incubation time, as known to those skilled in the art, without any specific modifications based on the method of this disclosure, using primer T m This depends on the polymerase used and the length of the amplified product.

[0124] Within the scope of these disclosed ligation-coupled PCR methods, indexing primers, used only as primers during PCR or as both ligation adapters and PCR primers, can be used at reaction concentrations ranging from 100 nM to 1 μM or higher, depending on the number of PCR cycles required. When assembling indexing primers or other long primers, sprint oligonucleotides are used at a lower molar ratio of 0.75-fold or a molar excess ratio of up to 1.5-fold relative to the oligonucleotide subunits, with primer subunits further 5' in the final primer assembly being at a molar concentration of 2-fold or higher compared to primer subunits further 3' in the final primer assembly. This prevents an excess of unligated 3' primer subunits that function as primers during PCR and truncate the completed adapter. Excess primer subunits at the 5' end of the final primer assembly do not degrade the final yield of the PCR product because they do not truncate the final library when functioning as individual primers. When linear or hairpin blockers are used, the molar concentration of the blocker is equal to or greater than the concentration of the indexing primer it inhibits during the ligation reaction, and the ratio of blocker to primer is 1:1, 1.5:1, 2:1, 4:1, 6:1, or greater. When hairpin truncated adapters are used, reaction concentrations of 50–200 nM or greater can be used, depending on the amount of substrate present in the reactant. If further portions are simultaneously ligated to the 3' end of the truncated 3' adapter on the 3' end of the amplicon substrate, similar oligonucleotide concentrations of 50–200 nM or greater are used.

[0125] Ligation-coupled PCR method - Both indexing primers contain a common 3' terminal sequence complementary to the first common nucleotide sequence. In some embodiments, (i) provide a partially double-stranded DNA substrate comprising a first strand and a second strand, comprising a first 3' overhang, a double-stranded portion and a second 3' overhang, wherein the first strand comprises a first 5' end, a first portion and a second portion in the 5'-to-3' direction, and the second strand comprises a second 5' end, a third portion and a fourth portion in the 5'-to-3' direction, wherein the first portion of the first strand and the third portion of the second strand are complementary and form a double-stranded portion, the second portion of the first strand forms a first 3' overhang, and the fourth portion of the second strand (ii) The portion forms a second 3' overhang, and the second portion of the first strand and the fourth portion of the second strand each contain a first common nucleotide sequence located at the 5' end of the first 3' overhang and the second 3' overhang, respectively; (ii) A first reaction mixture is produced by adding a plurality of first indexing primers, a plurality of second indexing primers, a ligase, a DNA polymerase, and deoxynucleotide triphosphates (dNTPs) to a partially double-stranded DNA substrate, wherein each of the plurality of first indexing primers is a first (iii) each of a plurality of second indexing primers contains a first 3' terminal portion complementary to the common nucleotide sequence, and each of a plurality of second indexing primers contains a second 3' terminal portion complementary to the first common nucleotide sequence, (iii) the first reaction mixture is incubated under a first set of conditions including a ligation temperature over a ligation duration, the first set of conditions being: 1) the first 3' terminal portions of the plurality of first indexing primers anneal to the first common nucleotide sequence, and 2) a ligase anneals one of the plurality of first indexing primers to the first chain (iv) The second reaction mixture is sufficient to produce a second reaction mixture comprising: (iv) 1) ligating one of the multiple first indexing primers to the second 5' end of the second strand of the first strand of the first reaction mixture to the second 5' end of the second strand of the first strand of the first reaction mixture to produce a second reaction mixture comprising a third strand containing one of the multiple first indexing primers, a first portion and a second portion in the 5' to 3' direction, and a fourth strand containing one of the multiple first indexing primers, a third portion and a fourth portion in the 5' to 3' direction, (iv) the second reaction mixture 1) inactivates ligases, denatures double-stranded DNA, and activates DNA polymerase as necessary,2) The second 3' end portion of one of the multiple second indexing primers anneals to the first common nucleotide sequence of the second portion of the third strand, and one of the multiple second indexing primers anneals to the first common nucleotide sequence of the fourth portion of the fourth strand, and 3) DNA polymerase anneals to one of the multiple second indexing primers annealed to the first common nucleotide sequence of the second portion of the third strand, and to one of the multiple second indexing primers annealed to the first common nucleotide sequence of the fourth portion of the fourth strand. -Extending one of the chains to produce a third reaction mixture comprising a third chain, a fourth chain, a fifth chain and a sixth chain, wherein the fifth chain comprises, in the 5' to 3' direction, one of a plurality of second indexing primers, a third portion and one of a plurality of first indexing primers in a reverse complement, and the sixth chain comprises, in the 5' to 3' direction, one of a plurality of second indexing primers, a first portion and one of a plurality of first indexing primers in a reverse complement, sufficient to produce a third reaction mixture with a first denaturation duration over a first denaturation duration (v) Incubating under a second set of conditions including temperature, a first annealing temperature over a first annealing duration, and a first extension temperature over a first extension duration; (v) The third reaction mixture 1) denatures double-stranded DNA; 2) one of a plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers on the fifth or sixth strand; and 3) DNA polymerase anneals to the plurality of first indexing primers that have annealed to the reverse complement of one of the plurality of first indexing primers on the fifth strand. A fourth reaction mixture comprising a fifth chain, a sixth chain, a seventh chain and an eighth chain, wherein the seventh chain comprises, in the 5' to 3' direction, one of the plurality of first indexing primers, a first portion, and the reverse complement of one of the plurality of second indexing primers, and the eighth chain comprises, in the 5' to 3' direction, one of the plurality of first indexing primers, a third portion,A method is provided comprising (vi) incubating under a third set of conditions including a second denaturation temperature over a second denaturation duration, a second annealing temperature over a second annealing duration, and a second extension temperature over a second extension duration, which is sufficient to produce a fourth reaction mixture comprising a reverse complement of one of a plurality of second indexing primers, wherein the seventh chain is complementary to the fifth chain and the eighth chain is complementary to the sixth chain; and (vi) incubating under a fourth set of conditions including a third denaturation temperature over a third denaturation duration, a third annealing temperature over a third annealing duration, and a third extension temperature over a third extension duration, which is sufficient to amplify at least a portion of the plurality of first indexing primers and at least a portion of the plurality of second indexing primers into a fifth oligonucleotide and a seventh oligonucleotide or a sixth oligonucleotide and an eighth oligonucleotide.

[0126] In any of the embodiments described above, steps (i) to (vi) can be carried out in a single sealed tube. In any of the embodiments described above, the method may not include any purification steps between steps (i) to (vi).

[0127] In any of the embodiments described above, the partially double-stranded DNA substrate can have a length of about 24 to about 6000 bases. For example, without limitation, the partially double-stranded DNA substrate can be about 24 to about 6000 bases, about 24 to about 5500 bases, about 24 to about 5000 bases, about 24 to about 4500 bases, about 24 to about 4000 bases, about 24 to about 3500 bases, about 24 to about 3000 bases, about 24 to about 2500 bases, about 24 to about 2000 bases, about 24 to about 1500 bases, about 24 to about 1000 bases, about 24 to about 750 bases, about 24 to about 500 bases, or about 24 to about 250 bases. , approximately 24 bases to approximately 200 bases, approximately 24 bases to approximately 100 bases, approximately 24 bases to approximately 50 bases, approximately 100 bases to approximately 6000 bases, approximately 100 bases to approximately 5500 bases, approximately 100 bases to approximately 5000 bases, approximately 100 bases to approximately 4500 bases, approximately 100 bases to approximately 4000 bases, approximately 100 bases to approximately 3500 bases, approximately 100 bases to approximately 3000 bases, approximately 100 bases to approximately 2500 bases, approximately 100 bases to approximately 2000 bases, approximately 100 bases to approximately 1500 bases, approximately 100 bases to approximately 1000 bases, approximately 100 bases to approximately 750 bases, approximately 1 00 bases to approximately 500 bases, approximately 100 bases to approximately 250 bases, approximately 100 bases to approximately 200 bases, approximately 200 bases to approximately 6000 bases, approximately 200 bases to approximately 5500 bases, approximately 200 bases to approximately 5000 bases, approximately 200 bases to approximately 4500 bases, approximately 200 bases to approximately 4000 bases, approximately 200 bases to approximately 3500 bases, approximately 200 bases to approximately 3000 bases, approximately 200 bases to approximately 2500 bases, approximately 200 bases to approximately 200 bases, approximately 200 bases to approximately 1500 bases, approximately 200 bases to approximately 1000 bases, approximately 200 bases to approximately 750 bases, approximately 200 bases to approximately 500 bases, approximately 200 bases to approximately 250 bases, approximately 250 bases to approximately 6000 bases, approximately 250 bases to approximately 5500 bases, approximately 250 bases to approximately 5000 bases, approximately 250 bases to approximately 4500 bases, approximately 250 bases to approximately 4000 bases, approximately 250 bases to approximately 3500 bases, approximately 250 bases to approximately 3000 bases, approximately 250 bases to approximately 250 bases, approximately 250 bases to approximately 2000 bases, approximately 250 bases to approximately 1500 bases, approximately 250 bases to approximately 1000 bases, approximately 250 bases to approximately 750 bases, approximately 250 bases to approximately 500 bases,Approximately 500 bases to approximately 6000 bases, approximately 500 bases to approximately 5500 bases, approximately 500 bases to approximately 5000 bases, approximately 500 bases to approximately 4500 bases, approximately 500 bases to approximately 4000 bases, approximately 500 bases to approximately 3500 bases, approximately 500 bases to approximately 3000 bases, approximately 500 bases to approximately 2500 bases, approximately 500 bases to approximately 2000 bases, approximately 500 bases to approximately 1500 bases, approximately 500 bases to approximately 1000 bases, approximately 500 bases to approximately 750 bases, approximately 750 bases to approximately 6000 bases, approximately 750 bases to approximately 5500 bases, approximately 750 bases to approximately 5000 bases, approximately 750 bases to approximately 4 500 bases, approximately 750 bases to approximately 4000 bases, approximately 750 bases to approximately 3500 bases, approximately 750 bases to approximately 3000 bases, approximately 750 bases to approximately 2500 bases, approximately 750 bases to approximately 2000 bases, approximately 750 bases to approximately 1500 bases, approximately 750 bases to approximately 1000 bases, approximately 1000 bases to approximately 6000 bases, approximately 1000 bases to approximately 5500 bases, approximately 1000 bases to approximately 5000 bases, approximately 1000 bases to approximately 4500 bases, approximately 1000 bases to approximately 4000 bases, approximately 1000 bases to approximately 3500 bases, approximately 1000 bases to approximately 3000 bases, approximately 1000 bases to approximately 25 00 bases, approximately 1000 bases to approximately 2000 bases, approximately 1000 bases to approximately 1500 bases, approximately 1500 bases to approximately 6000 bases, approximately 1500 bases to approximately 5500 bases, approximately 1500 bases to approximately 5000 bases, approximately 1500 bases to approximately 4500 bases, approximately 1500 bases to approximately 4000 bases, approximately 1500 bases to approximately 3500 bases, approximately 1500 bases to approximately 3000 bases, approximately 1500 bases to approximately 2500 bases, approximately 1500 bases to approximately 2000 bases, approximately 2000 bases to approximately 6000 bases, approximately 2000 bases to approximately 5500 bases, approximately 2000 bases to approximately 5000 bases, approximately 2000 salt Approximately 4500 bases from the base, approximately 4000 bases from approximately 2000 bases, approximately 3500 bases from approximately 2000 bases, approximately 3000 bases from approximately 2000 bases, approximately 2500 bases from approximately 2500 bases, approximately 6000 bases from approximately 2500 bases, approximately 5500 bases from approximately 2500 bases, approximately 5000 bases from approximately 2500 bases, approximately 4500 bases from approximately 2500 bases, approximately 4000 bases from approximately 2500 bases, approximately 3500 bases from approximately 2500 bases, approximately 3000 bases from approximately 3000 bases, approximately 6000 bases from approximately 3000 bases, approximately 5500 bases from approximately 3000 bases,Approximately 3000 bases to approximately 4500 bases, approximately 3000 bases to approximately 4000 bases, approximately 3000 bases to approximately 3500 bases, approximately 3500 bases to approximately 6000 bases, approximately 3500 bases to approximately 5500 bases, approximately 3500 bases to approximately 5000 bases, approximately 3500 bases to approximately 4500 bases, approximately 3500 bases to approximately 4000 bases, approximately 4000 bases to approximately 6000 bases, approximately 4000 bases to approximately 5500 bases, approximately 4000 bases to approximately 5000 bases, approximately 4000 bases to approximately 4500 bases, approximately 4500 bases The lengths can range from approximately 6000 bases, from approximately 4500 bases to approximately 5500 bases, from approximately 4500 bases to approximately 5000 bases, from approximately 5000 bases to approximately 6000 bases, from approximately 5000 bases to approximately 5500 bases, from approximately 5500 bases to approximately 6000 bases, and from approximately 24, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 bases. It should be understood that the lengths of partially double-stranded DNA substrates are illustrative, and that other sizes are also within the scope of this disclosure. It should be understood that the length of a partially double-stranded DNA substrate refers to the length of the first or second strand of the partially double-stranded DNA substrate, i.e., the length from the first 5' end to the 3' end of the first 3' overhang or the length from the second 5' end to the 3' end of the second 3' overhang.

[0128] In any of the embodiments described above, the first part of the first strand and the first part of the second strand and the third part of the second strand can have lengths ranging from about 20 bases to about 6000 bases, respectively. For example, without limitation, the first part of the first oligonucleotide and the third part of the second oligonucleotide can have lengths ranging from about 20 bases to about 6000 bases, from about 20 bases to about 5500 bases, from about 20 bases to about 5000 bases, from about 20 bases to about 4500 bases, from about 20 bases to about 4000 bases, from about 20 bases to about 3500 bases, from about 20 bases to about 3000 bases, from about 20 bases to about 2500 bases, from about 20 bases to about 2000 bases, from about 20 bases to about 1500 bases, from about 20 bases to about 1000 bases, and from about 20 bases or Approximately 750 bases, approximately 20 to approximately 500 bases, approximately 20 to approximately 250 bases, approximately 20 to approximately 200 bases, approximately 20 to approximately 100 bases, approximately 20 to approximately 50 bases, approximately 100 to approximately 6000 bases, approximately 100 to approximately 5500 bases, approximately 100 to approximately 5000 bases, approximately 100 to approximately 4500 bases, approximately 100 to approximately 4000 bases, approximately 100 to approximately 3500 bases, approximately 100 to approximately 3000 bases, approximately 100 to approximately 2500 bases, approximately 100 to approximately 2000 bases, approximately 100 to approximately 1 500 bases, approximately 100 to 1000 bases, approximately 100 to 750 bases, approximately 100 to 500 bases, approximately 100 to 250 bases, approximately 100 to 200 bases, approximately 200 to 6000 bases, approximately 200 to 5500 bases, approximately 200 to 5000 bases, approximately 200 to 4500 bases, approximately 200 to 4000 bases, approximately 200 to 3500 bases, approximately 200 to 3000 bases, approximately 200 to 2500 bases, approximately 200 to 2000 bases, approximately 200 salt Approximately 1500 bases from the base, approximately 1000 bases from approximately 200 bases, approximately 750 bases from approximately 200 bases, approximately 500 bases from approximately 200 bases, approximately 250 bases from approximately 200 bases, approximately 6000 bases from approximately 250 bases, approximately 5500 bases from approximately 250 bases, approximately 5000 bases from approximately 250 bases, approximately 4500 bases from approximately 250 bases, approximately 4000 bases from approximately 250 bases, approximately 3500 bases from approximately 250 bases, approximately 3000 bases from approximately 250 bases, approximately 250 bases from approximately 250 bases, approximately 2000 bases from approximately 250 bases, approximately 1500 bases from approximately 250 bases,Approximately 250 bases to approximately 1000 bases, approximately 250 bases to approximately 750 bases, approximately 250 bases to approximately 500 bases, approximately 500 bases to approximately 6000 bases, approximately 500 bases to approximately 5500 bases, approximately 500 bases to approximately 5000 bases, approximately 500 bases to approximately 4500 bases, approximately 500 bases to approximately 4000 bases, approximately 500 bases to approximately 3500 bases, approximately 500 bases to approximately 3000 bases, approximately 500 bases to approximately 2500 bases, approximately 500 bases to approximately 2000 bases, approximately 500 bases to approximately 1500 bases, approximately 500 bases to approximately 1000 bases, approximately 500 bases to approximately 750 bases, approximately 750 bases to approximately 6 000 bases, approximately 750 bases to approximately 5500 bases, approximately 750 bases to approximately 5000 bases, approximately 750 bases to approximately 4500 bases, approximately 750 bases to approximately 4000 bases, approximately 750 bases to approximately 3500 bases, approximately 750 bases to approximately 3000 bases, approximately 750 bases to approximately 2500 bases, approximately 750 bases to approximately 2000 bases, approximately 750 bases to approximately 1500 bases, approximately 750 bases to approximately 1000 bases, approximately 1000 bases to approximately 6000 bases, approximately 1000 bases to approximately 5500 bases, approximately 1000 bases to approximately 5000 bases, approximately 1000 bases to approximately 4500 bases, approximately 1000 bases to approximately 4000 bases Bases, approximately 1000 bases to approximately 3500 bases, approximately 1000 bases to approximately 3000 bases, approximately 1000 bases to approximately 2500 bases, approximately 1000 bases to approximately 2000 bases, approximately 1000 bases to approximately 1500 bases, approximately 1500 bases to approximately 6000 bases, approximately 1500 bases to approximately 5500 bases, approximately 1500 bases to approximately 5000 bases, approximately 1500 bases to approximately 4500 bases, approximately 1500 bases to approximately 4000 bases, approximately 1500 bases to approximately 3500 bases, approximately 1500 bases to approximately 3000 bases, approximately 1500 bases to approximately 2500 bases, approximately 1500 bases to approximately 2000 bases, approximately 2000 bases From approximately 6000 bases, from approximately 2000 bases to approximately 5500 bases, from approximately 2000 bases to approximately 5000 bases, from approximately 2000 bases to approximately 4500 bases, from approximately 2000 bases to approximately 4000 bases, from approximately 2000 bases to approximately 3500 bases, from approximately 2000 bases to approximately 3000 bases, from approximately 2000 bases to approximately 2500 bases, from approximately 2500 bases to approximately 6000 bases, from approximately 2500 bases to approximately 5500 bases, from approximately 2500 bases to approximately 5000 bases, from approximately 2500 bases to approximately 4500 bases, from approximately 2500 bases to approximately 4000 bases, from approximately 2500 bases to approximately 3500 bases, from approximately 2500 bases to approximately 3000 bases,Approximately 3000 bases to approximately 6000 bases, approximately 3000 bases to approximately 5500 bases, approximately 3000 bases to approximately 5000 bases, approximately 3000 bases to approximately 4500 bases, approximately 3000 bases to approximately 4000 bases, approximately 3000 bases to approximately 3500 bases, approximately 3500 bases to approximately 6000 bases, approximately 3500 bases to approximately 5500 bases, approximately 3500 bases to approximately 5000 bases, approximately 3500 bases to approximately 4500 bases, approximately 3500 bases to approximately 4000 bases, approximately 4000 bases to approximately 6000 bases, approximately 4000 bases to approximately 5500 bases, approximately 4000 bases to approximately 5000 bases The lengths can range from approximately 4000 to approximately 4500 bases, approximately 4500 to approximately 6000 bases, approximately 4500 to approximately 5500 bases, approximately 4500 to approximately 5000 bases, approximately 5000 to approximately 6000 bases, approximately 5000 to approximately 5500 bases, approximately 5500 to approximately 6000 bases, and approximately 24, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 bases. It should be understood that the lengths of the first portion of the first strand and the third portion of the second strand are illustrative, and that other sizes are also within the scope of this disclosure.

[0129] In any of the embodiments described above, the second portion of the first strand and the fourth portion of the second strand, i.e., the first 3' overhang and the second 3' overhang, each may contain approximately 4 to approximately 100 bases. For example, without limitation, the second portion of the first strand and the fourth portion of the second strand may contain approximately 4 to approximately 100 bases, approximately 4 to approximately 90 bases, approximately 4 to approximately 80 bases, approximately 4 to approximately 75 bases, approximately 4 to approximately 70 bases, approximately 4 to approximately 60 bases, approximately 4 to approximately 50 bases, approximately 4 to approximately 40 bases, approximately 4 to approximately 30 bases, approximately 4 to approximately 25 bases, approximately 4 to approximately 20 bases, approximately 4 to approximately 15 bases, approximately 4 to approximately 10 bases, approximately 4 to approximately 5 bases, and approximately 5 to approximately 100 salts. Base, approximately 5 to 90 bases, approximately 5 to 80 bases, approximately 5 to 75 bases, approximately 5 to 70 bases, approximately 5 to 60 bases, approximately 5 to 55 bases, approximately 5 to 50 bases, approximately 5 to 40 bases, approximately 5 to 30 bases, approximately 5 to 25 bases, approximately 5 to 20 bases, approximately 5 to 15 bases, approximately 5 to 10 bases, approximately 10 to 100 bases, approximately 10 to 90 bases, approximately 10 to 80 bases, approximately 10 to 75 bases, approximately 10 to 70 bases , approximately 10 to 60 bases, approximately 10 to 50 bases, approximately 10 to 40 bases, approximately 10 to 30 bases, approximately 10 to 25 bases, approximately 10 to 20 bases, approximately 10 to 15 bases, approximately 15 to 100 bases, approximately 15 to 90 bases, approximately 15 to 80 bases, approximately 15 to 75 bases, approximately 15 to 70 bases, approximately 15 to 60 bases, approximately 15 to 50 bases, approximately 15 to 40 bases, approximately 15 to 30 bases, approximately 15 to 25 bases, Approximately 15 to 20 bases, approximately 20 to 100 bases, approximately 20 to 90 bases, approximately 20 to 80 bases, approximately 20 to 75 bases, approximately 20 to 70 bases, approximately 20 to 60 bases, approximately 20 to 50 bases, approximately 20 to 40 bases, approximately 20 to 30 bases, approximately 20 to 25 bases, approximately 25 to 100 bases, approximately 25 to 90 bases, approximately 25 to 80 bases, approximately 25 to 75 bases, approximately 25 to 70 bases, approximately 25 to 60 bases,Approximately 25 to 50 bases, approximately 25 to 40 bases, approximately 25 to 30 bases, approximately 30 to 100 bases, approximately 30 to 90 bases, approximately 30 to 80 bases, approximately 30 to 75 bases, approximately 30 to 70 bases, approximately 30 to 60 bases, approximately 30 to 50 bases, approximately 30 to 40 bases, approximately 40 to 100 bases, approximately 40 to 90 bases, approximately 40 to 80 bases, approximately 40 to 75 bases, approximately 40 to 70 bases, approximately 40 to 60 bases, approximately 40 to 50 bases, approximately 50 to 100 bases, approximately 50 to 90 bases, approximately 50 to 80 bases, approximately 50 to 75 bases, approximately 50 to 7 It may contain 0 bases, approximately 50 to 60 bases, approximately 60 to 100 bases, approximately 60 to 90 bases, approximately 60 to 80 bases, approximately 60 to 75 bases, approximately 60 to 70 bases, approximately 70 to 100 bases, approximately 70 to 90 bases, approximately 70 to 80 bases, approximately 70 to 75 bases, approximately 75 to 100 bases, approximately 75 to 90 bases, approximately 75 to 80 bases, approximately 80 to 100 bases, approximately 80 to 90 bases, approximately 90 to 100 bases, and approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 33, 40, 50, 60, 70, 75, 80, 90, or 100 bases.

[0130] In any of the embodiments described above, the first common nucleotide sequence may include approximately 1 to 50 bases. For example, without limitation, the first common nucleotide sequence may include approximately 1 to 50 bases, approximately 1 to 45 bases, approximately 1 to 40 bases, approximately 1 to 35 bases, approximately 1 to 30 bases, approximately 1 to 25 bases, approximately 1 to 20 bases, approximately 1 to 15 bases, approximately 1 to 10 bases, approximately 5 to 50 bases, approximately 5 to 45 bases, approximately 5 to 40 bases, and approximately 5 to Approximately 35 bases, approximately 5 to approximately 30 bases, approximately 5 to approximately 25 bases, approximately 5 to approximately 20 bases, approximately 5 to approximately 15 bases, approximately 5 to approximately 10 bases, approximately 10 to approximately 50 bases, approximately 10 to approximately 45 bases, approximately 10 to approximately 40 bases, approximately 10 to approximately 35 bases, approximately 10 to approximately 30 bases, approximately 10 to approximately 25 bases, approximately 10 to approximately 20 bases, approximately 10 to approximately 15 bases, approximately 15 to approximately 50 bases Bases, approximately 15 to 45 bases, approximately 15 to 40 bases, approximately 15 to 35 bases, approximately 15 to 30 bases, approximately 15 to 25 bases, approximately 15 to 20 bases, approximately 20 to 50 bases, approximately 20 to 45 bases, approximately 20 to 40 bases, approximately 20 to 35 bases, approximately 20 to 30 bases, approximately 25 to 30 bases, approximately 30 to 50 bases, approximately 30 to 50 bases, approximately 30 to 45 bases, approximately 20 to 35 bases, approximately 20 to 30 bases, approximately 25 to 3 It may include 45 bases, about 30 to about 40 bases, about 30 to about 35 bases, about 35 to about 50 bases, about 35 to about 45 bases, about 35 to about 40 bases, about 40 to about 50 bases, about 40 to about 45 bases, about 45 to about 50 bases, and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 33, 35, 40, or 50 bases. Preferably, the first common nucleotide sequence contains 13 bases. In some embodiments, the first common nucleotide sequence contains the sequence of SEQ ID NO: 127 (5'-AGATCGGAAGAGC-3'). If the first common nucleotide sequence contains the sequence of SEQ ID NO: 127, then the first 3' end and the second 3' end can each contain SEQ ID NO: 126 (5'-GCTCTTCCGATCT-3').

[0131] In any of the embodiments described above, the second portion of the first chain and the fourth portion of the second chain may further include a second common nucleotide sequence located 3' relative to the first common nucleotide sequence. In a particular embodiment, the second common nucleotide sequence may have a length of about 3 to about 100 bases. For example, without limitation, the second common nucleotide sequence may be about 1 to about 100 bases, about 1 to about 90 bases, about 1 to about 80 bases, about 1 to about 75 bases, about 1 to about 70 bases, about 1 to about 60 bases, about 1 to about 50 bases, about 1 to about 40 bases, about 1 to about 30 bases, about 1 to about 25 bases, about 1 to about 20 bases, about 1 to about 15 bases, about 1 to about 10 bases, about 1 to about 5 bases, about 5 to about 100 bases, about 5 salts Approximately 90 bases from the base, approximately 80 bases from approximately 5 bases, approximately 75 bases from approximately 5 bases, approximately 70 bases from approximately 5 bases, approximately 60 bases from approximately 5 bases, approximately 40 bases from approximately 5 bases, approximately 30 bases from approximately 5 bases, approximately 25 bases from approximately 5 bases, approximately 20 bases from approximately 5 bases, approximately 15 bases from approximately 5 bases, approximately 10 bases from approximately 5 bases, approximately 100 bases from approximately 10 bases, approximately 90 bases from approximately 10 bases, approximately 80 bases from approximately 10 bases, approximately 75 bases from approximately 10 bases, approximately 70 bases from approximately 10 bases, approximately 10 bases Approximately 60 bases, approximately 10 to approximately 50 bases, approximately 10 to approximately 40 bases, approximately 10 to approximately 30 bases, approximately 10 to approximately 25 bases, approximately 10 to approximately 20 bases, approximately 10 to approximately 15 bases, approximately 15 to approximately 100 bases, approximately 15 to approximately 90 bases, approximately 15 to approximately 80 bases, approximately 15 to approximately 75 bases, approximately 15 to approximately 70 bases, approximately 15 to approximately 60 bases, approximately 15 to approximately 50 bases, approximately 15 to approximately 40 bases, approximately 15 to approximately 30 bases, approximately 15 to approximately 25 bases Bases, approximately 15 to 20 bases, approximately 20 to 100 bases, approximately 20 to 90 bases, approximately 20 to 80 bases, approximately 20 to 75 bases, approximately 20 to 70 bases, approximately 20 to 60 bases, approximately 20 to 50 bases, approximately 20 to 40 bases, approximately 20 to 30 bases, approximately 20 to 25 bases, approximately 25 to 100 bases, approximately 25 to 90 bases, approximately 25 to 80 bases, approximately 25 to 75 bases, approximately 25 to 70 bases,Approximately 25 to 60 bases, approximately 25 to 50 bases, approximately 25 to 40 bases, approximately 25 to 30 bases, approximately 30 to 100 bases, approximately 30 to 90 bases, approximately 30 to 80 bases, approximately 30 to 75 bases, approximately 30 to 70 bases, approximately 30 to 60 bases, approximately 30 to 50 bases, approximately 30 to 40 bases, approximately 40 to 100 bases, approximately 40 to 90 bases, approximately 40 to 80 bases, approximately 40 to 75 bases, approximately 40 to 70 bases, approximately 40 to 60 bases, approximately 40 to 50 bases, approximately 50 to 100 bases, approximately 50 to 90 bases, approximately 50 to 80 bases, approximately 50 to 75 bases The base can have lengths of approximately 50 to 70 bases, approximately 50 to 60 bases, approximately 60 to 100 bases, approximately 60 to 90 bases, approximately 60 to 80 bases, approximately 60 to 75 bases, approximately 60 to 70 bases, approximately 70 to 100 bases, approximately 70 to 90 bases, approximately 70 to 80 bases, approximately 70 to 75 bases, approximately 75 to 100 bases, approximately 75 to 90 bases, approximately 75 to 80 bases, approximately 80 to 100 bases, approximately 80 to 90 bases, approximately 90 to 100 bases, and approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, or 100 bases.

[0132] In embodiments where the second portion of the first chain and the fourth portion of the second chain further include a second common nucleotide sequence, each of the plurality of second indexing primers may include a first 5' portion located at 5' relative to the second 3' terminal portion and complementary to the second common nucleotide sequence. In such embodiments, the second set of conditions in step (iv)(b) may be sufficient for the second 3' terminal portion and the first 5' portion of one of the plurality of second indexing primers to anneal to at least the first and second common nucleotide sequences on the second portion of the third chain or the fourth portion of the fourth chain. In such embodiments, the melting temperature from the first 3' terminal sequence to the first common nucleotide sequence may be lower than the first annealing temperature. As further examples, without limitation, the melting temperature from the first 3' terminal sequence to the first common nucleotide sequence may be lower than the first annealing temperature, the second annealing temperature, and the third annealing temperature. In such embodiments, each of the first indexing primers may not contain a sequence complementary to the second common nucleotide sequence. In such embodiments, the melting temperature between the first and second common nucleotide sequences and the second 3' and first 5' portions of each of the second indexing primers may be higher than the first annealing temperature. In such embodiments, the melting temperatures of each of the multiple second indexing primers derived from the first and second common nucleotide sequences may be higher than the first and third annealing temperatures.

[0133] In any of the embodiments described above, the melting temperature between each of the plurality of first indexing primers and the second or fourth portion is lower than the melting temperature between each of the plurality of second indexing primers and the second or fourth portion.

[0134] In any of the embodiments described above, each of the plurality of first indexing primers may have a length of about 20 to about 100 bases. For example, without limitation, each of the plurality of first indexing primers may have a length of about 20 to about 100 bases, about 20 to about 90 bases, about 20 to about 80 bases, about 20 to about 70 bases, about 20 to about 60 bases, about 20 to about 50 bases, about 20 to about 40 bases, about 20 to about 30 bases, about 25 to about 100 bases, and about 25 to about 90 bases. Bases, approximately 25 to 80 bases, approximately 25 to 70 bases, approximately 25 to 60 bases, approximately 25 to 50 bases, approximately 25 to 40 bases, approximately 25 to 30 bases, approximately 30 to 100 bases, approximately 30 to 90 bases, approximately 30 to 80 bases, approximately 30 to 70 bases, approximately 30 to 60 bases, approximately 30 to 50 bases, approximately 30 to Approximately 40 bases, approximately 40 to approximately 100 bases, approximately 40 to approximately 90 bases, approximately 40 to approximately 80 bases, approximately 40 to approximately 70 bases, approximately 40 to approximately 60 bases, approximately 40 to approximately 50 bases, approximately 50 to approximately 100 bases, approximately 50 to approximately 90 bases, approximately 50 to approximately 80 bases, approximately 50 to approximately 70 bases, approximately 50 to approximately 60 bases, approximately 60 to approximately 100 bases, approximately 6 They can have lengths of 0 to approximately 90 bases, approximately 60 to approximately 80 bases, approximately 60 to approximately 70 bases, approximately 70 to approximately 100 bases, approximately 70 to approximately 90 bases, approximately 70 to approximately 80 bases, approximately 80 to approximately 100 bases, approximately 80 to approximately 90 bases, approximately 90 to approximately 100 bases, and approximately 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 bases.

[0135] In any of the embodiments described above, each of the plurality of second indexing primers may have a length of about 20 to about 100 bases. For example, without limitation, each of the plurality of second indexing primers may have a length of about 20 to about 100 bases, about 20 to about 90 bases, about 20 to about 80 bases, about 20 to about 70 bases, about 20 to about 60 bases, about 20 to about 50 bases, about 20 to about 40 bases, about 20 to about 30 bases, about 25 to about 100 bases, and about 25 to about 90 bases. Bases, approximately 25 to 80 bases, approximately 25 to 70 bases, approximately 25 to 60 bases, approximately 25 to 50 bases, approximately 25 to 40 bases, approximately 25 to 30 bases, approximately 30 to 100 bases, approximately 30 to 90 bases, approximately 30 to 80 bases, approximately 30 to 70 bases, approximately 30 to 60 bases, approximately 30 to 50 bases, approximately 30 to Approximately 40 bases, approximately 40 to approximately 100 bases, approximately 40 to approximately 90 bases, approximately 40 to approximately 80 bases, approximately 40 to approximately 70 bases, approximately 40 to approximately 60 bases, approximately 40 to approximately 50 bases, approximately 50 to approximately 100 bases, approximately 50 to approximately 90 bases, approximately 50 to approximately 80 bases, approximately 50 to approximately 70 bases, approximately 50 to approximately 60 bases, approximately 60 to approximately 100 bases, approximately 6 They can have lengths of 0 to approximately 90 bases, approximately 60 to approximately 80 bases, approximately 60 to approximately 70 bases, approximately 70 to approximately 100 bases, approximately 70 to approximately 90 bases, approximately 70 to approximately 80 bases, approximately 80 to approximately 100 bases, approximately 80 to approximately 90 bases, approximately 90 to approximately 100 bases, and approximately 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 bases.

[0136] In any of the embodiments described above, each of the plurality of first indexing primers may further include the sequence of SEQ ID NO: 87. In any of the embodiments described above, each of the plurality of second indexing primers may further include the sequence of SEQ ID NO: 78.

[0137] In any of the embodiments described above, the first common nucleotide sequence and the first 3' terminal portion have a melting temperature (T) higher than the ligation temperature.m ) may have the following characteristics. For example, without limitation, the melting temperature of the first common nucleotide sequence and the first 3' terminal portion may be 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C higher than the ligation temperature. As a further example, without limitation, the first common nucleotide sequence and the first 3' terminal portion are 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C , 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃ or higher than 80℃, or approximately 40℃ to approximately 80℃, approximately 40℃ to approximately 70℃, approximately 40℃ to approximately 60℃, approximately 40℃ to approximately 50℃, approximately 50℃ to approximately 80℃, approximately 50℃ to approximately 70℃, approximately 50℃ to approximately 60℃, approximately 60℃ to approximately 80℃, approximately 60℃ to approximately 70℃ or approximately 70℃ to approximately 80℃ m It may have a first common nucleotide sequence and a first 3' terminal portion below the first, second, and third denaturation temperatures. m It should be understood that it has this characteristic.

[0138] In any of the embodiments described above, the ligation temperature may be any temperature sufficient for the ligase to ligate one of the plurality of first indexing primers to the first 5' end of the first strand or the second 5' end of the second strand. The ligation temperature is the T of a partially double-stranded DNA substrate. m It should be further understood that it cannot be any higher. Ligation temperature is partially the T of double-stranded DNA substrate. mAt higher temperatures, multiple primary indexing primers may act as primers rather than ligated 5' adapters. As an example, without limitation, ligation temperatures may range from approximately 25°C to approximately 40°C. As further examples, without limitation, ligation temperatures may range from approximately 25°C to approximately 40°C, approximately 25°C to approximately 37°C, approximately 25°C to approximately 35°C, approximately 25°C to approximately 30°C, approximately 30°C to approximately 40°C, approximately 30°C to approximately 35°C, approximately 35°C to approximately 40°C, approximately 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0139] In any of the embodiments described above, the ligation duration may be any time sufficient to ligate one of the plurality of first indexing primers to the first 5' end of the first chain or the second 5' end of the second chain. In any of the embodiments described above, the ligation duration may range from about 5 minutes to about 60 minutes. For example, without limitation, the duration of ligation could be approximately 5 to 60 minutes, approximately 5 to 50 minutes, approximately 5 to 40 minutes, approximately 5 to 30 minutes, approximately 5 to 20 minutes, approximately 5 to 10 minutes, approximately 10 to 60 minutes, approximately 10 to 50 minutes, approximately 10 to 40 minutes, approximately 10 to 30 minutes, approximately 10 to 20 minutes, approximately 20 to 60 minutes, approximately 20 to 50 minutes, approximately 20 to 40 minutes, approximately 20 to 30 minutes, approximately 30 to 60 minutes, approximately 30 to 50 minutes, approximately 30 to 40 minutes, approximately 40 to 60 minutes, approximately 40 to 50 minutes, approximately 50 to 60 minutes, or approximately 5, 10, 15, 20, 25, 30, 40, 50, or 60 minutes.

[0140] In any of the embodiments described above, any suitable ligase can be used. The ligase should be a thermally unstable ligase that can be ligated in a low-magnesium buffer and should be inactivated at a first denaturation temperature over a first denaturation duration. The low-magnesium buffer conditions should be suitable for PCR. For example, without limitation, the ligase may be a T3 DNA ligase. In any of the embodiments described above, for example, without limitation, the ligase may be added at a rate of about 30 to about 300 enzyme units per μL of the first reaction mixture.

[0141] In any of the embodiments described above, any suitable polymerase can be used. In some embodiments, the polymerase is inactive at the ligation temperature. For example, without limitation, the polymerase may further comprise a hot-start antibody or aptamer. In any of the embodiments described above, the polymerase may be a hot-start polymerase having an activation temperature, which is lower than the first denaturation temperature. For example, without limitation, the activation temperature may be lower than the first, second, and third denaturation temperatures. For example, without limitation, the polymerase may be a thermostable DNA polymerase having 3'-5' exonuclease proofreading activity, selected from the group consisting of Kapa HiFi DNA Polymerase (Roche), NEB Q5 DNA Polymerase (NEB), PrimeStar GXL DNA Polymerase (Takara), or High Fidelity DNA Polymerase (Qiagen). In any of the embodiments described above, the DNA polymerase may further comprise a hot-start antibody or aptamer that increases the activation temperature of the DNA polymerase. In such embodiments, the DNA polymerase may be Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 Hot Start DNA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara), or High Fidelity Hot Start DNA Polymerase (Qiagen).

[0142] In any of the embodiments described above, the first denaturation duration, the second denaturation duration, and the third denaturation duration may each range from about 30 seconds to about 2 minutes. For example, without limitation, the first denaturation duration, the second denaturation duration, and the third denaturation duration may each range from about 30 seconds to about 2 minutes, about 30 seconds to about 1.5 minutes, about 30 seconds to about 1 minute, about 1 minute to about 2 minutes, about 1 minute to about 1.5 minutes, about 1.5 minutes to about 2 minutes, about 30 seconds, 45 seconds, 1 minute, 1.5 minutes, or 2 minutes.

[0143] In any of the embodiments described above, the first, second, and third denaturation temperatures may be approximately 95°C to approximately 98°C, respectively. It should be understood that any suitable temperature for denaturing the double-stranded DNA annealed in each PCR cycle may be used. It should be understood that the first denaturation temperature should be sufficiently high to be above the melting temperatures of the first and second strands. Those skilled in the art can easily determine the temperature and required time through ordinary experimentation and / or knowledge in the art.

[0144] In any of the embodiments described above, the first annealing temperature, the second annealing temperature, and the third annealing temperature may each be about 55°C to about 65°C. For example, without limitation, the first annealing temperature, the second annealing temperature, and the third annealing temperature may each be about 55°C to about 65°C, 55°C to about 60°C, about 60°C to about 65°C, about 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C.

[0145] In any of the embodiments described above, the first annealing duration, the second annealing duration, and the third annealing duration may each range from approximately 10 seconds to approximately 60 seconds. It should be understood that the first annealing temperature, the second annealing temperature, and the third annealing temperature, as well as the first annealing duration, the second annealing duration, and the third annealing duration, should be sufficient to perform the annealing required for each PCR step. Those skilled in the art can readily determine these temperatures and required times through ordinary experimentation and / or knowledge in the art.

[0146] In any of the embodiments described above, the first elongation temperature, the second elongation temperature, and the third elongation temperature may each range from about 60°C to about 72°C. For example, without limitation, the first elongation temperature, the second elongation temperature, and the third elongation temperature may be about 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, or 72°C.

[0147] In any of the embodiments described above, the first extension duration, the second extension duration, and the third extension duration may each range from approximately 30 seconds to approximately 5 minutes. It should be understood that the first extension temperature, the second extension temperature, and the third extension temperature, as well as the first extension duration, the second extension duration, and the third extension duration, should be sufficient to perform the extension required for each PCR step. Those skilled in the art can readily determine these temperatures and required times through ordinary experimentation and / or knowledge in the art.

[0148] In any of the embodiments described above, a plurality of first indexing primers and a plurality of second indexing primers can be added to the first reaction mixture at concentrations ranging from about 100 nM to about 1 μM. For example, without limitation, a plurality of first indexing primers and a plurality of second indexing primers can be added to the first reaction mixture at concentrations ranging from about 100 nM to about 200 nM.

[0149] In any of the embodiments described above, the partially double-stranded DNA substrate may be derived from genomic DNA, cDNA obtained by reverse transcription of RNA, whole-genome amplification (WGA), multiplex PCR, or synthetic DNA.

[0150] How to use blocker oligonucleotides In any of the embodiments described above, step (ii) is to add a blocker oligonucleotide to the first reaction mixture, wherein the blocker oligonucleotide comprises a 5' portion at least partially complementary to at least a portion of the second 3' terminal portion of each of the plurality of second indexing primers, and the melting temperature of the blocker oligonucleotide and each of the plurality of second indexing primers is higher than the ligation temperature and lower than the first annealing temperature and the third annealing temperature. In such embodiments, the blocker oligonucleotide may be added in an amount sufficient to suppress the ligation of each of the plurality of second indexing primers to the first 5' terminal of the first chain and the second 5' terminal of the second chain. In some embodiments, the blocker oligonucleotide comprises a 5' portion that is fully complementary to at least a portion of the second 3' terminal portion of each of the plurality of second indexing primers.

[0151] In any of the embodiments described above, the melting temperature of each of the blocker oligonucleotide and the plurality of second indexing primers may be higher than the melting temperature of each of the blocker oligonucleotide and the plurality of first indexing primers.

[0152] In any of the embodiments described above, the blocker oligonucleotide can be added in an amount equal to 1, 1.5, 2, 4, or 6 times the amount of the multiple second indexing primers added in step (ii). For example, without limitation, the blocker oligonucleotide can be added in an amount equal to 1 to about 6 times, about 1.5 to about 6 times, about 2 to about 6 times, about 4 to about 6 times, 1 to 4 times, about 1.5 to about 4 times, about 2 to about 4 times, 1 to about 2 times, 1 to about 1.5 times, or about 1.5 to about 2 times the amount of the multiple second indexing primers added in step (ii). Alternatively, the multiple second indexing primers can be pre-annealed to the blocker oligonucleotide before adding the multiple second indexing primers in step (ii).

[0153] In any of the embodiments described above, the blocker oligonucleotide may further include a first further portion located at 3' relative to the 5' portion, which is complementary to each of the plurality of second indexing primers but not complementary to each of the plurality of first indexing primers. In any of the embodiments described above, the blocker oligonucleotide may contain about 14 to about 200 bases. For example, without limitation, blocker oligonucleotides can have lengths of approximately 14 to 200 bases, approximately 14 to 150 bases, approximately 14 to 100 bases, approximately 14 to 50 bases, approximately 14 to 25 bases, approximately 25 to 200 bases, approximately 25 to 150 bases, approximately 25 to 100 bases, approximately 25 to 50 bases, approximately 50 to 200 bases, approximately 50 to 150 bases, approximately 50 to 100 bases, approximately 100 to 200 bases, approximately 100 to 150 bases, approximately 150 to 200 bases, and approximately 14, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 bases. It should be understood that these lengths are illustrative and that blocker oligonucleotides may be of any preferred length.

[0154] In any of the embodiments described above, the melting temperature between the first further portion and each of the plurality of second indexing primers may be approximately the same as or higher than the melting temperature between the 5' portion and each of the plurality of first indexing primers and each of the plurality of second indexing primers. For example, without limitation, the melting temperature between the first further portion and each of the plurality of second indexing primers may be at least 1°C higher than the melting temperature between the 5' portion and each of the plurality of first indexing primers and each of the plurality of second indexing primers. For example, without limitation, the melting temperature between the first further portion and each of the plurality of second indexing primers may be at least 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, or 20°C higher than the melting temperature between the 5' portion and each of the plurality of first indexing primers and each of the plurality of second indexing primers.

[0155] In any of the embodiments described above, the blocker oligonucleotide may include a second further portion located between the 5' portion and the first further portion, the second further portion being not complementary to any of the plurality of first indexing primers and not complementary to any of the plurality of second primers. For example, without limitation, the second further portion may have a length of about 1 to about 30 bases. For example, without limitation, the second further portion may have a length of about 1 to about 30 bases, about 1 to about 25 bases, about 1 to about 20 bases, about 1 to about 15 bases, about 1 to about 10 bases, about 1 to about 5 bases, about 5 to about 30 bases, about 5 to about 25 bases, about 5 to about 20 bases, about 5 to about 15 bases, about 5 to about 10 bases, and about 10 to about 30 bases. They can have lengths of approximately 10 to 25 bases, approximately 10 to 20 bases, approximately 10 to 15 bases, approximately 15 to 30 bases, approximately 15 to 25 bases, approximately 15 to 20 bases, approximately 20 to 30 bases, approximately 20 to 25 bases, approximately 25 to 30 bases, and approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 bases.

[0156] In any of the embodiments described above, the blocker oligonucleotide may contain mismatches in its sequence that make it partially complementary to each of the multiple second indexing primers. This can be used to lower the melting temperatures of the blocker oligonucleotide and each of the multiple second indexing primers so that the blocker does not become active during the PCR steps, for example, steps (iv) to (iv).

[0157] In any of the embodiments described above, the blocker oligonucleotide may include sequences that are not complementary to each of the plurality of second indexing primers. The sequences that are not complementary to each of the plurality of second indexing primers may have a length of about 1 to about 30 bases. For example, without limiting, the sequences that are not complementary to each of the plurality of second indexing primers may have a length of about 1 to about 30 bases. For further example, without limiting, the second further portion may have a length of about 1 to about 30 bases, about 1 to about 25 bases, about 1 to about 20 bases, about 1 to about 15 bases, about 1 to about 10 bases, about 1 to about 5 bases, about 5 to about 30 bases, about 5 to about 25 bases, about 5 to about 20 bases, about 5 to about 15 bases, about 5 to about 10 bases, and about 10 to about 30 bases. It can have lengths of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 bases.

[0158] In any of the embodiments described above, the blocker oligonucleotide may further include a 3' modification to inhibit polymerase elongation, provided it does not include a hairpin portion. For example, without limitation, the 3' modification may be a DNA sequence that is not complementary to the adjacent primer sequence, such as a C3 carbon spacer, hexanediol, spacer 9, spacer 18, phosphate, RNA nucleotides such as 2',3'-dideoxynucleosides ddA, ddT, ddC and ddG, 3'-deoxynucleosides 3'-A, 3'-T, 3'-C and 3'-G, rU, 3-O-methylnucleotide, or poly-T, poly-A, poly-C and poly-G, and further includes a nuclease-resistant binding to prevent proofreading polymerase 3'-5' exonuclease activity from removing the DNA sequence that is not complementary to the adjacent primer sequence.

[0159] In any of the embodiments described above, the melting temperature between each of the blocker oligonucleotides and the plurality of first indexing primers is lower than the melting temperature between each of the blocker oligonucleotides and the plurality of second indexing primers. For example, but not limited to, the melting temperature between each of the blocker oligonucleotides and the plurality of first indexing primers may be at least 5°C lower than the melting temperature between each of the blocker oligonucleotides and the plurality of second indexing primers. For example, but not limited to, the melting temperature between each of the blocker oligonucleotides and the plurality of first indexing primers may be at least 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C or higher than the melting temperature between each of the blocker oligonucleotides and the plurality of second indexing primers.

[0160] In any of the embodiments described above, the 5' portion of the blocker oligonucleotide may contain the sequence of SEQ ID NO: 127. In such cases, each of the multiple second indexing primers may contain the sequence of SEQ ID NO: 126.

[0161] In any of the embodiments described above, the blocker oligonucleotide may further include a hairpin portion located at 3' of the first further portion, the hairpin portion comprising a first hairpin arrangement and a second hairpin arrangement, the first hairpin arrangement being located at 5' of the second hairpin arrangement, the first and second hairpin arrangements being complementary, and the hairpin portion having a melting temperature higher than the first, second, and third annealing temperatures. In such embodiments, the blocker oligonucleotide further includes a 3' hydroxyl group.

[0162] In any of the embodiments described above, the hairpin portion may further include a third hairpin sequence between the first and second hairpin sequences. In such embodiments, the third sequence can form a loop sufficient to enable the formation of a stable stem-loop structure by the hairpin portion and the first further portion. In such embodiments, the third hairpin sequence may have a length of about 4 bases to about 20 bases or more. It should be understood that the third hairpin sequence can form a loop sufficient to enable the formation of a stable stem-loop structure by the first and second hairpin sequences.

[0163] In any of the embodiments described above, the melting temperature of the hairpin portion may be higher than the melting temperature between the 5' portion and each of the multiple second indexing primers, between the further portions and each of the multiple second indexing primers, or both.

[0164] In any of the embodiments described above, the second set of conditions may be even more sufficient to cause the DNA polymerase to extend the 3' end of the blocker (hairpin) oligonucleotide to produce an extended hairpin blocker. In such embodiments, the extended hairpin blocker may have a stabilized secondary structure that provides it with a melting temperature higher than the second and third annealing temperatures. For example, but not limited to, the melting temperature of the extended hairpin blocker may be at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, or 30°C higher than the second and third annealing temperatures.

[0165] In any of the embodiments described above, the blocker oligonucleotide may further contain a dU base, step (ii) further comprises adding uracil DNA glycosylase to the first reaction mixture, the first set of conditions in step (iii) is further sufficient for UDG to excise the dU base and create an abasic site in the blocker oligonucleotide, and in step (iv), the second set of conditions is further sufficient to inactivate the UDG enzyme.

[0166] In any of the embodiments described above, the method may further include sequencing the fifth and seventh chains or the sixth and eighth chains.

[0167] In any of the embodiments described above, the first common nucleotide sequence and the first 3' terminal portion may have a melting temperature higher than the ligation temperature, but lower than the first annealing temperature. In such embodiments, the melting temperature of the first common nucleotide sequence and the first 3' terminal portion may be at least 1°C higher than the ligation temperature. For example, but not limited to, the melting temperature of the first common nucleotide sequence and the first 3' terminal portion may be at least 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, or 20°C higher than the ligation temperature. For example, but not limited to, the melting temperature of the first common nucleotide sequence and the first 3' terminal portion may be at least 1°C lower than the first annealing temperature, and optionally the second and third annealing temperatures. As a further example, but not limited to, the melting temperatures of the first common nucleotide sequence and the first 3' terminal portion may be at least 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, or 20°C lower than the first annealing temperature and, optionally, the second or third annealing temperature.

[0168] In some embodiments, the first 3' terminal portion and the second 3' terminal portion may have a melting temperature lower than the first annealing temperature. In such embodiments, the second and fourth portions may further include a second common nucleotide sequence, and each of the plurality of second indexing primers may be complementary to a second nucleotide sequence having a melting temperature between the second indexing primer and the first common nucleotide sequence, the second common nucleotide sequence having a melting temperature higher than the first annealing temperature.

[0169] In any of the embodiments described above, each of the blocker oligonucleotides and the plurality of second indexing primers may have a melting temperature lower than the melting temperature of each of the plurality of second indexing primers and the partially double-stranded DNA substrate.

[0170] Method for ligation-coupled PCR - neither indexing primer contains a common 3' end sequence; a hairpin adapter is used. In some embodiments, a method for ligation-coupled PCR provides a partially double-stranded DNA substrate comprising a first strand and a second strand, comprising a first 3' overhang, a double-stranded portion and a second 3' overhang, wherein the first strand comprises a first 5' end, a first portion and a second portion in the 5'-to-3' direction, and the second strand comprises a second 5' end, a third portion and a fourth portion in the 5'-to-3' direction, wherein the first portion of the first strand and the third portion of the second strand are complementary and form a double-stranded portion, and the second portion of the first strand is A 3' overhang is formed by the first, and the fourth portion of the second strand forms the second 3' overhang; the second portion of the first strand and the fourth portion of the second strand each contain a first common nucleotide sequence located at the 5' end of the first 3' overhang and the second 3' overhang, respectively; the second portion of the first strand and the fourth portion of the second strand each contain a second common nucleotide sequence located at 3' relative to the first common nucleotide sequence; a plurality of first indexing primers, a plurality of second indexing primers, a plurality of 5' adductors The first reaction mixture is produced by adding a primer, ligase, DNA polymerase, and deoxynucleotide triphosphate (dNTP) to a partially double-stranded DNA substrate, wherein each of a plurality of first indexing primers contains a first 3' terminal portion that is not complementary to the first common nucleotide sequence, each of a plurality of second indexing primers contains a second 3' terminal portion that is not complementary to the first common nucleotide sequence, the second 3' terminal sequence is complementary to the second common nucleotide sequence, and each of a plurality of 5' adapters contains the first common nucleotide The 5' adapter comprises a third 3' terminal sequence complementary to the creotide sequence, a first 5' portion located at 5' of the third 3' terminal sequence, a second 5' portion located at 5' relative to the first 5' portion and complementary to the first 5' portion, and a replication blocker capable of inhibiting DNA polymerase located at the 5' end of the first 5' portion, wherein the 5' adapter can form a hairpin formed by annealing the first 5' portion to the second 5' portion;The first reaction mixture undergoes a ligation duration sufficient to produce a second reaction mixture, which includes: 1) a third 3' terminal portion annealing to the first common nucleotide sequence; and 2) a ligase ligating one of the multiple 5' adapters to the first 5' end of the first strand and the second 5' end of the second strand, resulting in a third strand containing one of the multiple 5' adapters, the first and second portions, in the 5'-to-3' direction, and a fourth strand containing one of the multiple 5' adapters, the third and fourth portions, in the 5'-to-3' direction. Incubate under a first set of conditions including ligation temperature; the second reaction mixture a) inactivates ligase, denatures double-stranded DNA, and activates DNA polymerase as needed; b) the second 3' end portion of one of several second indexing primers anneals to the second common nucleotide sequence of the second portion of the third strand, and one of several second indexing primers anneals to the second common nucleotide sequence of the fourth portion of the fourth strand; and c) DNA polymerase anneals to the second of the third strand The third, fourth, and fifth strands are formed by extending one of the multiple second indexing primers annealed to the second common nucleotide sequence of the second portion of the fourth strand, and extending one of the multiple second indexing primers annealed to the second common nucleotide sequence of the fourth portion of the fourth strand, with the fifth strand containing one of the multiple second indexing primers, the third portion, the reverse complement of the first common nucleotide sequence, and the reverse complement of the first 5' portion of one of the multiple 5' adapters in the 5' to 3' direction. The mixture is incubated under a second set of conditions, including a first denaturation temperature over a first denaturation duration, a first annealing temperature over a first extension duration, and a first extension temperature over a first extension duration, to produce a third reaction mixture comprising a sixth chain containing one of several second indexing primers, a first portion, a reverse complement of the first common nucleotide sequence, and a reverse complement of the first 5' portion of one of several 5' adapters, in the 5' to 3' direction, and the sixth chain is formed by incubation;The third reaction mixture a) denatures any double-stranded DNA, b) the first 3' end portion of one of the multiple first indexing primers anneals to the reverse complement of one of the multiple 5' adapters of the fifth strand, and one of the multiple first indexing primers anneals to the reverse complement of one of the multiple 5' adapters of the sixth strand, and c) DNA polymerase extends one of the multiple first indexing primers that annealed to the reverse complement of one of the multiple 5' adapters of the fifth strand, and extends one of the multiple first indexing primers that annealed to the reverse complement of one of the multiple 5' adapters of the sixth strand, thereby extending the fifth Incubating under a third set of conditions sufficient to produce a fourth reaction mixture comprising a second denaturation temperature over a second denaturation duration, a second annealing temperature over a second annealing duration, and a second extension temperature over a second extension duration, wherein the seventh chain comprises one of a plurality of first indexing primers, a first common nucleotide sequence, a first portion, and a reverse complement of one of a plurality of second indexing primers in the 5'-3' direction, and the eighth chain comprises one of a plurality of first indexing primers, a first common nucleotide sequence, a third portion, and a reverse complement of one of a plurality of second indexing primers in the 5'-3' direction;The fourth reaction mixture a) denatures any double-stranded DNA, b) one of the multiple second indexing primers anneals to the reverse complement of one of the multiple second indexing primers on the seventh strand, one of the multiple second indexing primers anneals to the reverse complement of one of the multiple second indexing primers on the eighth strand, and c) DNA polymerase anneals to the multiple second indexins annealed to the seventh and eighth strands, respectively. The method involves extending one of the indexing primers and incubating it under a fourth set of conditions including a third denaturation temperature over a third denaturation duration, a third annealing temperature over a third annealing duration, and a third extension temperature over a third extension duration, sufficient to produce a fifth reaction mixture including a seventh, eighth, ninth, and tenth chain, wherein the ninth chain is in the 5' to 3' direction, one of a plurality of second indexing primers, a third portion, and a first common The fifth reaction mixture includes a reverse complement of a nucleotide sequence and a reverse complement of one of a plurality of first indexing primers, wherein the tenth strand includes one of a plurality of second indexing primers, a first portion, a reverse complement of a first common sequence, and a reverse complement of one of a plurality of first indexing primers in the 5'-3' direction, wherein the seventh and ninth strands are complementary and the eighth and tenth strands are complementary; and the fifth reaction mixture may include incubating at least a portion of a plurality of first indexing primers, at least a portion of a plurality of second indexing primers, and DNA polymerase under a fifth set of conditions including a fourth denaturation temperature over a fourth denaturation duration, a fourth annealing temperature over a fourth annealing duration, and a fourth extension temperature over a fourth extension duration, sufficient to amplify the seventh and ninth strands and the eighth and tenth strands. ;

[0171] In the embodiments described above, it should be understood that the second common nucleotide sequence may be different from the first common nucleotide sequence, or it may overlap with the first common nucleotide sequence or be its 3' portion. Therefore, in some embodiments, one of the multiple second indexing primers may not be able to ligate to either the first or second strand, but each of the multiple second indexing primers may be able to anneal to the 3' end of the first common nucleotide sequence so that it can still function as a primer. Alternatively, in some embodiments, the second common nucleotide sequence may be different from the first common nucleotide sequence.

[0172] In any of the embodiments above, steps (i) to (vii) can be carried out in a single sealed tube. In any of the embodiments above, the method may not include any purification steps between steps (i) to (vii).

[0173] In any of the embodiments described above, if multiple 5' adapters are used for 5' adapter ligation, the first 5' portion and the second 5' portion may each have a length of about 12 to about 20 bases.

[0174] In any of the embodiments described above, if multiple 5' adapters are used for 5' adapter ligation, each of the multiple 5' adapters may further include an intervening sequence between the first 5' portion and the second 5' portion. For example, but not limited to, the intervening sequence may have a length of about 4 to about 20 bases.

[0175] In any of the embodiments described above, if multiple 5' adapters are used for 5' adapter ligation, the replication block can be selected from a stable debase site, a C3 spacer, a hexanediol, a spacer 9, a spacer 18, three or more rU bases, and a 2'-O-methylRNA base.

[0176] In any of the embodiments described above, if multiple 5' adapters are used for 5' adapter ligation, each of the multiple 5' adapters may have a length of about 25 to about 100 bases. As an example, but not limited to, each of the multiple 5' adapters may have a length of about 25 to about 100 bases, about 30 to about 100 bases, about 40 to about 100 bases, about 50 to about 100 bases, about 60 to about 100 bases, about 70 to about 100 bases, about 80 to about 100 bases, about 90 to about 100 bases, or about 25, 30, 40, 50, 60, 70, 80, 90, or 100 bases.

[0177] In any of the embodiments above, when multiple 5' adapters are used for 5' adapter ligation, the partially double-stranded DNA substrate can have lengths ranging from about 24 to about 6000 bases. For example, but not limited to, partially double-stranded DNA substrates can range in length from about 24 to about 6000 bases, about 24 to about 5500 bases, about 24 to about 5000 bases, about 24 to about 4500 bases, about 24 to about 4000 bases, about 24 to about 3500 bases, about 24 to about 3000 bases, about 24 to about 2500 bases, about 24 to about 2000 bases, about 24 to about 1500 bases, about 24 to about 1000 bases, about 24 to about 750 bases, and about 24 to about 500 bases. , approximately 24 bases to approximately 250 bases, approximately 24 bases to approximately 200 bases, approximately 24 bases to approximately 100 bases, approximately 24 bases to approximately 50 bases, approximately 100 bases to approximately 6000 bases, approximately 100 bases to approximately 5500 bases, approximately 100 bases to approximately 5000 bases, approximately 100 bases to approximately 4500 bases, approximately 100 bases to approximately 4000 bases, approximately 100 bases to approximately 3500 bases, approximately 100 bases to approximately 3000 bases, approximately 100 bases to approximately 2500 bases, approximately 100 bases to approximately 2000 bases, approximately 100 bases to approximately 1500 bases, approximately 100 bases to Approximately 1000 bases, approximately 100 to approximately 750 bases, approximately 100 to approximately 500 bases, approximately 100 to approximately 250 bases, approximately 100 to approximately 200 bases, approximately 200 to approximately 6000 bases, approximately 200 to approximately 5500 bases, approximately 200 to approximately 5000 bases, approximately 200 to approximately 4500 bases, approximately 200 to approximately 4000 bases, approximately 200 to approximately 3500 bases, approximately 200 to approximately 3000 bases, approximately 200 to approximately 2500 bases, approximately 200 to approximately 2000 bases, approximately 200 to approximately 150 0 bases, approximately 200 bases to approximately 1000 bases, approximately 200 bases to approximately 750 bases, approximately 200 bases to approximately 500 bases, approximately 200 bases to approximately 250 bases, approximately 250 bases to approximately 6000 bases, approximately 250 bases to approximately 5500 bases, approximately 250 bases to approximately 5000 bases, approximately 250 bases to approximately 4500 bases, approximately 250 bases to approximately 4000 bases, approximately 250 bases to approximately 3500 bases, approximately 250 bases to approximately 3000 bases, approximately 250 bases to approximately 2500 bases, approximately 250 bases to approximately 2000 bases, approximately 250 bases to approximately 1500 bases,Approximately 250 bases to approximately 1000 bases, approximately 250 bases to approximately 750 bases, approximately 250 bases to approximately 500 bases, approximately 500 bases to approximately 6000 bases, approximately 500 bases to approximately 5500 bases, approximately 500 bases to approximately 5000 bases, approximately 500 bases to approximately 4500 bases, approximately 500 bases to approximately 4000 bases, approximately 500 bases to approximately 3500 bases, approximately 500 bases to approximately 3000 bases, approximately 500 bases to approximately 2500 bases, approximately 500 bases to approximately 2000 bases, approximately 500 bases to approximately 1500 bases, approximately 500 bases to approximately 1000 bases, approximately 500 bases to approximately 750 bases, approximately 750 bases to approximately 6 000 bases, approximately 750 bases to approximately 5500 bases, approximately 750 bases to approximately 5000 bases, approximately 750 bases to approximately 4500 bases, approximately 750 bases to approximately 4000 bases, approximately 750 bases to approximately 3500 bases, approximately 750 bases to approximately 3000 bases, approximately 750 bases to approximately 2500 bases, approximately 750 bases to approximately 2000 bases, approximately 750 bases to approximately 1500 bases, approximately 750 bases to approximately 1000 bases, approximately 1000 bases to approximately 6000 bases, approximately 1000 bases to approximately 5500 bases, approximately 1000 bases to approximately 5000 bases, approximately 1000 bases to approximately 4500 bases, approximately 1000 bases to approximately 4000 bases Bases, approximately 1000 bases to approximately 3500 bases, approximately 1000 bases to approximately 3000 bases, approximately 1000 bases to approximately 2500 bases, approximately 1000 bases to approximately 2000 bases, approximately 1000 bases to approximately 1500 bases, approximately 1500 bases to approximately 6000 bases, approximately 1500 bases to approximately 5500 bases, approximately 1500 bases to approximately 5000 bases, approximately 1500 bases to approximately 4500 bases, approximately 1500 bases to approximately 4000 bases, approximately 1500 bases to approximately 3500 bases, approximately 1500 bases to approximately 3000 bases, approximately 1500 bases to approximately 2500 bases, approximately 1500 bases to approximately 2000 bases, approximately 2000 bases From approximately 6000 bases, from approximately 2000 bases to approximately 5500 bases, from approximately 2000 bases to approximately 5000 bases, from approximately 2000 bases to approximately 4500 bases, from approximately 2000 bases to approximately 4000 bases, from approximately 2000 bases to approximately 3500 bases, from approximately 2000 bases to approximately 3000 bases, from approximately 2000 bases to approximately 2500 bases, from approximately 2500 bases to approximately 6000 bases, from approximately 2500 bases to approximately 5500 bases, from approximately 2500 bases to approximately 5000 bases, from approximately 2500 bases to approximately 4500 bases, from approximately 2500 bases to approximately 4000 bases, from approximately 2500 bases to approximately 3500 bases, from approximately 2500 bases to approximately 3000 bases,Approximately 3000 bases to approximately 6000 bases, approximately 3000 bases to approximately 5500 bases, approximately 3000 bases to approximately 5000 bases, approximately 3000 bases to approximately 4500 bases, approximately 3000 bases to approximately 4000 bases, approximately 3000 bases to approximately 3500 bases, approximately 3500 bases to approximately 6000 bases, approximately 3500 bases to approximately 5500 bases, approximately 3500 bases to approximately 5000 bases, approximately 3500 to approximately 4500 bases, approximately 3500 to approximately 4000 bases, approximately 4000 bases to approximately 6000 bases, approximately 4000 bases to approximately 5500 bases, approximately 4000 bases to approximately 5000 bases, The lengths can range from approximately 4000 to 4500 bases, 4500 to 6000 bases, 4500 to 5500 bases, 4500 to 5000 bases, 5000 to 6000 bases, 5000 to 5500 bases, 5500 to 6000 bases, and approximately 24, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 bases. It should be understood that the lengths of partially double-stranded DNA substrates are illustrative, and other sizes are within the scope of this disclosure. It should be understood that the length of a partially double-stranded DNA substrate refers to the length of the first or second strand of the partially double-stranded DNA substrate, i.e., from the first 5' end to the 3' end of the first 3' overhang, or from the second 5' end to the 3' end of the second 3' overhang.

[0178] In any of the embodiments above, if multiple 5' adapters are used for 5' adapter ligation, the first and third portions of the first and second strands, respectively, can have lengths ranging from about 20 bases to about 6000 bases. For example, but not limited to, the first portion of the first oligonucleotide and the third portion of the second oligonucleotide may have lengths ranging from about 20 bases to about 6000 bases, about 20 bases to about 5500 bases, about 20 bases to about 5000 bases, about 20 bases to about 4500 bases, about 20 bases to about 4000 bases, about 20 bases to about 3500 bases, about 20 bases to about 3000 bases, about 20 bases to about 2500 bases, about 20 bases to about 2000 bases, about 20 bases to about 1500 bases, and about 20 bases to about 1000 bases. , approximately 20 bases to approximately 750 bases, approximately 20 bases to approximately 500 bases, approximately 20 bases to approximately 250 bases, approximately 20 bases to approximately 200 bases, approximately 20 bases to approximately 100 bases, approximately 20 bases to approximately 50 bases, approximately 100 bases to approximately 6000 bases, approximately 100 bases to approximately 5500 bases, approximately 100 bases to approximately 5000 bases, approximately 100 bases to approximately 4500 bases, approximately 100 bases to approximately 4000 bases, approximately 100 bases to approximately 3500 bases, approximately 100 bases to approximately 3000 bases, approximately 100 bases to approximately 2500 bases, approximately 100 bases to approximately 20 00 bases, approximately 100 bases to approximately 1500 bases, approximately 100 bases to approximately 1000 bases, approximately 100 bases to approximately 750 bases, approximately 100 bases to approximately 500 bases, approximately 100 bases to approximately 250 bases, approximately 100 bases to approximately 200 bases, approximately 200 bases to approximately 6000 bases, approximately 200 bases to approximately 5500 bases, approximately 200 bases to approximately 5000 bases, approximately 200 bases to approximately 4500 bases, approximately 200 bases to approximately 4000 bases, approximately 200 bases to approximately 3500 bases, approximately 200 bases to approximately 3000 bases, approximately 200 bases to approximately 2500 bases Base, approximately 200 bases to approximately 2000 bases, approximately 200 bases to approximately 1500 bases, approximately 200 bases to approximately 1000 bases, approximately 200 bases to approximately 750 bases, approximately 200 bases to approximately 500 bases, approximately 200 bases to approximately 250 bases, approximately 250 bases to approximately 6000 bases, approximately 250 bases to approximately 5500 bases, approximately 250 bases to approximately 5000 bases, approximately 250 bases to approximately 4500 bases, approximately 250 bases to approximately 4000 bases, approximately 250 bases to approximately 3500 bases, approximately 250 bases to approximately 3000 bases, approximately 250 bases to approximately 2500 bases,Approximately 250 bases to approximately 2000 bases, approximately 250 bases to approximately 1500 bases, approximately 250 bases to approximately 1000 bases, approximately 250 bases to approximately 750 bases, approximately 250 bases to approximately 500 bases, approximately 500 bases to approximately 6000 bases, approximately 500 bases to approximately 5500 bases, approximately 500 bases to approximately 5000 bases, approximately 500 bases to approximately 4500 bases, approximately 500 bases to approximately 4000 bases, approximately 500 bases to approximately 3500 bases, approximately 500 bases to approximately 3000 bases, approximately 500 bases to approximately 2500 bases, approximately 500 bases to approximately 2000 bases, approximately 500 bases to approximately 1500 bases, approximately 500 bases to approximately 1000 bases, approximately 500 to 750 bases, approximately 750 to 6000 bases, approximately 750 to 5500 bases, approximately 750 to 5000 bases, approximately 750 to 4500 bases, approximately 750 to 4000 bases, approximately 750 to 3500 bases, approximately 750 to 3000 bases, approximately 750 to 2500 bases, approximately 750 to 2000 bases, approximately 750 to 1500 bases, approximately 750 to 1000 bases, approximately 1000 to 6000 bases, approximately 1000 to 5500 bases, approximately 1000 to 5000 salts Base, approximately 1000 bases to approximately 4500 bases, approximately 1000 bases to approximately 4000 bases, approximately 1000 bases to approximately 3500 bases, approximately 1000 bases to approximately 3000 bases, approximately 1000 bases to approximately 2500 bases, approximately 1000 bases to approximately 2000 bases, approximately 1000 bases to approximately 1500 bases, approximately 1500 bases to approximately 6000 bases, approximately 1500 bases to approximately 5500 bases, approximately 1500 bases to approximately 5000 bases, approximately 1500 bases to approximately 4500 bases, approximately 1500 bases to approximately 4000 bases, approximately 1500 bases to approximately 3500 bases, approximately 1500 bases to approximately 3000 bases, approximately 1500 bases From approximately 2500 bases, from approximately 1500 bases to approximately 2000 bases, from approximately 2000 bases to approximately 6000 bases, from approximately 2000 bases to approximately 5500 bases, from approximately 2000 bases to approximately 5000 bases, from approximately 2000 bases to approximately 4500 bases, from approximately 2000 bases to approximately 4000 bases, from approximately 2000 bases to approximately 3500 bases, from approximately 2000 bases to approximately 3000 bases, from approximately 2000 bases to approximately 2500 bases, from approximately 2500 bases to approximately 6000 bases, from approximately 2500 bases to approximately 5500 bases, from approximately 2500 bases to approximately 5000 bases, from approximately 2500 bases to approximately 4500 bases, from approximately 2500 bases to approximately 4000 bases,Approximately 2500 bases to approximately 3500 bases, approximately 2500 bases to approximately 3000 bases, approximately 3000 bases to approximately 6000 bases, approximately 3000 bases to approximately 5500 bases, approximately 3000 bases to approximately 5000 bases, approximately 3000 bases to approximately 4500 bases, approximately 3000 bases to approximately 4000 bases, approximately 3000 bases to approximately 3500 bases, approximately 3500 bases to approximately 6000 bases, approximately 3500 bases to approximately 5500 bases, approximately 3500 bases to approximately 5000 bases, approximately 3500 bases to approximately 4500 bases, approximately 3500 to approximately 4000 bases, approximately 4000 bases to approximately 6000 bases, approximately 4000 bases to approximately 5500 bases, They can have lengths of approximately 4000 to 5000 bases, approximately 4000 to 4500 bases, approximately 4500 to 6000 bases, approximately 4500 to 5500 bases, approximately 4500 to 5000 bases, approximately 5000 to 6000 bases, approximately 5000 to 5500 bases, approximately 5500 to 6000 bases, and approximately 24, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 bases, respectively. It should be understood that the lengths of the first part of the first chain and the third part of the second chain are illustrative, and other sizes are withi...

Claims

1. (i) To provide a partially double-stranded DNA substrate comprising a first strand and a second strand, comprising a first 3' overhang, a double-stranded portion and a second 3' overhang, The first chain includes a first 5' end, a first portion, and a second portion in the direction from 5' to 3', The second strand includes, in the 5' to 3' direction, a second 5' end, a third portion, and a fourth portion of the partially double-stranded DNA substrate. The first portion of the first chain and the third portion of the second chain are complementary, forming the double-stranded portion. The second portion of the first chain forms the first 3' overhang. The fourth portion of the second chain forms the second 3' overhang. The second portion of the first strand and the fourth portion of the second strand each include a first common nucleotide sequence located at the 5' end of the first 3' overhang and the 5' end of the second 3' overhang, The second portion of the first chain and the fourth portion of the second chain each contain a second common nucleotide sequence located at 3' with respect to the first common nucleotide sequence. (ii) Providing a plurality of first indexing primers, a plurality of second indexing primers, a ligase, a DNA polymerase, and a deoxynucleotide triphosphate (dNTP) to the partially double-stranded DNA substrate to produce a first reaction mixture, wherein each of the plurality of first indexing primers includes a first 3' terminal portion complementary to the first common nucleotide sequence, and each of the plurality of second indexing primers includes a second 3' terminal portion complementary to the first common nucleotide sequence, and a first 5' portion located 5' relative to the second 3' terminal portion and complementary to the second common nucleotide sequence. (iii) The first reaction mixture is incubated under a first set of conditions including a ligation temperature over the duration of the ligation, the first set of conditions being a) The first 3' terminal portion anneals to the first common nucleotide sequence, b) The ligase ligates one of the plurality of first indexing primers to the first 5' end of the first chain, and one of the plurality of first indexing primers to the second 5' end of the second chain, In the direction from 5' to 3', a third chain comprising one of the plurality of first indexing primers, the first portion and the second portion, In the direction from 5' to 3', a fourth chain including one of the plurality of first indexing primers, the third portion and the fourth portion It is sufficient to generate a second reaction reaction containing, (iv) The second reaction mixture is incubated under a second set of conditions comprising a first denaturation temperature over a first denaturation duration, a first annealing temperature over a first annealing duration, and a first extension temperature over a first extension duration, wherein the second set of conditions is a) Inactivate the ligase, denature the double-stranded DNA, and activate the DNA polymerase as necessary. b) The second 3' terminal portion and the first 5' portion of one of the plurality of second indexing primers anneal to at least the first common nucleotide sequence and the second common nucleotide sequence of the second portion of the third chain, and the second 3' terminal portion and the first 5' portion of one of the plurality of second indexing primers anneal to at least the first common nucleotide sequence and the second common nucleotide sequence of the fourth portion of the fourth chain, c) The DNA polymerase extends one of the plurality of second indexing primers annealed to the first common nucleotide sequence and the second common nucleotide sequence of the second portion of the third strand, and the DNA polymerase extends one of the plurality of second indexing primers annealed to the first common nucleotide sequence and the second common nucleotide sequence of the fourth portion of the fourth strand, thereby extending the third strand, the fourth strand, the fifth strand A third reaction mixture comprising a fifth chain and a sixth chain, wherein the fifth chain comprises, in the 5' to 3' direction, one of the plurality of second indexing primers, the third portion, and the reverse complement of one of the plurality of first indexing primers, and the sixth chain is sufficient to produce a third reaction mixture comprising, in the 5' to 3' direction, one of the plurality of second indexing primers, the first portion, and the reverse complement of one of the plurality of first indexing primers, (v) The third reaction mixture is incubated under a third set of conditions comprising a second denaturation temperature over a second denaturation duration, a second annealing temperature over a second annealing duration, and a second extension temperature over a second extension duration, wherein the third set of conditions is a) Denaturing double-stranded DNA, b) One of the plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers of the fifth chain, and one of the plurality of first indexing primers anneals to the reverse complement of one of the plurality of first indexing primers of the sixth chain, c) The DNA polymerase extends one of the plurality of first indexing primers annealed to the reverse complement of one of the plurality of first indexing primers of the fifth strand, and one of the plurality of first indexing primers annealed to the reverse complement of one of the plurality of first indexing primers of the sixth strand, thereby forming a fourth reaction mixture comprising the fifth strand, the sixth strand, the seventh strand, and the eighth strand, wherein the seventh strand is 5' to 3 Sufficient to produce a fourth reaction mixture comprising, in the direction of ', one of the plurality of first indexing primers, the first portion, and the reverse complement of one of the plurality of second indexing primers, and the eighth chain comprising, in the direction of 5' to 3', one of the plurality of first indexing primers, the third portion, and the reverse complement of one of the plurality of second indexing primers, with the seventh chain being complementary to the fifth chain and the eighth chain being complementary to the sixth chain, and (vi) The fourth reaction mixture is incubated under a fourth set of conditions including a third denaturation temperature over a third denaturation duration, a third annealing temperature over a third annealing duration, and a third extension temperature over a third extension duration, wherein the fourth set of conditions is sufficient to amplify at least a portion of the plurality of first indexing primers and at least a portion of the plurality of second indexing primers, the fifth and seventh chains and the sixth and eighth chains. A ligation-coupled polymerase chain reaction (PCR) method, including [specific details omitted].

2. The method according to claim 1, wherein steps (i) to (vi) are carried out in a single sealed tube.

3. The method according to claim 1, wherein the partially double-stranded DNA substrate has a length of about 24 bases to about 6,000 bases, the first strand and the first and third portions of the second strand each have a length of about 20 bases to about 6,000 bases, the second portion of the first strand and the fourth portion of the second strand each have a length of about 6 bases to about 100 bases, the first common nucleotide sequence has a length of about 1 base to about 50 bases, the second common nucleotide sequence has a length of about 5 bases to about 30 bases, each of the plurality of first indexing primers has a length of about 20 bases to about 100 bases, and each of the plurality of second indexing primers has a length of about 20 bases to about 100 bases.

4. The first common nucleotide sequence and the first 3' terminal portion have a melting temperature (T) higher than the ligation temperature. ｍ ) has the ligation temperature being the melting temperature (T) of the partially double-stranded DNA substrate. ｍ The melting temperature (T) of the third and fourth chains is lower than ). ｍ ) is lower than the first denaturation temperature, and the first common nucleotide sequence, the first 3' terminal portion, and the second 3' terminal portion are lower than the first annealing temperature T ｍ The method according to claim 1, comprising:

5. The method according to claim 1, wherein the ligation temperature is approximately 25°C to approximately 40°C.

6. The method according to claim 1, wherein the duration of ligation is approximately 5 minutes to approximately 60 minutes.

7. The method according to claim 1, wherein the ligase is a thermally unstable ligase capable of ligation in a low-magnesium buffer solution.

8. The method according to claim 1, wherein the ligase is a T3 DNA ligase.

9. The method according to claim 8, wherein the ligase is added in an enzyme unit of about 30 to about 300 units per 50 μL of the first reaction mixture.

10. The method according to claim 1, wherein the ligase is temperature-sensitive, and the first denaturation temperature over the first denaturation duration in step (iv)(a) is sufficient to inactivate the ligase.

11. The method according to claim 1, wherein the DNA polymerase is a thermally stable DNA polymerase having 3'-5' exonuclease proofreading activity.

12. The method according to claim 1, wherein the DNA polymerase is inactive at the ligation temperature.

13. The method according to claim 12, wherein the DNA polymerase further comprises a hot-start antibody or aptamer, the hot-start antibody or aptamer raising the activation temperature of the DNA polymerase.

14. The method according to claim 1, wherein the DNA polymerase is a hot-start polymerase, and the first denaturation temperature over the first denaturation duration in step (iv)(a) is sufficient to activate the hot-start polymerase.

15. The melting temperature (T) of each of the plurality of second indexing primers, and of the first chain and the second or fourth portion of the second chain. ｍ ) each of the plurality of first indexing primers, and the T of the second or fourth portion of the first and second chains. ｍ A higher method according to claim 1.

16. The method according to claim 1, wherein the first denaturation temperature, the second denaturation temperature, and the third denaturation temperature are each independently about 95°C to about 98°C, and the first denaturation duration, the second denaturation duration, and the third denaturation duration are each independently about 30 seconds to about 2 minutes.

17. The method according to claim 1, wherein the first annealing temperature, the second annealing temperature, and the third annealing temperature are each independently about 55°C to about 65°C, and the first annealing duration, the second annealing duration, and the third annealing duration are each independently about 10 seconds to about 60 seconds.

18. The method according to claim 1, wherein the first elongation temperature, the second elongation temperature, and the third elongation temperature are each independently about 62°C to about 72°C, and the first elongation duration, the second elongation duration, and the third elongation duration are each independently about 30 seconds to about 5 minutes.

19. The method according to claim 1, wherein the plurality of first indexing primers and the plurality of second indexing primers are each independently added to the first reaction mixture in a concentration of about 100 nM to about 1 μM.

20. The method according to claim 1, further comprising sequencing the fifth and seventh chains or the sixth and eighth chains.

21. Each of the plurality of first indexing primers comprises a first 5' terminal portion, each of the plurality of second indexing primers further comprises a second 5' terminal portion, each of the first 5' terminal portion and the second 5' terminal portion comprises, in the 5' to 3' direction, a first sequence containing two or more deoxynucleotides and a second sequence containing three or more ribonucleotides, wherein the DNA polymerase has 3' to 5' exonuclease activity, and the fifth and sixth strands However, the fifth and sixth chains may further include the second 5' end portion at their 5' ends, the seventh and eighth chains may further include the first 5' end portion at their 5' ends, the fifth and seventh chains may form a first double-stranded product having a first 5' overhang and a second 5' overhang, the sixth and eighth chains may form a second double-stranded product having a third 5' overhang and a fourth 5' overhang, and the method may be The method involves adding a sufficient amount of probes complementary to each of the first, second, third, and fourth 5' overhangs, as well as a second ligase, to generate a target molar amount of the fifth, sixth, seventh, and eighth chains, wherein a greater amount of the fifth, sixth, seventh, and eighth chains is present than the target molar amount, and the probes include modifications to obtain resistance to digestion by enzymes having 3'-exonuclease activity. The fifth chain, sixth chain, seventh chain, eighth chain, second ligase, and probe are incubated under conditions sufficient to generate a pre-normalized reaction mixture, by ligating the probe to the target molar amounts of the fifth chain, sixth chain, seventh chain, and eighth chain. Adding an enzyme having 3'-exonuclease activity to the pre-normalization reaction mixture, and The pre-normalization reaction mixture and the enzyme having exonuclease activity are incubated under conditions sufficient to allow the enzyme having 3'-exonuclease activity to digest the fifth, sixth, seventh, and eighth chains that have not ligated to the probe, thereby generating a normalized next-generation sequencing (NGS) library. The method according to claim 1, further comprising:

22. The method according to claim 21, further comprising sequencing the normalized NGS library.

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