Controlled strand displacement for double-ended sequencing

Abstract

The invention relates to methods and compositions for double-terminal sequencing. The method comprises providing a DNA array comprising a surface on which DNA concatemers are immobilized. For each of a plurality of DNA concatemers on the array, the method includes annealing a first reading primer to a primer binding site on the DNA concatemer, extending at least some of the first reading primer to incorporate dNTP or dNTP analogs, thereby producing a first reading strand, where each incorporated dNTP or dNTP analog is identified as producing a first read segment, and extending at least some of the first reading primer to incorporate dNTP or dNTP analog to produce a second read segment. Performing controlled MDA by extending at least some of the first read strands with a polymerase having strand displacement activity to produce a plurality of second strands, each second strand comprising a moiety that hybridizes to the DNA concatemer and an unhybridized single strand branch; and annealing the second reading primer to single chain branches of the plurality of second chains, and extending the second reading primer to produce a second read.

Description

This application is a divisional application of the invention patent application with application number 202080089628.7, application date December 23, 2020, and applicant Shenzhen BGI Intelligent Manufacturing Technology Co., Ltd., and the invention name is "Controlled chain displacement for double-end sequencing". CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] The present invention relates to the fields of DNA sequencing, genomics and molecular biology. Background Art

[0003] 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. Patent No. 10,227,647. Compared to single-end sequencing, paired-end sequencing produces twice as many reads in library preparation with the same amount of time and effort. Using the read pairs generated in paired-end sequencing allows accurate read alignment and detection of mutations (e.g., insertion / deletion variants) that are difficult to detect with single-end sequencing. Summary of the Invention

[0004] The double-end sequencing method disclosed herein uses the controlled production of a chain complementary to the DNA template (first chain) so that they remain associated with the DNA template. These complementary chains are called second chains. The nucleotide sequence of the 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 chain. Also disclosed is a method for synchronizing the generation of the second chain and maximizing the number of the second chain at a suitable length to generate a second read.

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

[0006] In some aspects, the methods disclosed herein include providing a DNA array comprising a surface having immobilized thereon at least 1,000, at least 10,000, at least 105 , at least 10 6 or at least 10 7 DNA concatemers. The number of DNA concatemers can range from 1,000 to 10 13 , 10 4 and 10 12 , 10 4 to 10 10 or 10 5 to 10 8 For each of the multiple DNA concatemers on the array, a first reading primer is annealed to a primer binding site on the DNA concatemer, and at least some of the first reading primers are extended to incorporate dNTPs or dNTP analogs, thereby generating a first reading chain. Each of the incorporated dNTPs or dNTP analogs is identified to generate a first read segment. The method further includes performing controlled MDA by extending at least some of the first reading chains with a polymerase having strand displacement activity to generate a plurality of second chains, each second chain comprising a portion hybridized to the DNA concatemer and unhybridized single-stranded branches. The method may also include annealing a second reading primer to the single-stranded branches of the plurality of second chains, and extending the second reading primer to generate a second read segment.

[0007] In some aspects, the methods disclosed herein include extending a first reading primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of an excisable nucleotide to produce a first read segment of the plurality of single-stranded DNA concatemers, wherein the extension produces a first reading strand incorporating the excisable nucleotide. The method may also include one or more of the following: cutting the first reading strand at the position of the excisable base to produce a fragment of the first reading strand having an extendable 3' end; performing controlled MDA by extending the fragment of the first reading primer to produce a plurality of second strands, and wherein the controlled MDA produces 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 reading primer hybridized to the single-stranded branch of the plurality of second strands to produce a second read segment.

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

[0009] On the other hand, the array is a support comprising an array of discrete areas, wherein a plurality of areas 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 of which comprises 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, and at least one adapter hybridizes to a primer with a reversible 3' blocking group and a primer with an extendable 3' end, and the primer with the reversible 3' blocking group is located upstream of the primer with the extendable 3' end.

[0010] The present disclosure also provides a test kit comprising a mixture of multiple sequencing primers, a non-displacement DNA polymerase, a mixture of reversible termination nucleotides for SBS, a strand displacement DNA polymerase, and dNTPs. The test kit also comprises one or more of: i) a magnesium-free buffer ("magnesium-free buffer") and ii) an extension inhibitor (e.g., EDTA, excess salts including KCl and NaCl, ionic detergents such as sodium decyclate, sodium lauryl sarcosine (sarkosyl) and SDS, ethanol and isopropanol). In some embodiments, the nucleotide mixture can also include uracil. In some embodiments, the ratio of uracil to thymidine in the mixture is in the range of 1:2 to 1:10, such as 1:3 to 1:8, or 1:4 to 1:5. In some embodiments, the test kit can further include a mixture of a primer blocked at 3' (i.e., a 3' blocked primer) and an unblocked primer. In some embodiments, the ratio of blocked primer to unblocked primer is in the range of 1:1 to 1:5, such as 1:2 to 1:4. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 An embodiment of the paired-end sequencing method in the present disclosure is illustrated.

[0012] Figure 2 An embodiment of the present disclosure is illustrated in which a multiple displacement amplification (MDA) primer having a reversible blocking group (indicated by "*" in the figure) is hybridized to a DNA concatemer upstream of a first reading primer. After removal of the reversible blocking group, both the MDA primer and the first reading strand are extended in the MDA to produce multiple second strands.

[0013] Figure 3The embodiment of the disclosed method is illustrated.After producing the first reading strand during the first reading order-checking, the strand displacement DNA polymerase is combined with the DNA concatemer and starts MDA.With high concentration, the DNA polymerase is introduced into the reaction so that excessive DNA polymerase molecules in the reaction are not combined with the DNA concatemer and the first reading strand.After MDA starts, but before completing, unbound DNA polymerase molecules are removed, and MDA continues to produce the second chain.

[0014] Figure 4 An embodiment of the method of the present disclosure is shown. After forming a first read strand from the first read sequencing, a strand-displacing DNA polymerase is added to the reaction under extension-blocking conditions. The polymerase binds to the concatemer as well as the first read strand, but does not extend the primer under these conditions. An asterisk (*) in the figure indicates that the primer cannot be extended due to extension-blocking conditions.

[0015] Figure 5 An embodiment of the method of the present disclosure is shown in which excisable nucleotides are incorporated into the first reading strand during the first reading sequencing. The first reading strand is then cut at the position where these excisable nucleotides are present to generate fragments with extendable 3' ends. These fragments are used as extension primers in MDA to generate the second strand. DETAILED DESCRIPTION 1. Overview

[0016] The application relates to the double-ended sequencing method of the improvement using DNA chain.In one mode, take steps to maximize the quantity of the second chain with the length optimized for sequencing, for example, by including MDA primer to carry out.In some cases, primer is cut to produce a plurality of fragments, and each fragment is all used as primer to produce extra second chain.In some cases, can take steps to optimize the production of synchronous second chain, for example, use extension to prevent condition to carry out.

[0017] In some aspects of the present disclosure, the first read of double-end sequencing is generated by extending a first reading primer that hybridizes to a DNA concatemer fixed on an array ("first read sequencing"). The first read sequencing produces a first reading strand that is complementary to the DNA concatemer. An extension primer is then used to perform controlled multiple displacement amplification (MDA) and produce a second strand. The extension primer can be an additional MDA primer, the first reading strand, or both. Each second strand comprises a sequence that hybridizes to a DNA concatemer and an unhybridized single-stranded branch having one or more priming sequences that are complementary to the second reading primer. The first reading strand can be further extended to produce a second strand and become part of the second strand. The second read is obtained by extending a second reading primer that hybridizes to a primer binding sequence on the second strand, a process referred to as "second read sequencing" in the present disclosure.

[0018] In some embodiments, the method includes various features that can increase the yield of the second chain (i.e., the number of second chains suitable for second read sequencing) and the efficiency of second chain production. In some embodiments, the double-end sequencing method disclosed herein uses excisable nucleotides, for example, uracil, which are incorporated during the extension process of the first read primer. The first read primer or the first read chain containing these excisable nucleotides can be cut at the position where these excisable nucleotides are present to produce multiple fragments with extendable 3' ends, and then the multiple fragments are extended to produce more second chains.

[0019] In some ways, described method comprises the feature that makes the generation synchronization of multiple second chains, makes the second chain produced have similar length (for example, being suitable for the length of second reading order-checking).Multiple ways can be used for realizing this synchronization.In one embodiment, after completing the first reading order-checking, introduce high concentration polymerase to combine under extension and stop the primer (that is, MDA primer or the first reading chain) that hybridizes with DNA concatemer.Extension stops this initial step under condition and maximizes the combination of archaeal dna polymerase and DNA template and primer.Then reversal extends and stops condition to allow the synchronous MDA from primer.In some ways, after carrying out the MDA of the first period, redundant archaeal dna polymerase molecule (that is, polymerase molecule is free in reaction, not combined with primer and DNA template) is removed, and MDA continues to exist when there is no unbound archaeal dna polymerase molecule.This process will exceed the initial combination period in the interaction between free polymerase molecule and the newly formed second chain and the interaction between free polymerase molecule and DNA template minimize, thereby promote the synchronous extension and generation of the second chain.

[0020] The drawings accompanying this application illustrate certain embodiments of the present invention. Figure 1 Shown is a DNA concatemer comprising multiple monomers, each monomer comprising an adapter sequence and a target DNA sequence. The first reading primer is annealed to the primer binding sequence in the adapter of the DNA concatemer and extended to generate the first reading by sequencing while synthesis (SBS). Extending the first reading primer produces the first reading strand. After the last cycle of SBS, the 3' blocking group of the terminal nucleotide in the first reading strand is removed, and a chain displacement polymerase is added. The first reading strand is extended to generate the second chain in MDA by a chain displacement polymerase. Control MDA so that the second chain is partially hybridized with the DNA concatemer, and each second chain comprises an unhybridized branch. The second reading primer is then annealed to the branch (for example, by hybridizing with the second reading primer binding sequence on the adapter sequence on the branch). These second reading primers are then extended to produce the second read segment.

[0021] Figure 2Shown are MDA primers and first reading primers with 3' blocking groups hybridizing to DNA concatemers. 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 in each cycle of SBS, the blocking groups on the MDA primers are retained throughout the SBS sequencing process. The first reading primer is extended to produce the first read and produce the first reading strand. After the first reading sequencing is completed, the blocking group on the MDA primer is removed, and the blocking group of the last nucleotide added in the last sequencing cycle is also removed. In a controlled MDA reaction, in the presence of a strand-displacing DNA polymerase, both the MDA primer and the first reading strand are extended to produce a second strand that partially hybridizes to the DNA concatemer. The second read can be generated in the same manner as described above.

[0022] Figure 3 The array of the present invention is used for the preparation of the array of nucleic acid sequence of the present invention.The first nucleic acid sequence of the present invention is read and is read by the ...Shown that after completing the first reading order-checking, the first reading chain formed by the first reading order-checking is still hybridized with the DNA concatemer.High concentration of the archaeal dna polymerase is added in the array so that the first reading chain is extended in the initial period.Owing to have excessive archaeal dna polymerase molecule in the reaction, so some (but not all) archaeal dna polymerase molecules are combined with concatemer, and remaining archaeal dna polymerase molecule is free, namely is not combined with the DNA concatemer and does not participate in extension reaction.Then clean the array to remove excessive archaeal dna polymerase molecule.The buffer with Nucleotide (but not comprising archaeal dna polymerase) is added in the reaction, so that MDA continues to produce the second chain partially hybridized with the DNA concatemer.

[0023] Figure 4 Shown after completing the first reading order-checking, the first reading chain formed by the first reading order-checking keeps hybridizing with the DNA concatemer.Under extension stopping condition, chain displacement DNA polymerase is added in the reaction, for example, chain displacement DNA polymerase is positioned at the buffer that lacks at least one composition (for example magnesium) required for extension.Under these conditions, chain displacement DNA polymerase is combined with the DNA concatemer but does not extend.After array maintains the initial period under these conditions, these conditions are reversed to allow extension, for example, the composition (for example, magnesium) omitted is added back in the reaction with the concentration that is applicable to MDA.Then start MDA to produce the second chain.

[0024] Figure 5 Shown that the first reading sequencing process has produced the first reading strand comprising excisable nucleotides (e.g., uracil). These first reading strands are cut by an enzyme that recognizes excisable nucleotides, and this cutting causes multiple fragments to have extendable 3' ends and release excisable nucleotides. These fragments extend in MDA to form multiple second chains.

[0025] The contents of US Patent No. 10,227,647 are incorporated herein by reference in their entirety for all purposes. 2. Definition

[0026] As used herein, "primer" refers to an oligonucleotide that, after forming a duplex with a polynucleotide template, can serve as a starting point for nucleic acid synthesis and extend from its 3' end along the template to form an extended duplex. A primer can comprise a natural sequence or a synthetic sequence. A primer can also comprise non-natural nucleotides. The nucleotide sequence added during the extension process is determined by the sequence of the template polynucleotide. Primers are typically extended by DNA polymerase.

[0027] As used herein, "random primers" refer to primers having random nucleotide sequences.

[0028] As used herein, MDA or Multiple Displacement Amplification refers to DNA amplification based on strand displacement replication by multiple primers.

[0029] As used herein, "polynucleotide" can be used interchangeably with the term "nucleic acid" to refer to DNA, RNA, and hybrid and synthetic nucleic acids, and can be single-stranded or double-stranded. An "oligonucleotide" is a short polynucleotide having a length of between about 6 and about 300 nucleotides. A "complementary polynucleotide" refers to a polynucleotide that is complementary to a target nucleic acid.

[0030] As used herein, the term "strand displacement activity" describes the ability of the downstream DNA encountered in the synthesis process to displace DNA.Strand displacement activity is described in U.S. Patent Publication No. 20120115145 (which is incorporated herein by reference), as follows: "strand displacement activity" refers to the phenomenon that a biological, chemical or physical agent (e.g., DNA polymerase) causes a paired nucleic acid to dissociate from its complementary strand in the direction from 5' to 3', binds to and approaches template-dependent nucleic acid synthesis.Strand displacement begins at the 5' end of the paired nucleic acid sequence, so the enzyme immediately performs nucleic acid synthesis in the 5' of the displacement site. The newly synthesized nucleic acid and the replaced nucleic acid generally have the same nucleotide sequence complementary to the template nucleic acid strand.Strand displacement activity can be located on the same molecule as the molecule that imparts the activity of nucleic acid synthesis (particularly DNA synthesis), or it can be a separate and independent activity.DNA polymerases, such as Escherichia coli DNA polymerase I, the Klenow fragment of DNA polymerase I, T7 or T5 phage DNA polymerases, and HIV virus reverse transcriptase, are enzymes with both polymerase activity and strand displacement activity. Reagents such as helicases can be used in combination with inducing agents that do not have strand displacement activity to produce a strand displacement effect, that is, the displacement of nucleic acids is coupled to the synthesis of nucleic acids of the same sequence. Similarly, together with other inducing agents, proteins (such as Rec A or single-stranded binding proteins from Escherichia coli or from another organism) can be used to produce or promote strand displacement (Kornberg and Baker, 1992, DNA Replication, Second Edition, pp 113-225, Freeman, NY). In one embodiment, the polymerase is Phi29 polymerase. Phi29 polymerase has a strong displacement activity at moderate temperatures (e.g., 20-37°C). In one embodiment, Bst DNA polymerase is used, large fragment (e.g., NEB#MO275, available from New England Biolabs (Ipswich, MA)). Bst DNA polymerase is active at elevated temperatures (~65°C).

[0031] The term "first strand" refers to the single-stranded DNA template used in paired-end sequencing.

[0032] The term "second strand" refers to the single strand of DNA that is complementary to the first strand.

[0033] The term "MDA primer" refers to an extension primer that hybridizes to a DNA template and is extended to produce a second strand in a displacement-extension reaction. The MDA primer disclosed in this application is not a sequencing primer (e.g., a first read primer). In some embodiments, the MDA primer and the first read primer have different sequences.

[0034] The term "first read primer" refers to a primer that hybridizes to a DNA template and is used to generate the first read for paired-end sequencing.

[0035] The term "first read" refers to nucleotide sequence information obtained by sequencing a DNA template using a first reading primer.

[0036] The term "first-read sequencing" refers to the sequencing process used to obtain the first read.

[0037] The term "first reading strand" refers to a single-stranded polynucleotide generated by first reading sequencing (i.e., by extending the first reading primer). The first reading strand is also referred to as an extended first reading primer. The first reading strand can be further extended to form a second strand.

[0038] The term "second reading primer" refers to a primer that hybridizes to the second strand, ie, the strand complementary to the DNA template. In some embodiments, the second strand primer hybridizes to the second strand that is partially hybridized to the DNA template.

[0039] The term "second read" refers to the nucleotide sequence information obtained by sequencing the second strand.

[0040] The term "second-read sequencing" refers to the sequencing process used to obtain the second read.

[0041] The term "second read strand" refers to a single-stranded polynucleotide generated by second-read sequencing (ie, by extending a second-read primer).

[0042] The term "excisable nucleotide" refers to a nucleotide that can be removed from a DNA chain, and the removal results in the DNA chain breaking into two DNA fragments. An exemplary method for removing nucleotides from a DNA chain is by enzymes. An exemplary excisable nucleotide is uracil.

[0043] The term "reversible blocking group" for reversible terminator nucleotides may also be referred to as a "removable blocking group," "blocking moiety," "blocking group," "reversible terminator blocking group," and the like. A reversible blocking group is a chemical moiety attached to a nucleotide sugar (e.g., deoxyribose), typically located at the 3'-OH position of the sugar moiety, which prevents polymerases from adding nucleotides at that position. A reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), a chemical reaction, heat, light, or the like to provide a hydroxyl group at the 3'-OH position of a nucleoside or nucleotide, thereby allowing addition of nucleotides by a polymerase to occur.

[0044] As used herein, "dNTPs" include naturally occurring deoxyribonucleotide triphosphates and analogs thereof, including analogs having a 3'-O cleavable blocking group.

[0045] The terms "solid support" and "support" are used interchangeably to refer to a material or group of materials having a rigid or semi-rigid surface. A microarray typically comprises at least one planar solid support, such as a glass microscope slide.

[0046] As used herein, the term "simultaneous" or "synchronized" with respect to primer extension reactions refers to the simultaneous extension of multiple primers or the simultaneous initiation of multiple extension primers.

[0047] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" refers to one reagent or a mixture of such reagents, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the devices, compositions, formulations, and methodologies that are described in the publications and that might be used in connection with the presently described invention.

[0049] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range (to the tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value in that range are encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limitations in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0050] As used herein, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details.

[0051] While the invention has been described primarily with reference to specific embodiments, it is contemplated that other embodiments will become apparent to those skilled in the art upon reading this disclosure, and it is intended that such embodiments be encompassed within the methods of the present invention. 3. DNA template

[0052] In some ways, 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 comprising multiple copies of the same DNA sequence ("monomers" or "subunits" linked in series). The DNA concatemer can comprise at least two, at least three, at least four, at least 10, at least 25 monomers, at least 50 monomers, at least 200 monomers, or at least 500 monomers. In some ways, the DNA concatemer comprises 25-1000 monomers, such as 50-800 monomers or 300-600 monomers).

[0053] In one embodiment, the monomer of the concatemer comprises an adapter sequence and a target DNA sequence. Due to the tandem connection of the monomers, the target DNA sequence will be flanked by two adapter sequences. In some ways, the target DNA sequence in the monomer is flanked by two "half adapter" sequences, so that each target sequence flanked by two adapters linked in series in the concatemer. In some ways, the monomer unit includes one, two, three or four or more adapters. In some ways, all adapters of the monomer (and concatemer) have the same sequence. In other embodiments, the adapter can have different sequences, such as two, three or four different sequences. It should be recognized that a single monomer can include more than one DNA template sequence. Exemplary DNA concatemer structures are described in Table 1 of U.S. Patent No. 10,227,647.

[0054] The DNA concatemers used in the methods described herein can 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 in U.S. Patent No. 8,592,150 to Drmanac et al. The entire contents of both references are incorporated herein by reference.

[0055] DNA nanoballs (DNBs) are single-stranded copies of DNA sequences connected into linear DNA structures. Typically, DNBs are produced by using a chain displacement polymerase (such as phi29 polymerase or Bst polymerase) to replicate single-stranded circular DNA in a process called rolling circle replication. The polymerase starts with the extension of a primer hybridized with a single-stranded ring and produces a reverse complementary chain hybridized with the ring. Once a complete cycle is completed around the ring, the polymerase continues to extend by displacing the newly formed chain before the direction of travel. As the polymerase continues to extend the chain around the ring, multiple reverse complementary copies are produced, which are connected to each other in a linear manner. This strategy creates a target with many probes or primer binding sites, resulting in a signal intensity higher than that obtained using a single copy ring subunit. These single-stranded DNA concatemers of sufficient length typically form random coils that fill a roughly spherical volume in a solution (e.g., SSC buffer at room temperature). In some ways, DNA nanoballs typically have a diameter of about 100 to 300 nm. Typically, each monomer comprises at least one target DNA sequence.

[0056] DNA concatemers (including DNA nanoballs) can be prepared by any suitable method. In one embodiment, a single genomic fragment is used to generate a single-stranded circular DNA having an adapter inserted into the genome adjacent to or close to the target sequence. The circular DNA construct can be amplified enzymatically (e.g., by rolling circle replication, or by connecting monomers to each other). For illustration and not limitation, DNA nanoballs can be prepared according to the methods described in U.S. Patent No. 8,445,194 and U.S. Patent No. 8,592,150.

[0057] Amplification of DNA by rolling circle replication has several advantages: 1) amplification is linear, which prevents mutant copies from overrepresenting the original template sequence, 2) all copies are localized to a single molecule, making it ideal for microscopic analysis of fluorescent probes or reporter genes, and 3) replication of the circle can be performed isothermally, making it easier to automate.

[0058] The target DNA portion can be derived from any source, including naturally occurring sequences (e.g., genomic DNA, cDNA, mitochondrial DNA, free DNA, etc.), artificial sequences (e.g., synthetic sequences, products of gene shuffling or molecular evolution, etc.), or combinations thereof. The target DNA can be derived from organisms or cells (e.g., from plants, animals, viruses, bacteria, fungi, humans, mammals, insects), forensic sources, and the like. The target DNA sequence can be derived from a population of organisms, such as a population of enteric bacteria. The target DNA sequence can be obtained directly from a sample, or can be the product of an amplification reaction, a fragmentation reaction, or the like.

[0059] In some embodiments, the target DNA can have a length within a specific size range, for example, a length of 50 to 600 nucleotides. Other exemplary size ranges include a length of 25 to 2000, 50 to 1000, 100 to 600, 50-100, 50-300, 100-300 and 100-400 nucleotides. In the DNA template polynucleotides with two or more different target DNAs, the target DNA can have the same length or different lengths. In the library of DNA template polynucleotides, the members of the library can have similar lengths (for example, all within the scope of 25 to 2000 nucleotides or within another scope) in some ways.

[0060] In one approach, target DNA can be prepared by fragmenting a larger source DNA (e.g., genomic DNA) to produce fragments within a desired size range. In some methods, a size selection step is used to obtain a pool of fragments within a specific size range.

[0061] The DNA templates described herein may include two or more adapter sequences. The adapter may comprise an element for fixing the DNA template polynucleotide on a substrate, an element for binding to an oligonucleotide used in sequence determination (e.g., a binding site for a primer extended in sequencing by synthesis and / or a binding site for a probe for cPAL or other ligation-based sequencing methods, etc.), or an element for both fixing and sequencing. The adapter may include additional features such as, but not limited to, a restriction endonuclease recognition site, an extension primer hybridization site (for analysis), a barcode sequence, a unique molecular identifier sequence, and a polymerase recognition sequence.

[0062] The adapter sequence can have a length, structure, and other properties suitable for a particular sequencing platform and intended use. For example, the adapter can be single-stranded, double-stranded, or partially double-stranded and can have a length suitable for the intended use. For example, the adapter can be in the range of 10-200 nucleotides, 20-100 nucleotides, 40-100 nucleotides, or 50-80 nucleotides in length. In some embodiments, the adapter can comprise one or more modified nucleotides containing modifications to bases, sugars, and / or phosphate moieties.

[0063] It will be understood by those skilled in the art that different members of a library will typically comprise a common adapter sequence, although different species or subclasses within a library may have unique characteristics, such as subgenus-specific barcodes.

[0064] In some embodiments, the adapter sequence of the present invention can include multiple functionally different subsequences. For example, as discussed in detail in the present disclosure, a single adapter sequence can include two or more primer binding sequences (which can be recognized by different complementary primers or probes). The functionally different sequences in the adapter can be overlapping or non-overlapping. For illustration, given an adapter of 40 bases in length, in one embodiment, bases 1-20 are the first primer binding site, and bases 21-40 are the second primer binding site. In different embodiments, bases 1-15 are the first primer binding site, and bases 21-40 are the second primer binding site. In different embodiments, bases 5-25 are the first primer binding site, and bases 15-35 are the second primer binding site. Similarly, given an adapter of 40 bases in length, bases 1-20 can be an immobilized sequence, and bases 21-40 can be a primer binding site. Different primer sequences in the same or different adapters of the DNA template polynucleotide can have the same or different lengths.

[0065] An adapter (e.g., a first adapter, a second adapter, a third adapter, etc.) may comprise 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 may be specifically bound by two different primers. In one embodiment, the two primer binding sequences in the same adapter overlap, i.e., share a portion of the nucleotide sequence. In some embodiments, the overlapping region does not exceed 50%, or 40%, or 30%, or 20%, or 10% or 5% of any one of the two overlapping primer sequences. In one embodiment, more than one primer binding sequence is non-overlapping. In some embodiments, non-overlapping primer sequences are adjacent to each other; in some other embodiments, non-overlapping primer sequences are separated by 1-10, 10-20, 30-40, or 40-50 nucleotides.

[0066] It will be apparent that in a given DNA template polynucleotide, different adapters may have the same sequence or different sequences, and may have the same primer binding sequence or different primer sequences. Figure 1 ) to illustrate the present invention, but the use of representations of linkers such as cross-hatching should not be constructed as indicating sequence identity. 4. Primers

[0067] The primer used in the methods described herein has enough length to allow primer hybridization, and its accurate length and sequence depend on the expected function (for example, extension primer, index sequence etc.) of primer.The length of primer sequence is generally at least 10, at least 12, at least 15 or at least 18 bases.In some embodiments, the length range of primer sequence is 8 to 60 nucleotide, for example, 10 to 25 nucleotide, or 40 to 60 nucleotide is long.Selection or design for primer of the present invention is fully within the capability of those of ordinary skill in the art.

[0068] It will be understood that primers and probes can be fully or partially complementary to the primer binding sequence in the adaptor to which they hybridize. For example, a primer can have at least 85%, 90%, 95% or 100% identity to the sequence to which it hybridizes.

[0069] The primer can also contain another 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 can be a length that does not interfere with the hybridization between the primer and its primer binding sequence. Typically, the non-complementary portion is 1 to 100 nucleotides long. In some embodiments, the non-complementary portion is 4 to 8 nucleotides long. The primer can comprise a DNA and / or RNA portion, and in some ways, the primer used in the present invention can also have one or more modified nucleotides containing modifications to bases, sugars and / or phosphate moieties.

[0070] A "sequencing oligonucleotide" or "sequencing primer" may be an extension primer for a sequencing-by-synthesis reaction (also known as "sequencing-by-extension"). A "sequencing oligonucleotide" may be an oligonucleotide used in a sequencing-by-ligation method such as the "combined probe-anchor ligation reaction" (cPAL) (including singleplex, duplex, and multiplex cPAL) described in U.S. Patent Publication No. 20140213461, which is incorporated herein by reference for all purposes.

[0071] In some cases, extension primers are also used as sequencing primers, such as the first reading primer and the second reading primer. In some cases, extension primers can also be the product of primer extension reaction, such as the first reading strand that can be further extended. The first reading primer is a sequencing primer that hybridizes with the DNA concatemer and extends to produce the first read and forms the first reading strand. As mentioned above, in some cases, the first reading strand is used as an extension primer and is extended to produce the second strand. In some cases, an extra extension primer (called an MDA primer) is used to hybridize with the DNA concatemer and extend to produce the second strand in a displacement extension reaction. In some embodiments, MDA primers and the first reading primer have different sequences. In some embodiments, MDA primers can hybridize with the DNA concatemer upstream of the first reading primer, that is, MDA primers are located at 5' of the first reading primer. In some embodiments, one or more MDA primers and the first reading primer are bound to the identical adapter of the monomer of the DNA concatemer, and one or more MDA primers are located at 5' of the first reading primer. In some embodiments, MDA primers include random primers. In some embodiments, excess random primers (ie, random primers that have not hybridized to the DNA concatemers) are removed prior to initiating MDA to prevent excess random primers from binding and priming the second strand.

[0072] In some cases, the primer used in the method (for example, the first reading primer, the first reading strand or the MDA primer) can comprise excisable nucleotides, and these primers can be cut to form new extendable 3 ' ends at the position of the excisable nucleotides. This cutting causes the release of the excisable nucleotides. In some cases, these excisable nucleotides are core bases except any one of A, G, T or C. Alternatively or additionally, these excisable nucleotides can be incorporated with required spacing during sequencing reaction. Selecting excisable nucleotides is for their ability to excise and remove from the DNA chain to produce 3 ' exposed ends that can be used for extension. An exemplary excisable nucleotide that can be used for the method is uracil, which can be incorporated during the extension reaction to replace thymidine. When incorporated into single-stranded DNA, uracil can be removed by a uracil-specific excision reagent (for example, USER SEQ ID NO: 1). TM ) identification. USER TM It consists of a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-cleaving endonuclease VIII, which together first catalyze the excision of uracil to form an abasic site and then break the phosphodiester bond at the abasic site, resulting in single-strand DNA breaks and the release of uracil. See, Bitinaite et al., USER TMFriendly DNA engineering and cloning method by uracil excision, Nucleic Acids Research, Vol. 35, Issue 6, 15 March 2007, pp 1992-2002. Primers containing these excisable nucleotides first bind to the DNA template and then are cleaved at the position where the excisable nucleotide appears to allow simultaneous extension of multiple second strands.

[0073] In some cases, one or more MDA primers comprise a blocking group that prevents them from extending at their 3' ends. The quantity of the 3' blocking primers can be controlled to control the properties and density of the chain that each DNB independently displaces. In some embodiments, the quantity of the 3' extendable primers is controlled by maintaining the primer at a suitable concentration during hybridization. In some embodiments, the quantity of the 3' extendable primers is controlled by mixing the 3' extendable primer and the 3' blocking primer in a suitable ratio.

[0074] In some cases, the extension primers (i.e., MDA primers) used in the methods disclosed herein may contain a reversible blocking group at their 3' end, and these extension primers will only extend when the blocking group is removed. In some embodiments, one or more MDA primers bind to the DNA template upstream of the first read primer, and after the first read is generated, the reversible blocking group of the MDA primer is removed to allow extension of multiple second strands starting from the MDA primer. See Figure 2 In some embodiments, the reversible blocking group at the 3' end of the MDA primer can be removed under conditions that are different from the blocking group added to the first read primer during each sequencing cycle, such that the blocking group of the MDA primer will remain throughout the first read sequencing. In some embodiments, the reversible blocking group at the 3' end of the MDA primer is different from the blocking group of the nucleotide added to the first read primer during each sequencing cycle, such that the blocking group of the MDA primer will remain unchanged throughout the first read sequencing process. In some cases, the 3' blocking group is a 3' phosphate, which can be removed by a phosphatase.

[0075] In the method described in the present disclosure, the generation of the second chain is to start by hybridizing a primer comprising a sequence as the reverse complement of a DNA template (such as a DNA concatemer). In some cases, primers are hybridized with the adapter of a DNA concatemer monomer. In some cases, the synthesis of the second chain can also start from extending random oligonucleotides, i.e., using an MDA primer with a random nucleotide sequence. In this way, one group of different primers can be used to hybridize with the DNA concatemers in an array. In some ways, these sequences are 5 to 10 bases, for example, oligonucleotides of random sequences of 6 to 10 bases or 5 to 8 base lengths. Each sequence in the random pool will hybridize with the complementary sequence in the DNA concatemer. 4.1. Primer Linking

[0076] In some aspects, the primers used in the above methods and compositions (e.g., first reading primers, or MDA primers, or both) are linked to form a link pair. Term "link" refers to non-covalent and covalent interactions, and two nucleic acid molecules are combined together by this interaction. In some cases, the link is by DNA hybridization, chemical bond or both. The primers of these links can link continuous or discontinuous monomers of DNA concatemers, thereby stabilizing DNA concatemers.

[0077] Primers can be linked in various ways. In some embodiments, they are connected by chemical linkage. In some embodiments, two primers are connected by hybridization between two complementary sequences (called "hybridization sequences"), and each primer has a complementary sequence. In some embodiments, the hybridization sequence is a non-palindromic sequence. In some embodiments, the hybridization sequence is a palindromic sequence, that is, a sequence in which half of the sequence is complementary to the other half of the sequence. The method for linking primers and the composition of the linked primers are described in PCT application number PCT / CN2020 / 124338, the entire contents of which are incorporated herein by reference for all purposes.

[0078] The length of the hybridization sequence can 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 linked primer pairs can remain hybridized throughout the assay. In some embodiments, the length of the hybridization sequence can be in the range of 20 to 150 nucleotides, for example, in the range of 40 to 120 nucleotides, 50 to 100 nucleotides.

[0079] In some embodiments, the hybridizing sequence is 5' relative to the sequence on the primer that is complementary to the DNA template. 5. Generate the first read segment

[0080] As mentioned above, in some embodiments, the first reading primer is used as a sequencing primer and is used to determine the sequence of the DNA template by producing the first reading section. The first reading primer itself is a part for the second chain, and further produces more in the second chain with nucleotide extension of the first reading primer. Nucleotide extension can occur so that the modification of nucleotide is only allowed to add one nucleotide, and then the modified nucleotide is detected from one group of A, C, G and T bases to identify the nucleotide added. For example, a nucleotide added can contain a 3'-O-blocking group, such as a 3'-O-azidomethyl group, at the 3'-OH position of A, C, G or T. In order to determine the next base in the series, the modified nucleotide is converted into an extendable form, to allow another base to be added in another sequence determination cycle. In this sequencing method, the first chain primer of extension serves as the primer that each wheel nucleotide adds. After the last cycle of the nucleotide added for sequencing, the first reading primer (also referred to as " the first reading chain ") of extension is not blocked, and then use a polymerase that can be the same or different from the polymerase for producing the first reading chain to further extend. Extension of the first reading primer can be performed by any DNA polymerase, including a polymerase with strand displacement activity, a polymerase lacking strand displacement activity, or a mixture of polymerases. In some embodiments, the DNA polymerase is one that does not have any or almost no strand displacement activity, such as BG9 DNA polymerase. 6. Controlled MDA

[0081] In some embodiments, the first reading strand is separated from the DNA concatemer and removed from the array, and a new extension primer is added and extended in the MDA. In some embodiments, the 3' blocking group of the terminal nucleotide of the first reading strand (added in the last cycle of the first reading sequencing) is removed, and these first reading strands are used as extension primers in the MDA. The MDA method disclosed herein is controlled so that the second chain remains partially hybridized with the DNA concatemer, that is, the second chain remains bound to the DNA template by the sequence hybridized with the DNA template. The second chain also includes a single-stranded branch, which includes a primer binding site for a second reading primer. The second reading primer can be used as a sequencing primer to produce a second read segment.

[0082] An illustrative example is Figure 1 As shown in FIG. A first reading primer anneals to the DNA concatemer and is extended to produce a first read. The first reading sequence produces a first reading strand. Controlled MDA is then performed by extending the first reading strand with a polymerase having strand displacement activity to produce multiple second strands. Each second strand contains a sequence that hybridizes to the concatemer and unhybridized single-stranded branches. A second reading primer ① then hybridizes to the single-stranded branches of the multiple second strands and is extended to produce a second read. 6.1. Strand-displacing DNA polymerases

[0083] The MDA in the method requires a DNA polymerase with strand displacement activity. In one approach, the present invention uses a DNA polymerase with strong 5'→3' strand displacement activity. The polymerase preferably does not have 5'→3' exonuclease activity. However, when the activity does not prevent the implementation of the method of the present invention, for example, by using reaction conditions that inhibit exonuclease activity, a DNA polymerase with 5'-3' exonuclease activity can be used. In one approach, the polymerase is Phi29 polymerase. Phi29 polymerase has strong displacement activity at moderate temperatures (e.g., 20-37°C). In one approach, Bst DNA polymerase, large fragment (e.g., NEB#MO275, available from New England BioLabs, Ipswich, MA) is used. Bst DNA polymerase is active at elevated temperatures (~65°C). In one embodiment, the polymerase is Deep-VentR DNA polymerase (eg, NEB #MO258) (Hommelsheim et al., Scientific Reports 4:5052 (2014)).

[0084] In addition to strand-displacing polymerases, other strand-displacing mechanisms can also be used to assist strand displacement, such as helicases, using strand denaturants or agents that can lower the melting temperature, commonly referred to as Tm-lowering agents (e.g., formamide, betaine, proline, 1,2-propanediol, and trehalose). The reaction temperature can also be adjusted to promote chain melting and aid polymerase extension. In some cases, the temperature can be varied within the range of 25°C to 40°C, such as 28°C to 35°C, or approximately 30°C.

[0085] The double-end sequencing in the present disclosure requires that at least some of the second strand remain partially hybridized with the DNA template. This allows the sequence reads generated by the second strand sequencing to be paired with the first strand to construct the sequence information of the DNA template. That is, complete displacement that causes the second strand to dissociate from the first strand should be avoided or minimized. This can be achieved by controlling the reaction process in the following ways: selecting a polymerase with a suitable polymerization rate or other characteristics, and using various reaction parameters, including (but not limited to) reaction temperature, reaction duration, primer composition, DNA polymerase, primer and nucleotide concentrations, additives and buffer composition. Optimal conditions can be determined empirically.

[0086] One way to control the extended displacement reaction to avoid complete displacement is to use a DNA polymerase with suitable strand displacement activity to generate the second strand. Known DNA polymerases (e.g., Phi29, Bst DNA polymerase, Klenow fragment of DNA polymerase I, and Deep-VentR DNA polymerase) have strand displacement activities of varying strengths. See Kornberg and Baker (1992, DNA Replication, Second Edition, pp. 113-225, Freeman, NY). It is within the capabilities of those of ordinary skill in the art to select a DNA polymerase suitable for use in the present invention.

[0087] In another embodiment, the extension-displacement reaction is controlled to avoid complete displacement by using an appropriate concentration of a DNA polymerase having strand displacement activity, or an appropriate concentration of dNTPs, or an appropriate concentration of a second primer.

[0088] In some embodiments, the extension reaction is carried out by including in the reaction buffer an agent that affects duplex formation between the extension primer and the template DNA (e.g., DMSO (e.g., 1%-2%), betaine (e.g., 0.5 M), glycerol (e.g., 10%-20%), Gene32 single-stranded DNA binding protein from T4 bacteriophage (T4 G32 SSB) (e.g., 10-20 ng / ul), and a size exclusion agent.

[0089] The reaction temperature can also be selected to allow for appropriate rates of polymerization and chain displacement. Higher temperatures generally result in a greater degree of chain displacement. In some embodiments, the reaction temperature is maintained in 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.

[0090] In some ways, the extension reaction is controlled by using a mixture of conventional (extendable) primers and non-extendable primers (i.e., 3' end blocking primers). In some embodiments, the non-extendable primer is blocked and extended by a chemical blocking group that prevents DNA polymerase polymerization. By mixing these two different primers in different proportions, the length of the double-stranded (hybridization) portion of the newly synthesized complementary DNA chain (subsequent fragment) can be controlled. For example, in one approach, a mixture of the first primers is used, of which 50-70% is non-extendable ("blocked") and 30-50% can be extendable ("unblocked"). Many types of non-extendable primers are known in the art and are suitable for use in the present invention. 6.2.Synchronous production of multiple second chains

[0091] It is desirable to synchronize the extension and displacement of the multiple second strands to avoid a situation where some strands are partially hybridized while others are fully displaced and others have not yet been extended far enough. Synchronization can be achieved in a variety of ways. 6.2.1 Two-step second chain production

[0092] In the method for producing the second strand in a two-step method, the second strand can be prepared in two steps. The first reaction step includes performing MDA in a high concentration of strand-displacing DNA polymerase, so that free DNA polymerase molecules are present in 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 are retained in solution. In some embodiments, the DNA polymerase is at a high concentration so that the number of DNA polymerase molecules is greater than the number of adapters in the DNA concatemers on the array, so that there is free DNA polymerase that is not bound to the array. The concentration of DNA polymerase suitable for this method can be determined empirically by those skilled in the art. The first reaction step can last 1-15 minutes, for example 1-3 or 2-5 or 3-10 minutes. The second step includes removing the reaction components including unbound enzyme and unincorporated nucleotides, and adding fresh buffer and reaction components, but excluding any DNA polymerase, to allow the MDA reaction to continue. In these methods, polymerases with strong associative properties will remain bound to the extended strand, while free polymerases will be removed after the first incubation step. This two-step reaction process minimizes the interaction between the free polymerase and the newly formed second strand, as well as the interaction between the free polymerase and the first strand beyond the initial binding period, and promotes synchronous extension of the second strand primer by the polymerase. Figure 3 . 6.2.2. Improved two-step second-strand production

[0093] In the method for improved two-step method second chain production, after completing the first reading sequencing, use high concentration of polymerase in the initial period to extend and prevent conditions in conjunction with primer (i.e. MDA primer or the first reading chain) and DNA concatemer." extension and prevention conditions" are conducive to polymerase and template binding rather than the condition of extending primer by polymerase. In some embodiments, extension and prevention conditions are conditions that refer to that primer extension cannot be carried out. This initial step under the improved two-step method second chain production mode maximizes the combination of archaeal dna polymerase and DNA template and primer. Then reversal and extension prevent conditions to allow the synchronous extension of primers.

[0094] Extension blocking conditions can be produced by removing nucleotides or magnesium from the reaction, by adding inhibitors (such as EDTA) to the reaction, or by both. The concentration of EDTA can vary in the range of 0.5mM to 5mM, for example, in the range of 0.8mM to 4mM, in the range of 1mM to 3mM or about 1mM. Therefore, in some embodiments, extension blocking conditions include reaction buffers that do not contain independent nucleotides or magnesium or both, so that MDA does not occur. In some embodiments, extension conditions include using primers that are blocked at 3'. The term "does not contain" or "essentially does not contain" refers to that magnesium concentration or nucleotide concentration in the reaction mixture are less than 10%, less than 5%, less than 3%, less than 2%, less than 1% of the concentration of magnesium required for the extension reaction or the concentration of nucleotides. The buffer that does not contain magnesium is also referred to as magnesium-free buffer. The buffer that does not contain unincorporated nucleotides is also referred to as nucleotide-free buffer. In some cases, the extension blocking condition comprises the reaction buffer comprising magnesium that is less than 0.2mM, less than 0.1mM, less than 0.05mM, less than 0.02mM, less than 0.01mM or less than 0.005mM. In some cases, the concentration of every type of Nucleotide (A, T, C or G) is less than 0.02mM, less than 0.01mM, less than 0.005mM or less than 0.001mM. Reducing the initial reaction temperature also can be used for changing the ratio of binding event than extension event. Can promote in initial period (for example 1-5 minute or 2-10 minute) in conjunction with rather than extending. Then extension subsequently can continue 10-20,20-30,30-60 minute again.This two-step process allows synchronous production of multiple second chains.

[0095] An illustrative embodiment of the method is as follows Figure 4 After obtaining a first read by extending a first read primer hybridized on a DNA concatemer, controlled MDA is performed by contacting the array with a strand-displacing DNA polymerase under extension-blocking conditions, such that the strand-displacing DNA polymerase binds to the first read strand (and / or the MDA primer) but does not extend the first read strand. After an initial period of placing the array under extension-blocking conditions, the extension-blocking conditions are reversed, such that the first read strand and / or the MDA primer are synchronously extended to produce a plurality of second strands. 6.2.3. Second Strand Generation Using Modified Primers

[0096] As described above, the use of modified first reading primers (and / or MDA primers), such as those containing uracil instead of thymidine, can also promote synchronization of second chain production. In some cases, the first chain primer is extended in the presence of a nucleotide mixture containing uracil to produce modified first chain primers that contain uracil instead of thymidine at different positions. These modified first chain primers or extended first chain primers are then cut to allow multiple second chains to be extended simultaneously.

[0097] An illustrative example of this approach is shown in Figure 5 In a method for sequencing a first read strand, wherein the first read strand generated by extending the first read primer comprises an excisable nucleotide. The first read primer is cut at the position of the excisable nucleotide to produce a fragment with an extendable 3' end. Each fragment serves as a primer to generate multiple second strands by controlled MDA, each second strand comprising a sequence that hybridizes with one of the multiple DNA concatemers and an unhybridized single-stranded branch. The second read primer ① then hybridizes with the second strand and is extended in the second read sequencing. 6.2.4. Second Strand Generation Using Additional MDA Primers

[0098] In some embodiments, in order to generate a greater number of second strands, additional MDA primers are added to the MDA process. These MDA primers bind to regions of the DNA concatemer that are not hybrid structures (single strands) and can therefore serve as binding sites for the added primers. These MDA primers, along with the first reading strand, are extended, and together they generate multiple second strands. The introduction of these MDA primers can increase the yield of second strands (strand strands), thereby increasing the amount of sequencing data. In some embodiments, these MDA primers are introduced at the same time as the first reading primer, and the MDA primers hybridize upstream of the first reading primer. See Figure 2 The MDA primers used in these embodiments typically contain a reversible 3' blocking group so that they are not extended during the first read sequencing step. After the first read sequencing is completed, the 3' blocking group is removed from the MDA primer to allow controlled MDA using these MDA primers.

[0099] Suitable reversible blocking groups for use are known in the art. In some embodiments, suitable blocking groups are those that can be removed by chemical or enzymatic treatment, and the treatment produces a 3'-OH group. The chemical treatment should not significantly degrade the template or primer extension chain. Various molecular moieties for 3' blocking groups of reversible terminators have been described, such as 3'-O-allyl (Ju et al., Proc. Natl. Acad. Sci. USA 103: 19635-19640, 2006), 3'-O-azidomethyl-dNTPs (Guo et al., Proc. Natl Acad. Sci. USA 105, 9145-9150, 2008), aminoalkoxy (Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29: 879-895, 2010), and 3'-O-(2-cyanoethyl) groups (Knapp et al., Chem. Eur. J., 17, 2903-2915, 2011). Exemplary RT blocking groups include -O-azidomethyl and -O-cyanovinyl. Non-limiting examples of reversible protecting groups that can be used are disclosed in PCT / US2018 / 012425, the relevant portions of which are incorporated herein by reference in their entirety.

[0100] In some embodiments, the methods disclosed herein employ one or more of the above-described features to increase the efficiency of second-strand production and paired-end sequencing processes. 7. Sequence Determination

[0101] The sequence of the DNA template can be determined by combining the sequence reads generated from sequencing the first strand (DNA template) and the sequence reads generated from sequencing the second strand. As described above, the first strand can be sequenced while extending the first strand primer, that is, each nucleotide incorporated into the extended first strand primer is identified 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 an adapter of a monomer of the DNA concatemer (e.g., Figure 1 In some embodiments, the branches of the second strand are sequenced by extending the second reading primer. The sequence reads generated by sequencing the second strand are paired with the sequence reads generated by sequencing the DNA template to determine the entire target DNA sequence.

[0102] It will be appreciated that any of the primers described above may be used as sequencing oligonucleotides.

[0103] Any suitable sequence determination method can be used to determine the sequence of the first and second chains, such as SBS, pyrophosphate sequencing, ligation sequencing, etc. In some ways, more than one sequencing method is used. For example, a method (e.g., SBS) can be used to sequence the DNA template strand, and a different method (e.g., cPAL) can be used to sequence the second strand. In one approach, affinity reagents are used for sequencing, for example, as described in U.S. Patent No. 10,851,410, which is incorporated herein by reference.

[0104] In some embodiments, the first read sequencing is performed by sequencing by synthesis. In some embodiments, the second read sequencing is performed using a plurality of second read primers as primers for primer extension (e.g., sequencing by synthesis reaction), or extension products of such primers, or oligonucleotides that can serve as anchors for ligation sequencing, or ligation products of such oligonucleotides and labeled probes (e.g., labeled cPAL probes). In one embodiment, the second primer comprises a portion complementary to the adapter sequence and can be extended for sequencing the second strand.

[0105] SBS can rely on DNA polymerase activity to perform chain extension during the sequencing reaction step. SBS is well known in the art. See, for example, U.S. Patent Nos. 6,787,308 and 8,241,573B2. Sequencing of DNA nanoballs can be performed by a variety of processes. In one approach, the circle used to generate DNBs is prepared with a DNA region (adapter) of known sequence and an adjacent sequence of unknown identity to be determined. The adaptor provides a primer hybridization site so that if a polymerase is used to extend, the extension of the primer will result in the addition of nucleotides to the "unknown" or "to be determined" region. If the nucleotide is reversibly blocked at the 3' position, one position is added each time and is complementary to the base position in the DNB. After removing the 3' blocking group, the additional position can be read in the next cycle. The fluorescent part characteristic of the base type is used to detect the incorporated base, thus revealing the base at that position in the DNB.

[0106] Alternatively, sequencing by ligation can be used. Primers or anchors can be extended by ligating fluorescent oligonucleotides that extend into the unknown sequence. In this sequencing method, a fluorescent oligonucleotide with a degenerate base is attached to the starting anchor, but one base of the oligonucleotide is defined and associated with a fluorescent moiety. The ligation of the oligoprobe to the anchor produces stable fluorescence after washing the excess probe, which depends on the recognition of a defined base that is complementary to the base at the same position as the DNB. Sequencing by ligation is described, for example, in Shendure et al., 2005, Science, 309: 1728-1739.)

[0107] Other sequencing methods can also be used, such as pyrosequencing (see, e.g., Ronaghi et al., Anal. Biochem. (1996) 242:84-89) and sequencing by hybridization (see, e.g., Drmanac et al, Advances in Biochemical Engineering / Biotechnology (2002) 77:75-101).

[0108] It will be apparent to the reader that variations of the specific embodiments outlined herein may 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 one approach, the extension primer and the sequencing oligonucleotide bind to the same portion of the adapter sequence (e.g., the complement of the portion of the adapter sequence used for extension and the same portion of the adapter sequence used for sequencing). 8. Base and septum

[0109] In some applications, the DNA template polynucleotide is fixed on a substrate. Typically, fixation occurs before the synthesis of the above-mentioned second chain. Exemplary substrates can be substantially flat (e.g., a glass slide, a hole, a flow cell) or non-flat and single or formed by multiple different units (e.g., beads). Exemplary materials include glass, ceramics, silica, silicon, metal, elastomers (e.g., siloxanes), polyacrylamide (e.g., polyacrylamide hydrogels; see WO 2005 / 065814). In some ways, the substrate comprises an ordered or disordered array of fixed sites or holes. In some methods, the target DNA polynucleotide is fixed on a substantially flat substrate, such as a substrate comprising an ordered or disordered array of fixed sites or holes. In some methods, the target DNA polynucleotide is fixed on a bead.

[0110] Polynucleotide can be fixed on substrate by the multiple technology that comprises covalent and non-covalent attachment.Polynucleotide can be fixed on substrate by various technology.In one embodiment, surface can comprise the capture probe that forms complex (for example double-stranded duplex) with the assembly (such as adapter oligonucleotide) of polynucleotide molecule.In another embodiment, surface can have the reactive functional group with the complementary functional group reaction on polynucleotide molecule to form covalent bond.DNA molecule also can be effectively connected to hydrophobic surface (for example, the clean glass surface of various reactive functional groups (for example-OH group) with low concentration).In another embodiment, polynucleotide molecule can be by with the non-specific interaction on surface or by non-covalent interaction (for example hydrogen bond, van der Waals force etc.) adsorbed onto surface.

[0111] For example, DNA nanospheres can be fixed to discrete, spaced-apart regions as described in U.S. Patent No. 8,609,335. In one approach, DNA nanospheres are fixed to a substrate by hybridization with fixed probe sequences, and solid-phase nucleic acid amplification methods are used to generate clonal clusters comprising DNA template polynucleotides. See, for example, WO 98 / 44151 and WO 00 / 18957.

[0112] In some approaches, the DNA template polynucleotide is compartmentalized in an emulsion, droplets, beads, and / or microwells prior to the primer extension step (Margulies et al., “Genome sequencing in microfabricated high-density picoliter reactors.” Nature 437:7057 (2005); Shendure et al., “Accurate multiplexpolony sequencing of an evolved bacterial genome” Science 309, 1728-1732 (2005)).

[0113] Typically, DNA nanospheres are arranged on a substrate in an ordered or random array. In many applications, adsorption to the substrate is mediated by substrate-protein-DNA interactions. In addition, to achieve a stable nanosphere array through sequencing cycles, post-attachment deposition of a protein layer can improve the stability of the DNA array, see WO2013066975A1, the entire disclosure of which is incorporated herein by reference. 9. Test kit

[0114] In some aspects, the method for producing the second chain for sequencing the DNA template can be performed using a test kit. The test kit can include a mixture of one or more DNA polymerases (including strand displacement DNA polymerases), nucleotides (including A, C, T, G) and primers. The test kit can also include a non-displacement DNA polymerase and the dNTPs of the reversible termination that can be used for sequencing while synthesizing. The test kit can also include a buffer solution that does not contain magnesium ("magnesium-free buffer"). The test kit can also include an extension inhibitor (for example, EDTA, excess salt (including KCl and NaCl), an ion detergent (for example sodium decyclate, sarkosyl and SDS), ethanol and isopropanol). In some embodiments, the nucleotide mixture can also include uracil. In some embodiments, the ratio of uracil to thymidine in the mixture is in the range of 1:2 to 1:10, for example 1:3 to 1:8, or 1:4 to 1:5. In some embodiments, the test kit can also include a mixture of a primer (that is, a 3'-sealed primer) and an unsealed primer that is blocked at 3'. In some embodiments, the ratio of blocked primer to unblocked primer is in the range of 1:1 to 1:5, such as 1:2 to 1:4. 10. Arrays of DNA Complexes

[0115] In one aspect, the present invention includes DNA complex arrays. In one aspect, the array is a support comprising an array of discrete areas, wherein a plurality of areas comprise clone clusters of single-stranded DNA templates and a plurality of primers. In some 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 some embodiments, each of some of the plurality of primers comprises an excisable nucleotide, and the primer can be cut at a position where an excisable nucleotide is present to produce two or more fragments with an extendable 3' end.

[0116] On the other hand, the array is a support comprising an array of discrete areas, wherein a plurality of areas 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 of which comprises 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, and at least one adapter hybridizes to a primer with a reversible 3' blocking group and a primer with an extendable 3' end, and the primer with the reversible 3' blocking group is located upstream of the primer with the extendable 3' end.

[0117] On the one hand, disclosed herein is a reaction mixture comprising a discrete area array, wherein multiple regions comprise clone clusters of single-stranded DNA concatemers, multiple primers and a DNA polymerase. The single-stranded concatemer comprises multiple monomers, and each monomer comprises an adapter sequence and a DNA target sequence. Each primer comprises a primer sequence that is complementary to the adapter sequence of the DNA template and hybridizes. The array is located in a reaction mixture that is in an extension blocking condition so that the DNA polymerase is combined with the DNA template, but the primer can not be extended. In some embodiments, the extension blocking condition is that the reaction mixture comprises an extension inhibitor (e.g., EDTA, salts including KCl and NaCl, ionic detergents (e.g., sodium decyclate, sarkosyl and SDS), ethanol and isopropanol), a buffer lacking magnesium, or both.

[0118] It should be understood that the DNA complexes of the array may include any of the properties of the complexes described herein or prepared according to the methods described herein. In addition, the complexes may have any combination of one or more of the following features: (i) the array comprises at least 10 6 The invention further comprises a plurality of discrete regions, (ii) wherein the DNA is single-stranded, (iii) wherein the second primer comprises at least 10 bases, preferably at least 12 bases, optionally at least 15 bases of the adaptor sequence, and (iv) the second primer is fully complementary to the second DNA strand to which it hybridizes. 11. Implementation Plan

[0119] The following are exemplary embodiments of the methods and compositions disclosed in this application.

[0120] Embodiment 1. A double-end sequencing method comprising: (f) providing a DNA array comprising at least 1,000 DNA concatemers immobilized on a surface thereof, (g) for each of the plurality of DNA concatemers on the array, (iii) annealing a first reading primer to the primer binding site on the DNA concatemer, (iv) extending at least some of the first read primers to incorporate dNTPs or dNTP analogs, thereby generating a first read strand, wherein each of the incorporated dNTPs or dNTP analogs is identified to generate a first read, (h) performing controlled MDA by extending at least some of the first read strands with a polymerase having strand displacement activity to generate a plurality of second strands, each second strand comprising a portion hybridized to the DNA concatemer and unhybridized single-stranded branches; and (i) annealing a second reading primer to the single-stranded branch of the plurality of second strands, (j) extending the second read primer to generate a second read.

[0121] Embodiment 2. The method according to embodiment 1, wherein the DNA concatemer comprises a plurality of monomers, each monomer comprising an adaptor and a target sequence.

[0122] Embodiment 3. The method of embodiment 2, wherein the first reading primer hybridizes to the adaptor of each monomer of the DNA concatemer.

[0123] Embodiment 4. The method of any one of embodiments 1-3, wherein the first reading strand produced in step (a) comprises a 3' blocking group that prevents the first reading strand from being further extended, and wherein the 3' blocking group from the first reading strand is removed before performing the controlled MDA in step (b).

[0124] Embodiment 5. The method according to any one of embodiments 1 to 4, wherein a plurality of MDA primers are hybridized to the DNA concatemer prior to step (b)(ii), wherein the MDA primer comprises a reversible blocking group to prevent extension of the MDA primer, and the reversible blocking group is removed after step (b)(ii) but before step (c), and Wherein step (c) comprises extending the plurality of MDA primers and the first reading strand.

[0125] Embodiment 6. The method of embodiment 5, wherein the plurality of MDA primers comprises random primers.

[0126] Embodiment 7. The method of embodiment 5 or 6, wherein the DNA concatemer comprises an adaptor hybridized to at least one MDA primer and at least one first reading primer, and wherein the at least one MDA primer is upstream of the at least one first reading primer.

[0127] Embodiment 8. The method of any one of embodiments 5-7, wherein the MDA primer has a length of 5-10 nucleotides.

[0128] Embodiment 9. The method of any of the preceding embodiments, wherein the controlled MDA in step (c) comprises: (iii) performing MDA for a first period of time in the presence of a strand-displacing DNA polymerase at a concentration such that excess molecules of the DNA polymerase do not bind to the first reading strand, and, (iv) removing the excess molecules of the strand-displacing DNA polymerase from the array at the end of the first period and continuing the MDA to generate the second strands, each of the second strands comprising a portion hybridized to the at least one DNA concatemer and the unhybridized single-stranded branches.

[0129] Embodiment 10. The method of embodiment 9, wherein removing the excess molecules of the strand-displacing DNA polymerase is performed by washing the array, and Wherein said step (ii) comprises washing the array and adding fresh buffer containing unincorporated nucleotides but without DNA polymerase.

[0130] Embodiment 11. A method according to embodiment 9 or 10, wherein the ratio of the length of the first period to the length of the period during which the controlled MDA is completed in step (c) is in the range of 1:30 to 1:2.

[0131] Embodiment 12. The method according to any one of embodiments 9-11, wherein the time for completing the controlled MDA in step (c) is 10-90 minutes.

[0132] Embodiment 13. The method of any one of embodiments 9-12, wherein the first period of time is 1-10 minutes.

[0133] Embodiment 14. The method of any one of embodiments 1-13, wherein step (c) of performing the controlled MDA comprises: (iii) contacting the array with the strand-displacing DNA polymerase under extension-blocking conditions, and then (iv) reversing the extension-blocking condition such that the first read strand is simultaneously extended to generate a plurality of second strands, each second strand comprising a portion hybridized to the at least one DNA concatemer and the unhybridized single-stranded branch.

[0134] Embodiment 15. The method of embodiment 14, wherein the extension-blocking condition is a reaction buffer that does not contain magnesium or unincorporated nucleotides.

[0135] Embodiment 16. The method of embodiment 14, wherein the extension-blocking condition is a reaction buffer containing a polymerization inhibitor.

[0136] Embodiment 17. The method of embodiment 16, wherein the polymerization inhibitor is EDTA.

[0137] Embodiment 18. The method of embodiment 14, wherein the extension-preventing condition is a temperature below 20°C.

[0138] Embodiment 19. The method of embodiment 14, wherein the concentration of the strand-displacing DNA polymerase in (i) is such that excess molecules of the DNA polymerase do not bind to the first reading primer, and wherein the method further comprises removing unbound molecules of the strand-displacing DNA polymerase from the array between step (i) and step (ii).

[0139] Embodiment 20. The method of embodiment 14, wherein the concentration of the strand-displacing DNA polymerase in (i) is such that excess molecules of the DNA polymerase do not bind to the first reading primer, wherein step (ii) comprises extending the first reading 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, and MDA is continued in the reaction without excess molecules of DNA polymerase.

[0140] Embodiment 21. The method of embodiment 20, wherein the ratio of the first period to the period for completing the controlled MDA in step (c) ranges from 1:30 to 1:2.

[0141] Embodiment 22. The method according to embodiment 20 or 21, wherein the time period for completing the MDA in step (c) is 10-90 minutes.

[0142] Embodiment 23. The method of any one of Embodiments 20-22, wherein the first period of time is 1-10 minutes.

[0143] Embodiment 24. A method for paired-end sequencing, comprising: (e) 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 segment of the plurality of single-stranded DNA concatemers, wherein the extending generates a first read strand incorporating the excisable nucleotide; (f) cleaving the first reading strand where the excisable base is present to generate fragments of the first reading strand having extendable 3' ends, (g) performing controlled MDA by extending the segment of the first read strand to generate a plurality of second strands, and wherein the controlled MDA generates a plurality of second strands, each second strand comprising a sequence hybridized to one of the plurality of single-stranded DNA concatemers and an unhybridized single-stranded branch; and (h) extending a second read primer that hybridizes to the single-stranded branch of the plurality of second strands to generate the second read.

[0144] Embodiment 25. The method of embodiment 24, wherein the removable nucleotide is uracil.

[0145] Embodiment 26. A method for paired-end sequencing, comprising: (c) extending a first reading primer hybridized to the plurality of single-stranded DNA concatemers immobilized on the array to generate a first read segment of the plurality of single-stranded DNAs, wherein the extending generates a first read strand; (d) removing the first reading 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 the second strands comprising a sequence hybridized to the plurality of single-stranded DNA concatemers and unhybridized single-stranded branches; and (d) extending a second reading primer that hybridizes to the single-stranded branch of the plurality of second strands to generate the second read.

[0146] Embodiment 27. The method of embodiment 26, wherein the plurality of MDA primers comprises random primers.

[0147] Embodiment 28. The method of any one of embodiments 1-27, wherein extending the first reading primer in step (a) and extending the first reading strand in step (b) are performed by using a single DNA polymerase.

[0148] Embodiment 29. The method of any one of embodiments 1-27, wherein extending the first strand primer in step (a) and extending the first reading strand in step (b) are performed by using different DNA polymerases.

[0149] Embodiment 30. The method of any one of embodiments 1-27, wherein extending the first strand primer in step (a) is performed by a non-displacing DNA polymerase.

[0150] Embodiment 31. A method according to any one of embodiments 1-30, wherein the first read is determined by sequencing by synthesis.

[0151] Embodiment 32. The method of any one of Embodiments 1-31, wherein the extension of the first reading primer continues for a period of 1-15 minutes.

[0152] Embodiment 33. A method according to any of the above embodiments, further comprising combining the first read and the second read to determine the sequence of the target DNA sequence.

[0153] Embodiment 34. An array comprising a plurality of single-stranded DNA concatemers and a plurality of primers, Each single-stranded concatemer contains multiple monomers. Each monomer comprises an adapter sequence and a DNA target sequence. wherein each primer comprises a primer sequence that is complementary to and hybridizes with the adapter sequence of the DNA concatemer, wherein at least one primer comprises a removable nucleotide, and Wherein, the at least one primer can be cleaved to release the excisable nucleotide and generate two or more fragments having extendable 3' ends.

[0154] Embodiment 35. A kit comprising Multiple sequencing primers, DNA polymerase without strand displacement activity, A mixture of reversible terminator nucleotides for SBS, strand-displacing DNA polymerase, and dNTP mixture.

[0155] Embodiment 36. The kit of embodiment 35, wherein the mixture of reversibly terminating nucleotides is uracil, and the kit further comprises a cleavage agent capable of removing uracil from a DNA strand.

[0156] Embodiment 37. The kit of embodiment 35 or 36, wherein the kit further comprises a magnesium-free buffer. ***

[0157] All publications and patent documents cited herein are hereby incorporated by reference to the same extent as if each such publication or document was specifically and individually indicated to be incorporated by reference. Although the present invention has been described primarily with reference to specific embodiments, it is contemplated that other embodiments will become apparent to those skilled in the art upon reading this disclosure, and it is intended that such embodiments be included in the methods of the present invention.

Claims

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

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

4. The method of claim 1 , wherein the first read strand generated in step (b) comprises a 3′ blocking group that prevents the first read strand from being further extended, and wherein the 3′ blocking group from the first read strand is removed before performing the controlled MDA in step (c).

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

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

8. The method according to claim 1, wherein The time for completing the controlled MDA in step (c) is 10-90 minutes.

9. A double-end sequencing method comprising: (a) extending a first reading primer hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of a nucleotide comprising a reversible blocking group to generate a first read segment of the plurality of single-stranded DNA concatemers, wherein the extension generates a first reading strand having a reversible blocking group nucleotide at its 3′ end; (b) cutting the first reading strand having a reversible blocking group nucleotide at its 3′ end to generate a first reading strand having an extendable 3′ end, (c) performing controlled MDA by extending the first read strand to generate a plurality of second strands, and wherein the controlled MDA generates a plurality of second strands comprising a sequence hybridized to one of the plurality of single-stranded DNA concatemers and unhybridized single-stranded branches; as well as (d) extending the second reading primer hybridized to the plurality of second strands to generate a second reading strand.

10. The method of claim 9, wherein extending the first reading primer in step (a) and extending the first reading strand in step (c) are performed by using different DNA polymerases.

11. The method of claim 10, wherein extending the first reading primer in step (a) is performed by a non-displacing DNA polymerase.

12. An array comprising a plurality of single-stranded DNA concatemers and a plurality of primers, in, Each single-stranded concatemer contains multiple monomers. Each monomer comprises an adapter sequence and a DNA target sequence. wherein each primer comprises a primer sequence that is complementary to and hybridizes with the adapter sequence of the DNA concatemer, wherein the 3' end of at least one primer comprises a nucleotide having a reversible blocking group, and The at least one primer can be cleaved to release the reversible blocking group and generate an extendable 3' end.

13. A kit comprising the array according to claim 12, and Multiple sequencing primers, DNA polymerase without strand displacement activity, a mixture of reversible terminating nucleotides for generating a first read strand, strand-displacing DNA polymerase, and mixture of dNTPs, The kit further comprises a cleavage agent capable of removing the reversible blocking group from the DNA chain.

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