Controlled strand displacement for paired-end sequencing

Abstract

Provides methods used for paired-end sequencing. [Solution] For each of a plurality of DNA concatemers on the array, the method includes annealing a first read primer to the primer binding site; extending at least a portion of the first read primer to incorporate a dNTP or a dNTP analog to generate a first read strand, wherein each of the incorporated dNTPs or dNTP analogs is identified to generate the first read; performing controlled MDA to extend a portion of the first read strand with a polymerase having strand displacement activity to generate a plurality of second strands, wherein the second strands include a portion hybridized to the DNA concatemer and unhybridized single-stranded branches; and annealing a second read primer to the single-stranded branches of the plurality of second strands and extending the second read primer to generate a second read strand.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 952,713, filed December 23, 2019, which is incorporated herein by reference for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to the fields of DNA sequencing, genomics and molecular biology. [Background technology]

[0003] background Paired-end sequencing allows sequencing of both ends of a DNA fragment. It can be performed by sequencing a DNA template and its complementary strand. See U.S. Pat. No. 10,227,647. Paired-end sequencing generates twice the number of reads as single-end sequencing in the same time and effort. The read pairs generated by paired-end sequencing enable accurate read alignment and detection of mutations (e.g., insertion-deletion mutations) that are difficult to detect by single-end sequencing. Summary of the Invention

[0004] Brief Summary of the Invention The paired-end sequencing method described herein uses controlled production of a strand (first strand) complementary to a DNA template so that it remains bound to 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 generating a second read from the second strand. Also disclosed is a method for synchronizing the generation of second strands and maximizing the number of second strands of suitable length for generating second reads.

[0005] In one aspect, the methods described herein comprise extending a first read primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array to generate a plurality of single-stranded DNA first reads, wherein the extension generates a first read strand; performing controlled multiple displacement amplification (MDA) by extending the first read strand, or a portion thereof, with a polymerase having strand displacement activity to generate a plurality of second strands, each comprising (i) a sequence hybridized to one of the plurality of DNA concatemers, and (ii) an unhybridized single-stranded branch; and extending a second read primer that hybridizes to the single-stranded branch of the plurality of second strands to generate second reads.

[0006] In some aspects, the methods described herein involve the generation of at least 1,000, at least 10,000, at least 10 5 Pieces, at least 10 6 or at least 10 7 The method further comprises providing a DNA array having a surface on which DNA concatemers are immobilized. The number of DNA concatemers is preferably between 1,000 and 10. 13 , 10 4 ~10 12 , 10 4 ~10 10 or 10 5 ~10 8For each of the plurality of DNA concatemers on the array, a first read primer is annealed to the primer binding site on the DNA concatemer, and at least a portion of the first read primer is extended to incorporate a dNTP or a dNTP analog, thereby generating a first read strand. Each of the incorporated dNTPs or dNTP analogs is identified to generate a first read. The method further includes performing controlled MDA by extending at least a portion of the first read strand with a polymerase having strand displacement activity to generate a plurality of second strands, each of which comprises a portion that hybridizes to the DNA concatemer and a single-stranded branch that does not hybridize. The method may further include annealing a second read primer to the single-stranded branches of the plurality of second strands and extending the second read primer to generate a second read.

[0007] In one aspect, a method described herein comprises extending a first read primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of an excisable nucleotide to generate first reads of the plurality of single-stranded DNA concatemers, wherein the extension generates a first read strand incorporating the excisable nucleotide. The method may further include one or more of the following steps: cleaving the first read strand at the position of the excisable base to generate fragments of the first read strand having extendable 3' ends; performing controlled MDA by extending the fragments of the first read primer to generate a plurality of second strands, wherein the controlled MDA generates a plurality of 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 read primer hybridized to the single-stranded branch of the plurality of second strands to generate second reads.

[0008] Also provided is 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, each monomer comprising an adapter sequence and a DNA target sequence, each primer comprising a primer sequence that is complementary to and hybridizes to the adapter sequence of the DNA concatemer, at least one primer comprising a excisable nucleotide, and at least one primer can be cleaved to release the excisable nucleotide and generate two or more fragments having extendable 3' ends.

[0009] In another aspect, the array is a support comprising an array of discrete regions, wherein a plurality of regions contain clonal clusters of single-stranded DNA concatemers, a plurality of primers with reversible 3' blocking groups, and a plurality of primers with extendable 3' ends. The single-stranded concatemers comprise a plurality of monomers, each monomer comprising an adapter sequence and a DNA target sequence. Each primer comprises a primer sequence that is complementary to and hybridizes to an adapter sequence on a DNA template, and at least one adapter hybridizes to one primer with a reversible 3' blocking group and one primer with an extendable 3' end, the primer with the reversible 3' blocking group being upstream of the primer with the extendable 3' end.

[0010] The present invention also provides kits comprising multiple sequencing primers, a non-displacing DNA polymerase, a mixture of reversibly terminated nucleotides for SBS, a strand-displacing DNA polymerase, and a mixture of dNTPs. The kits further comprise one or more of the following: i) a magnesium-free buffer ("magnesium-free buffer") and ii) an elongation inhibitor (e.g., EDTA, excess salts such as KCl and NaCl, ionic detergents such as sodium desoxycholate, sarkosyl, and SDS, ethanol, isopropanol, etc.). In certain embodiments, the mixture of nucleotides 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, e.g., 1:3 to 1:8, or 1:4 to 1:5. In some embodiments, the kits may further comprise a mixture of 3'-blocked primers (i.e., 3'-blocked primers) and unblocked primers. In some embodiments, the ratio of blocked to unblocked primers is in the range of 1:1 to 1:5, for example, in the range of 1:2 to 1:4. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows one embodiment of the paired-end sequencing method of the present invention. [Figure 2] 2 illustrates one embodiment of the methods described herein, in which a multiple displacement amplification (MDA) primer bearing a reversible blocking group (indicated by an "*" in the figure) is hybridized onto a DNA concatemer upstream of a first lead primer. After removal of the reversible blocking group, both the MDA primer and the first lead strand are extended with MDA to generate multiple second strands. [Figure 3]Figure 3 illustrates one embodiment of the method described herein. After the first lead strand is generated during first lead sequencing, a strand-displacing DNA polymerase binds to the DNA concatemers and initiates MDA. The DNA polymerase is introduced into the reaction at a high concentration so that excess DNA polymerase molecules in the reaction do not bind to the DNA concatemers and the first lead strand. After initiation and before completion of MDA, unbound DNA polymerase molecules are removed, and MDA continues to generate the second strand. [Figure 4] Figure 4 shows one embodiment of the method described herein. After the first read sequence forms a first read strand, a strand-displacing DNA polymerase is added to the reaction under extension-blocking conditions. This polymerase binds to the concatemers and the first read strand, but does not extend the primer under these conditions. The asterisk (*) in this figure indicates that the primer cannot be extended due to the extension-blocking conditions. [Figure 5] 5 shows one embodiment of the method described herein, in which excisable nucleotides are incorporated into the first read strand during first read sequencing.The first read strand is then cleaved at the positions where these excisable nucleotides exist to generate fragments with extendable 3' ends.This fragment is used as an extension primer for MDA to generate the second strand. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Invention 1. Overview The present invention relates to an improved paired-end sequencing method using DNA strands. In one approach, multiple steps are taken to maximize the number of second strands with lengths optimized for sequencing, for example, by including MDA primers. In some cases, the primers are cleaved to generate multiple fragments, each of which serves as a primer for generating additional second strands. In other cases, multiple steps are taken to optimize synchronous second strand generation, for example, using extension-blocking conditions.

[0013] In one aspect of the present invention, a first read of a paired-end sequence is generated by extending a first read primer hybridized to DNA concatemers immobilized on an array ("first read sequencing"). The first read sequencing generates a first read strand complementary to the DNA concatemer. Controlled multiple displacement amplification (MDA) is then performed using the extension primer to generate a second strand. 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 one or more unhybridized single-stranded branches having one or more priming sequences complementary to the second read primer. The first read strand can be further extended to generate a 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, a process referred to herein as "second read sequencing."

[0014] In some approaches, the methods include various features that can increase the second strand yield (i.e., the number of second strands suitable for second read sequencing) and the efficiency of second strand production. In some approaches, the paired-end sequencing methods described herein use excisable nucleotides, such as uracil, that are incorporated during extension of the first read primer. The first read primer or first read strand containing these excisable nucleotides is cleaved at the locations where these excisable nucleotides are present to generate multiple fragments with extendable 3' ends, which can then be extended to generate additional second strands.

[0015] In some approaches, the method includes a feature of synchronizing the generation of multiple second strands so that the generated second strands have similar lengths (e.g., lengths suitable for second read sequencing). Various approaches are available for achieving such synchronization. In one embodiment, after the first read sequencing is completed, a high concentration of polymerase is introduced to bind to a primer (i.e., an MDA primer or the first read strand) hybridized to the DNA concatemer under extension-blocking conditions. This initial step under extension-blocking conditions maximizes the binding of the DNA polymerase to the DNA template and primer. The extension-blocking conditions can then be reversed to synchronize MDA from the primer. In some approaches, after a first period of MDA, excess DNA polymerase molecules (i.e., polymerase molecules free in the reaction and not bound to the primer and DNA template) are removed, and MDA is continued in the absence of unbound DNA polymerase molecules. This process minimizes interactions between the free polymerase molecule and the newly forming second strand, and between the free polymerase molecule and the DNA template beyond the initial binding period, facilitating synchronous extension and generation of the second strand.

[0016] The drawings of the present application illustrate specific embodiments of the present invention. Figure 1 shows a DNA concatemer containing multiple monomers, each containing an adapter sequence and a target DNA sequence. A first read primer is annealed to the primer binding sequence in the adapter of the DNA concatemer and extended to generate a first read by sequencing by synthesis (SBS). Extending the first read primer generates a first read strand. After the final cycle of SBS, the 3' blocking group of the terminal nucleotide of the first read strand is removed and a strand-displacing polymerase is added. The strand-displacing polymerase extends the first read strand, generating a second read strand by MDA. MDA is controlled so that the second strand partially hybridizes to the DNA concatemer, and each second strand contains an unhybridized branch. Next, a second read primer is annealed to the branch (e.g., by hybridizing to the second read primer binding sequence on the adapter sequence on the branch). These secondary read primers are then extended to generate secondary reads.

[0017] Figure 2 shows that an MDA primer with a 3' blocking group and a first lead primer hybridize to a DNA concatemer. The blocking groups on these MDA primers are different from the blocking groups on the nucleotides incorporated during SBS. Unlike the blocking groups on the nucleotides that are removed during each cycle of SBS, the blocking groups on the MDA primers are retained throughout the SBS sequencing process. The first lead primer is extended to generate the first read, generating the first read strand. Once sequencing of the first read is complete, the blocking group on the MDA primer is removed, and the blocking group on the last nucleotide added in the final sequencing cycle is also removed. Both strands of the MDA primer and the first read are extended in a controlled MDA reaction in the presence of a strand-displacing DNA polymerase to generate a second strand that partially hybridizes to the DNA concatemer. The second read can be generated in a similar manner to that described above.

[0018] Figure 3 shows that after the first read sequencing is complete, the first read strand formed by the first read sequencing remains hybridized to the DNA concatemers. A high concentration of DNA polymerase is added to the array to extend the first read strand for a first period of time. During this time, due to the excess DNA polymerase molecules in the reaction, some, but not all, of the DNA polymerase molecules bind to the concatemers, while the remaining DNA polymerase molecules are free, i.e., they do not bind to the DNA concatemers and do not participate in the extension reaction. The array is then washed to remove the excess DNA polymerase molecules. A buffer containing nucleotides (but not DNA polymerase) is added to the reaction to allow the MDA to continue generating second strands that partially hybridize to the DNA concatemers.

[0019] Figure 4 shows that after the first read sequencing is completed, the first read strand formed by the first read sequencing remains hybridized to the DNA concatemer. A strand-displacing DNA polymerase is added to the reaction under extension-blocking conditions, e.g., the strand-displacing DNA polymerase is in a buffer lacking at least one component required for extension, e.g., magnesium. Under these conditions, the strand-displacing DNA polymerase binds to the DNA concatemer but does not extend it. After the array is maintained under these conditions for a first period, these conditions are reversed to allow extension. For example, the removed component (e.g., magnesium) is added back to the reaction at a concentration appropriate for MDA. MDA is then initiated to generate the second strand.

[0020] 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 such cleavage results in multiple fragments with extendable 3' ends, leading to the release of the excisable nucleotide. These fragments are extended in MDA to form multiple second strands.

[0021] The disclosure of US Pat. No. 10,227,647 is incorporated by reference in its entirety for all purposes.

[0022] 2.Definition As used herein, "primer" refers to an oligonucleotide that serves as a starting point for nucleic acid synthesis when forming a double strand using a polynucleotide as a template, and is extended from its 3' end along the template to form an extended double strand. A primer may contain a natural sequence or a synthetic sequence. A primer may also contain non-natural nucleotides. The sequence of nucleotides added during the extension process is determined by the base sequence of the template polynucleotide. A primer is generally extended by a DNA polymerase.

[0023] As used herein, "random primer" refers to a primer having a random nucleotide sequence.

[0024] As used herein, MDA or multiple displacement amplification refers to DNA amplification based on strand displacement replication with multiple primers.

[0025] As used herein, "polynucleotide" is used interchangeably with the term "nucleic acid" to refer to DNA, RNA, and hybrid and synthetic nucleic acids, which may be single-stranded or double-stranded. "Oligonucleotide" is a short polynucleotide having a length of about 6 to about 300 nucleotides. "Complementary polynucleotide" refers to a polynucleotide that is complementary to a target nucleic acid.

[0026] As used herein, the term "strand displacement activity" refers to the ability to displace downstream DNA occurring during synthesis. Strand displacement activity is described in U.S. Patent Publication No. 2010115145 (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, dissociates a paired nucleic acid from its complementary strand in a 5 to 3 direction in association with and near template-dependent nucleic acid synthesis. Strand displacement begins at the 5' end of the paired nucleic acid sequence, and the enzyme synthesizes nucleic acid immediately 5' from the displacement site. The newly synthesized nucleic acid and the displaced nucleic acid generally have the same base sequence and are complementary to the template nucleic acid strand. Strand displacement activity may be located on the same molecule as that which confers nucleic acid synthesis activity, particularly DNA synthesis activity, or may be a separate, independent activity. E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T7 or T5 bacteriophage DNA polymerase, and HIV reverse transcriptase are enzymes that possess both polymerase and strand displacement activity. Agents such as helicases can be used in combination with inducers that lack strand displacement activity to exert a strand displacement effect, i.e., the combined effect of nucleic acid displacement and synthesis of identical nucleic acids. Similarly, proteins such as Rec A or single-strand binding protein from E. coli or another organism can be used in combination with other inducers to generate or promote strand displacement (Kornberg and Baker, 1992, DNA Replication, 2nd Edition, pp. 113-225, Freeman, NY). In one approach, the polymerase is Phi29 polymerase, which has strong strand displacement activity at moderate temperatures (e.g., 20-37°C). One approach uses Bst DNA polymerase, large fragment (e.g., NEB#MO275 available from New England Biolabs, Ipswich, Mass.) Bst DNA polymerase is active at high temperatures (-65°C).

[0027] The term "first strand" refers to the single-stranded DNA template used in paired-end sequencing.

[0028] The term "second strand" refers to a single strand of DNA that is complementary to the first strand.

[0029] The term "MDA primer" refers to an extension primer that hybridizes to a DNA template and is extended to generate a second strand in a displacement-extension reaction. The MDA primers described herein are not sequencing primers (e.g., first lead primers). In some embodiments, the MDA primer and the first lead primer have different sequences.

[0030] The term "first read primer" refers to a primer that hybridizes to a DNA template and is used to generate a first read for paired-end sequencing.

[0031] The term "first read" refers to the nucleotide sequence information obtained by sequencing a DNA template using a first read primer.

[0032] The term "first read sequencing" refers to the sequencing process used to obtain the first read.

[0033] The term "first read strand" refers to a single-stranded polynucleotide generated by first read sequencing, i.e., by extending a first read primer. The first read strand is also referred to as an extended first read primer. The first read strand can be further extended to form a second strand.

[0034] The term "second lead primer" refers to a primer that hybridizes to the second strand, i.e., the strand that is complementary.

[0035] The term "second read" refers to the nucleotide sequence information obtained by sequencing the second strand.

[0036] The term "second read sequencing" refers to the sequencing process used to obtain a second read.

[0037] The term "second read strand" refers to a single-stranded polynucleotide generated by second read sequencing, i.e., by extending a second read primer.

[0038] The term "excisable nucleotide" refers to a nucleotide in a DNA strand that can be removed, such that the DNA strand is broken into two DNA fragments. One exemplary method for removing a nucleotide from a DNA strand is via an enzyme. One exemplary excisable nucleotide is uracil.

[0039] The term "reversible blocking group" of a reversible terminator nucleotide may also be referred to as a "removable blocking group," "blocking site," "blocking group," "reversible terminator blocking group," etc. A reversible blocking group is a chemical moiety attached to a nucleotide sugar (e.g., deoxyribose), usually at the 3'-OH position of the sugar moiety, that prevents addition of a nucleotide by a polymerase at that position. The reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), chemical reaction, heat, light, etc., to provide a hydroxyl group at the 3'-OH position of the nucleoside or nucleotide so that addition of a nucleotide by a polymerase can occur.

[0040] As used herein, "dNTP" includes both naturally occurring deoxyribonucleotide triphosphates and their analogs, including analogs having a 3'-O cleavable blocking group.

[0041] 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(s). Microarrays generally include at least one planar solid support, such as a glass microscope slide.

[0042] As used herein, the terms "synchronized" or "synchronous" in reference to a primer extension reaction means extending multiple primers or multiple extension primers that are initiated at the same time.

[0043] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" includes reference to one agent or a mixture of such agents, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art.

[0044] Unless otherwise defined, 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 by reference for the purpose of describing and disclosing the devices, compositions, formulations, and methods that are described in the publications and that may be used in connection with the inventions described herein.

[0045] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, as well as any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges are independently encompassed within the invention, also encompassed within the smaller ranges, except for any specifically excluded limit within the stated range. When the stated range includes one or both of the limits, ranges excluding either both of those included limits are also encompassed within the invention.

[0046] Throughout this specification, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details.

[0047] Although the present invention will be described primarily with reference to particular embodiments, it is anticipated that other embodiments will become apparent to those skilled in the art upon reading this specification, and such embodiments are intended to be encompassed within the methods of the present invention.

[0048] 3. DNA Template In some approaches, 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 containing multiple copies of the same DNA sequence ("monomers" or "subunits" linked in series). A DNA concatemer can contain at least 2 monomers, at least 3 monomers, at least 4 monomers, at least 10 monomers, at least 25 monomers, at least 50 monomers, at least 200 monomers, or at least 500 monomers. In some approaches, a DNA concatemer contains 25 to 1000 monomers, e.g., 50 to 800 monomers or 300 to 600 monomers).

[0049] In one embodiment, each monomer of a concatemer contains one adapter sequence and one target DNA sequence. Because the monomers are ligated in tandem, the target DNA sequence is flanked by two adapter sequences. In some approaches, the target DNA sequence in the monomer is flanked by two "half adapter" sequences, such that each tandemly ligated target sequence in the concatemer is flanked by two adapters. In some approaches, a monomer unit contains one, two, three, four, or more adapters. In some approaches, all of the adapters in the monomer (and concatemer) have the same sequence. In other embodiments, the adapters may have different sequences, such as two, three, or four different sequences. It will be appreciated that an individual monomer may contain more than one DNA template sequence. Exemplary DNA concatemer structures are described in Table 1 of U.S. Pat. No. 10,227,647.

[0050] The DNA concatemers used in the methods described herein may be DNA nanoballs, or "DNBs." 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 Drmanac et al. U.S. Patent No. 8,592,150, the entire contents of both of which are incorporated herein by reference.

[0051] DNA nanoballs (DNBs) are single-stranded copies of DNA sequences linked to a linear DNA structure. DNBs are typically produced by copying 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 begins by extending a primer that hybridizes to the single-stranded circle, generating a reverse-complementary strand that hybridizes to the circle. After completing one full rotation around the circle, the polymerase continues extending the newly created strand by shifting it forward in the direction of travel. As the polymerase continues to extend the strand around the circle, multiple reverse-complementary strands are created, which linearly bind to each other. This strategy creates targets with many probe or primer binding sites, allowing for higher signal intensity than can be achieved with a single copy of the circular subunit. Single-stranded DNA of sufficient length typically forms random coils, filling an approximately spherical volume in solution (e.g., in SSC buffer at room temperature). In some approaches, the DNA nanoballs typically have a diameter of about 100-300 nm, and each monomer typically contains at least one target DNA sequence.

[0052] DNA concatemers (including DNA nanoballs) can be produced by any suitable method. In one approach, a single genome fragment is used to generate single-stranded circular DNA with adapters interposed between consecutive or adjacent target sequences in the genome. Circular DNA constructs can be enzymatically amplified, for example, by rolling circle replication or by ligation of monomers. By way of example and not limitation, DNA nanoballs can be prepared according to the methods described in U.S. Pat. Nos. 8,445,194 and 8,592,150.

[0053] Amplifying DNA by rolling circle replication has several advantages: 1) amplification is linear, preventing mutated copies from over-representing the original template sequence, 2) all copies are localized to one single molecule, making it ideal for microscopic analysis with fluorescent probes or reporters, and 3) circle replication can proceed under isothermal conditions, making it easy to automate.

[0054] The target DNA may be from any source, including a naturally occurring sequence (e.g., genomic DNA, cDNA, mitochondrial DNA, cell-free DNA, etc.), an artificial sequence (e.g., a synthetic sequence, a product of gene shuffling or molecular evolution, etc.), or a combination thereof. The target DNA may be derived from an organism or cell (e.g., a plant, an animal, a virus, a bacterium, a fungus, a human, a mammal, an insect), a forensic source, etc. The target DNA sequence may be obtained from a population of organisms, such as a population of enteric bacteria. The target DNA sequence may be obtained directly from a sample or may be the product of an amplification reaction, a fragmentation reaction, etc.

[0055] The target DNA may have a length within a particular size range, such as 50 to 600 nucleotides in length. Other exemplary size ranges include lengths of 25 to 2000 nucleotides, 50 to 1000 nucleotides, 100 to 600 nucleotides, 50 to 100 nucleotides, 100 to 300 nucleotides, and 100 to 400 nucleotides in length. In DNA template polynucleotides with two or more different target DNAs, the target DNAs may be the same length or different lengths. In a library of DNA template polynucleotides, the library members may, in some approaches, have similar lengths (e.g., all in the 25 to 2000 nucleotide length range, or another range).

[0056] In one approach, target DNA can be prepared by fragmenting a larger DNA source (e.g., genomic DNA) to generate fragments of a desired size range. In some approaches, a size selection step is used to obtain a pool of fragments within a specific size range.

[0057] The DNA templates described herein may contain two or more adapter sequences. The adapters may include 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 methods and / or probes for cPAL or other ligation-based sequencing methods), or both elements for immobilization and sequencing. The adapters may also include additional features, such as, but not limited to, restriction endonuclease recognition sites, extension primer hybridization sites (for use in analysis), barcode sequences, unique molecular identifier sequences, and polymerase recognition sequences.

[0058] Adapter sequences can have a length, structure, and other properties appropriate for a particular sequencing platform and intended use. For example, adapters can be single-stranded, double-stranded, or partially double-stranded and can be of a length appropriate for the intended use. For example, adapters can have lengths ranging from 10 to 200 nucleotides, 20 to 100 nucleotides, 40 to 100 nucleotides, or 50 to 80 nucleotides. In some approaches, adapters can include one or more modified nucleotides, including modifications to the base, sugar, and / or phosphate moieties.

[0059] It will be understood by those skilled in the art that while different members of a library will generally contain a common adapter sequence, different species or subgenera in the library may have unique features, such as subgenus-specific barcodes.

[0060] An individual adapter sequence may contain multiple functionally distinct subsequences. For example, as described in detail herein, a single adapter sequence may contain two or more primer sequences (which can be recognized by different complementary primers or probes). Functionally distinct sequences within an adapter may or may not overlap. For illustrative purposes, considering a 40-base-long adapter, in one embodiment, bases 1-20 are a first primer binding site and bases 21-40 are a second primer binding site. In a different embodiment, bases 1-15 are a first primer binding site and bases 21-40 are a second primer binding site. In a different embodiment, bases 5-25 are a first primer binding site and bases 15-35 are a second primer binding site. Similarly, given a 40-base-long adapter, bases 1-20 may be a fixed sequence and bases 21-40 may be primer binding sites. Different primer sequences in the same or different adapters of a DNA template polynucleotide may have the same or different lengths.

[0061] An adapter (e.g., a first adapter, a second adapter, a third adapter, etc.) may be composed of one, two, or more primer binding sequences. A primer binding sequence is functionally defined as a site or sequence to which a primer (or oligonucleotide) specifically binds. For example, an adapter having two primer binding sequences can be specifically bound by two different primers. In some approaches, the two primer binding sequences in the same adapter overlap, i.e., share a portion of their nucleotide sequence. In some approaches, the overlapping region does not exceed 50%, 40%, 30%, 20%, 10%, or 5% of either of the two overlapping primer sequences. In some approaches, the two or more primer binding sequences do not overlap. In some approaches, the non-overlapping primer sequences are immediately adjacent to each other. In some other embodiments, the non-overlapping primer sequences are separated by 1-10 nucleotides, 10-20 nucleotides, 30-40 nucleotides, or 40-50 nucleotides.

[0062] It may be apparent that within a given DNA template polynucleotide, different adapters may have the same or different sequences, or may have the same primer binding sequence or different primer sequences. Although certain figures (e.g., Figure 1) are provided to illustrate the invention, the representation of adapters using similar cross-hatching or the like should not be construed as indicating sequence identity.

[0063] 4. Primer Primers used in the methods described herein are of sufficient length to allow hybridization of the primer, with the exact length and sequence varying depending on the primer's intended function (e.g., extension primer, index sequence, etc.). Primer sequences are often at least 10 bases long, at least 12 bases long, at least 15 bases long, or at least 18 bases long. In some embodiments, primer sequences have lengths ranging from 8 to 60 nucleotides, e.g., 10 to 25 nucleotides long, or 40 to 60 nucleotides long. Selecting or designing primers for use in the present invention is well within the capabilities of one of ordinary skill in the art.

[0064] It will be appreciated that primers and probes may be fully or partially complementary to the primer binding sequence in the adapter to which they hybridize. For example, a primer may have at least 85%, 90%, 95%, or 100% sequence identity to the sequence to which it hybridizes.

[0065] The primer may also contain an additional sequence at the 5' end of the primer that is not complementary to the primer binding sequence in the adapter. The non-complementary portion of the primer may be of any length that does not interfere with hybridization between the primer and its primer binding sequence. Generally, 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 may contain a DNA portion and / or an RNA portion, and in some approaches, the primer used in the present invention may have one or more modified nucleotides, including modifications to the base, sugar, and / or phosphate moieties.

[0066] A "sequencing oligonucleotide" or "sequencing primer" can be an extension primer used in a sequencing-by-synthesis reaction (also called "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, double, and multiple cPAL), described in U.S. Patent Publication No. 20140213461, which is incorporated herein by reference for all purposes.

[0067] In some cases, the extension primers are also used as sequencing primers, such as the first and second read primers. In some cases, the extension primers can also be the product of a primer extension reaction, such as the first read strand, which can be further extended. The first read primer is a sequencing primer that hybridizes to the DNA concatemer and is extended to generate the first read, forming the first read strand. As described above, in some cases, the first read strand functions as an extension primer and is extended to generate the second strand. In some cases, an additional extension primer, called an MDA primer, is used to hybridize to the DNA concatemer and is extended to generate the 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 5' to the first read primer. In some embodiments, one or more MDA primers and the first lead primer bind to the same adapter of the monomer of the DNA concatemer, and the one or more MDA primers are located 5' to the first lead primer. In some embodiments, the MDA primers include random primers. In some embodiments, excess random primers (i.e., random primers that do not hybridize to the DNA concatemer) can be removed (e.g., by washing the array) before MDA begins to prevent excess random primers from binding to and priming the second strand.

[0068] In some cases, the primers used in the method (e.g., the first lead primer, the first lead strand, or the MDA primer) may contain a excisable nucleotide, and these primers can be cleaved at the position of the excisable nucleotide to form a new, extendable 3-prime end. Such cleavage releases the excisable nucleotide. In some cases, these excisable nucleotides are nucleobases other than A, G, T, or C. Alternatively, or in addition, these excisable nucleotides can be incorporated at desired intervals during the sequencing reaction. The excisable nucleotides are selected for their ability to be excised and removed from the DNA strand, generating an exposed 3-prime end available for extension. An exemplary excisable nucleotide that can be used in this method is uracil, which can be incorporated in place of thymidine during the extension reaction. Uracil incorporated into single-stranded DNA can be excised using a uracil-specific excision reagent, such as USER. (商標) USER (商標) USER consists of a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-clease endonuclease VIII, which together first catalyze the excision of uracil to form an abasic site (abasic site), and then cleave the phosphodiester bond at the abasic site to generate a break in single-stranded DNA and release uracil. Bitinaite et al., USER TM See "A friendly 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 are first bound to a DNA template and then cleaved at the location where the excisable nucleotide appears, allowing for the simultaneous extension of multiple second strands.

[0069] In some cases, one or more MDA primers contain a blocking group at their 3' end to prevent them from extending. The number of 3'-blocked primers can be controlled to control the nature and density of independently displaced strands per DNB. In some embodiments, the number of 3'-extendable primers is controlled by maintaining the primers at an appropriate concentration during hybridization. In some embodiments, the number of 3'-extendable primers is controlled by mixing 3'-extendable primers and 3'-blocked primers in an appropriate ratio.

[0070] In some cases, the extension primers (i.e., MDA primers) used in the methods described herein may contain reversible blocking groups at their 3' ends, and these extension primers are only extended when the blocking groups are removed. In some embodiments, one or more MDA primers bind to a DNA template upstream of a first lead primer, and after a first read is generated, the reversible blocking groups of the MDA primers are removed to allow multiple second strands to initiate extension from the MDA primers. See Figure 2. In some embodiments, the 3' reversible blocking group of the MDA primer can be removed under different conditions than the blocking group added to the first lead primer during each cycle of sequencing, so that the blocking group of the MDA primer remains throughout first read sequencing. In some embodiments, the 3' reversible blocking group of the MDA primer is different from the blocking group of the nucleotide added to the first lead primer during each cycle of sequencing, so that the blocking group of the MDA primer remains throughout first read sequencing. In some cases, the 3' blocking group is a 3' phosphate, which can be removed by a phosphatase.

[0071] In the methods described herein, second-strand generation is initiated by hybridizing a primer containing a sequence that is the reverse complement of a DNA template, e.g., a DNA concatemer. In some cases, the primer hybridizes to an adapter of a monomer of the DNA concatemer. In some cases, second-strand synthesis can also be initiated from the extension of a random oligonucleotide, i.e., using an MDA primer with a random nucleotide sequence. In this approach, a diverse set of primers can be used to hybridize to the sequenced DNA concatemer. In some approaches, these sequences are oligonucleotides of random sequence, 5-10 bases in length, e.g., 6-10 bases, or 5-8 bases in length. Individual sequences within the random pool can hybridize to complementary sequences within the DNA concatemer.

[0072] 4.1. Primer Binding In some aspects, the primers (e.g., the first lead primer or the MDA primer, or both) used in the above methods and compositions are linked to form a linked pair. The term "link" refers to both non-covalent and covalent interactions that hold two nucleic acid molecules together. In some cases, the linkage is achieved through DNA hybridization, chemical bonding, or both. These linked primer pairs can link consecutive or non-consecutive monomers of a DNA concatemer, thereby stabilizing the DNA concatemer.

[0073] Primers can be linked via various means. In some embodiments, they are linked via chemical bonds. In some embodiments, two primers are linked via hybridization between two complementary sequences (referred to as "hybridization sequences"), one in each primer. 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. Methods for linking primers and the composition of the linked primers are described in PCT application PCT / CN2020 / 124338, the entire contents of which are incorporated herein by reference for all purposes.

[0074] The length of the hybridization sequence may vary. The length of the hybridization sequence is selected so that the Tm of the stapler sequence is between 50°C and 72°C. This ensures that the bound primer pair can remain hybridized throughout the assay. In some embodiments, the length of the hybridization sequence can range from 20 to 150 nucleotides, for example, from 40 to 120 nucleotides, or from 50 to 100 nucleotides.

[0075] In certain embodiments, the hybridization sequence is 5' to the sequence on the primer that is complementary to the DNA template.

[0076] 5. Generate first leads As described above, in some embodiments, the first read primer functions 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 by extending the first read primer with nucleotides, many of the second strands are further generated. The extension with nucleotides is performed so that only one nucleotide can be added by modifying the 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 may contain a 3'-O-blocking group, such as a 3'-O-azide methyl group, at the 3'-OH position of A, C, G, or T. To determine the next base in a series of sequencing cycles, the modified nucleotide is converted into an extendable form, and another base is added in another cycle to determine the sequence. In such a sequencing method, the extended first strand primer functions as a primer for nucleotide addition in each round. After the final cycle of nucleotide addition for sequencing, the extended first lead primer (also referred to as the "first lead strand") is unblocked and then further extended using a polymerase that is the same as or different from the polymerase used to generate the first lead strand. Extension of the first lead primer can be performed by any DNA polymerase, including polymerases with strand displacement activity, polymerases lacking strand displacement activity, or a mixture of polymerases. In some approaches, the DNA polymerase has no or little strand displacement activity, such as BG9 DNA polymerase.

[0077] 6. Controlled MDA 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 by MDA. In some embodiments, the 3' blocking group (added in the final cycle of first read sequencing) of the terminal nucleotide of the first read strand is removed, and these first read strands are used as extension primers in MDA. The MDA method described herein is controlled so that the second strand remains partially hybridized to the DNA concatemer, i.e., the second strand remains bound to the DNA template via the sequence that hybridizes to the DNA template. The second strand also contains a single-stranded branch containing a primer binding site for the second read primer. The second read primer can function as a sequencing primer to generate the second read.

[0078] One illustrative example is shown in Figure 1. A first read primer is annealed to a DNA concatemer and extended to generate a first read strand. First read sequencing generates the first read strand. Controlled MDA is then performed by extending the first read strand with a polymerase with strand displacement activity to generate multiple second strands. Each second strand contains a sequence that hybridizes to the concatemer and an unhybridized single-stranded branch. A second read primer (1) is then hybridized to the single-stranded branches of the multiple second strands and extended to generate second reads.

[0079] 6.1. Strand-displacing DNA polymerase The MDA in this method requires a DNA polymerase with strand displacement activity. In one approach, the present invention uses a DNA polymerase with strong 5' to 3' strand displacement activity. Preferably, the polymerase does not have 5' to 3' exonuclease activity. However, a DNA polymerase with 5'-3' exonuclease activity can be used if the activity does not interfere with the performance of the method of the present invention, for example, by using reaction conditions that inhibit the exonuclease activity. In one approach, the polymerase is Phi29 polymerase. Phi29 polymerase has strong displacement activity at moderate temperatures (e.g., 20 to 37°C). In another 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 high temperatures (up to 65°C). In one approach, the polymerase is Deep-VentR DNA polymerase (e.g., NEB#MO258) (Hommelsheim et al., Scientific Reports 4:5052 (2014)).

[0080] In addition to strand-displacing polymerases, other strand-displacing mechanisms can be used to aid strand displacement, such as with helicase enzymes, using reagents that can lower the melting temperature, commonly referred to as strand-denaturing agents or Tm-lowering agents (e.g., formamide, betaine, proline, 1,2-propanediol, and trehalose). The temperature of the reaction can also be adjusted to facilitate strand melting and support polymerase extension. In some cases, the temperature can range from 25°C to 40°C, for example, 28°C to 35°C, or about 30°C.

[0081] Paired-end sequencing herein 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 sequence information for the DNA template. That is, complete displacement of the second strand from the first strand must be avoided or minimized. This can be achieved by selecting a polymerase(s) with appropriate polymerization rate or other properties and controlling the progress of the reaction using various reaction parameters, including but not limited to, reaction temperature, reaction time, primer composition, DNA polymerase, primer and nucleotide concentrations, additives, and buffer composition. Optimal conditions can be determined empirically.

[0082] One approach to control the extension-displacement reaction and avoid complete displacement is to use a DNA polymerase with appropriate strand displacement activity to generate the second strand: Phi29, Bst DNA polymerase, Klenow fragment of DNA polymerase I, and Deep-Vent (登録商標) DNA polymerases, such as DNA polymerase, are known to have different strengths of strand displacement activity. See Kornberg and Baker (1992, DNA Replication, Second Edition, pp. 113-225, Freeman, NY). Selecting a DNA polymerase suitable for the present invention is within the capabilities of one of ordinary skill in the art.

[0083] In another approach, the extension-displacement reaction can be 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.

[0084] In some embodiments, the extension reaction is controlled by including in the reaction buffer an agent that affects duplex formation between the extension primer and the template DNA, such as DMSO (e.g., 1% to 2%), betaine (e.g., 0.5 M), glycerol (e.g., 10% to 20%), Gene 32 single-stranded DNA binding protein from T4 phage (T4 G32 SSB) (e.g., 10 to 20 ng / ul), or a volume exclusion agent.

[0085] The reaction temperature can also be selected to allow for an appropriate rate of polymerization and strand displacement. Higher temperatures generally result in a higher frequency of strand displacement. In some embodiments, the reaction temperature is maintained within the range of 20°C to 37°C, e.g., 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C, to avoid complete displacement.

[0086] In some approaches, the extension reaction is controlled by using a mixture of conventional (extendable) and non-extendable primers, i.e., 3'-blocked primers. In some embodiments, the non-extendable primers are blocked from extension via a chemical blocking group that prevents polymerization by DNA polymerase. By mixing these two different primers in different ratios, the length of the duplex (hybridized) portion (continuation fragment) of the newly synthesized complementary DNA strand can be controlled. For example, one approach uses a mixture of first primers that are 50-70% non-extendable ("blocked") and 30-50% extendable ("unblocked"). Many types of non-extendable primers are known in the art and may be suitable for the present invention.

[0087] 6.2. Synchronizing the Generation of Multiple Second Strands To avoid a scenario in which some strands are partially hybridized, while others are fully displaced, and still others are not fully extended, it is desirable to synchronize the extension and displacement of multiple second strands. Synchronization can be achieved by a variety of approaches.

[0088] 6.2.1 Two-step second strand formation In the two-step second strand generation approach, the second strand can be prepared in two steps. The first reaction step involves performing MDA with a strand-displacing DNA polymerase at a high concentration so that free DNA polymerase molecules are present during the reaction. As used herein, "free polymerase" or "free polymerase molecules" refers to DNA polymerase molecules that are not bound to the DNA concatemers on the array and remain in solution. In some embodiments, the DNA polymerase is at a high concentration so that the number of DNA polymerase molecules exceeds the number of adapters in the DNA concatemers on the array, and free DNA polymerase is present that is not bound to the array. The appropriate DNA polymerase concentration for this method can be determined empirically by one of skill in the art. The first reaction step may last 1 to 15 minutes, e.g., 1 to 3 minutes, 2 to 5 minutes, or 3 to 10 minutes. The second step involves removing reaction components, including unbound enzyme and unincorporated nucleotides, and adding fresh buffer and reaction components, excluding any DNA polymerase, to allow the MDA reaction to continue. In these approaches, the tightly bound polymerase remains bound to the elongating strand, and the free polymerase can be removed after the first incubation step. This two-step reaction process minimizes interactions between the free polymerase and the newly formed second strand and between the free polymerase and the first strand beyond the initial binding period, facilitating synchronized polymerase extension of the second strand primer. See Figure 3.

[0089] 6.2.2. Modified Two-Step Second Strand Generation In the modified two-step second strand generation approach, after the first lead sequencing is completed, a high concentration of polymerase is used to bind to the primer (i.e., MDA primer or first lead strand) and DNA concatemers under extension-blocking conditions for a first period of time. "Extension-blocking conditions" are conditions that favor the binding of the polymerase to the template over the extension of the primer by the polymerase. In some embodiments, extension-blocking conditions refer to conditions under which primer extension cannot be performed. This first step in the modified two-step second strand generation approach maximizes the binding of the DNA polymerase to the DNA template and primer. The extension-blocking conditions are then reversed to allow synchronous extension of the primer.

[0090] Extension-blocking conditions can be achieved by removing nucleotides or magnesium from the reaction, by adding an inhibitor such as EDTA to the reaction, or both. The concentration of EDTA may vary from 0.5 mM to 5 mM, e.g., 0.8 mM to 4 mM, 1 mM to 3 mM, or about 1 mM. Thus, in some embodiments, extension-blocking conditions include a reaction buffer that does not contain individual nucleotides or magnesium, or both, so that MDA cannot occur. In some embodiments, extension conditions include using 3'-blocked primers. The terms "free" or "essentially free" refer to a magnesium or nucleotide concentration in the reaction mixture that is less than 10%, 5%, 3%, 2%, or 1% of the magnesium or nucleotide concentration required for the extension reaction. A magnesium-free buffer is also referred to as a magnesium-free buffer. A buffer that does not contain 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 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. Initially lowering the reaction temperature can also be used to alter the ratio of binding and extension events. Binding can be accelerated during extension for an initial period, such as 1-5 minutes or 2-10 minutes. Extension can then continue for an additional 10-20 minutes, 20-30 minutes, or 30-60 minutes. This two-step process allows for synchronized production of multiple second strands.

[0091] One exemplary embodiment of the method is shown in Figure 4. After a first read primer hybridized to a DNA concatemer is extended to obtain a first read, a strand-displacing DNA polymerase is contacted with the array under extension-blocking conditions so that the polymerase binds to the first read strand (and / or the MDA primer) but does not extend the first read strand, thereby performing controlled MDA. After an initial period in which the array is under extension-blocking conditions, the extension-blocking conditions are reversed, and the first read strand and / or the MDA primer are synchronously extended to generate multiple second strands.

[0092] 6.2.3. Second Strand Generation Using Modified Primers As described above, the use of modified first lead primers (and / or MDA primers) to contain uracil instead of thymidine can also facilitate synchronization of second strand production. In some cases, extension of a first strand primer in the presence of a nucleotide mixture containing uracil generates modified first strand primers that contain uracil instead of thymidine at various positions. These modified or extended first strand primers are then cleaved to allow simultaneous extension of multiple second strands.

[0093] One illustrative example of this approach is shown in Figure 5, in which the first read strand generated by extending the first read primer contains a cleavable nucleotide. The first read primer is cleaved at the position of the cleavable nucleotide to generate fragments with extendable 3' ends. Each fragment is used as a primer to generate multiple second strands by controlled MDA. Each second strand contains a sequence that hybridizes to one of the multiple DNA concatemers and a single-stranded branch that does not hybridize. A second read primer (1) hybridizes to the second strand and is extended in second read sequencing.

[0094] 6.2.4. Second Strand Generation Using Additional MDA Primers In some embodiments, additional MDA primers are added to the MDA step to generate more second strands. These MDA primers bind to regions of the unhybridized (single-stranded) DNA concatemers and can therefore serve as binding sites for additional primers. These MDA primers are extended in the same manner as the first read strand, generating multiple second read strands in total. Introducing these MDA primers can increase strand generation and therefore the amount of sequencing data. In some embodiments, these MDA primers are introduced simultaneously with the first read primer, and the MDA primers hybridize upstream of the first read primer. See Figure 2. The MDA primers used in these embodiments generally contain a reversible 3' blocking group to prevent extension during the first read sequencing step. After completion of the first read sequencing, the 3' blocking group is removed from the MDA primers, allowing for controlled MDA using these MDA primers.

[0095] Suitable reversible blocking groups 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. Preferably, the chemical treatment does not significantly degrade the template or primer extension strand. Examples of 3'-blocking groups in reversible terminators include a 3'-O-allyl group (Ju et al., Proc. Natl. Acad. Sci. USA 103: 19635-19640, 2006), a 3'-O-azidemethyl-dNTP (Guo et al., Proc. Natl Acad. Sci. USA 105, 9145-9150, 2008), an aminoalkoxyl group (Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29:879-895, 2010), and a 3'-O-(2-cyanoethyl) group (Knapp et al., Chem. Eur. J., 17, 2903-2915, 2011). Exemplary RT blocking groups include -O-azidemethyl and -O-cyanoethenyl. Non-limiting examples of reversible blocking groups that can be used are disclosed in PCT / US2018 / 012425, the relevant portions of which are incorporated herein by reference in their entirety.

[0096] In certain embodiments, the methods described herein employ one or more of the above features to improve the efficiency of the second strand generation and paired-end sequencing process.

[0097] 7. Sequencing 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, sequencing the first strand can be performed while the first strand primer is being extended, i.e., while each nucleotide incorporated into the extended first strand primer is determined, so that the sequence of the portion of the first strand that is 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 adapter of the monomer of the DNA concatemer (e.g., "(1)" in Figure 1) 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 read 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.

[0098] It will be appreciated that any of the above primers can be used as a sequencing oligonucleotide.

[0099] To determine the sequence of the first strand and the second strand, any suitable sequencing method can be used, such as SBS, pyrosequencing, ligation sequencing, and other methods.In some approaches, one or more sequencing methods are used.For example, the DNA template strand can be sequenced using one method (e.g., SBS), and the second strand can be sequenced using a different method (e.g., cPAL).In one approach, sequencing is carried out using affinity reagents, for example, as described in U.S. Patent No. 10,851,440, the contents of which are incorporated herein by reference.

[0100] In some approaches, the first lead sequencing is performed by sequencing by synthesis. In some approaches, the second lead sequencing is performed using multiple second lead primers as primers for primer extension (e.g., sequencing by synthesis reaction), or the extension products of such primers, or oligonucleotides that can act as anchors for sequencing by ligation, or the ligation products of such oligonucleotides with labeled probes (e.g., labeled cPAL probes). In one approach, the second primer contains a portion complementary to the adapter sequence and can be extended to sequence the second strand.

[0101] SBS can rely on DNA polymerase activity to perform chain extension during the sequencing reaction. SBS is well known in the art. See, for example, U.S. Patent Nos. 6,787,308 and 8,241,573 B2. Sequencing on DNA nanoballs can be performed through a variety of processes. In one approach, the circles used to generate DNBs are prepared with a DNA region of known sequence (the adapter) and an adjacent sequence of unknown identity to be determined. The adapter provides a primer hybridization site such that, when a polymerase is used for extension, nucleotides are added to the "unknown" or "to be determined" region by primer extension. Nucleotides are added one position at a time when the 3' position is reversibly blocked and complementary to the base position of the DNB. After removing the 3' blocking group, additional positions can be read in the next cycle. A fluorescent substance characteristic of the base type is used to detect the incorporated base, revealing the base at that position of the DNB.

[0102] Alternatively, sequencing by ligation can also be used.Primer or anchor can be extended by ligating fluorescent oligonucleotide that extends to unknown sequence.In this sequencing method, base-degenerated fluorescent oligonucleotide is ligated to the starting anchor, but one base of the oligonucleotide is defined and is linked to a fluorescent moiety.The ligation of oligo probe to anchor produces stable fluorescence after washing away excess probe, and is determined by the recognition that the defined base is complementary to the base at the same position of DNB.Sequencing by ligation is described, for example, in Shendure et al., 2005, Science, 309: 1728-1739.

[0103] Other sequencing methods, such as pyrosequencing (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), can also be used.

[0104] It may be clear to the reader that variations of the specific embodiments outlined herein can be used. In one approach, the extension primer (e.g., first strand primer) and the sequencing oligonucleotide (e.g., second strand oligonucleotide) bind to different portions of the adapter sequence. In another approach, the extension primer and the sequencing oligonucleotide bind to the same portion of the adapter sequence (e.g., a portion of the adapter sequence for extension and the complement of the same portion of the adapter sequence for sequencing).

[0105] 8. Substrates and Compartments The DNA template polynucleotide is immobilized on a substrate. Generally, immobilization is performed before the second strand synthesis described above. Exemplary substrates can be substantially planar (e.g., slides, wells, flow cells) or non-planar, and can be formed from a single or multiple different units (e.g., beads). Exemplary materials include glass, ceramic, silica, silicon, metal, elastomers (e.g., silicon), polyacrylamide (e.g., polyacrylamide hydrogel; see WO2005 / 065814), and the like. In some approaches, the substrate comprises an ordered or disordered array of immobilization sites or wells. In some approaches, the target DNA polynucleotide is immobilized on a substantially planar substrate, such as a substrate comprising an ordered or disordered array of immobilization sites or wells. In some approaches, the target DNA polynucleotide is immobilized on beads.

[0106] Polynucleotides can be immobilized on substrates by a variety of techniques, including covalent and non-covalent bonding. Polynucleotides can be immobilized on substrates by a variety of techniques. In one embodiment, the surface may contain a capture probe that forms a complex, e.g., a double-stranded duplex, with a component of the polynucleotide molecule, such as an adapter oligonucleotide. In another embodiment, the surface may have reactive functional groups that react with complementary functional groups on the polynucleotide molecule to form covalent bonds. DNA molecules can also be efficiently attached to hydrophobic surfaces, such as clean glass surfaces, which have a low concentration of various reactive functional groups, such as -OH groups. In yet another embodiment, polynucleotide molecules can be adsorbed to the surface through non-specific interactions with the surface or non-covalent interactions, such as hydrogen bonding or van der Waals forces.

[0107] For example, DNA nanoballs can be immobilized in spaced apart regions, as described in U.S. Patent No. 8,609,335. In one approach, DNA nanoballs are immobilized on a substrate by hybridization to immobilized probe sequences, and solid-phase nucleic acid amplification is used to produce clonal clusters containing DNA template polynucleotides. See, for example, WO98 / 44151 and WO00 / 18957.

[0108] In some approaches, DNA template polynucleotides are compartmentalized in emulsions, droplets, on beads, and / or in microwells prior to the primer extension step (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)).

[0109] Generally, DNA nanoballs are arranged on a substrate in either an ordered or random array. In many applications, adsorption to the substrate is mediated by substrate-protein-DNA interactions. Furthermore, to achieve a stable nanoball array throughout the sequencing cycle, post-deposition accumulation of a protein layer can improve the stability of the DNA array. See WO2013066975A1 (the entire contents of which are incorporated herein by reference).

[0110] 9. Kit In some aspects, the method for producing a second strand for sequencing a DNA template can be carried out using a kit. The kit may include one or more DNA polymerases, including a strand-displacing DNA polymerase; a mixture of nucleotides including A, C, T, and G; and a primer. The kit may further include a non-displacing DNA polymerase and reversibly terminating dNTPs that can be used for sequencing by synthesis. The kit may further include a magnesium-free buffer ("magnesium-free buffer"). The kit may further include an elongation inhibitor (e.g., EDTA, excess salt including KCl and NaCl, sodium dehydrogenase, ionic detergents such as sarcosyl and SDS, ethanol, and isopropanol). In some embodiments, the mixture of nucleotides may further include uracil. In some embodiments, the ratio of uracil to thymidine in the mixture ranges from 1:2 to 1:10, e.g., 1:3 to 1:8, or 1:4 to 1:5. In some embodiments, the kit may further include a mixture of 3'-blocked primers (i.e., 3'-blocked primers) and unblocked primers, in which the ratio of blocked primers to unblocked primers is in the range of 1:1 to 1:5, e.g., 1:2 to 1:4.

[0111] 10. Array of DNA Complexes In one aspect, the present invention includes an array of DNA complexes. In one aspect, the array is a support comprising an array of discrete regions, wherein the plurality of regions comprises clonal clusters of single-stranded DNA templates and a plurality of primers. In certain embodiments, the DNA template is a single-stranded concatemer comprising a plurality of monomers, each monomer comprising an adapter sequence and a DNA target sequence. Each of the plurality of primers comprises a primer sequence that is complementary to and hybridizes with the adapter sequence of the DNA template. In certain embodiments, each of some of the plurality of primers comprises a excisable nucleotide, and the primer can be cleaved at the position where the excisable nucleotide is present to generate two or more fragments with extendable 3' ends.

[0112] In another aspect, the array is a support comprising an array of discrete regions, wherein a plurality of regions comprise clonal clusters of single-stranded DNA concatemers, a plurality of primers with reversible 3' blocking groups, and a plurality of primers with extendable 3' ends. The single-stranded concatemers comprise a plurality of monomers, each monomer comprising an adapter sequence and a DNA target sequence. Each primer comprises a primer sequence that is complementary to and hybridizes to an adapter sequence on a DNA template, and at least one adapter hybridizes to one primer with a reversible 3' blocking group and one primer with an extendable 3' end, the primer with the reversible 3' blocking group being upstream of the primer with the extendable 3' end.

[0113] In one aspect, the present specification discloses a reaction mixture containing an array of discrete regions, wherein the multiple regions comprise clonal clusters of single-stranded DNA concatemers, multiple primers, and a DNA polymerase. The single-stranded DNA concatemers comprise multiple monomers, each monomer comprising an adapter sequence and a DNA target sequence. Each primer comprises a primer sequence that is complementary to and hybridizes with the adapter sequence of the DNA template. The array is in a reaction mixture under extension-blocking conditions such that the DNA polymerase binds to the DNA template but cannot extend the primers. In one embodiment, the extension-blocking conditions are such that the reaction mixture contains an extension inhibitor (e.g., EDTA, salts including KCl and NaCl, ionic detergents such as sodium desoxycholate, sarkosyl and SDS, ethanol and isopropanol), a buffer lacking magnesium, or both.

[0114] It will be appreciated that the DNA complexes of the array may comprise any of the properties of the complexes described herein or may be produced according to the methods described herein. Furthermore, the complexes may have any combination of one or more of the following characteristics: (i) the array comprises at least 10 6 (ii) the DNA is single-stranded; (iii) the second primer comprises at least 10 bases, preferably at least 12 bases, and optionally at least 15 bases of the adapter sequence; and (iv) the second primer is perfectly complementary to the second DNA strand to which it hybridizes. [Example]

[0115] 11. Aspects The following provide exemplary embodiments of the methods and compositions described herein.

[0116] Embodiment 1. (a) providing a DNA array comprising a surface having at least 1,000 DNA concatemers immobilized thereon; (b) for each of a plurality of DNA concatemers on the array, (i) annealing a first read primer to a primer binding site on the DNA concatemer; (ii) extending at least a portion of the first lead primer to incorporate a dNTP or a dNTP analog, thereby generating a first read strand, wherein each of the incorporated dNTPs or dNTP analogs is identified to generate the first read; (c) performing controlled MDA by extending at least a portion of the first leading strand with a polymerase having strand displacement activity to generate a plurality of second strands, each second strand comprising a portion hybridized to a DNA concatemer and an unhybridized single-stranded branch; (d) annealing a second read primer to the single-stranded branches of the plurality of second strands; and (e) extending the second read primer to generate a second read. A paired-end sequencing method comprising:

[0117] Embodiment 2. The method of embodiment 1, wherein the DNA concatemer comprises a plurality of monomers, each monomer comprising an adaptor and a target sequence.

[0118] Embodiment 3. The method of embodiment 2, wherein a first lead primer is hybridized to an adapter of each monomer of the DNA concatemer.

[0119] Embodiment 4. The method of any of embodiments 1 to 3, wherein the first leading strand produced in step (a) comprises a 3' blocking group that prevents said first leading strand from being further extended, and wherein removal of the 3' blocking group from the first leading strand is performed prior to performing controlled MDA in step (b).

[0120] Embodiment 5. A plurality of MDA primers are hybridized to the DNA concatemers prior to step (b)(ii), wherein the MDA primer comprises a reversible blocking group to prevent extension of the MDA primer, the reversible blocking group being removed after step (b)(ii) and before step (c); and 5. The method of any of embodiments 1 to 4, wherein step (c) comprises extending the plurality of MDA primers and the first reading strand.

[0121] Embodiment 6 The method of embodiment 5, wherein the plurality of MDA primers comprises random primers.

[0122] Embodiment 7. The method of embodiment 5 or 6, wherein the DNA concatemer comprises an adaptor that hybridizes to at least one MDA primer and at least one first lead primer, wherein the at least one MDA primer is upstream of the at least one first lead primer.

[0123] Embodiment 8. The method of any one of embodiments 5 to 7, wherein the MDA primer has a length in the range of 5 to 10 nucleotides in length.

[0124] Embodiment 9. The controlled MDA in step (c) is (i) performing MDA for a first period of time in the presence of a strand-displacing DNA polymerase concentration such that excess molecules of the DNA polymerase do not bind to the first leading strand; and (ii) removing excess molecules of the strand-displacing DNA polymerase from the array at the end of the first period and continuing MDA to generate second strands, each containing a portion hybridized to at least one DNA concatemer and an unhybridized single-stranded branch. 9. The method of any of embodiments 1 to 8, comprising:

[0125] Embodiment 10. The method of embodiment 9, wherein removing excess molecules of the strand-displacing DNA polymerase is carried out by washing the array, wherein step (ii) comprises washing the array and adding fresh buffer comprising unincorporated nucleotides but no DNA polymerase.

[0126] Embodiment 11. The method of embodiment 9 or 10, wherein the ratio of the length of the first period to the length of the period completing controlled MDA in step (c) ranges from 1:30 to 1:2.

[0127] Embodiment 12. The method of any one of embodiments 9 to 11, wherein the period of time for completing the controlled MDA in step (c) is 10 to 90 minutes.

[0128] Embodiment 13. The method of any one of embodiments 9 to 12, wherein the first period of time is 1 to 10 minutes.

[0129] Embodiment 14. Performing the controlled MDA in step (c) comprises: (i) contacting the array with a strand-displacing DNA polymerase under extension-blocking conditions; and (ii) reversing the extension blocking conditions so that the first leading strand is simultaneously extended to generate multiple second strands, each of which comprises a portion hybridized to at least one DNA concatemer and an unhybridized single-stranded branch. 14. The method of any of aspects 1 to 13, comprising:

[0130] Embodiment 15. The method of embodiment 14, wherein the extension-blocking conditions comprise using a magnesium-free reaction buffer or non-incorporated nucleotides.

[0131] Embodiment 16 The method of embodiment 14, wherein the elongation-blocking conditions are a reaction buffer containing a polymerization inhibitor.

[0132] Embodiment 17 The method of embodiment 16, wherein the polymerization inhibitor is EDTA.

[0133] Embodiment 18. The method of embodiment 14, wherein the elongation-inhibiting conditions are temperatures below 20°C.

[0134] Embodiment 19. The method of embodiment 14, wherein the strand-displacing DNA polymerase in (i) is at a concentration such that excess molecules of the DNA polymerase do not bind to the first read primer, and wherein the method further comprises removing unbound molecules of the strand-displacing DNA polymerase from the array between steps (i) and (ii).

[0135] Embodiment 20. The method of embodiment 14, wherein the strand-displacing DNA polymerase of (i) is at a concentration such that excess molecules of the DNA polymerase do not bind to the first read primer, and wherein step (ii) comprises extending the first read strand for a first period of time, removing excess molecules of the strand-displacing DNA polymerase from the array at the end of the first period of time, and continuing MDA in a reaction that does not contain excess molecules of the DNA polymerase.

[0136] Embodiment 21. The method of embodiment 20, wherein the ratio of the first period to the period completing controlled MDA in step (c) ranges from 1:30 to 1:2.

[0137] Embodiment 22. The method according to embodiment 20 or 21, wherein the period for completing MDA in step (c) is 10 to 90 minutes.

[0138] Embodiment 23. The method of any one of embodiments 20 to 22, wherein the first period of time is 1 to 10 minutes.

[0139] Embodiment 24. (a) extending a first read primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of an excisable nucleotide to generate first reads of the plurality of single-stranded DNA concatemers, wherein the extension generates a first read strand incorporating the excisable nucleotide; (b) cleaving the first reading strand where the excisable base is present to generate a fragment of the first reading strand having an extendable 3' end; (c) performing controlled MDA by extending fragments of the first leading strand to generate a plurality of second strands, wherein the controlled MDA obtains a plurality of second strands each comprising a sequence that hybridizes to one of the plurality of single-stranded DNA concatemers and a non-hybridizing single-stranded branch; and (d) extending second read primers that hybridize to the single-stranded branches of the plurality of second strands to generate second reads. A paired-end sequencing method comprising:

[0140] Embodiment 25. The method of embodiment 24, wherein the excisable nucleotide is uracil.

[0141] Embodiment 26. (a) extending a first read primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array to generate a plurality of single-stranded DNA first reads, wherein the extension generates a first read strand; (b) removing the first leading strand; (c) performing control 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 which contains a sequence that hybridizes to one of the plurality of single-stranded DNA concatemers and a non-hybridizing single-stranded branch; and (d) extending second read primers that hybridize to the single-stranded branches of the plurality of second strands to generate second reads. A paired-end sequencing method comprising:

[0142] Embodiment 27 The method of embodiment 26, wherein the plurality of MDA primers comprises random primers.

[0143] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the step of extending the first read primer in step (a) and the step of extending the first read strand in step (b) are carried out by using a single DNA polymerase.

[0144] Embodiment 29. The method of any one of embodiments 1 to 27, wherein the step of extending the first strand primer in step (a) and the step of extending the first leading strand in step (b) are carried out by using different DNA polymerases.

[0145] Embodiment 30. The method of any one of embodiments 1 to 27, wherein the extension of the first strand primer in step (a) is with a non-displacing DNA polymerase.

[0146] Embodiment 31 The method of any one of embodiments 1 to 30, wherein the first read is determined by sequencing by synthesis.

[0147] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the extension step of the first lead primer lasts for 1 to 15 minutes.

[0148] Embodiment 33 The method of any one of embodiments 1 to 32, further comprising combining the first read and the second read to determine the sequence of the target DNA sequence.

[0149] Embodiment 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, each monomer comprises an adapter sequence and a DNA target sequence; each primer comprises a primer sequence that is complementary to and hybridizes to an adapter sequence of the DNA concatemer; At least one primer comprises an excisable nucleotide, and at least one primer can be cleaved to release an excisable nucleotide and generate two or more fragments with extendable 3' ends; array.

[0150] 35. A plurality of sequencing primers. DNA polymerases that do not have strand displacement activity, a mixture of reversibly terminated nucleotides for SBS; strand-displacing DNA polymerase, and dNTP mixture Includes a kit.

[0151] Embodiment 36. The kit according to embodiment 35, wherein the mixture of reversibly terminated nucleotides comprises uracil, and the kit further comprises a cleavage agent capable of removing uracil from a DNA strand.

[0152] Embodiment 37. The kit according to embodiment 35 or 36, wherein the kit further comprises a magnesium-free buffer.

[0153] All publications and patent documents cited herein are incorporated by reference as if each such publication or document were specifically and individually indicated to be incorporated by reference. Although the present invention has been described primarily with reference to particular embodiments, it is anticipated that different embodiments will become apparent to those skilled in the art upon reading the description herein, and such embodiments are intended to be encompassed by the methods of the present invention.

Claims

1. (a) providing a DNA array comprising a surface having at least 1,000 DNA concatemers immobilized thereon; (b) for each of a plurality of DNA concatemers on the array, (i) annealing a first read primer to a primer binding site on the DNA concatemer; and (ii) extending at least a portion of the first lead primer to incorporate a dNTP or a dNTP analog, thereby generating a first lead strand, wherein each of the incorporated dNTPs or dNTP analogs is identified to generate the first lead strand; (c) performing controlled MDA by extending at least a portion of the first leading strand with a polymerase having strand displacement activity to generate a plurality of second strands, wherein each of the second strands comprises a portion hybridized to a DNA concatemer and an unhybridized single-stranded branch; (d) annealing a second read primer to the single-stranded branches of the plurality of second strands; and (e) extending the second read primer to generate a second read strand. A paired-end sequencing method comprising:

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

3. 3. The method of claim 2, wherein the first read primer hybridizes to an adapter of each monomer of the DNA concatemer.

4. The first leading strand produced in step (a) comprises a 3' blocking group that prevents further extension of the first leading strand, wherein: The method of claim 1, wherein the 3' blocking group is removed from the first leading strand before performing the controlled MDA in step (b).

5. A method of hybridizing a plurality of MDA primers to DNA concatemers prior to step (b)(ii), comprising: 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) and before step (c); and 2. The method of claim 1, wherein step (c) comprises extending a plurality of MDA primers and the first leading strand.

6. The method of claim 5 , wherein the plurality of MDA primers comprises random primers.

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

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

9. The controlled MDA in step (c) (i) performing MDA for a first period of time in the presence of a strand-displacing DNA polymerase concentration such that excess molecules of the DNA polymerase do not bind to the first leading strand; and (ii) removing excess molecules of the strand-displacing DNA polymerase from the array at the end of the first period of time and continuing the MDA to generate second strands each comprising a portion that hybridizes to at least one DNA concatemer and a non-hybridizing single-stranded branch.

9. The method of claim 1, comprising:

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

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

2.

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

13. 10. The method of claim 9, wherein the first period of time is from 1 to 10 minutes.

14. Conducting the controlled MDA in step (c) (i) contacting the array with a strand-displacing DNA polymerase under extension-blocking conditions; and (ii) reversing the extension blocking conditions so that the first leading strand is simultaneously extended to generate multiple second strands, each of which comprises a portion that hybridizes to at least one DNA concatemer and a non-hybridizing single-stranded branch. The method of claim 1 , comprising:

15. 15. The method of claim 14, wherein the extension-blocking conditions comprise using a magnesium-free reaction buffer or unincorporated nucleotides.

16. 15. The method of claim 14, wherein the elongation-blocking conditions comprise using a reaction buffer containing a polymerization inhibitor.

17. 17. The method of claim 16, wherein the polymerization inhibitor is EDTA.

18. 15. The method of claim 14, wherein the elongation-inhibiting condition is a temperature below 20°C.

19. The strand-displacing DNA polymerase in (i) is at a concentration such that excess molecules of the DNA polymerase do not bind to the first read primer, and the method further comprises removing unbound molecules of the strand-displacing DNA polymerase from the array between steps (i) and (ii).

15. The method of claim 14, comprising:

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

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

2.

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

23. 21. The method of claim 20, wherein the first period of time is from 1 to 10 minutes.

24. (a) extending a first read 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 said extension generates a first read strand incorporating the excisable nucleotide; (b) cleaving the first reading strand where the excisable base occurs to generate a fragment of the first reading strand having an extendable 3' end; (c) performing controlled MDA by extending fragments of the first leading strand to generate a plurality of second strands, wherein the controlled MDA results in a plurality of second strands, each of which comprises a sequence that hybridizes to one of the plurality of single-stranded DNA concatemers and a non-hybridizing single-stranded branch; and (d) extending second read primers that hybridize to the single-stranded branches of the plurality of second strands to generate second reads. A paired-end sequencing method comprising:

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

26. (a) extending a first read primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array to generate a plurality of first reads of single-stranded DNA, wherein said extension generates a first read strand; (b) removing the first leading strand; (c) performing controlled MDA by extending a plurality of MDA primers hybridized to the DNA concatemers with a polymerase having strand displacement activity to generate a plurality of second strands, each of which contains a sequence that hybridizes to one of the plurality of single-stranded DNA concatemers and a non-hybridizing single-stranded branch; and (d) extending second read primers that hybridize to the single-stranded branches of the plurality of second strands to generate second reads. A paired-end sequencing method comprising:

27. 27. The method of claim 26, wherein the plurality of MDA primers comprises random primers.

28. 2. The method of claim 1, wherein the extension of the first read primer in step (a) and the extension of the first read strand in step (b) are carried out using a single DNA polymerase.

29. 2. The method of claim 1, wherein the extension of the first strand primer in step (a) and the extension of the first leading strand in step (b) are carried out by using different DNA polymerases.

30. 2. The method of claim 1, wherein the extension of the first strand primer in step (a) is carried out by a non-displacing DNA polymerase.

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

32. 2. The method of claim 1, wherein the extension of the first lead primer continues for 1 to 15 minutes.

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

34. 1. 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; each monomer comprises an adapter sequence and a DNA target sequence; each primer comprises a primer sequence that is complementary to and hybridizes to an adapter sequence of the DNA concatemer; At least one primer comprises an excisable nucleotide, and An array in which at least one primer can be cleaved to release an excisable nucleotide and generate two or more fragments with extendable 3' ends.

35. multiple sequencing primers, a DNA polymerase that does not have strand displacement activity; a mixture of reversibly terminated nucleotides for SBS; a strand-displacing DNA polymerase, and dNTP mixture Includes a kit.

36. 36. The kit of claim 35, wherein the mixture of reversibly terminating nucleotides comprises uracil, and the kit further comprises a cleaving agent capable of removing uracil from a DNA strand.

37. 36. The kit of claim 35, further comprising a magnesium-free buffer.

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