Controlled Strand Displacement for Double-Ended Sequencing

Abstract

The present application relates to methods and compositions for dual - strand sequencing. The method includes providing a DNA array comprising a surface to which DNA concatemers are immobilized. For each of a plurality of DNA concatemers on the array, the method includes annealing a first read primer to a primer binding site on the DNA concatemer, extending at least some of the first read primer to incorporate dNTPs or dNTP analogs, thereby generating a first read strand, wherein each incorporated dNTP or dNTP analog is identified as generating a first read, performing controlled MDA by extending at least some of the first read strand with a polymerase having strand displacement activity to generate multiple second strands, each second strand comprising a portion hybridized to the DNA concatemer and an unhybridized single - stranded branch; and annealing a second read primer to the single - stranded branches of the multiple second strands and extending the second read primer to generate a second read.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Application No. 62 / 952,713, filed on December 23, 2019. The provisional application is incorporated herein by reference for all purposes. Technical Field

[0003] The present invention relates to the fields of DNA sequencing, genomics, and molecular biology. Background Art

[0004] Paired - end sequencing allows for sequencing of both ends of a DNA fragment. It can be performed by sequencing a DNA template and its complementary strand. See U.S. Patent No. 10,227,647. Compared to single - end sequencing, paired - end sequencing produces twice as many reads in library preparation for the same amount of time and effort. Using the reads generated in paired - end sequencing allows for accurate read alignment and detection of mutations (e.g., indel variants) that are difficult to detect by single - end sequencing. Summary of the Invention

[0005] The paired - end sequencing method disclosed herein uses controlled production of strands complementary to a DNA template (first strand) such that they remain associated with the DNA template. These complementary strands are called second strands. The nucleotide sequence of a DNA template (e.g., a DNA concatemer) can be determined by generating a first read from the DNA template and a second read from the second strand. Methods are also disclosed for synchronizing the production of second strands and maximizing the number of second strands of a suitable length to generate second reads.

[0006] In some aspects, the method disclosed herein includes extending a first reading primer hybridized to a plurality of single - stranded DNA concatemers immobilized on an array to generate a first read of a plurality of single - stranded DNAs, wherein the extension generates a first reading strand; performing controlled multiple displacement amplification (MDA) by extending the first reading strand or a portion thereof with a polymerase having strand displacement activity to generate multiple second strands, each second strand comprising (i) a sequence hybridized to one of the plurality of DNA concatemers, and (ii) an unhybridized single - stranded branch; and extending a second reading primer hybridized to the single - stranded branches of the multiple second strands to generate second reads.

[0007] In some aspects, the method disclosed herein includes providing a DNA array that includes a surface on which at least 1,000, at least 10,000, at least 10 5 、at least 10 6 or at least 10 7 DNA concatemers are immobilized. The number of DNA concatemers can be from 1,000 to 10 13 、104 and 10 12 、10 4 to 10 10 or 10 5 to 10 8 within the range of. For each of a plurality of DNA concatemers on the array, a first reading primer is annealed to a primer binding site on the DNA concatemer, and at least some of the first reading primer are extended to incorporate dNTP or dNTP analogs, thereby generating a first reading strand. Each of the incorporated dNTP or dNTP analogs is identified to generate a first read. The method further includes performing controlled MDA by extending at least some of the first reading strands with a polymerase having strand displacement activity to generate multiple second strands, each second strand comprising a portion hybridized to the DNA concatemer and an unhybridized single-stranded branch. The method may further include annealing a second reading primer to the single-stranded branches of the multiple second strands, and extending the second reading primer to generate a second read.

[0008] In some aspects, the methods disclosed herein include: extending a first reading primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of an excisable nucleotide to generate a first read of the plurality of single-stranded DNA concatemers, wherein the extension generates a first reading strand incorporating the excisable nucleotide. The method may further include one or more of the following: cleaving the first reading strand at the position of the excisable base to generate a fragment of the first reading strand having an extendable 3' end; performing controlled MDA by extending a fragment of the first reading primer to generate multiple second strands, and wherein the controlled MDA generates multiple second strands, each second strand comprising a sequence hybridized to one of the plurality of DNA concatemers and an unhybridized single-stranded branch; and extending a second reading primer hybridized to the single-stranded branches of the multiple second strands to generate a second read.

[0009] An array comprising a plurality of single-stranded DNA concatemers and a plurality of primers is also provided, wherein each single-stranded concatemer comprises a plurality of monomers, wherein each monomer comprises an adaptor sequence and a DNA target sequence, wherein each primer comprises a primer sequence complementary to and hybridized to the adaptor sequence of the DNA concatemer, wherein at least one primer comprises an excisable nucleotide, and wherein the at least one primer is cleavable to release the excisable nucleotide and generate two or more fragments having extendable 3' ends.

[0010] In another aspect, an array is a support comprising an array of discrete regions, where multiple regions contain clusters of clones of single-stranded DNA polynucleotides, multiple primers having reversible 3'-blocking groups, and multiple primers having extendable 3'-termini. The single-stranded polynucleotides comprise multiple monomers, each monomer comprising an adaptor sequence and a DNA target sequence. Each primer comprises a primer sequence that is complementary to and hybridizes with the adaptor sequence of the DNA template, and at least one adaptor hybridizes with one primer having a reversible 3'-blocking group and one primer having an extendable 3'-terminus, and the primer having the reversible 3'-blocking group is upstream of the primer having the extendable 3'-terminus.

[0011] The present disclosure also provides a kit that comprises multiple sequencing primers, a non-displacing DNA polymerase, a mixture of reversible terminator nucleotides for SBS, a strand-displacing DNA polymerase, and a mixture of dNTPs. The kit further comprises one or more of the following: i) a magnesium-free buffer (“magnesium-free buffer”) and ii) an extension inhibitor (e.g., EDTA, excess salts including KCl and NaCl, ionic detergents such as deoxycholate, sodium sarcosyl, and SDS, ethanol, and isopropanol). In some embodiments, the nucleotide mixture may further comprise uracil. In some embodiments, the ratio of uracil to thymidine in the mixture is in the range of 1:2 to 1:10, such as 1:3 to 1:8, or 1:4 to 1:5. In some embodiments, the kit may further comprise a mixture of primers blocked at the 3'-end (i.e., 3'-blocked primers) and unblocked primers. In some embodiments, the ratio of blocked primers to unblocked primers is in the range of 1:1 to 1:5, such as 1:2 to 1:4. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Illustrates an embodiment of the double-ended sequencing method in the present disclosure.

[0013] Figure 2 Illustrates an embodiment of the present disclosure, in which multiple displacement amplification (MDA) primers having reversible blocking groups (represented by “*” in the figure) hybridize to a DNA polynucleotide upstream of a first reading primer. After removal of the reversible blocking groups, both the MDA primers and the first reading strand are extended in MDA to generate multiple second strands.

[0014] Figure 3Embodiments of the method of the present disclosure are illustrated. After the first read strand is generated during the first read sequencing, the strand displacement DNA polymerase binds to the DNA concatemer and initiates MDA. The DNA polymerase is introduced into the reaction at a high concentration so that the excess DNA polymerase molecules in the reaction do not bind to the DNA concatemer and the first read strand. After MDA is initiated but before completion, the unbound DNA polymerase molecules are removed and MDA continues to generate the second strand.

[0015] Figure 4 Embodiments of the method of the present disclosure are shown. After the first read strand is formed from the first read sequencing, the strand displacement DNA polymerase is added to the reaction under extension-blocking conditions. The polymerase binds to the concatemer and the first read strand, but does not extend the primer under these conditions. The asterisk (*) in the figure indicates that the primer cannot be extended due to the extension-blocking conditions.

[0016] Figure 5 Embodiments of the method of the present disclosure are shown, in which excisable nucleotides are incorporated into the first read strand during the first read sequencing. The first read strand is then cleaved at the positions where these excisable nucleotides are present to generate fragments having extendable 3' ends. These fragments are used as extension primers in MDA to generate the second strand. Detailed Description

[0017] I. Overview

[0018] This application relates to an improved paired-end sequencing method using DNA strands. In one approach, steps are taken to maximize the number of second strands having lengths optimized for sequencing, for example, by including MDA primers. In certain cases, the primers are cleaved to produce multiple fragments, each fragment being used as a primer to produce additional second strands. In certain cases, steps are taken to optimize the synchronous production of second strands, for example, by using extension-blocking conditions.

[0019] In some aspects of the present disclosure, the first read of paired-end sequencing is generated by extending a first read primer hybridized to a DNA concatemer immobilized on an array ("first read sequencing"). The first read sequencing generates a first read strand complementary to the DNA concatemer. Then, controlled multiple displacement amplification (MDA) is performed using an extension primer and the second strand is generated. The extension primer can be an additional MDA primer, the first read strand, or both. Each second strand contains a sequence hybridized to one DNA concatemer and an unhybridized single-stranded branch having one or more primer sequences complementary to the second read primer. The first read strand can be further extended to generate the second strand and become part of the second strand. The second read is obtained by extending a second read primer hybridized to the primer-binding sequence on the second strand, which process is referred to as "second read sequencing" in the present disclosure.

[0020] In some ways, the method includes various features that can increase the yield of the second strand (i.e., the number of second strands suitable for second-read sequencing) and the efficiency of second-strand production. In some ways, the paired-end sequencing methods disclosed herein use excisable nucleotides, e.g., uracil, which are incorporated during the extension of the first-read primer. The first-read primer or the first-read strand containing these excisable nucleotides can be cleaved at the positions where these excisable nucleotides are present to generate a plurality of fragments with extendable 3'-ends, and then the plurality of fragments are extended to generate more second strands.

[0021] In some ways, the method includes features that synchronize the generation of multiple second strands such that the generated second strands have similar lengths (e.g., lengths suitable for second-read sequencing). Multiple ways can be used to achieve this synchronization. In one embodiment, after completion of the first-read sequencing, a high concentration of polymerase is introduced to bind to the primers (i.e., MDA primers or first-read strands) hybridized to the DNA concatemer under extension-blocking conditions. This initial step under extension-blocking conditions maximizes the binding of DNA polymerase to the DNA template and the primers. Then the extension-blocking conditions are reversed to allow synchronous MDA from the primers. In some ways, after a first period of MDA, the excess DNA polymerase molecules (i.e., polymerase molecules that are free in the reaction and not bound to the primers and DNA template) are removed, and MDA continues in the absence of unbound DNA polymerase molecules. This process minimizes the interaction between free polymerase molecules and newly formed second strands as well as the interaction between free polymerase molecules and the DNA template beyond the initial binding period, thereby facilitating the synchronous extension and generation of second strands.

[0022] The accompanying drawings in this application illustrate certain embodiments of the present invention. Figure 1 A DNA concatemer containing multiple monomers is shown, with each monomer containing an adapter sequence and a target DNA sequence. The first-read primer anneals to the primer-binding sequence in the adapter of the DNA concatemer and is extended by sequencing by synthesis (SBS) to generate the first read. Extension of the first-read primer generates the first-read strand. After the last cycle of SBS, the 3'-blocking group of the terminal nucleotide in the first-read strand is removed, and a strand-displacement polymerase is added. The first-read strand is extended by the strand-displacement polymerase to generate the second strand in MDA. The MDA is controlled such that the second strand partially hybridizes to the DNA concatemer, and each second strand contains an unhybridized branch. Then the second-read primer is annealed to the branch (e.g., by hybridizing with the second-read primer-binding sequence on the adapter sequence on the branch). Then these second-read primers are extended to generate the second reads.

[0023] Figure 2Shows hybridization of MDA primers with 3' blocking groups and first read primers to DNA concatemers. The blocking groups on these MDA primers are different from the blocking groups on the nucleotides incorporated during SBS. Different from the blocking groups on the nucleotides removed in each cycle of SBS, the blocking groups on the MDA primers are retained throughout the SBS sequencing process. The first read primer is extended to generate a first read and a first read strand. After completion of the first read sequencing, the blocking group on the MDA primer is removed, and the blocking group of the last nucleotide added in the last sequencing cycle is also removed. In a controlled MDA reaction, in the presence of strand-displacing DNA polymerase, both the MDA primer and the first read strand are extended to generate a second strand that hybridizes to a portion of the DNA concatemer. A second read can be generated in the same manner as described above.

[0024] Figure 3 Shows that after completion of the first read sequencing, the first read strand formed by the first read sequencing remains hybridized to the DNA concatemer. A high concentration of DNA polymerase is added to the array to extend the first read strand during an initial period. Since there are excess DNA polymerase molecules in the reaction, some (but not all) of the DNA polymerase molecules bind to the concatemer, while the remaining DNA polymerase molecules are free, i.e., do not bind to the DNA concatemer and do not participate in the extension reaction. The array is then washed to remove the excess DNA polymerase molecules. A buffer with nucleotides (but not including DNA polymerase) is added to the reaction to allow MDA to continue generating a second strand that hybridizes to a portion of the DNA concatemer.

[0025] Figure 4 Shows that after completion of the first read sequencing, the first read strand formed by the first read sequencing remains hybridized to the DNA concatemer. Under extension-blocking conditions, a strand-displacing DNA polymerase is added to the reaction, e.g., the strand-displacing DNA polymerase is in a buffer lacking at least one component (such as magnesium) required for extension. Under these conditions, the strand-displacing DNA polymerase binds to the DNA concatemer but does not extend. After the array is maintained under these conditions for an initial period, these conditions are reversed to allow extension, e.g., the omitted component (such as magnesium) is added back to the reaction at a concentration suitable for MDA. Then MDA is initiated to generate a second strand.

[0026] Figure 5 Shows that the first read sequencing process generates first read strands containing excisable nucleotides (e.g., uracil). These first read strands are cleaved by an enzyme that recognizes the excisable nucleotide, and this cleavage results in multiple fragments having extendable 3' ends and releases the excisable nucleotide. These fragments are extended in MDA to form multiple second strands.

[0027] For all purposes, the entire contents of U.S. Patent No. 10,227,647 are hereby incorporated by reference.

[0028] 2. Definitions

[0029] As used herein, a "primer" refers to an oligonucleotide that, after forming a duplex with a polynucleotide template, is capable of serving as an initiation point for nucleic acid synthesis and extending from its 3' end along the template to form an extended duplex. A primer can comprise a natural sequence or a synthetic sequence. A primer can also comprise non-natural nucleotides. The nucleotide sequence added during extension is determined by the sequence of the template polynucleotide. A primer is typically extended by a DNA polymerase.

[0030] As used herein, a "random primer" refers to a primer having a random nucleotide sequence.

[0031] As used herein, MDA or multiple displacement amplification refers to DNA amplification based on strand displacement replication by multiple primers.

[0032] As used herein, the term "polynucleotide" can be used interchangeably with the term "nucleic acid" to refer to DNA, RNA, and hybrid and synthetic nucleic acids, and can be single-stranded or double-stranded. An "oligonucleotide" is a short polynucleotide having a length between about 6 and about 300 nucleotides. A "complementary polynucleotide" refers to a polynucleotide that is complementary to a target nucleic acid.

[0033] As used herein, the term "strand displacement activity" describes the ability to displace downstream DNA encountered during synthesis. Strand displacement activity is described in U.S. Patent Publication No. 20120115145, which is incorporated herein by reference, as follows: "Strand displacement activity" refers to the phenomenon in which a biological, chemical, or physical agent (such as a DNA polymerase) causes paired nucleic acids to dissociate from their complementary strands in the 5' to 3' direction, bind, and approach template-dependent nucleic acid synthesis. Strand displacement begins at the 5' end of the paired nucleic acid sequence, so the enzyme immediately performs nucleic acid synthesis in the 5' of the displacement site. The newly synthesized nucleic acid and the displaced nucleic acid typically have the same nucleotide sequence complementary to the template nucleic acid strand. Strand displacement activity can be located on the same molecule as the activity that confers nucleic acid synthesis (especially DNA synthesis), or it can be a separate and independent activity. DNA polymerases, such as Escherichia coli DNA polymerase I, the Klenow fragment of DNA polymerase I, T7 or T5 bacteriophage DNA polymerases, and HIV virus reverse transcriptase, are enzymes that have both polymerase activity and strand displacement activity. Reagents such as helicases can be used in combination with inducers that do not have strand displacement activity to produce a strand displacement effect, that is, the displacement of nucleic acids is coupled with the synthesis of nucleic acids of the same sequence. Similarly, together with other inducers, proteins (such as Rec A or single-stranded binding protein from Escherichia coli or from another organism) can be used to produce or promote strand displacement (Kornberg and Baker, 1992, DNA Replication, Second Edition, pp 113-225, Freeman, NY). In one approach, the polymerase is Phi29 polymerase. Phi29 polymerase has strong displacement activity at moderate temperatures (e.g., 20-37 °C). In one approach, Bst DNA polymerase, large fragment (e.g., NEB#MO275, available from New England Biolabs (Ipswich, MA)) is used. Bst DNA polymerase is active at elevated temperatures (~65 °C).

[0034] The term "first strand" refers to the single-stranded DNA template used in paired-end sequencing.

[0035] The term "second strand" refers to a single DNA strand complementary to the first strand.

[0036] The term "MDA primer" refers to an extension primer that hybridizes to a DNA template and is extended to produce a second strand in a displacement-extension reaction. The MDA primers disclosed in this application are not sequencing primers (e.g., first-read primers). In some embodiments, the MDA primer and the first-read primer have different sequences.

[0037] The term "first-read primer" refers to a primer that hybridizes to a DNA template and is used to produce a first read for paired-end sequencing.

[0038] The term "first read segment" refers to nucleotide sequence information obtained by sequencing a DNA template using a first reading primer.

[0039] The term "first reading sequencing" refers to the sequencing process used to obtain the first read segment.

[0040] The term "first reading strand" refers to a single-stranded polynucleotide produced by first reading sequencing (i.e., by extending the first reading primer). The first reading strand is also referred to as the extended first reading primer. The first reading strand can be further extended to form a second strand.

[0041] The term "second reading primer" refers to a primer that hybridizes to the second strand, i.e., the strand complementary to the DNA template. In some embodiments, the second strand primer hybridizes to the second strand that hybridizes partially to the DNA template.

[0042] The term "second read segment" refers to nucleotide sequence information obtained by sequencing the second strand.

[0043] The term "second reading sequencing" refers to the sequencing process used to obtain the second read segment.

[0044] The term "second reading strand" refers to a single-stranded polynucleotide produced by second reading sequencing (i.e., by extending the second reading primer).

[0045] The term "excisable nucleotide" refers to a nucleotide in a DNA strand that can be removed, and the removal results in the DNA strand being cleaved into two DNA fragments. An exemplary method of removing a nucleotide from a DNA strand is by an enzyme. An exemplary excisable nucleotide is uracil.

[0046] The term "reversible blocking group" of a reversible terminator nucleotide may also be referred to as "removable blocking group", "blocking moiety", "blocking group", "reversible terminator blocking group", etc. A reversible blocking group is a chemical moiety attached to the nucleotide sugar (e.g., deoxyribose), typically at the 3'-OH position of the sugar moiety, which prevents the polymerase from adding a nucleotide at that position. The reversible blocking group can be cleaved by an enzyme (e.g., phosphatase or esterase), chemical reaction, heat, light, etc. to provide a hydroxyl group at the 3'-OH position of the nucleoside or nucleotide, thereby allowing nucleotide addition by the polymerase to occur.

[0047] As used herein, "dNTP" includes naturally occurring deoxyribonucleotide triphosphates and their analogs, including analogs having a 3'-O cleavable blocking group.

[0048] The terms "solid support" and "support" are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface. A microarray typically comprises at least one planar solid support, such as a glass microscope slide.

[0049] As used herein, the term "simultaneous" or "simultaneously" with respect to primer extension reactions refers to the extension of multiple primers at the same time or multiple extended primers initiated at the same time.

[0050] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" refers to a reagent or a mixture of such reagents, and reference to "the method" includes reference to equivalent steps and / or methods known to those of ordinary skill in the art.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the devices, compositions, formulations, and methods described in the publications and that may be used in connection with the presently described invention.

[0052] Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) as well as any other stated value or intermediate value within that range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specific exclusions recited in the range. Where the range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0053] As used herein, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details.

[0054] Although the invention has been described primarily with reference to specific embodiments, it is contemplated that other embodiments will be apparent to those of ordinary skill in the art upon reading the present disclosure, and it is intended that such embodiments be included within the methods of the present invention.

[0055] 3. DNA Template

[0056] In some embodiments, the DNA template used in the present invention is a DNA concatemer. As used in this context, the term "concatemer" or "DNA concatemer" refers to a DNA molecule that contains multiple copies of the same DNA sequence (tandemly linked "monomers" or "subunits"). A DNA concatemer can contain at least two, at least three, at least four, at least 10, at least 25, at least 50, at least 200, or at least 500 monomers. In some embodiments, the DNA concatemer contains 25 - 1000 monomers, such as 50 - 800 monomers or 300 - 600 monomers).

[0057] In one embodiment, the monomer of the concatemer contains a linker sequence and a target DNA sequence. Since the monomers are tandemly linked, the target DNA sequences will be flanked by two linker sequences. In some embodiments, the target DNA sequence in the monomer is flanked by two "half - linker" sequences such that each target sequence tandemly linked in the concatemer is flanked by two linkers. In some embodiments, the monomer unit includes one, two, three, or four or more linkers. In some embodiments, all of the linkers of the monomer (and concatemer) have the same sequence. In other embodiments, the linkers can have different sequences, such as two, three, or four different sequences. It should be recognized that a single monomer can contain more than one DNA template sequence. An exemplary DNA concatemer structure is described in Table 1 of U.S. Patent No. 10,227,647.

[0058] The DNA concatemer used in the methods described herein can be a DNA nanoball, or "DNB". Without intending to limit the present invention in any way, DNA nanoballs are described in Drmanac et al., 2010, Science 327:5961, pp. 78 - 81 and U.S. Patent No. 8,592,150 to Drmanac et al. The entire contents of both references are incorporated herein by reference.

[0059] A DNA nanoball (DNB) is a single-stranded copy of a DNA sequence that is ligated into a linear DNA structure. Typically, DNBs are generated by replicating single-stranded circular DNA using a strand-displacing polymerase (such as phi29 polymerase or Bst polymerase) in a process called rolling circle replication. The polymerase starts with the extension of a primer hybridized to the single-stranded loop and generates a reverse complementary strand that hybridizes to the loop. Once a complete circle around the loop is completed, the polymerase continues the extension by displacing the newly formed strand ahead of the direction of travel. As the polymerase continues to extend the strand around the loop, multiple reverse complementary copies are generated and ligated to each other in a linear fashion. This strategy creates a target with many probe or primer binding sites, resulting in a higher signal intensity than that obtained using single-copy circular subunits. These single-stranded DNA concatemers of sufficient length typically form random coils that fill a roughly spherical volume in solution (e.g., SSC buffer at room temperature). In some ways, DNA nanoballs typically have a diameter of about 100 to 300 nm. Typically, each monomer contains at least one target DNA sequence.

[0060] DNA concatemers (including DNA nanoballs) can be prepared by any suitable method. In one way, a single genomic fragment is used to generate a single-stranded circular DNA with adapters inserted between target sequences that are adjacent or close together in the genome. The circular DNA construct can be amplified enzymatically (e.g., by rolling circle replication or by ligating monomers to each other). By way of illustration and not limitation, DNA nanoballs can be prepared according to the methods described in U.S. Patent No. 8,445,194 and U.S. Patent No. 8,592,150.

[0061] Amplifying DNA by rolling circle replication has several advantages: 1) the amplification is linear, which prevents mutant copies from over-representing the original template sequence, 2) all copies are localized to a single molecule and are thus ideal for microscopic analysis with fluorescent probes or reporter genes, and 3) the replication of the loop can be carried out under isothermal conditions, making automation easier.

[0062] The target DNA portion can be from any source, including naturally occurring sequences (such as genomic DNA, cDNA, mitochondrial DNA, cell-free DNA, etc.), artificial sequences (such as synthetic sequences, products of gene shuffling or molecular evolution, etc.) or combinations thereof. The target DNA can be from sources such as organisms or cells (e.g., from plants, animals, viruses, bacteria, fungi, humans, mammals, insects), forensic sources, etc. The target DNA sequence can be from a biological population, such as the gut bacterial population. The target DNA sequence can be obtained directly from a sample or can be the product of an amplification reaction, fragmentation reaction, etc.

[0063] The target DNA can have a length within a specific size range, e.g., a length of 50 to 600 nucleotides. Other exemplary size ranges include lengths of 25 to 2000, 50 to 1000, 100 to 600, 50 - 100, 50 - 300, 100 - 300, and 100 - 400 nucleotides. In a DNA template polynucleotide having two or more different target DNAs, the target DNAs can have the same length or different lengths. In a library of DNA template polynucleotides, the members of the library can have similar lengths in some ways (e.g., all within the range of 25 to 2000 nucleotides or another range).

[0064] In one way, the target DNA can be prepared by fragmenting a larger source DNA (e.g., genomic DNA) to produce fragments within the desired size range. In some methods, a size selection step is used to obtain a pool of fragments within a specific size range.

[0065] The DNA templates described herein can include two or more adaptor sequences. The adaptor can contain elements for immobilizing the DNA template polynucleotide on a substrate, elements for binding oligonucleotides used in sequencing (e.g., binding sites for primers to be extended in sequencing by synthesis and / or binding sites for probes in cPAL or other ligation-based sequencing methods, etc.) or elements for both immobilization and sequencing. The adaptor can include additional features such as, but not limited to, restriction endonuclease recognition sites, primer extension hybridization sites (for analysis), barcode sequences, unique molecular identifier sequences, and polymerase recognition sequences.

[0066] The adaptor sequence can have a length, structure, and other properties suitable for a particular sequencing platform and intended use. For example, the adaptor can be single-stranded, double-stranded, or partially double-stranded and can have a length suitable for the intended use. For example, the length of the adaptor can be within the range of 10 - 200 nucleotides, 20 - 100 nucleotides, 40 - 100 nucleotides, or 50 - 80 nucleotides. In some ways, the adaptor can contain one or more modified nucleotides containing modifications to the base, sugar, and / or phosphate moieties.

[0067] Those skilled in the art will understand that different members of the library will generally contain a common adaptor sequence, although different species or subcategories within the library can have unique features such as genus-specific barcodes.

[0068] Individual linker sequences can include multiple subsequences with different functions. For example, as discussed in detail in this disclosure, a single linker sequence can contain more than two primer binding sequences (which can be recognized by different complementary primers or probes). The functionally different sequences within a linker can be overlapping or non-overlapping. By way of illustration, given a 40-base-long linker, in one embodiment, bases 1-20 are the first primer binding site and bases 21-40 are the second primer binding site. In different embodiments, bases 1-15 are the first primer binding site and bases 21-40 are the second primer binding site. In different embodiments, bases 5-25 are the first primer binding site and bases 15-35 are the second primer binding site. Similarly, given a 40-base-long linker, bases 1-20 can be the immobilization sequence and bases 21-40 can be the primer binding site. Different primer sequences in the same or different linkers of a DNA template polynucleotide can have the same or different lengths.

[0069] Linkers (e.g., first linker, second linker, third linker, etc.) can contain one, two, or more than two primer binding sequences. A primer binding sequence is functionally defined as the site or sequence to which a primer (or oligonucleotide) specifically binds. For example, a linker with two primer binding sequences can be specifically bound by two different primers. In one way, the two primer binding sequences in the same linker are overlapping, i.e., sharing a part of the nucleotide sequence. In some ways, the overlapping region does not exceed 50%, or 40%, or 30%, or 20%, or 10% or 5% of either of the two overlapping primer sequences. In one way, more than one primer binding sequence is non-overlapping. In some ways, the non-overlapping primer sequences are adjacent to each other; in some other embodiments, the non-overlapping primer sequences are separated by 1-10, 10-20, 30-40, or 40-50 nucleotides.

[0070] Obviously, in a given DNA template polynucleotide, different linkers can have the same sequence or different sequences, and can have the same primer binding sequences or different primer sequences. Although certain drawings (e.g., Figure 1 ) are provided to illustrate the present invention, the representation of linkers using, for example, similar cross-hatching, etc. should not be construed as indicating sequence identity.

[0071] 4. Primer

[0072] The primers used in the methods described herein have sufficient length to allow primer hybridization, and their exact length and sequence depend on the intended function of the primer (e.g., extension primer, index sequence, etc.). The length of the primer sequence is typically at least 10, at least 12, at least 15, or at least 18 bases. In some embodiments, the length of the primer sequence ranges from 8 to 60 nucleotides, e.g., 10 to 25 nucleotides, or 40 to 60 nucleotides in length. Selecting or designing primers for use in the present invention is well within the capabilities of one of ordinary skill in the art.

[0073] It should be understood that the primers and probes can be fully or partially complementary to the primer binding sequence in the adaptor to which they hybridize. For example, the primer can have at least 85%, 90%, 95%, or 100% identity to the sequence to which it hybridizes.

[0074] The primer can also contain additional sequences at the 5' end of the primer that are not complementary to the primer binding sequence in the adaptor. The non-complementary portion of the primer can be a length that does not interfere with hybridization between the primer and its primer binding sequence. Typically, the non-complementary portion is 1 to 100 nucleotides in length. In some embodiments, the non-complementary portion is 4 to 8 nucleotides in length. The primer can comprise DNA and / or RNA moieties, and in some instances, the primers used in the present invention can also have one or more modified nucleotides containing modifications to the base, sugar, and / or phosphate moieties.

[0075] A "sequencing oligonucleotide" or "sequencing primer" can be an extension primer for use in a sequencing-by-synthesis reaction (also known as "sequencing-by-extension"). A "sequencing oligonucleotide" can be an oligonucleotide used in a sequencing-by-ligation method such as the "combinatorial probe-anchor ligation reaction" (cPAL) (including single, dual, and multiplex cPAL) described in, for example, U.S. Patent Publication No. 20140213461, which is incorporated herein by reference for all purposes.

[0076] In some cases, the extension primer also serves as a sequencing primer, such as the first read primer and the second read primer. In certain cases, the extension primer can also be the product of a primer extension reaction, such as the first read strand that can be further extended. The first read primer is a sequencing primer that hybridizes to a DNA concatemer and extends to generate a first read and form a first read strand. As described above, in certain cases, the first read strand serves as an extension primer and is extended to generate a second strand. In certain cases, an additional extension primer (referred to as the MDA primer) is used to hybridize to the DNA concatemer and extend to generate a second strand in a displacement extension reaction. In some embodiments, the MDA primer and the first read primer have different sequences. In some embodiments, the MDA primer can hybridize to the DNA concatemer upstream of the first read primer, i.e., the MDA primer is located at the 5' of the first read primer. In some embodiments, one or more MDA primers and the first read primer bind to the same adaptor of the monomer of the DNA concatemer, and one or more MDA primers are located at the 5' of the first read primer. In some embodiments, the MDA primer includes random primers. In some embodiments, excess random primers (i.e., random primers that have not hybridized to the DNA concatemer) are removed prior to initiating MDA to avoid excess random primers binding and priming the second strand.

[0077] In some cases, the primers used in the method (e.g., the first read primer, the first read strand, or the MDA primer) can contain excisable nucleotides, and these primers can be cleaved at the position of the excisable nucleotides to form a new extendable 3' end. This cleavage results in the release of the excisable nucleotides. In some cases, these excisable nucleotides are nucleobases other than any of A, G, T, or C. Alternatively or additionally, these excisable nucleotides can be incorporated at a desired spacing during the sequencing reaction. The excisable nucleotides are selected for their ability to be excised and removed from the DNA strand to produce a 3' exposed end that can be used for extension. An exemplary excisable nucleotide that can be used in the method is uracil, which can be incorporated during the extension reaction in place of thymidine. When incorporated into single-stranded DNA, uracil can be recognized by a uracil-specific excision reagent (such as USER TM ) TM consisting of a mixture of uracil DNA glycosylase (UDG) and endonuclease VIII, which together first catalyze the excision of uracil to form an abasic site and then break the phosphodiester bond of the abasic site, resulting in a single-stranded DNA break and the release of uracil. See, Bitinaite et al., USER TMFriendly DNA engineering and cloning method by uracil excision, Nucleic Acids Research, Vol. 35, Issue 6, 15 March 2007, pp 1992–2002. Primers containing these excisable nucleotides first bind to the DNA template and then are cleaved at the positions where the excisable nucleotides occur to allow the simultaneous extension of multiple second strands.

[0078] In some cases, one or more MDA primers contain a blocking group at their 3' end that prevents their extension. The number of 3'-blocked primers can be controlled to control the nature and density of the strands independently displaced by each DNB. In some embodiments, the number of 3'-extensible primers is controlled by maintaining the primers at an appropriate concentration during hybridization. In some embodiments, the number of 3'-extensible primers is controlled by mixing 3'-extensible primers and 3'-blocked primers in an appropriate ratio.

[0079] In some cases, the extension primers (i.e., MDA primers) used in the methods disclosed herein can contain a reversible blocking group at their 3' end, and these extension primers will only extend when the blocking group is removed. In some embodiments, one or more MDA primers bind to the DNA template upstream of the first read primer, and after the first read is generated, the reversible blocking group of the MDA primer is removed to allow the extension of multiple second strands starting from the MDA primer. See Figure 2 . In some embodiments, the reversible blocking group at the 3' of the MDA primer can be removed under conditions different from the blocking group added to the first read primer during each sequencing cycle, such that the blocking group of the MDA primer will be retained throughout the first read sequencing. In some embodiments, the reversible blocking group at the 3' of the MDA primer is different from the blocking group of the nucleotide added to the first read primer during each sequencing cycle, such that the blocking group of the MDA primer will remain unchanged throughout the first read sequencing process. In certain cases, the 3'-blocking group is a 3'-phosphate, which can be removed by a phosphatase.

[0080] In the methods described in the present disclosure, the generation of the second strand is initiated by hybridizing primers that contain reverse complementary sequences as a DNA template (e.g., a DNA concatemer). In some cases, the primer hybridizes to the adaptor of the DNA concatemer monomer. In some cases, second strand synthesis can also start from the extension of a random oligonucleotide, i.e., starting with an MDA primer having a random nucleotide sequence. In this way, a set of different primers can be used to hybridize to an array of DNA concatemers. In some ways, these sequences are oligonucleotides of random sequences that are 5 to 10 bases in length, such as 6 to 10 bases or 5 to 8 bases in length. Each sequence in the random pool will hybridize to the complementary sequence in the DNA concatemer.

[0081] 4.1. Linking of Primers

[0082] In some aspects, the primers (e.g., the first reading primer, or the MDA primer, or both) used in the above methods and compositions are linked to form a linked pair. The term "linked" refers to non-covalent and covalent interactions through which two nucleic acid molecules bind together. In some cases, the linking is through DNA hybridization, chemical bonds, or both. These linked primer pairs can link consecutive or non-consecutive monomers of the DNA concatemer, thereby stabilizing the DNA concatemer.

[0083] The primers can be linked in various ways. In some embodiments, they are joined by chemical linking. In some embodiments, two primers are linked by hybridization between two complementary sequences (referred to as "hybridization sequences"), each primer having a complementary sequence. In some embodiments, the hybridization sequence is a non-palindromic sequence. In some embodiments, the hybridization sequence is a palindromic sequence, i.e., a sequence in which one half of the sequence is complementary to the other half of the sequence. The methods for linking primers and the compositions of the linked primers are described in PCT application number PCT / CN2020 / 124338, the entire content of which is incorporated herein by reference for all purposes.

[0084] The length of the hybridization sequence can vary. The length of the hybridization sequence is selected such that the Tm of the stapler sequence is between 50°C and 72°C. This ensures that the linked primer pair can remain hybridized throughout the assay. In some embodiments, the length of the hybridization sequence can range from 20 to 150 nucleotides, such as in the range of 40 to 120 nucleotides, 50 to 100 nucleotides.

[0085] In some embodiments, the hybridization sequence is 5' relative to the sequence on the primer that is complementary to the DNA template.

[0086] 5. Generation of the First Read

[0087] As described above, in some embodiments, the first read primer serves as a sequencing primer and is used to determine the sequence of a DNA template by generating a first read. The first read primer itself is part of the second strand, and extending the first read primer with nucleotides further generates more of the second strand. Nucleotide extension can occur such that modification of the nucleotide allows addition of only one nucleotide, and then the modified nucleotide is detected from a set of A, C, G, and T bases to identify the added nucleotide. For example, the added nucleotide can contain a 3'-O-blocking group, such as a 3'-O-azidomethyl group, at the 3'-OH position of A, C, G, or T. To determine the next base in the series, the modified nucleotide is converted to an extendable form to allow addition of another base in another sequence determination cycle. In this sequencing method, the extended first strand primer serves as the primer for each round of nucleotide addition. After the last cycle of nucleotide addition for sequencing, the extended first read primer (also referred to as the "first read strand") is unblocked and then further extended using a polymerase that can be the same as or different from the polymerase used to generate the first read strand. Extension of the first read primer can be performed by any DNA polymerase, including a polymerase with strand displacement activity, a polymerase lacking strand displacement activity, or a mixture of polymerases. In some ways, the DNA polymerase is one that does not have any or has very little strand displacement activity, such as BG9 DNA polymerase.

[0088] 6. Controlled MDA

[0089] In some embodiments, the first read strand is separated from the DNA concatemer and removed from the array, and a new extension primer is added and extended in MDA. In some embodiments, the 3'-blocking group of the terminal nucleotide of the first read strand (added in the last cycle of first read sequencing) is removed, and these first read strands are used as extension primers in MDA. The MDA method disclosed herein is controlled such that the second strand remains partially hybridized to the DNA concatemer, i.e., the second strand remains bound to the DNA template through the sequence hybridized to the DNA template. The second strand also contains a single-stranded branch that contains a primer binding site for a second read primer. The second read primer can serve as a sequencing primer to generate a second read.

[0090] An illustrative example is shown in Figure 1 . The first read primer anneals to the DNA concatemer and is extended to generate a first read. First read sequencing generates a first read strand. Controlled MDA is then performed by extending the first read strand with a polymerase having strand displacement activity to generate multiple second strands. Each second strand contains a sequence hybridized to the concatemer and an unhybridized single-stranded branch. The second read primer ① then hybridizes to the single-stranded branches of the multiple second strands and is extended to generate a second read.

[0091] 6.1 Strand displacement DNA polymerase

[0092] MDA in the method requires a DNA polymerase with strand displacement activity. In one aspect, the present invention uses a DNA polymerase with strong 5'→3' strand displacement activity. The polymerase preferably does not have 5'→3' exonuclease activity. However, when the activity does not prevent the practice of the methods of the present invention, for example, by using reaction conditions that inhibit exonuclease activity, a DNA polymerase with 5'-3' exonuclease activity can be used. In one aspect, the polymerase is Phi29 polymerase. Phi29 polymerase has strong displacement activity at moderate temperatures (e.g., 20 - 37°C). In one aspect, Bst DNA polymerase, large fragment (e.g., NEB#MO275, available from New England BioLabs, Ipswich, MA) is used. Bst DNA polymerase is active at elevated temperatures (∼65°C). In one aspect, the polymerase is Deep-VentR DNA polymerase (e.g., NEB#MO258) (Hommelsheim et al., Scientific Reports 4:5052 (2014)).

[0093] In addition to strand displacement polymerases, other strand displacement mechanisms can also be used to assist strand displacement, such as helicases, using strand denaturants or reagents capable of reducing the melting temperature, commonly referred to as Tm-reducing reagents (e.g., formamide, betaine, proline, 1,2-propanediol, and trehalose). The reaction temperature can also be adjusted to promote strand melting and assist polymerase extension. In some cases, the temperature can vary in the range of 25°C to 40°C, such as 28°C to 35°C, or approximately 30°C.

[0094] Dual-strand sequencing in the present disclosure requires that at least some of the second strand remain partially hybridized to the DNA template. This allows the sequence reads generated from sequencing the second strand to be paired with the first strand to construct the sequence information of the DNA template. That is, complete displacement that causes dissociation of the second strand from the first strand should be avoided or minimized. This can be achieved by controlling the reaction process by: selecting a polymerase with a suitable polymerization rate or other properties, and using various reaction parameters, which include (but are not limited to) reaction temperature, reaction duration, primer composition, DNA polymerase, primer and nucleotide concentrations, additives, and buffer composition. The optimal conditions can be determined empirically.

[0095] One way to control the strand displacement reaction to avoid complete displacement is to use a DNA polymerase with appropriate strand displacement activity to generate the second strand. DNA polymerases are known (e.g., Phi29, Bst DNA polymerase, the Klenow fragment of DNA polymerase I, and Deep-VentR DNA polymerase) to have different strengths of strand displacement activity. See Kornberg and Baker (1992, DNA Replication, Second Edition, pp. 113-225, Freeman, N.Y.). Selecting a DNA polymerase suitable for the present invention is within the ability of one of ordinary skill in the art.

[0096] In another way, the extension-displacement reaction is controlled to avoid complete displacement by using an appropriate concentration of a DNA polymerase with strand displacement activity, or an appropriate concentration of dNTPs, or an appropriate concentration of a second primer.

[0097] In some embodiments, the extension reaction is carried out by including in the reaction buffer a reagent that affects the formation of the duplex between the extension primer and the template DNA (e.g., DMSO (e.g., 1%-2%), betaine (e.g., 0.5 M), glycerol (e.g., 10%-20%), the Gene32 single-stranded DNA binding protein from bacteriophage T4 (T4 G32 SSB) (e.g., 10-20 ng / ul), and a size exclusion agent.

[0098] The reaction temperature can also be selected to allow an appropriate rate of polymerization and strand displacement. Higher temperatures generally result in a greater degree of strand displacement. In some embodiments, the reaction temperature is maintained in the range of 20°C - 37°C, such as 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C, to avoid complete displacement.

[0099] In some ways, the extension reaction is controlled by using a mixture of a conventional (extendable) primer and a non-extendable primer (i.e., a 3'-end blocked primer). In some embodiments, the non-extendable primer is blocked from extension by a chemical blocking group that prevents DNA polymerase polymerization. By mixing these two different primers in different ratios, the length of the double-stranded (hybridized) portion of the newly synthesized complementary DNA strand (subsequent fragment) can be controlled. For example, in one way, a mixture of a first primer is used, where 50 - 70% is non-extendable ("blocked") and 30 - 50% can be extendable ("unblocked"). Many types of non-extendable primers are known in the art and are suitable for the present invention.

[0100] 6.2. Synchronous production of multiple second strands

[0101] Ideally, the extension and displacement of multiple second strands are synchronized to avoid a situation where some strands hybridize partially while others have been completely displaced and still others have not been extended far enough. Synchronization can be achieved in various ways.

[0102] 6.2.1 Two-step second-strand production

[0103] In the method of two-step second-strand production, the second strand can be prepared in two steps. The first reaction step involves performing MDA in the presence of a high concentration of strand-displacing DNA polymerase such that free DNA polymerase molecules are present in the reaction. As used herein, "free polymerase" or "free polymerase molecule" refers to a DNA polymerase molecule that is not bound to the DNA concatemer on the array and remains in solution. In some embodiments, the DNA polymerase is at a high concentration such that the number of DNA polymerase molecules is greater than the number of adapters in the DNA concatemer on the array, such that free DNA polymerase that is not bound to the array is present. The DNA polymerase concentration suitable for this approach can be determined empirically by those skilled in the art. The first reaction step can last for 1 - 15 minutes, such as 1 - 3 or 2 - 5 or 3 - 10 minutes. The second step involves removing the reaction components including unbound enzyme and unincorporated nucleotides and adding fresh buffer and reaction components, except for any DNA polymerase, to allow the MDA reaction to continue. In these ways, the polymerase with strong associative properties will remain bound to the extended strand, while the free polymerase will be removed after the first-step incubation. This two-step reaction process minimizes the interaction between the free polymerase and the newly formed second strand and, beyond the initial binding period, between the free polymerase and the first strand, and promotes the synchronization of polymerase extension of the second-strand primer. See Figure 3 .

[0104] 6.2.2 Improved two-step second-strand production

[0105] In the method of improved two-step second-strand production, after completion of the first-pass sequencing, a high concentration of polymerase is used to bind the primer (i.e., the MDA primer or the first-pass strand) and the DNA concatemer under extension-blocking conditions for an initial period. "Extension-blocking conditions" are conditions that favor polymerase binding to the template rather than primer extension by the polymerase. In some embodiments, the extension-blocking conditions are conditions under which primer extension cannot occur. This initial step in the improved two-step second-strand production mode maximizes the binding of the DNA polymerase to the DNA template and the primer. Then the extension-blocking conditions are reversed to allow synchronous extension of the primer.

[0106] Extension blocking conditions can be created by removing nucleotides or magnesium from the reaction, by adding an inhibitor (e.g., EDTA) to the reaction, or by both. The concentration of EDTA can vary in the range of 0.5 mM to 5 mM, such as in the range of 0.8 mM to 4 mM, 1 mM to 3 mM, or about 1 mM. Thus, in some embodiments, the extension blocking conditions include a reaction buffer that is free of individual nucleotides or magnesium or both, such that MDA does not occur. In some embodiments, the extension conditions include using a primer that is blocked at the 3'. The terms "free of" or "substantially free of" mean that the magnesium concentration or nucleotide concentration in the reaction mixture is less than 10%, less than 5%, less than 3%, less than 2%, less than 1% of the magnesium concentration or nucleotide concentration required for the extension reaction. A buffer free of magnesium is also referred to as a magnesium-free buffer. A buffer free of unincorporated nucleotides is also referred to as a nucleotide-free buffer. In some cases, the extension blocking conditions include a reaction buffer containing less than 0.2 mM, less than 0.1 mM, less than 0.05 mM, less than 0.02 mM, less than 0.01 mM, or less than 0.005 mM of magnesium. In some cases, the concentration of each type of nucleotide (A, T, C, or G) is less than 0.02 mM, less than 0.01 mM, less than 0.005 mM, or less than 0.001 mM. Lowering the initial reaction temperature can also be used to alter the ratio of binding events to extension events. Binding can be promoted over extension during an initial period (e.g., 1 - 5 minutes or 2 - 10 minutes). Then subsequent extension can continue for 10 - 20, 20 - 30, 30 - 60 minutes. This two-step process allows for the synchronous production of multiple second strands.

[0107] An illustrative embodiment of the method is as Figure 4 shown. After obtaining a first read by extending a first read primer hybridized to a DNA concatemer, controlled MDA is performed by contacting the array with a strand-displacing DNA polymerase in extension blocking conditions such that the strand-displacing DNA polymerase binds to the first read strand (and / or MDA primer), but does not extend the first read strand. After an initial period of placing the array in extension blocking conditions, the extension blocking conditions are reversed such that the first read strand and / or MDA primer are synchronously extended to produce multiple second strands

[0108] 6.2.3. Generating second strands using modified primers

[0109] As described above, the use of modified first read primers (and / or MDA primers), such as those containing uracil instead of thymidine, can also facilitate the synchronization of second strand production. In some cases, extending the first strand primer in the presence of a nucleotide mixture containing uracil produces modified first strand primers that contain uracil instead of thymidine at different positions. These modified first strand primers or extended first strand primers are then cleaved to allow for the simultaneous extension of multiple second strands.

[0110] An illustrative example of this approach is shown in Figure 5 where the first read strand generated by extending the first read primer contains excisable nucleotides. The first read primer is cleaved at the position of the excisable nucleotide to produce a fragment with an extendable 3'-end. Each fragment serves as a primer to generate multiple second strands by controlled MDA, each second strand containing a sequence hybridized to one of a plurality of DNA concatemers and an unhybridized single-stranded branch. A second read primer ① then hybridizes to the second strand and is extended in second read sequencing.

[0111] 6.2.4. Generating Second Strands Using Additional MDA Primers

[0112] In some embodiments, to generate a greater number of second strands, additional MDA primers are added to the MDA process. These MDA primers bind to regions of the DNA concatemer that are not hybridized structures (single strands) and can thus serve as binding sites for the added primers. These MDA primers, along with the first read strand, are extended and together they generate multiple second strands. Introducing these MDA primers can increase the yield of second strands, thereby increasing the amount of sequencing data. In some embodiments, these MDA primers are introduced at the same time as the first read primer, and the MDA primers hybridize upstream of the first read primer. See Figure 2 , and the MDA primers used in these embodiments typically contain reversible 3'-blocking groups such that they do not extend during the first read sequencing step. After completion of the first read sequencing, the 3'-blocking groups are removed from the MDA primers to allow the use of these MDA primers to control MDA.

[0113] Reversible blocking groups suitable for use are known in the art. In some embodiments, suitable blocking groups are those that can be removed by chemical or enzymatic treatment, and the treatment generates a 3'-OH group. The chemical treatment should not significantly degrade the template or the primer extension strand. Various molecular moieties for 3'-blocking groups of reversible terminators have been described, such as 3'-O-allyl (Ju et al., Proc. Natl. Acad. Sci. USA 103:19635–19640, 2006), 3'-O-azidomethyl-dNTPs (Guo et al., Proc. Natl Acad. Sci. USA 105, 9145–9150, 2008), aminoalkoxy (Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29:879–895, 2010), and 3'-O-(2-cyanoethyl) group (Knapp et al., Chem. Eur. J., 17, 2903–2915, 2011). Exemplary RT blocking groups include -O-azidomethyl and -O-cyanoethenyl. Non-limiting examples of reversible protecting groups that can be used are disclosed in PCT / US2018 / 012425, the relevant portions of which are incorporated herein by reference in their entirety.

[0114] In some embodiments, the methods disclosed herein employ one or more of the above features to improve the efficiency of second-strand production and the double-stranded sequencing process.

[0115] 7. Sequence determination

[0116] The sequence of a DNA template can be determined by combining the sequence reads generated from sequencing the first strand (DNA template) and the sequence reads generated from sequencing the second strand. As described above, the first strand can be sequenced while extending the first-strand primer, i.e., identifying each nucleotide incorporated into the extended first-strand primer such that the sequence of the portion of the first strand complementary to the extended second strand can be determined. Sequencing the second strand can include hybridizing a sequencing oligonucleotide to a sequence in the second strand that is complementary to at least a portion of the adaptor of the monomer of the DNA concatemer (e.g., Figure 1 "①" in), and determining the nucleotide sequence of the branch of the second strand. In some embodiments, the branch of the second strand is sequenced by extending a second reading primer. The sequence reads generated from sequencing the second strand are paired with the sequence reads generated from sequencing the DNA template to determine the entire target DNA sequence.

[0117] It should be understood that any of the primers described above can be used as a sequencing oligonucleotide.

[0118] Any suitable sequence determination method can be used to determine the sequences of the first strand and the second strand, such as SBS, pyrosequencing, ligation sequencing, etc. In some ways, more than one sequencing method is used. For example, one method (e.g., SBS) can be used to sequence the DNA template strand, and a different method (e.g., cPAL) can be used to sequence the second strand. In one way, sequencing is performed using an affinity reagent, for example, as described in U.S. Patent No. 10,851,410, which is incorporated herein by reference.

[0119] In some ways, the first read sequencing is performed by sequencing by synthesis. In some ways, the second read sequencing is performed using multiple second read primers as primers for primer extension (e.g., sequencing by synthesis reaction), or an extension product of such primers, or an oligonucleotide capable of serving as an anchor for ligation sequencing, or a ligation product of such oligonucleotide and a labeled probe (e.g., a labeled cPAL probe). In one way, the second primer contains a portion complementary to the adapter sequence and can be extended for sequencing the second strand.

[0120] SBS can rely on DNA polymerase activity to perform strand extension during the sequencing reaction step. SBS is well known in the art. See, for example, U.S. Patent Nos. 6,787,308 and 8,241,573B2. Sequencing of DNA nanoballs can be performed by a variety of processes. In one way, the circles used to generate DNBs are prepared with a DNA region of known sequence (adapter) and an adjacent sequence of unknown identity to be determined. The adapter provides a primer hybridization site such that if a polymerase is used for extension, the extension of the primer will result in nucleotide addition to the "unknown" or "to be determined" region. If the nucleotides are reversibly blocked at the 3' position, one position is added at a time and is complementary to the base position in the DNB. After removal of the 3' blocking group, additional positions can be read in the next cycle. The fluorescent moiety characteristics of the base type are used to detect the incorporated base, thus revealing the base at that position in the DNB.

[0121] Alternatively, sequencing by ligation can be used. The primer or anchor can be extended by ligating a fluorescent oligonucleotide that extends into the unknown sequence. In this sequencing method, a fluorescent oligonucleotide with degenerate bases is ligated to the starting anchor, however, one base of the oligonucleotide is defined and is associated with a fluorescent moiety. The ligation of the oligoprobe to the anchor produces a stable fluorescence after washing away the excess probe, which depends on the recognition of the defined base complementary to the base at the same position in the DNB. Sequencing by ligation is described in, for example, Shendure et al., 2005, Science, 309:1728 - 1739.)

[0122] Other sequencing methods can also be used, such as pyrosequencing (see, e.g., Ronaghi et al., Anal. Biochem. (1996) 242:84–89) and sequencing by hybridization (see, e.g., Drmanac et al, Advances in Biochemical Engineering / Biotechnology (2002) 77:75-101).

[0123] It will be apparent to the reader that variants of the specific embodiments outlined herein can be used. In one manner, the extension primer (e.g., the first strand primer) and the sequencing oligonucleotide (e.g., the second strand oligonucleotide) bind to different portions of the adapter sequence. In one manner, the extension primer and the sequencing oligonucleotide bind to the same portion of the adapter sequence (e.g., the portion of the adapter sequence for extension and the complement of the same portion of the adapter sequence for sequencing).

[0124] 8. Substrates and Compartments

[0125] In some applications, the DNA template polynucleotide is immobilized on a substrate. Generally, the immobilization occurs prior to the synthesis of the second strand described above. Exemplary substrates can be substantially flat (e.g., slides, wells, flow cells) or non-flat and single or formed of multiple different units (e.g., beads). Exemplary materials include glass, ceramics, silica, silicon, metals, elastomers (e.g., siloxanes), polyacrylamides (e.g., polyacrylamide hydrogels; see WO2005 / 065814). In some manners, the substrate contains an ordered or disordered array of immobilization sites or pores. In some methods, the target DNA polynucleotide is immobilized on a substantially flat substrate, such as a substrate containing an ordered or disordered array of immobilization sites or pores. In some methods, the target DNA polynucleotide is immobilized on beads.

[0126] The polynucleotide can be immobilized on the substrate by a variety of techniques including covalent and non-covalent linkages. The polynucleotide can be immobilized on the substrate by various techniques. In one embodiment, the surface can include capture probes that form a complex (e.g., a duplex) with components of the polynucleotide molecule, such as adapter oligonucleotides. In another embodiment, the surface can have reactive functional groups that react with complementary functional groups on the polynucleotide molecule to form covalent bonds. DNA molecules can also be effectively linked to a hydrophobic surface (e.g., a clean glass surface with a low concentration of various reactive functional groups (e.g., -OH groups)). In another embodiment, the polynucleotide molecule can be adsorbed to the surface by non-specific interactions with the surface or by non-covalent interactions (e.g., hydrogen bonds, van der Waals forces, etc.).

[0127] For example, DNA nanoballs can be immobilized to discrete, spaced-apart regions as described in U.S. Patent No. 8,609,335. In one approach, DNBs are immobilized on a substrate by hybridization to immobilized probe sequences, and solid-phase nucleic acid amplification methods are used to generate a clonal cluster comprising a DNA template polynucleotide. See, e.g., WO 98 / 44151 and WO 00 / 18957.

[0128] In some approaches, prior to the primer extension step, the DNA template polynucleotide is compartmentalized in an emulsion, droplet, bead, and / or microwell (Margulies et al., "Genome sequencing in microfabricated high-density picolitre reactors." Nature 437:7057 (2005); Shendure et al., "Accurate multiplex polony sequencing of an evolved bacterial genome” Science 309, 1728–1732 (2005)).

[0129] Generally, DNA nanoballs are arranged on a substrate in an ordered or random array. In many applications, adsorption to the substrate is mediated by substrate-protein-DNA interactions. Additionally, to achieve a stable nanoball array through sequencing cycles, post-deposition of a protein layer can enhance the stability of the DNA array. See WO2013066975A1, the entire disclosure of which is incorporated herein by reference.

[0130] 9. Kit

[0131] In some aspects, methods for generating a second strand for sequencing a DNA template can be performed using a kit. The kit can include one or more DNA polymerases (including strand displacement DNA polymerases), a mixture of nucleotides (including A, C, T, G), and primers. The kit can also include a non-displacement DNA polymerase and reversibly terminating dNTPs that can be used for sequencing-by-synthesis. The kit can also include a magnesium-free buffer (“magnesium-free buffer”). The kit can also include extension inhibitors (e.g., EDTA, excess salts (including KCl and NaCl), ionic detergents (e.g., deoxycholate, sarcosyl, and SDS), ethanol, and isopropanol). In some embodiments, the nucleotide mixture can also include uracil. In some embodiments, the ratio of uracil to thymidine in the mixture is in the range of 1:2 to 1:10, such as 1:3 to 1:8, or 1:4 to 1:5. In some embodiments, the kit can also include a mixture of primers that are blocked at the 3′ end (i.e., 3′-blocked primers) and unblocked primers. In some embodiments, the ratio of blocked primers to unblocked primers is in the range of 1:1 to 1:5, such as 1:2 to 1:4.

[0132] 10. Array of DNA complexes

[0133] In one aspect, the invention includes an array of DNA complexes. In one aspect, the array is a support comprising an array of discrete regions, where multiple regions contain clusters of clones of single-stranded DNA templates and multiple primers. In some embodiments, the DNA template is a single-stranded multimer comprising multiple monomers, each monomer comprising an adaptor sequence and a DNA target sequence. Each of the multiple primers comprises a primer sequence that is complementary to and hybridizes with the adaptor sequence of the DNA template. In some embodiments, each of some of the multiple primers comprises an excisable nucleotide, and the primer can be cleaved at the position of the excisable nucleotide to produce two or more fragments having an extendable 3′ end.

[0134] In another aspect, the array is a support comprising an array of discrete regions, where multiple regions contain clusters of clones of single-stranded DNA multimers, multiple primers having reversible 3′-blocking groups, and multiple primers having an extendable 3′ end. The single-stranded multimer comprises multiple monomers, each monomer comprising an adaptor sequence and a DNA target sequence. Each primer comprises a primer sequence that is complementary to and hybridizes with the adaptor sequence of the DNA template, and at least one adaptor hybridizes with one primer having a reversible 3′-blocking group and one primer having an extendable 3′ end, and the primer having a reversible 3′-blocking group is upstream of the primer having an extendable 3′ end.

[0135] In one aspect, the present disclosure provides a reaction mixture comprising an array of discrete regions, where multiple regions contain clusters of clones of single-stranded DNA multimers, multiple primers, and a DNA polymerase. The single-stranded multimers contain multiple monomers, each monomer containing an adapter sequence and a DNA target sequence. Each primer contains a primer sequence that is complementary to and hybridizes with the adapter sequence of the DNA template. The array is located in the reaction mixture, which is under extension-blocking conditions such that the DNA polymerase binds to the DNA template but cannot extend the primer. In some embodiments, the extension-blocking conditions are that the reaction mixture contains an extension inhibitor (e.g., EDTA, salts including KCl and NaCl, ionic detergents (e.g., deoxycholate, sarcosyl, and SDS), ethanol, and isopropanol), a buffer lacking magnesium, or both.

[0136] It should be understood that the DNA complexes of the array can include any properties of the complexes described herein or prepared according to the methods described herein. In addition, the complexes can have any combination of one or more of the following characteristics: (i) the array contains at least 10 6 discrete regions, (ii) the DNA is single-stranded therein, (iii) the second primer contains at least 10 bases, preferably at least 12 bases, optionally at least 15 bases of the adapter sequence, and (iv) the second primer and the second DNA strand hybridizing therewith are fully complementary.

[0137] 11. Embodiments

[0138] The following are exemplary embodiments of the methods and compositions disclosed in this application.

[0139] Embodiment 1. A method for double-ended sequencing, comprising:

[0140] (f) providing a DNA array having at least 1,000 DNA multimers immobilized on a surface,

[0141] (g) for each of the multiple DNA multimers on the array,

[0142] (iii) annealing a first reading primer to a primer binding site on the DNA multimer,

[0143] (iv) extending at least some of the first reading primers to incorporate dNTPs or dNTP analogs, thereby generating a first reading strand, where each of the incorporated dNTPs or dNTP analogs is identified to generate a first read,

[0144] (h) performing controlled MDA by extending at least some of the first reading strands with a polymerase having strand displacement activity to generate multiple second strands, each second strand containing a portion hybridized to the DNA multimer and an unhybridized single-stranded branch; and

[0145] (i) Annealing a second reading primer to the single-stranded branches of the plurality of second strands,

[0146] (j) Extending the second reading primer to generate a second read.

[0147] Embodiment 2. The method according to Embodiment 1, wherein the DNA concatemer comprises a plurality of monomers, each monomer comprising an adaptor and a target sequence.

[0148] Embodiment 3. The method according to Embodiment 2, wherein the first reading primer hybridizes to the adaptor of each monomer of the DNA concatemer.

[0149] Embodiment 4. The method according to any one of Embodiments 1-3, wherein the first reading strand generated in step (a) comprises a 3'-blocking group that prevents further extension of the first reading strand, and wherein the 3'-blocking group from the first reading strand is removed before performing the controlled MDA in step (b).

[0150] Embodiment 5. The method according to any one of Embodiments 1-4, wherein a plurality of MDA primers are hybridized to the DNA concatemer before step (b)(ii),

[0151] wherein the MDA primer comprises a reversible blocking group to prevent extension of the MDA primer, and the reversible blocking group is removed after step (b)(ii) but before step (c), and

[0152] wherein step (c) comprises extending the plurality of MDA primers and the first reading strand.

[0153] Embodiment 6. The method according to Embodiment 5, wherein the plurality of MDA primers comprise random primers.

[0154] Embodiment 7. The method according to Embodiment 5 or 6, wherein the DNA concatemer comprises an adaptor that hybridizes to at least one MDA primer and at least one first reading primer, and

[0155] wherein the at least one MDA primer is upstream of the at least one first reading primer.

[0156] Embodiment 8. The method according to any one of Embodiments 5-7, wherein the MDA primer has a length of 5-10 nucleotides.

[0157] Embodiment 9. The method according to any one of the foregoing embodiments, wherein the controlled MDA in step (c) comprises:

[0158] (iii) Perform MDA for a first period of time in the presence of a strand displacement DNA polymerase at a certain concentration such that the excess molecules of the DNA polymerase do not bind to the first read strand, and,

[0159] (iv) Remove the excess molecules of the strand displacement DNA polymerase from the array at the end of the first period of time and continue the MDA to generate the second strand, each of the second strands comprising a portion hybridized to the at least one DNA polynucleotide and the unhybridized single-stranded branch.

[0160] Embodiment 10. The method according to embodiment 9, wherein removing the excess molecules of the strand displacement DNA polymerase is performed by washing the array, and

[0161] wherein step (ii) comprises washing the array and adding a fresh buffer containing unincorporated nucleotides but no DNA polymerase.

[0162] Embodiment 11. The method according to embodiment 9 or 10, wherein the ratio of the length of the first period of time to the length of the period for completing the controlled MDA in step (c) is in the range of 1:30 to 1:2.

[0163] Embodiment 12. The method according to any one of embodiments 9-11, wherein the time for completing the controlled MDA in step (c) is 10-90 minutes.

[0164] Embodiment 13. The method according to any one of embodiments 9-12, wherein the first period of time is 1-10 minutes.

[0165] Embodiment 14. The method according to any one of embodiments 1-13, wherein performing step (c) of the controlled MDA comprises:

[0166] (iii) Contact the array with the strand displacement DNA polymerase under extension-blocking conditions, and then

[0167] (iv) Reverse the extension-blocking conditions such that the first read strand is simultaneously extended to generate multiple second strands, each of the second strands comprising a portion hybridized to the at least one DNA polynucleotide and the unhybridized single-stranded branch.

[0168] Embodiment 15. The method according to embodiment 14, wherein the extension-blocking conditions are a reaction buffer without magnesium or unincorporated nucleotides.

[0169] Embodiment 16. The method according to embodiment 14, wherein the extension-blocking conditions are a reaction buffer containing a polymerization inhibitor.

[0170] Embodiment 17. The method according to embodiment 16, wherein the polymerization inhibitor is EDTA.

[0171] Embodiment 18. The method according to embodiment 14, wherein the extension blocking condition is a temperature below 20°C.

[0172] Embodiment 19. The method according to embodiment 14, wherein the concentration of the strand displacement DNA polymerase in (i) is such that an excess of DNA polymerase molecules does not bind to the first reading primer, and the method further comprises removing unbound molecules of the strand displacement DNA polymerase from the array between step (i) and step (ii).

[0173] Embodiment 20. The method according to embodiment 14, wherein the concentration of the strand displacement DNA polymerase in (i) is such that an excess of DNA polymerase molecules does not bind to the first reading primer, wherein step (ii) comprises extending the first reading strand for a first period, removing excess molecules of the strand displacement DNA polymerase from the array at the end of the first stage, and

[0174] continuing MDA in a reaction without excess molecules of DNA polymerase.

[0175] Embodiment 21. The method according to embodiment 20, wherein the ratio of the first period to the period for completing the controlled MDA in step (c) ranges from 1:30 to 1:2.

[0176] Embodiment 22. The method according to embodiment 20 or 21, wherein the period for completing the MDA in step (c) is 10 - 90 minutes.

[0177] Embodiment 23. The method according to any one of embodiments 20 - 22, wherein the first period is 1 - 10 minutes.

[0178] Embodiment 24. A double - end sequencing method, comprising:

[0179] (e) extending a first reading primer hybridized to a plurality of single - stranded DNA concatemers immobilized on an array in the presence of excisable nucleotides to generate first read segments of the plurality of single - stranded DNA concatemers, wherein the extension generates a first reading strand incorporating the excisable nucleotides;

[0180] (f) cleaving the first reading strand having the excisable base to generate a fragment of the first reading strand having an extendable 3' end,

[0181] (g) Performing controlled MDA by extending the fragment of the first read strand to generate multiple second strands, and wherein the controlled MDA generates multiple second strands, each of the second strands comprising a sequence hybridized to one of the multiple single-stranded DNA multimers and an unhybridized single-stranded branch; and

[0182] (h) Extending a second read primer hybridized to the single-stranded branches of the multiple second strands to generate the second read segments.

[0183] Embodiment 25. The method according to embodiment 24, wherein the excisable nucleotide is uracil.

[0184] Embodiment 26. A double-ended sequencing method, comprising:

[0185] (c) Extending a first read primer hybridized to multiple single-stranded DNA multimers immobilized on an array to generate first read segments of the multiple single-stranded DNAs, wherein the extension generates a first read strand;

[0186] (d) Removing the first read strand;

[0187] (c) Performing controlled MDA by extending multiple MDA primers hybridized to the DNA multimers with a polymerase having strand displacement activity to generate multiple second strands, each of the second strands comprising a sequence hybridized to the multiple single-stranded DNA multimers and an unhybridized single-stranded branch; and

[0188] (d) Extending a second read primer hybridized to the single-stranded branches of the multiple second strands to generate the second read segments.

[0189] Embodiment 27. The method according to embodiment 26, wherein the multiple MDA primers comprise random primers.

[0190] Embodiment 28. The method according to any one of embodiments 1-27, wherein the extension of the first read primer in step (a) and the extension of the first read strand in step (b) are performed using a single DNA polymerase.

[0191] Embodiment 29. The method according to any one of embodiments 1-27, wherein the extension of the first strand primer in step (a) and the extension of the first read strand in step (b) are performed using different DNA polymerases.

[0192] Embodiment 30. The method according to any one of embodiments 1-27, wherein the extension of the first strand primer in step (a) is performed by a non-displacing DNA polymerase.

[0193] Embodiment 31. The method according to any one of Embodiments 1-30, wherein the first read is determined by sequencing by synthesis.

[0194] Embodiment 32. The method according to any one of Embodiments 1-31, wherein the extension of the first reading primer lasts for a period of 1-15 minutes.

[0195] Embodiment 33. The method according to any one of the above embodiments, further comprising combining the first read and the second read to determine the sequence of the target DNA sequence.

[0196] Embodiment 34. An array comprising a plurality of single-stranded DNA concatemers and a plurality of primers,

[0197] wherein each single-stranded concatemer comprises a plurality of monomers,

[0198] wherein each monomer comprises an adaptor sequence and a DNA target sequence,

[0199] wherein each primer comprises a primer sequence that is complementary to and hybridizes with the adaptor sequence of the DNA concatemer,

[0200] wherein at least one primer comprises excisable nucleotides, and

[0201] wherein the at least one primer can be cleaved to release the excisable nucleotides and generate two or more fragments having an extendable 3' end.

[0202] Embodiment 35. A kit comprising

[0203] a plurality of sequencing primers,

[0204] a DNA polymerase without strand displacement activity,

[0205] a mixture of reversible terminator nucleotides for SBS,

[0206] a strand displacement DNA polymerase, and

[0207] a mixture of dNTPs.

[0208] Embodiment 36. The kit according to Embodiment 35, wherein the mixture of reversible terminator nucleotides contains uracil, and the kit further comprises a cleavage agent capable of removing uracil from the DNA strand.

[0209] Embodiment 37. The kit according to Embodiment 35 or 36, wherein the kit further comprises a magnesium-free buffer.

[0210] ***

[0211] All publications and patent documents cited herein are incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference. Although the invention has been described primarily with reference to specific embodiments, it is contemplated that other embodiments will be apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be included within the methods of the invention.

Claims

1. A double - ended sequencing method, which includes: (a) Provide a DNA array comprising at least 1,000 DNA concatemers immobilized on a surface, (b) For each of a plurality of DNA concatemers on the array, (i) Anneal a first reading primer to a primer binding site on the DNA concatemer, (ii) Extend at least some of the first reading primers to incorporate dNTPs or dNTP analogs, thereby generating first reading strands, wherein each of the incorporated dNTPs or dNTP analogs is identified to generate a first read segment, (c) Perform controlled MDA by extending at least some of the first reading strands with a strand-displacing polymerase to generate multiple second strands, each second strand comprising a portion hybridized to the DNA concatemer and an unhybridized single-stranded branch; And (d) Anneal a second reading primer to the single-stranded branches of the multiple second strands, (e) Extend the second reading primer to generate a second read segment.

2. The method according to claim 1, wherein the DNA concatemer comprises a plurality of monomers, and each monomer comprises an adaptor and a target sequence.

3. The method according to claim 2, wherein the first reading primer hybridizes to the adaptor of each monomer of the DNA concatemer.

4. The method according to claim 1, wherein the first reading strand generated in step (b) comprises a 3' blocking group, which prevents the first reading strand from being further extended, and wherein the 3' blocking group from the first reading strand is removed before performing the controlled MDA in step (c).

5. The method according to claim 1, wherein a plurality of MDA primers are hybridized to the DNA concatemer before step (b)(ii), wherein the MDA primer comprises a reversible blocking group to prevent the extension of the MDA primer, and the reversible blocking group is removed after step (b)(ii) but before step (c), and wherein step (c) includes extending the plurality of MDA primers and the first reading strand.

6. The method according to claim 5, wherein the plurality of MDA primers include random primers.

7. The method according to claim 5, wherein the DNA concatemer comprises an adaptor that hybridizes to at least one MDA primer and at least one first reading primer, and wherein the at least one MDA primer is upstream of the at least one first reading primer.

8. The method according to claim 5, wherein the MDA primer has a length of 5 - 10 nucleotides.

9. The method according to any one of the preceding claims, wherein the controlled MDA in step (c) includes: (i) Perform MDA for a first period in the presence of a certain concentration of strand-displacing DNA polymerase such that excess molecules of the DNA polymerase do not bind to the first reading strands, and, (ii) Remove the excess molecules of the strand-displacing DNA polymerase from the array at the end of the first period and continue the MDA to generate the second strands, each of the second strands comprising a portion hybridized to the at least one DNA concatemer and the unhybridized single-stranded branch.

10. The method according to claim 9, wherein removing the excess molecules of the strand displacement DNA polymerase is performed by washing the array, and wherein step (ii) comprises washing the array and adding fresh buffer containing unincorporated nucleotides but no DNA polymerase.

11. The method according to claim 9, wherein, The ratio of the length of the first period to the length of the period for completing the controlled MDA in step (c) is in the range of 1:30 to 1:

2.

12. The method according to claim 9, wherein, The time for completing the controlled MDA in step (c) is 10 - 90 minutes.

13. The method according to claim 9, wherein, The first period is 1 - 10 minutes.

14. The method according to claim 1, wherein performing step (c) of the controlled MDA comprises: (i) Contact the array with the strand-displacing DNA polymerase under extension-blocking conditions, and then (ii) Reverse the extension-blocking conditions such that the first reading strands are simultaneously extended to generate multiple second strands, each second strand comprising a portion hybridized to the at least one DNA concatemer and the unhybridized single-stranded branch.

15. The method according to claim 14, wherein the extension-blocking condition is a reaction buffer without magnesium or unincorporated nucleotides.

16. The method according to claim 14, wherein, The extension-blocking conditions are a reaction buffer containing a polymerization inhibitor.

17. The method according to claim 16, wherein, The polymerization inhibitor is EDTA.

18. The method according to claim 14, wherein, The extension-blocking conditions are a temperature below 20°C.

19. The method according to claim 14, wherein the concentration of the strand displacement DNA polymerase in (i) is such that excess molecules of the DNA polymerase do not bind to the first reading primer, and the method further comprises removing unbound molecules of the strand displacement DNA polymerase from the array between step (i) and step (ii).

20. The method according to claim 14, wherein the concentration of the strand displacement DNA polymerase in (i) is such that excess DNA polymerase molecules do not bind to the first reading primer, wherein step (ii) comprises extending the first reading strand for a first period of time and, at the end of the first period of time, removing the excess molecules of the strand displacement DNA polymerase from the array, and continuing the MDA in a reaction without excess molecules of DNA polymerase.

21. The method according to claim 20, wherein the ratio of the first period of time to the period of time for completing the controlled MDA in step (c) ranges from 1:30 to 1:

2.

22. The method according to claim 20, wherein, The period for completing the MDA in step (c) is 10 - 90 minutes.

23. The method according to claim 20, wherein, The first period is 1 - 10 minutes.

24. A paired-end sequencing method, comprising: (a) Extend a first reading primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of excisable nucleotides to generate first read segments of the plurality of single-stranded DNA concatemers, wherein the extension generates first reading strands incorporating the excisable nucleotides; (b) Cleave the first reading strands having the excisable bases to generate fragments of the first reading strands having extendable 3'-ends, (c) Perform controlled MDA by extending the fragments of the first reading strands to generate multiple second strands, and wherein the controlled MDA generates multiple second strands, each second strand comprising a sequence hybridized to one of the plurality of single-stranded DNA concatemers and an unhybridized single-stranded branch; And (d) Extend a second read primer that hybridizes to the single-stranded branches of the plurality of second strands to generate a second read.

25. The method according to claim 24, wherein the excisable nucleotide is uracil.

26. A paired-end sequencing method, comprising: (a) Extend a first read primer that hybridizes to a plurality of single-stranded DNA concatemers immobilized on an array to generate a first read of the plurality of single-stranded DNAs, wherein the extension generates a first read strand; (b) Remove the first read strand; (c) Perform controlled MDA by extending a plurality of MDA primers that hybridize to the DNA concatemers with a polymerase having strand displacement activity to generate a plurality of second strands, each of the second strands comprising a sequence hybridized to the plurality of single-stranded DNA concatemers and an unhybridized single-stranded branch; and (d) Extend a second read primer that hybridizes to the single-stranded branches of the plurality of second strands to generate a second read.

27. The method according to claim 26, wherein the plurality of MDA primers includes random primers.

28. The method according to claim 1, wherein extending the first reading primer in step (a) and extending the first reading strand in step (b) are performed by using a single DNA polymerase.

29. The method according to claim 1, wherein extending the first reading primer in step (a) and extending the first reading strand in step (b) are performed by using different DNA polymerases.

30. The method according to claim 1, wherein extending the first reading primer in step (a) is performed by a non-displacing DNA polymerase.

31. The method according to claim 1, wherein the first read is determined by sequencing by synthesis.

32. The method according to claim 1, wherein the extension of the first reading primer lasts for a period of 1 - 15 minutes.

33. The method according to claim 1, further comprising combining the first read and the second read to determine the sequence of the target DNA sequence.

34. An array comprising a plurality of single-stranded DNA concatemers and a plurality of primers, wherein, Each single-stranded concatemer comprises a plurality of monomers, wherein each monomer comprises an adaptor sequence and a DNA target sequence, wherein each primer comprises a primer sequence that is complementary to and hybridizes to the adaptor sequence of the DNA concatemer, wherein at least one primer comprises an excisable nucleotide, and wherein the at least one primer is cleavable to release the excisable nucleotide and generate two or more fragments having an extendable 3' end.

35. A kit, comprising a plurality of sequencing primers, a DNA polymerase without strand displacement activity, a mixture of reversible terminator nucleotides for SBS, a strand displacement DNA polymerase, and a mixture of dNTPs, wherein the mixture of reversible terminator nucleotides contains uracil, and the kit further comprises a cleavage agent capable of removing uracil from a DNA strand.

36. The kit according to claim 35, wherein the kit further comprises a magnesium-free buffer.

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