CN117813392A

CN117813392A - Single molecule seeding and amplification on surfaces

Info

Publication number: CN117813392A
Application number: CN202280048224.2A
Authority: CN
Inventors: 旻叡·理查德·沈; 库尔特·帕特森; 盖瑞·R·雅伯; 哈梅德·霍希尼贝
Original assignee: Pacific Biosciences of California Inc
Current assignee: Pacific Biosciences of California Inc
Priority date: 2021-05-10
Filing date: 2022-05-09
Publication date: 2024-04-02

Abstract

Methods, compositions, and systems for single molecule seeding and amplification on a flow cell are provided. In some embodiments, nucleic acids are isothermally inoculated and amplified on a flow-through cell comprising more than one binding region (e.g., pad), resulting in a collection of substantially identical amplified molecules on each binding region.

Description

Single molecule seeding and amplification on surfaces

Cross Reference to Related Applications

The present application claims the benefit of priority from U.S. provisional patent application Ser. No. 63/186,649, filed on 5/10 of 2021, and U.S. provisional patent application Ser. No. 63/283,877, filed on 11/29 of 2021. The contents of each of these related applications are incorporated herein by reference in their entirety.

Background

FIELD

The present application relates generally to molecular biology and more particularly to the amplification and sequencing of nucleic acids.

Description of the Related Art

Rolling Circle Amplification (RCA) is an efficient method of amplifying circular template nucleic acids to produce long single stranded linear nucleic acid molecules comprising tandem copies of the template nucleic acid sequence (concatenated copies). RCA has been used in many applications, such as nucleic acid sequencing.

SUMMARY

The disclosure herein includes methods, systems, and compositions for nucleic acid amplification. In some embodiments, the method comprises: (a) Providing a first circular DNA template comprising a first sequence, a second sequence, and a third sequence; (b) Providing a surface comprising more than one binding region, wherein each of the more than one binding region has attached more than one oligonucleotide a and more than one oligonucleotide B, wherein oligonucleotide a comprises a first capture sequence complementary to the first sequence and a second capture sequence complementary to the second sequence, and wherein oligonucleotide B comprises a third sequence; and (c) contacting the first circular DNA template with more than one oligonucleotide a attached to a first binding region of more than one binding region in the presence of a DNA polymerase to produce an amplified single-stranded concatemers (concatemers) of the first DNA template via Rolling Circle Amplification (RCA), and contacting the single-stranded, oligonucleotide a-primed concatemers with more than one oligonucleotide B to produce complementary concatemers of the first DNA template.

The method may, for example, further comprise providing a second circular DNA template comprising a first sequence, a second sequence, and a third sequence; and contacting the second circular DNA template with more than one oligonucleotide a attached to a second binding region that is more than one binding region in the presence of a DNA polymerase to produce an amplified single-stranded concatemer of the second DNA template via RCA, and contacting the single-stranded, oligonucleotide a-primed concatemer of the second DNA template with more than one oligonucleotide B to produce a complementary concatemer of the second DNA template.

In some embodiments, contacting the first circular DNA template with more than one oligonucleotide a attached to a first binding region of more than one binding region occurs simultaneously with contacting the second circular DNA template with more than one oligonucleotide a attached to a second binding region of more than one binding region in the presence of a DNA polymerase. In some embodiments, contacting the first circular DNA template with more than one oligonucleotide a attached to a first binding region of more than one binding region in the presence of a DNA polymerase to produce an amplified single-stranded concatemer of the first DNA template via RCA comprises: hybridizing a first circular DNA template to an oligonucleotide a attached to a first binding region that is more than one binding region; and extending the oligonucleotide a bound to the first circular DNA template along the first circular DNA template by a DNA polymerase, thereby producing an amplified single-stranded concatemer of the first DNA template.

In some embodiments, contacting the single-stranded, oligonucleotide a-primed concatemer with more than one oligonucleotide B to produce a complementary concatemer of the first DNA template comprises: hybridizing a single-stranded, oligonucleotide a-primed concatemer to an oligonucleotide B attached to a first binding region of more than one binding region; and extending oligonucleotide B bound to the single stranded, oligonucleotide a-initiated concatemer by a DNA polymerase, thereby producing a complementary concatemer of the first DNA template.

In some embodiments, extending oligonucleotide a bound to the first circular DNA template along the first circular DNA template by a DNA polymerase occurs simultaneously with extending oligonucleotide B bound to a single-stranded, oligonucleotide a-initiated concatemer.

In some embodiments, the method may include: contacting the complementary concatamer of the first DNA template with one or more of the more than one oligonucleotides a attached to the first binding region to produce an additional concatamer of the first DNA template. In some embodiments, the method comprises: contacting the complementary concatamer of the second DNA template with one or more of the more than one oligonucleotides a attached to the second binding region to produce an additional concatamer of the second DNA template.

The complementary concatamer of the first DNA template may be reverse-complementary to the single-stranded concatamer of the first DNA template. The first circular DNA template and the second circular DNA template may be provided in the same sample. In some embodiments, the first circular DNA template, the second circular DNA template, or both are single stranded DNA. In some embodiments, the first circular DNA template, the second circular DNA template, or both are circularized by a linear nucleic acid template. The surface may be a flow cell surface. In some embodiments, the RCA reaction is performed within a flow cell.

In some embodiments, the method may include: the RCA reaction is terminated by depleting oligonucleotide a, oligonucleotide B or both. In some embodiments, the method does not include terminating the RCA reaction by denaturing the DNA polymerase. In some embodiments, the method does not include terminating the RCA reaction by removing the DNA polymerase. In some embodiments, RCA is performed at about 37 ℃. The DNA polymerase may be, for example, phi29 DNA polymerase. In some embodiments, the first capture sequence is 5' to the second capture sequence on oligonucleotide a. In some embodiments, the second capture sequence is 5' to the first capture sequence on oligonucleotide a. More than one oligonucleotide a, more than one oligonucleotide B, or both, may be covalently conjugated to the first binding region, the second binding region, or both. More than one oligonucleotide a, more than one oligonucleotide B, or both may be non-covalently attached to the first binding region, the second binding region, or both. In some embodiments, more than one oligonucleotide a and more than one oligonucleotide B are each attached at or near the 5' end of oligonucleotide a or oligonucleotide B. In some embodiments, more than one oligonucleotide a and more than one oligonucleotide B are not reverse complementary to each other. In some embodiments, a single oligonucleotide a, a single oligonucleotide B, or both are attached to a binding region of more than one binding region. In some embodiments, the binding region of more than one binding region has attached at least 10,000 oligonucleotides a, at least 10,000 oligonucleotides B, or both.

The ratio of more than one oligonucleotide a and more than one oligonucleotide B attached to the binding region of more than one binding region may be about 100:1 to about 1:100. In some embodiments, the first binding region comprises a clonal population of first DNA templates. In some embodiments, the first binding region does not comprise a second DNA template. In some embodiments, the second binding region comprises a clonal population of second DNA templates. In some embodiments, the second binding region does not comprise the first DNA template. In some embodiments, at least 90% of the binding regions comprise clonal populations of no more than one nucleic acid template. In some embodiments, at least 90% of the binding regions comprise template nucleic acids that are different from each other. The first sequence and the second sequence may be adjacent to each other. The third sequence may be adjacent to the first sequence or the second sequence.

The concatemer of the first DNA template and the complementary concatemer of the first DNA template may be attached to the first binding region of more than one binding region via more than one oligonucleotide a and more than one oligonucleotide B attached to the first binding region. The concatemer of the second DNA template and the complementary concatemer of the second DNA template may be attached to the second binding region of more than one binding region via more than one oligonucleotide a and more than one oligonucleotide B attached to the second binding region.

The surface may comprise, for example, about 10 ⁴ From a binding area to about 10 ⁸ And binding regions. In some embodiments, the surface comprises at least 10,000 ordered binding regions separated by non-predetermined and/or randomly distributed discontinuities (discontinuities)Domain. In some embodiments, one or more, or each of the more than one binding regions has a circular shape. The dimensions of one, one or more, or each of the more than one bonding regions may vary, for example by about 10 ^-9 m to about 10 ^-4 m. In some embodiments, the dimension of one, one or more, or each of the more than one bonding regions is the width or radius of the bonding region. In some embodiments, the surface is a planar surface.

Provided are methods, compositions, and kits for amplifying nucleic acids. The method may include: (a) Providing a first circular DNA template comprising a first sequence, a second sequence, and a third sequence; (b) Providing a surface comprising more than one binding region, wherein each of the more than one binding region has attached more than one oligonucleotide a and more than one oligonucleotide B, wherein oligonucleotide a comprises a first capture sequence complementary to the first sequence and a second capture sequence complementary to the second sequence, and wherein oligonucleotide B comprises a third sequence; and (c) contacting the first circular DNA template with more than one oligonucleotide a attached to a first binding region of more than one binding region in the presence of a DNA polymerase to produce an amplified single stranded concatemer of the first DNA template via rolling circle amplification, and contacting the single stranded, oligonucleotide a-primed concatemer with more than one oligonucleotide B to produce a complementary concatemer of the first DNA template.

Brief Description of Drawings

FIG. 1 is a non-limiting schematic diagram showing the generation of single stranded DNA loops (ssDNA templates). The ssDNA template comprises an adapter a having sequence 101 and sequence 103 and an adapter B having sequence 102.

Fig. 2 is a non-limiting schematic diagram showing an exemplary flow cell surface having more than one binding region (e.g., pad), and the composition of a separate pad having primer a and primer B conjugated thereto. Primer a has a sequence region complementary (or substantially complementary) to sequence 101 of the single-stranded DNA circle (ssDNA template) shown in fig. 1, and another sequence region complementary (or substantially complementary) to sequence 102 of the single-stranded DNA circle (ssDNA template) shown in fig. 1. Primer B has a sequence region having the same (or substantially the same) sequence as the sequence 103 of the single-stranded DNA circles (ssDNA templates) shown in fig. 1.

FIG. 3 is a non-limiting schematic showing capture of single stranded DNA templates by primer A attached to a pad in the presence of a polymerase.

FIG. 4 is a non-limiting schematic diagram showing the generation of DNA concatemers via rolling circle amplification.

FIG. 5 is a non-limiting schematic diagram showing the annealing of primer B to a DNA concatemer.

Fig. 6A-6B are non-limiting schematic diagrams illustrating the following: (1) Generation of complementary DNA concatamers grown from priming with primer B via rolling circle amplification (fig. 6A shows complementary concatamers grown from surface primer B); and (2) initiation of second strand initiation with the generation of first strands (fig. 6B shows that as more first strands are generated, more second strand initiation begins at the surface).

FIG. 7 is a non-limiting schematic diagram showing the formation of clusters of DNA concatemers via rolling circle amplification. The first strand primer may anneal to a second strand scale (scales) for additional extension and surface primer consumption. In some embodiments, primer consumption may prevent additional library loops from annealing in the same pad.

FIG. 8 is a non-limiting schematic diagram showing the amplification products of first strand concatemer DNA and second strand concatemer DNA generated on more than one pad on a surface. Each pad may have a first strand concatemer DNA and a second strand concatemer DNA.

FIG. 9A shows a non-limiting schematic of generating circular template nucleic acids hybridized to immobilized primers.

FIG. 9B shows a non-limiting schematic of the extension of immobilized primers along a circular template nucleic acid via rolling circle amplification to produce an amplified concatemer.

Detailed description of the preferred embodiments

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of the disclosure herein.

All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

The disclosure herein includes methods, compositions, and systems for isothermal seeding and amplifying nucleic acids on surfaces (e.g., structured surfaces) comprising more than one binding region (e.g., pad) using Rolling Circle Amplification (RCA). Such seeding and amplification may produce more than one binding region (e.g., pad), each binding region comprising substantially the same collection of amplified molecules, and the collection of amplified molecules on a binding region (e.g., pad) is different between binding regions (e.g., pads) (i.e., the collection of amplified molecules on a given binding region is different from the collection of amplified molecules on any other pad of more than one binding region). The surface may be a flow cell surface. The surface may be planar or curved. The practice of the present disclosure allows for the simultaneous capture of circular library components on a surface and amplification of the library components at specific regions of the structured surface. Typically, capture and amplification occurs at a faster rate than secondary capture, such that a particular region is inoculated with no more than one library component. Thus, the nucleic acid population inoculated at that location is typically a homogeneous amplification of a single original library component (homogeneous amplification) in order to facilitate sequencing of the library components.

Rolling circle amplification produces linear concatemer nucleic acid molecules in the form of random coils, commonly referred to as "picospheres". The pico-sphere may be immobilized to a surface suitable for sequencing (e.g., via hybridization to universal capture oligonucleotides on the surface of a sequencing substrate). The universal capture oligonucleotides have sequences that are unrelated to any particular target sequence of interest, and thus can be used to capture any target sequence. The universal capture oligonucleotide may hybridize to a universal priming sequence in the pico-sphere. In some embodiments, the universal capture oligonucleotide is a barcode sequencing primer. In some embodiments, the pico sphere is attached to the surface by ionic interactions, via covalent linkages, or mediated by binding of attached ligands (e.g., biotin and streptavidin). In some embodiments, one or several sequencing primers are hybridized to the pico-ball before or after attachment to a surface for sequencing.

In the methods, compositions, and systems disclosed herein, a surface (e.g., a structured surface) having more than one binding region (e.g., a pad) may be provided, wherein oligonucleotide a and oligonucleotide B are immobilized on each of the more than one binding regions. Oligonucleotide a comprises a binding region that is complementary and capable of capturing library elements, and oligonucleotide B comprises a binding region that is identical (or substantially identical) to the sequence of library elements. For example, as shown in FIG. 1, an adapter A and an adapter B are located at each end of a member nucleic acid of a linear library, which member nucleic acid can hybridize to a splint (splint) and form a ssDNA loop by ligation. Adaptor a comprises sequence 101 and sequence 103, and adaptor B comprises sequence 102. When adapter A is ligated to adapter B, the linear library components are converted to circular molecules with the region of adapter A/adapter B ligation that is reverse complementary to a set of oligonucleotides on the binding site and is partially identical in sequence to the second primer on the binding site. Thus, the capture oligonucleotide can be used to prime rolling circle amplification of circularized library components, which in turn produces a linear concatemer that is reverse-complementary to the original library components and has a segment reverse-complementary to the second oligonucleotide on the surface. As shown in fig. 2, the flow cell surface comprises more than one binding region (e.g., pad), and each of the binding regions has primer a and primer B attached (e.g., immobilized) thereto. Primer a and primer B each comprise a sequence spanning the ligation event between adapter a and adapter B of the original library components. Primer a and primer B can function as primers in subsequent reactions. Primer A comprises sequence 102 'complementary (or substantially complementary) to sequence 102 of adapter B and sequence 101' complementary (or substantially complementary) to sequence 101 of adapter A and located 5 'to sequence 102'. Primer B comprises sequence 103 (or a sequence substantially identical to sequence 103). For primer A, sequence 101 'may be adjacent to sequence 102' with no intervening sequence, or the 3 'end of sequence 101' may be one nucleotide, two nucleotides, three nucleotides, or more from the 5 'end of sequence 102'. Because of sequence complementarity, primer a can bind to ssDNA loop templates to produce DNA concatamers in the presence of DNA polymerase. Primer B can then bind to the concatemer resulting from primer a extension bound to the circular template, resulting in a reverse complementary concatemer. However, primer a and primer B are not reverse complementary to each other, so that they do not form dimers on the surface.

An extension mixture comprising library member nucleic acids in the form of ssDNA circles (also referred to as template ssDNA) and DNA polymerase may be formed and provided to contact pads on which oligonucleotides a and B are immobilized for nucleic acid extension and amplification (fig. 3). Oligonucleotide a and oligonucleotide B may be used as capture and amplification oligonucleotides due to their sequence complementarity to the adaptors a and B, respectively, on the member nucleic acids of the library. In some embodiments, biochemical methods that allow simultaneous capture and amplification (e.g., rolling Circle Amplification (RCA) in the presence of, for example, phi29 polymerase, salts, nucleotides, buffers, or more) may be performed. For example, using oligonucleotide a as a primer, RCA can be performed and a concatemer copy of the template ssDNA generated (fig. 4). Oligonucleotide B can then bind to the concatemer DNA and act as a primer to allow RCA to produce a complementary concatemer (fig. 5 and 6). However, since primer a and primer B are not reverse complementary to each other, they do not form primer dimers on the surface. As more first chains are generated, more second chain priming is initiated on the binding region (e.g., pad) to continue RCA (fig. 6A-6B). As shown in fig. 7, the first strand primer may anneal to the second strand squamous for additional extension and surface primer consumption. In some embodiments, preventing additional library loops from annealing in the same pad may be advantageous for primer consumption. As a result of the first and second strand-initiated RCAs, the binding region (e.g., pad) will be associated with the first and second strand concatemer DNA. The flow cell surface may have more than one binding region (e.g., pad). The binding regions (e.g., pads) may be in a structured configuration on the flow cell surface, or in some embodiments, the pads are in an unstructured and/or predetermined configuration.

The method may include: (a) Providing a first circular DNA template comprising a first sequence, a second sequence, and a third sequence; (b) Providing a surface comprising more than one binding region, wherein each of the more than one binding region has attached more than one oligonucleotide a and more than one oligonucleotide B, wherein oligonucleotide a comprises a first capture sequence complementary to the first sequence and a second capture sequence complementary to the second sequence, and wherein oligonucleotide B comprises a third sequence; and (c) contacting the first circular DNA template with more than one oligonucleotide a and more than one oligonucleotide B attached to a first binding region of more than one binding region in the presence of a DNA polymerase to produce an amplified concatamer of the first DNA template and a complementary concatamer of the first DNA template. In some embodiments, some methods begin with a linear library component having adaptors a and adaptors B at the ends, such that contact with primer a results in the linear library component being positioned to circularize upon contact with ligase. The generation of the amplified concatemers of the first DNA template and the complementary concatemers of the first DNA template may be performed via a Rolling Circle Amplification (RCA) reaction, e.g. at a temperature in the range of about 25 ℃ to 65 ℃, or at a temperature in the range of 25 ℃ to 65 ℃, e.g. an RCA reaction performed under isothermal conditions at 37 ℃. The resulting complementary concatemer comprises a region complementary to primer B such that primer B can prime the synthesis of the reverse complementary concatemer or the reverse complementary strand of the primer a primer concatemer strand.

In some embodiments, the method comprises: (a) Providing more than one circular DNA template, each circular DNA template comprising a first sequence, a second sequence, and a third sequence; (b) Providing a surface comprising more than one binding region, wherein each of the more than one binding region has attached more than one oligonucleotide a and more than one oligonucleotide B, wherein oligonucleotide a comprises a first capture sequence complementary to the first sequence and a second capture sequence complementary to the second sequence, and wherein oligonucleotide B comprises a third sequence; and (c) hybridizing each of the more than one circular DNA templates with the more than one oligonucleotide a and the more than one oligonucleotide B, respectively, attached to the binding region of the more than one binding region in the presence of a DNA polymerase to produce an amplified concatemer of the DNA template and a complementary concatemer of the DNA template via Rolling Circle Amplification (RCA). Such seeding and amplification methods can produce more than one binding region, each binding region comprising substantially the same collection of amplified molecules, and the collection of amplified molecules on a binding region is different between binding regions (i.e., the collection of amplified molecules on a given binding region is different from the collection of amplified molecules on any other pad of more than one binding region).

Definition of the definition

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., singleton et al, dictionary of Microbiology and Molecular Biology, 2 nd edition, j.wiley & Sons (New York, NY 1994); sambrook et al Molecular Cloning, A Laboratory Manual, cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

As used herein, the term "immobilized" when used in reference to a molecule refers to the direct or indirect, covalent or non-covalent attachment of the molecule to a surface, such as the surface of a solid support. In some configurations, covalent attachment is preferred, but generally all that is required is that the molecule (e.g., nucleic acid) remain immobilized or attached to the surface under conditions where surface retention is desired.

As used herein, the term "nucleotide" may be used to refer to a natural nucleotide or an analog thereof. Examples include, but are not limited to, nucleotide Triphosphates (NTPs) such as ribonucleotide triphosphates (rtps), deoxyribonucleotide triphosphates (dntps), or unnatural analogs thereof such as dideoxyribonucleotide triphosphates (ddntps) or reversibly terminated nucleotide triphosphates (rtntps).

As used herein, the term "complementary" or "complementary" means that one nucleic acid can form hydrogen bonds with another nucleic acid based on traditional Watson-Crick base pairing rules. Complementarity may be complete or partial. Complete complementarity indicates that each nucleobase of one strand is capable of forming hydrogen bonds with a corresponding base in another antiparallel nucleic acid sequence according to Watson-Crick classical base pairing. Partial complementarity indicates that only a certain percentage of consecutive residues in one nucleic acid sequence can form Watson-Crick base pairing with the same number of consecutive residues in another antiparallel nucleic acid sequence. "substantially complementary" refers to a number or range of complementary percentages between about, at least, or at least about 70%, 80%, 90%, 100% or any two of these values.

As used herein, the term "polymerase" may be used to refer to enzymes that synthesize nucleic acids, including, but not limited to, DNA polymerases, RNA polymerases, reverse transcriptases, primer enzymes, and transferases. Typically, a polymerase has one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. The polymerase may catalyze the polymerization of nucleotides to the 3' end of the first strand of the double-stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of the next correct nucleotide to the 3' oxygen portion of the first strand of a double-stranded nucleic acid molecule via a phosphodiester bond, thereby covalently incorporating the nucleotide into the first strand of the double-stranded nucleic acid molecule. In some embodiments, the polymerase need not be capable of nucleotide incorporation under one or more conditions used in the methods set forth herein. For example, a mutant polymerase may be capable of forming a ternary complex, but not catalyzing nucleotide incorporation.

As used herein, the term "primer" refers to a nucleic acid having a sequence that binds to the nucleic acid at or near the template sequence. Typically, the primers bind in a configuration that allows for replication of the template, e.g., via polymerase extension of the primers. The primer can be a first portion of a nucleic acid molecule that binds to a second portion of the nucleic acid molecule, the first portion being a primer sequence and the second portion being a primer binding sequence (e.g., a hairpin primer). In some embodiments, the primer is a first nucleic acid molecule that binds to a second nucleic acid molecule having a template sequence. The primer may be composed of DNA, RNA or an analogue thereof. The primer may have an extendable 3 'end or a 3' end extended by a blocked primer.

As used herein, a "container" is a vessel used to separate one chemical process (e.g., a binding event; a doping reaction; etc.) from another chemical process, or to provide a space in which a chemical process may occur. Examples of containers useful in connection with the disclosed technology include, but are not limited to, flow cells, wells of multi-well plates; a microscope slide; a tube (e.g., a capillary tube); droplets, vesicles, tubes, trays, centrifuge tubes, features in arrays, tubing, channels in substrates, etc.

As used herein, the term "circular," when used in reference to a nucleic acid strand, means that the strand has no ends (i.e., the strand lacks a 3 'end and a 5' end). Thus, the 3 'oxygen and 5' phosphate moieties of each nucleotide monomer in the circular chain are covalently attached to adjacent nucleotide monomers in the chain. The circular DNA strand may be used as a template for generating a concatemer amplicon via Rolling Circle Amplification (RCA), wherein each sequence unit of the concatemer amplicon is the reverse complement of the circular nucleic acid strand. The circular nucleic acid may be double stranded. One or both strands of a double stranded nucleic acid may lack a 3 'end and a 5' end. One strand in a double-stranded nucleic acid may have a nick (at least one nucleotide monomer is not present relative to the other strand) or a nick (a phosphodiester bond is not present between two nucleotide monomers) as long as the other strand is circular.

As used herein, the term "concatemer," when used in reference to nucleic acid molecules, means a contiguous nucleic acid molecule comprising more than one copy of a common sequence connected in series. Similarly, the term "concatemer", when used in reference to nucleotide sequences, means a contiguous nucleotide sequence comprising more than one copy of a common sequence in tandem. Each copy of a sequence may be referred to as a "sequence unit" of the concatemer. The sequence units may have a length of at least 10 bases, 50 bases, 100 bases, 250 bases, 500 bases, or more. Concatemers can comprise at least 2, 5, 10, 50, 100, or more sequence units. The sequence unit may comprise a sub-region having any of a variety of functions, such as a primer binding region, a target sequence region, a tag region, a Unique Molecular Identifier (UMI), and the like.

As used herein, a "flow cell" is a reaction chamber that includes one or more channels that direct fluids in a predetermined manner to perform a desired reaction. The flow cell may be coupled to a detector such that a reaction occurring in the reaction chamber may be observed. For example, the flow-through cell may comprise primed template nucleic acid molecules, e.g., tethered to a solid support, to which nucleotides and auxiliary reagents are iteratively applied and washed away. The flow cell may comprise a transparent material that allows imaging of the sample after the desired reaction has occurred. For example, a flow cell may include a slide containing a small fluidic channel through which polymerase, dntps, and buffer may be pumped. The glass inside the channel is decorated with one or more primed template nucleic acid molecules to be sequenced. An external imaging system may be placed to detect molecules on the surface of the glass. Reagent exchange in the flow-through cell is accomplished by pumping, withdrawing or otherwise "flowing" different liquid reagents through the flow-through cell. Exemplary flow cells, methods of their manufacture, and methods of their use are described below: US patent application publications US 2010/011768 or US2012/0270305; or WO 05/065814, each of which is incorporated herein by reference.

As used herein, the term "cluster," when used in reference to a nucleic acid, refers to a population of nucleic acids attached to a solid support, e.g., at a binding region in an array of binding regions on a solid support.

As used herein, the term "clonal population" refers to a population of nucleic acids that is homogeneous with respect to a particular nucleic acid sequence. The homogenous sequence is typically at least 10 nucleotides long, but may be even longer, including for example at least 50, 100, 500, 1000 or 2500 nucleotides long. The clonal population may be derived from a single template nucleic acid. The clonal population may comprise at least 2, 10, 100, 500, or 1000 copies of a particular nucleic acid sequence. Copies may be present in a single nucleic acid molecule, e.g., as concatemers, or copies may be present on different nucleic acid molecules (e.g., different concatemers). Typically, all nucleic acids in a cluster will have the same nucleotide sequence. It will be appreciated that a negligible number of contaminating nucleic acids or mutations (e.g., due to amplification artifacts (amplification artifact)) may occur in a cluster without deviating from apparent clonality. Clusters may be at least 80%, 85%, 90%, 95% or 99% cloned. In some embodiments, the clusters may be 100% cloned.

Rolling circle amplification

Provided herein include methods, systems, and compositions for seeding and amplifying nucleic acids on a surface using rolling circle amplification. Typically, the RCA reaction involves extending the primer annealed to the circular template such that the polymerase produces a concatemer single stranded DNA comprising more than one tandem repeat around the circular template, each repeat being complementary to the circular template.

FIGS. 9A-9B provide non-limiting schematic illustrations of rolling circle amplification of nucleic acids. As shown in fig. 9A, nucleic acid primers (indicated by open rectangles and dashed rectangles) (e.g., oligonucleotide a or primer a in fig. 2) are attached to a solid support (indicated by dotted rectangles) via linkers (indicated by grey lines). The primer may be used to capture a target nucleic acid of the template nucleic acid via a primer binding site in the template nucleic acid that is complementary to the primer. For example, a primer can have a first capture sequence (e.g., indicated by open rectangles) that is complementary to a first primer binding region of a template nucleic acid (e.g., indicated by open rectangles) and a second capture sequence (e.g., indicated by underlined rectangles) that is complementary to a second primer binding region of the template nucleic acid (e.g., indicated by underlined rectangles).

In the exemplary configuration shown in fig. 9A, the immobilized primer can hybridize to portions of the primer binding site that are present at opposite ends of the target sequence (target sequence indicated by dashed lines and flanking primer binding site regions indicated by open and underlined rectangles, respectively). Thus, the immobilized primer acts as a splint bringing together the two ends of the template nucleic acid. The two ends may be joined, for example, by a ligase, while hybridizing to the splint nucleic acid to form a circular form of template nucleic acid. In another configuration shown in fig. 9A, the template nucleic acid is circularized prior to hybridization with the immobilized primer on the solid support. Thus, the template sequence may be a linear nucleic acid or a circular nucleic acid.

FIG. 9B provides a non-limiting schematic of single-stranded concatamers generated via rolling circle amplification of primed circular templates hybridized to immobilized primers. The primer is immobilized in such a way that the 3 'end is available for polymerase extension (e.g., the primer may be attached at or near its 5' end). The product of the first sub-step is shown as having progressed to the point where two copies (two sequence units) of the circular template have been produced and the circular template hybridizes to a portion of the third copy (third sequence unit) being replicated. Each sequence unit contains a region complementary to the target sequence (indicated by solid black lines) and a region complementary to the primer (indicated by open rectangles and dashed rectangles). The product of the second sub-step has progressed to the point where nearly six copies of the circular template are produced. Fig. 9B shows the product of the RCA reaction after the annular template is absent (e.g., has been removed) in the third sub-step. For illustration purposes, two regions of the final product are shown: regions of sequence units (indicating the concatemer primary structure of the amplified strand) and regions of unspecified number and conformation of sequence units (indicating the dynamic and variable secondary structure of the entire cluster) are depicted.

The RCA reaction may be terminated by denaturing the polymerase, for example by heating the sample at 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ or higher. The RCA reaction may also be terminated by removing one or more components of the RCA, such as a polymerase and/or dntps. In some embodiments, the RCA reaction may be terminated by exhausting the primer. The components of RCA may be removed by washing with a washing reagent, for example.

Inoculation and amplification of nucleic acids on surfaces

The disclosure herein includes a method of nucleic acid amplification, and in particular a method of seeding and amplifying nucleic acid on a surface comprising more than one binding region. In some embodiments, the method can include providing a nucleic acid template comprising one or more primer binding regions (e.g., a first sequence, a second sequence, and a third sequence). The nucleic acid template may be a circular nucleic acid or a linear nucleic acid circularized to form a circular nucleic acid. One or more primer binding regions can be adjacent to each other such as in a circular template nucleic acid). One or more primer binding regions may be located at opposite ends of the linear template nucleic acid. In some embodiments, the primer binding region is a region that was originally used to circularize a linear nucleic acid library component (see, e.g., fig. 1). The nucleic acid template further includes a target region containing a target nucleic acid to be amplified. The term "providing" as used herein refers to preparing and delivering one or more components (e.g., nucleic acid templates) to a container in which an RCA reaction occurs, such as the surface of a flow cell comprising more than one binding region.

The method further comprises contacting the nucleic acid template with more than one oligonucleotide a and more than one oligonucleotide B attached (e.g., covalently or non-covalently) to a binding region of the more than one binding region in the presence of a polymerase (e.g., a DNA polymerase) to produce more than one amplified single-stranded concatamer of the nucleic acid template via rolling circle amplification. The more than one amplified single-stranded concatemers generated from the RCAs described herein each comprise more than one copy of a nucleic acid template or reverse complement thereof.

In some embodiments, contacting the nucleic acid template with more than one oligonucleotide a and more than one oligonucleotide B attached to the binding region comprises: (i) Contacting the nucleic acid template with more than one oligonucleotide a to produce a single-stranded, oligonucleotide a-initiated concatemer (or a first strand concatemer) of the nucleic acid template via rolling circle amplification, and (ii) contacting the single-stranded, oligonucleotide a-initiated concatemer with more than one oligonucleotide B to produce a single-stranded, oligonucleotide B-initiated concatemer (or a second strand concatemer) via rolling circle amplification, the oligonucleotide B-initiated concatemer being reverse-complementary to the oligonucleotide a-initiated concatemer. As used herein, "oligonucleotide a-primed concatemer" or "first strand concatemer" refers to a concatemer produced by extending oligonucleotide a along, for example, a template nucleic acid. Similarly, "oligonucleotide B-primed concatemer" or "second strand concatemer" refers to a concatemer produced by extending oligonucleotide B along, for example, a first strand concatemer.

In some embodiments, the step of generating a first strand concatemer using more than one oligonucleotide a and the step of generating a second strand concatemer using more than one oligonucleotide B may occur sequentially or simultaneously. For example, in some cases, oligonucleotide B may prime and extend along the first strand concatemer while the first strand concatemer is still extending via the RCA reaction. In some other cases, oligonucleotide B may be initiated after extension of the first strand concatamer is terminated and extended along the first strand concatamer. As more first strand concatamers are generated, more second strand initiation begins at the surface.

The resulting first and second strand concatamers can then initiate additional rounds of replication by annealing to fresh immobilized oligonucleotides a and B (or unused primers) on the surface to form a series of immobilized concatamer strands, each comprising more than one sequence unit, each sequence unit being substantially complementary or substantially identical to the template nucleic acid. For example, the second strand concatemer may be contacted with one or more fresh oligonucleotides a and hybridized, thereby facilitating a new round of amplification to form additional first strand concatemers. Additional first strand concatamers may be contacted with one or more fresh oligonucleotides B and hybridized, thereby facilitating a new round of amplification to form additional second strand concatamers. Additional rounds of priming and amplification allow for the consumption of primers and synthesis of a large number of concatemer strands in a short period of time. Primer consumption may prevent additional template nucleic acids from annealing at the same binding region. In some embodiments, the template sequence may be subjected to replication of about, at least about, up to, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 rounds or rounds of numbers or ranges between any two of these values via rolling circle amplification.

The seeding and amplification described therein produces binding regions tethered to more than one concatemer nucleic acid molecule, wherein the concatemer strand can partially hybridize to more than one complementary concatemer strand. The concatemer strand and the complementary concatemer strand can undergo next generation sequencing. When the concatemer strand and the complementary concatemer strand partially hybridize to each other, they can be sequenced. Alternatively or additionally, the concatemer strand and the complementary concatemer strand may be separated by, for example, thermal denaturation such that the strands do not partially hybridize.

The rounds of amplification described herein can produce nucleic acid clusters comprising a population of nucleic acids (e.g., concatemer strands) attached to a surface, such as in the binding region of the surface (see fig. 8 for non-limiting examples). In some embodiments, the clusters of binding regions of the surface are homogenous with respect to a particular nucleic acid sequence such that about, at least, or at least about 80%, 90%, 95%, or 99% of the amplified nucleic acids in the clusters contain the same target sequence. Thus, the seeding and amplification methods described herein can produce a collection of substantially identical amplified molecules on each binding region.

In some embodiments described herein, the container in which the template nucleic acid and/or concatemer is contacted with the immobilized oligonucleotide is a flow-through cell. Thus, in some embodiments, the contacting step may be facilitated by the use of a flow cell. Flowing a liquid reagent (e.g., an extension mixture) through a flow cell may allow the reagents to mix and exchange. For example, contacting a nucleic acid template with more than one immobilized oligonucleotide may comprise flowing an extension mixture comprising a polymerase, dNTP mixture, template nucleic acid through a flow cell having a surface comprising more than one binding region. The extension mixture may also contain auxiliary reagents necessary to carry out the RCA reaction, such as salts, buffers, small molecules, cofactors, metals and ions, as will be apparent to the skilled person.

The extension mixture may be incubated with the immobilized primer at any temperature that favors polymerase activity. In some embodiments, the RCA reactions described herein do not require thermal cycling and can be performed at substantially isothermal reaction temperatures, e.g., temperatures that do not vary above or below a given temperature by about 2-3 ℃. The reaction temperature may be between 20 ℃ and 70 ℃, for example 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, or numbers or ranges between any two of these values. In some embodiments, the reaction temperature is between 20 ℃ and 60 ℃. In some embodiments, the reaction temperature is between 20 ℃ and 50 ℃. In some embodiments, the reaction temperature is about 30 ℃ (e.g., about 37 ℃).

The extension reaction initiated by oligonucleotide a and oligonucleotide B may be terminated by depletion of oligonucleotide a and oligonucleotide B on the binding region. In some embodiments, the extension reaction can be terminated, for example, by introducing a 3 'blocking nucleotide that can inhibit or prevent the 3' oxygen of the nucleotide from forming a covalent linkage with the next nucleotide during the nucleic acid polymerization reaction. In some embodiments, the extension reaction is not terminated by denaturing a polymerase (e.g., a DNA polymerase), by removing the polymerase, or by any chemical inactivation of the polymerase.

Generation of first strand concatemers

In some embodiments, step (i) of contacting the nucleic acid template with more than one oligonucleotide a to produce a single-stranded, oligonucleotide a-initiated concatemer of the nucleic acid template may comprise hybridizing the nucleic acid template to more than one oligonucleotide a (e.g., by complementary base pairing between the capture sequence of oligonucleotide a and the first and second sequences of the nucleic acid template) (e.g., in fig. 3). Oligonucleotide a is then subjected to an extension reaction to add more than one monomer unit of the nucleic acid template at its 3' end via rolling circle amplification. The result is a concatemer of the multimer of the original nucleic acid template tethered to the surface via oligonucleotide a as seen in fig. 4. Thus, the method comprises extending more than one oligonucleotide a bound to a nucleic acid template to produce an oligonucleotide a-primed concatemer (or first strand concatemer) of the nucleic acid template.

In the non-limiting exemplary embodiment set forth in FIGS. 3-4, one or more oligonucleotide A-primed concatamers (or first strand concatamers) may be generated. More than one oligonucleotide a (indicated by the dashed arrow) and oligonucleotide B (indicated by the gray solid arrow) are immobilized via a linker (indicated by the black and gray lines) on the binding region (indicated by the blue oval). As shown in fig. 3, the nucleic acid template is captured by oligonucleotide a via complementary base pairing between primer binding sequences of the nucleic acid template (e.g., first and second sequences indicated as green and red solid lines) and corresponding capture sequences in oligonucleotide a (indicated by green and red dashed arrows). The bound oligonucleotide a is then extended along the template nucleic acid in an RCA reaction by a polymerase, such as a polymerase having strand displacement activity, to produce a first strand concatemer (shown in fig. 4 as a black dashed line partially annealed to the template nucleic acid). The resulting first strand concatamer remains attached to the surface via primer a. Primer a can be used as a capture primer and an amplification primer. One or more nucleic acid templates may be captured and amplified simultaneously, producing more than one first strand concatamers of the nucleic acid templates. In some embodiments, one or at most one template nucleic acid is captured and amplified at a binding region of more than one binding region, where one or at most one first strand concatemer is generated. The numbers of oligonucleotides a and B immobilized on the binding region and the number of strands produced shown in the figure are for illustration only and are not intended to be limiting.

In some embodiments, the template nucleic acid does not hybridize to oligonucleotide B because the capture sequence in oligonucleotide B is substantially identical to the third sequence of the nucleic acid template and is not reverse complementary to the first and second sequences of the nucleic acid template.

Each sequence unit in the first strand concatemer comprises a region complementary to the target sequence and a region complementary to the primer binding sequence (e.g., the first sequence, the second sequence, and the third sequence). Thus, each sequence unit in the first strand concatemer comprises a segment that is reverse complementary to oligonucleotide B or a portion thereof.

Generation of second strand concatemers

After the first strand concatemer of the nucleic acid template is generated, the first strand concatemer is then used as a template for generating a second strand concatemer of the nucleic acid template, primed by oligonucleotide B, which may anneal to a portion of the primer binding region of the first strand concatemer, e.g., a portion of the primer binding region complementary to the sequence of oligonucleotide B.

Similarly, step (ii) of contacting the single stranded, oligonucleotide a-initiated concatemer with more than one oligonucleotide B to produce a single stranded, oligonucleotide B-initiated concatemer may comprise hybridizing the oligonucleotide a-initiated concatemer (or first strand concatemer) with more than one oligonucleotide B and extending the more than one oligonucleotide B bound to the oligonucleotide a-initiated concatemer to produce the oligonucleotide B-initiated concatemer (or second strand concatemer) of the nucleic acid template.

One or more oligonucleotide B-primed concatamers (or second strand concatamers) can be generated using more than one oligonucleotide B attached to the binding region in the non-limiting exemplary embodiments set forth in fig. 5-6. As shown in fig. 5, oligonucleotide B (indicated by grey arrows) can prime the first strand concatemer (indicated by black dashed lines) by hybridizing to a portion of the first strand concatemer and being extended along the first strand concatemer by a polymerase via RCA to produce a second strand concatemer. The resulting second strand concatamer (indicated by the solid black line in the figure) remains attached to the surface via oligonucleotide B. Each of the more than one first strand concatamers may be simultaneously primed by one or more oligonucleotides B, yielding more than one second strand concatamers of the nucleic acid template immobilized to the surface.

In some embodiments, one or more second strand concatemers can be generated from a single first strand concatemer. For example, one or more oligonucleotides B can anneal to a first strand concatemer (e.g., a different sequence unit of the first strand concatemer) to produce a nucleic acid cluster having a first strand concatemer hybridized to one or more extension oligonucleotides B, thereby producing one or more second strand concatemers complementary to at least a portion of the first strand concatemer. One or more second strand concatemers generated from the same first strand concatemers can have a variety of lengths (e.g., a variety of numbers of sequence units) and annealing patterns. In one example shown in fig. 6A, two oligonucleotides B hybridize to two different sequence units of the first strand concatemer and extend in the RCA reaction to produce two second strand concatemers (shown as black solid lines at least partially annealed to the first strand). The numbers of oligonucleotides a and B immobilized on the binding region and the numbers of first and second strands generated are shown for illustration only and are not intended to be limiting.

Multiplex vaccination and amplification

The methods described herein can be performed in multiplex format such that more than one different template nucleic acid can be inoculated and amplified in parallel on discrete binding regions using the steps set forth herein (see, e.g., fig. 8). Each of the more than one binding regions may generate more than one concatemer strand, each concatemer strand comprising more than one copy of a particular target sequence or its reverse complement (see, e.g., fig. 8). For example, more than one nucleic acid template (e.g., sequencing library) comprising different target sequences may be distributed over more than one binding region of a surface, such that different library components are captured and amplified in discrete binding regions via rolling circle amplification. Each binding region can comprise a colony having a first strand concatemer and a second strand concatemer tethered to a particular target nucleic acid of the binding region via oligonucleotide a and oligonucleotide B. In some embodiments, the methods disclosed herein can be configured to inoculate and amplify in parallelAbout, at least about, up to or up to about 2, 10, 100, 1X 10 ³ 、1×10 ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁹ And one or more different nucleic acids, thereby providing cost savings, time savings, and uniformity of conditions. Thus, the number of binding regions on the surface may be within the ranges illustrated herein for different nucleic acids.

For example, in some embodiments, a second nucleic acid template is provided to a second binding region that is different from the first binding region that captures and amplifies the first nucleic acid template. The second nucleic acid template can comprise a target nucleic acid having a different sequence than the target nucleic acid in the first nucleic acid template.

Thus, in some embodiments, the method can include providing a second nucleic acid template (e.g., a second circular DNA template) comprising a first sequence, a second sequence, and a third sequence, and contacting the second nucleic acid template with more than one oligonucleotide a and oligonucleotide B attached to a second binding region of more than one binding region in the presence of a polymerase to produce an amplified single-stranded concatemer of the second nucleic acid template via rolling circle amplification. Each of the more than one amplified single-stranded concatemers generated from the RCAs described herein comprises more than one copy of the second nucleic acid template or reverse complement thereof.

In some embodiments, contacting the second nucleic acid template with more than one oligonucleotide a and more than one oligonucleotide B attached to the second binding region comprises: (i) Contacting the second nucleic acid template with more than one oligonucleotide a to produce a single-stranded, oligonucleotide a-initiated concatemer (or first strand concatemer) of the second nucleic acid template via rolling circle amplification, and (ii) contacting the single-stranded, oligonucleotide a-initiated concatemer with more than one oligonucleotide B to produce a single-stranded, oligonucleotide B-initiated concatemer (or second strand concatemer) of the second nucleic acid template via rolling circle amplification, the oligonucleotide B-initiated concatemer being reverse-complementary to the oligonucleotide a-initiated concatemer.

Similar to the amplification of the first nucleic acid template on the first binding region, the step of generating a first strand concatemer of the second nucleic acid template using more than one oligonucleotide a and the step of generating a second strand concatemer of the second nucleic acid template using more than one oligonucleotide B may occur sequentially or simultaneously. For example, in some cases, oligonucleotide B may prime the first strand concatemer of the second nucleic acid template while the first strand concatemer is still extending via the RCA reaction. In some other cases, oligonucleotide B may prime the first strand concatemer after extension of the first strand concatemer is terminated.

In some embodiments, the first binding region may comprise a clonal population of first nucleic acid templates and the second binding region may comprise a clonal population of second nucleic acid templates. For example, in some embodiments, the first binding region does not comprise the second nucleic acid template, and the second binding region does not comprise the first nucleic acid template.

In some embodiments, more than one nucleic acid template may be provided to a surface comprising more than one binding region. Thus, in some embodiments, the method may comprise: (a) Providing more than one nucleic acid template (e.g., circular DNA templates), each nucleic acid template comprising a first sequence, a second sequence, and a third sequence; (b) Providing a surface comprising more than one binding region, wherein each of the more than one binding region has attached thereto more than one oligonucleotide a and more than one oligonucleotide B, wherein oligonucleotide a comprises a first capture sequence complementary to the first sequence and a second capture sequence complementary to the second sequence, and wherein oligonucleotide B comprises a third sequence; and (c) hybridizing each of the more than one circular DNA templates to the more than one oligonucleotide a and the more than one oligonucleotide B, respectively, attached to the binding region of the more than one binding region in the presence of a DNA polymerase to produce an amplified concatemer of the DNA template and a complementary concatemer of the DNA template via Rolling Circle Amplification (RCA). Amplified concatemers can be generated independently at each binding region where a different template nucleic acid is amplified.

Multiplex seeding and amplification can produce more than one binding region each comprising a nucleic acid cluster attached to the binding region. The nucleic acid clusters generated at or on the binding region may comprise no more than one nucleic acid template. For example, the percentage of more than one binding region that each comprises no more than one nucleic acid template may be below, about below, at least about below, up to below, or up to about below: 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or a number or range between any two of these values. For example, at least 90% of the binding region comprises no more than one nucleic acid template.

A clonal population of no more than one target nucleic acid may be generated on or at the binding region. In different embodiments, the percentage of more than one binding region comprising the clonal population may differ. In some embodiments, the percentage of more than one binding region each comprising a clonal population can be below, about below, at least about below, up to below, or up to about below: 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or a number or range between any two of these values. For example, at least 90% of the binding regions comprise a clonal population of no more than one target sequence.

The collection of amplified molecules on the binding regions differs between binding regions such that the collection of amplified molecules on a given binding region differs from the collection of amplified molecules on any other binding region in more than one binding region. For example, a first binding region of the more than one binding regions may comprise a collection of concatemers each comprising more than one copy of the first nucleic acid template or reverse complement thereof. The second binding region of the more than one binding regions may comprise a collection of concatemers each comprising more than one copy of the second nucleic acid template or reverse complement thereof. The first nucleic acid template is different from the second nucleic acid template.

The number of binding regions having more than one binding region of different template nucleic acids may be the following, about the following, at least the about the following, up to the following, or up to about the following: 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or a number or range between any two of these values. For example, at least 90% of the binding regions comprise template nucleic acids that are different from each other.

Seeding and amplification of different nucleic acid templates on discrete binding regions of more than one binding region may occur simultaneously. For example, a DNA library comprising more than one library component may be distributed over a surface comprising more than one binding region, to allow simultaneous capture of library components on different binding regions, and amplification of library components at specific binding regions of the surface. In some embodiments, initial capture and amplification can occur at a faster rate than secondary capture (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) such that a particular binding region is vaccinated with no more than one library component. Thus, the nucleic acid population inoculated at a particular binding region is typically a homogeneous amplification of a single original library component in order to facilitate sequencing of the library components.

Concatemer chain

The first strand concatemer, also known as an oligonucleotide a-primed concatemer, comprises more than one sequence unit each complementary to a template nucleic acid. The first strand concatemer may be generated by extending oligonucleotide a along, for example, a nucleic acid template. The second strand concatemer, also known as an oligonucleotide B-initiated concatemer, comprises more than one sequence unit that is substantially identical to the template nucleic acid. The second strand concatemer may be generated by extending oligonucleotide B along the first strand concatemer. The second strand concatemer is reverse-complementary to the first strand concatemer. In some embodiments, the second strand concatemer can also anneal to oligonucleotide a, producing a concatemer strand that is reverse-complementary to the second strand concatemer and thus substantially identical to the first strand concatemer. Thus, a first strand concatemer can also be generated by extending oligonucleotide a along a second strand concatemer. The number of first strand concatamers in a binding region may be the same or different than the number of second strand concatamers in the same binding region. In some embodiments, the number of first strand concatamers in a binding region exceeds the number of second strand concatamers in the same binding region. In some embodiments, the number of second strand concatamers in a binding region exceeds the number of first strand concatamers in the same binding region.

The concatemer chains in a cluster may comprise more than one copy of a tandem connected sequence unit. For example, a concatemer strand may comprise about, at least about, up to about 2, 10, 25, 100, or more sequence units. The number of sequence units in the concatemers generated by RCA varies with the number of times the polymerase completes one turn around the circular template during replication. The content of each sequence unit in the concatemer is substantially the same as or reverse complementary to the content of the circular template being replicated. In some embodiments, the concatemers have incomplete sequence units. Concatamers in nucleic acid clusters generated at the same binding region need not be of the same length (e.g., need not have the same number of sequence units). For example, two of the first strand concatamers and/or two of the second strand concatamers may be the same length or different lengths.

The number of concatemer strands on the binding region may depend on or be similar to the number of oligonucleotides a and B attached to the binding region. The binding region comprising more than one binding region of at least one pair of oligonucleotide a and oligonucleotide B may comprise at least two concatemer strands (e.g., a first strand and a second strand), each concatemer strand tethered to the binding region via oligonucleotide a or oligonucleotide B. For example, in some embodiments, the binding region can comprise at least one first strand concatemer and at least one second strand concatemer. In some particular configurations (e.g., a binding region to which a single oligonucleotide a and a single oligonucleotide B are attached), the binding region may comprise a single first strand concatemer and a single second strand concatemer.

In some embodiments, the number of concatemer chains on the binding region can be below, about below, at least about below, up to below, or up to about below: 2. 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or a number or range between any two of these values.

In some embodiments, the concatemer strands in a particular binding region comprise the same target sequence or its reverse complement, thereby forming a colony that can undergo next generation sequencing.

Oligonucleotide primers

Primers used in the disclosed methods, systems and compositions are oligonucleotides having a sequence complementary to a portion of a template nucleic acid. Each of the more than one binding regions on the surface has more than one first oligonucleotide (e.g., oligonucleotide a or primer a) and more than one second oligonucleotide (e.g., oligonucleotide B or primer B) attached. When compared to each other, the binding regions in the population of binding regions may have a common primer (oligonucleotide a and oligonucleotide B). For example, a population of binding regions may have universal primers attached such that the same primer sequence is present on more than one binding region in the population.

Oligonucleotide a may comprise at least one capture sequence. For example, oligonucleotide a may comprise a capture sequence that is substantially complementary to the first and second sequences of the nucleic acid template. In some embodiments, oligonucleotide a may comprise two capture sequences: the first capture sequence is substantially complementary to a first primer binding sequence (e.g., a first sequence) of the nucleic acid template and the second capture sequence is substantially complementary to a second primer binding sequence (e.g., a second sequence) of the nucleic acid template. The first capture sequence and the second capture sequence may be adjacent to each other without a spacer sequence in between. In some embodiments, the first capture sequence and the second capture sequence may have a spacer sequence of one nucleotide, two nucleotides, three nucleotides, or more. On oligonucleotide a, the first capture sequence may be 5' to the second capture sequence. In some embodiments, the second capture sequence may be 5' to the first capture sequence on oligonucleotide a. For example, for primer A shown in FIG. 2, sequence 101 'may be adjacent to sequence 102' with no intervening sequence in between, or the 3 'end of sequence 101' may be one nucleotide, two nucleotides, three nucleotides, or more from the 5 'end of sequence 102'. Sequence 101 'is complementary to first sequence 101 of fig. 1 and sequence 102' is complementary to second sequence 102 of fig. 1.

Oligonucleotide B may comprise a sequence substantially identical to the third sequence of the nucleic acid template. The term "substantially identical" indicates a sequence identity of about, at least, or at least about 70%, 75%, 80%, 85%, 90%, 95% or a number or range between any two of these values relative to another sequence. In some embodiments, oligonucleotide B may comprise a sequence having 100% sequence identity to a third sequence of a nucleic acid template. As used herein, "sequence identity" or "identity" in the context of two nucleic acid sequences refers to the same nucleotide base in the two sequences when aligned within a particular comparison window using any suitable sequence alignment algorithm to obtain maximum correspondence.

In some embodiments, the template nucleic acid is a library component (linear or circular) having two adapter regions (e.g., a 5 'adapter and a 3' adapter), and the sequences of oligonucleotide a and oligonucleotide B may be designed based on the sequences of the 5 'adapter and the 3' adapter or a portion thereof of the linear library component such that oligonucleotide a is complementary to the 5 'adapter and the 3' adapter or a portion thereof, and oligonucleotide B is substantially identical to a portion of the 5 'adapter or a portion of the 3' adapter (see, e.g., fig. 1).

Due to sequence complementarity, oligonucleotide a may bind to a portion of a nucleic acid template (e.g., a first sequence and a second sequence of a nucleic acid template) to generate a first strand concatemer by extending the oligonucleotide a bound to the nucleic acid template in the presence of a polymerase. Oligonucleotide B may then be bound to a portion of the generated first strand concatemer, generating a second strand concatemer reverse-complementary to the first strand concatemer by primer extension. Thus, oligonucleotide a and oligonucleotide B may function as capture primers and amplification primers. In some embodiments, oligonucleotide a and oligonucleotide B are not reverse complementary to each other, such that they do not form dimers on the surface even when they are in contact with each other.

In embodiments described herein, the binding region of more than one binding region has attached at least one oligonucleotide a and at least one oligonucleotide B. For example, the binding region may have a single oligonucleotide a and a single oligonucleotide B attached. In some embodiments, the binding region may have more than one oligonucleotide a and oligonucleotide B attached. For example, the binding region may be attached with 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000 or a number or range of between any two of these values of oligonucleotide a and/or oligonucleotide B.

In different embodiments, the density of oligonucleotide a and the density of oligonucleotide B on the binding region may be different or the same. In some embodiments, each of the density of oligonucleotide a and the density of oligonucleotide B on the binding region (e.g., pad) may be, independently, the following, about the following, at least the about the following, at most the following, or at most about the following: 1X 10 ¹⁰ 、2×10 ¹⁰ 、3×10 ¹⁰ 、4×10 ¹⁰ 、5×10 ¹⁰ 、6×10 ¹⁰ 、7×10 ¹⁰ 、8×10 ¹⁰ 、9×10 ¹⁰ 、1×10 ¹¹ 、2×10 ¹¹ 、3×10 ¹¹ 、4×10 ¹¹ 、5×10 ¹¹ 、6×10 ¹¹ 、7×10 ¹¹ 、8×10 ¹¹ 、9×10 ¹¹ 、1×10 ¹² 、2×10 ¹² 、3×10 ¹² 、4×10 ¹² 、5×10 ¹² 、6×10 ¹² 、7×10 ¹² 、8×10 ¹² 、9×10 ¹² 、1×10 ¹³ 、2×10 ¹³ 、3×10 ¹³ 、4×10 ¹³ 、5×10 ¹³ 、6×10 ¹³ 、7×10 ¹³ 、8×10 ¹³ 、9×10 ¹³ 、1×10 ¹⁴ 、2×10 ¹⁴ 、3×10 ¹⁴ 、4×10 ¹⁴ 、5×10 ¹⁴ 、6×10 ¹⁴ 、7×10 ¹⁴ 、8×10 ¹⁴ 、9×10 ¹⁴ 、1×10 ¹⁵ 、2×10 ¹⁵ 、3×10 ¹⁵ 、4×10 ¹⁵ 、5×10 ¹⁵ 、6×10 ¹⁵ 、7×10 ¹⁵ 、8×10 ¹⁵ 、9×10 ¹⁵ 、1×10 ¹⁶ 、2×10 ¹⁶ 、3×10 ¹⁶ 、4×10 ¹⁶ 、5×10 ¹⁶ 、6×10 ¹⁶ 、7×10 ¹⁶ 、8×10 ¹⁶ 、9×10 ¹⁶ Each primer/m ² Or a number or range between any two of these values.

Various separation distances (separation distance) or average separation distances between two adjacent primers on the binding region are contemplated herein. The or average separation distance between two adjacent primers on a binding region may be below, about below, at least about below, up to below, or up to about below: 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm, 75nm, 76nm, 77nm, 78nm, 79nm, 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91nm, 92nm, 93nm, 94nm, 95nm, 96nm, 97nm, 98nm, 99nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm values between any two of 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 800nm, 810nm, 820nm, 830nm, 840nm, 850nm, 860nm, 870nm, 890nm, 900nm, 910nm, 920nm, 940, 950nm, 960nm, 1000nm, or any two of these values.

The number of oligonucleotides a attached to a binding region may and need not be the same as the number of oligonucleotides B attached to (e.g., immobilized to) the same binding region. Various ratios of the number of oligonucleotides a and the number of oligonucleotides B are contemplated herein. The ratio of the number of oligonucleotides a to the number of oligonucleotides B may be the following, about the following, at least about the following, up to the following, or up to about the following: 1:100, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83, 1:82, 1:81, 1:80, 1:79, 1:78, 1:77, 1:76, 1:75, 1:91, 1:85 1:74, 1:73, 1:72, 1:71, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:59 1:74, 1:73, 1:72, 1:71, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, and 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 52:1, 56:1: 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1 or a number or range between any two of these values. In some embodiments, oligonucleotide a and oligonucleotide B are provided in more than one primer set, each primer set comprising oligonucleotide a and oligonucleotide B.

Oligonucleotide a and oligonucleotide B as used herein may be of the same length or of different lengths. The length of the oligonucleotide primers (or two or more primers in a type or population, or each primer in a type or population) may be the following, about the following, at least the following, at most about the following: 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides.

Oligonucleotide a and oligonucleotide B as used herein may have one or more modified nucleotides (nucleotide analogs). Nucleotide analogs are some type of modified nucleotide that contain pairs of bases, sugars, and/or moieties as will be understood by the skilled artisan. In some embodiments, modifications introduced to the primer may alter certain chemical properties of the primer, such as increasing stability of primer hybridization and/or increasing binding specificity.

More than one oligonucleotide a and more than one oligonucleotide B may be attached to a binding region of more than one binding region via covalent or non-covalent bonds. For example, the binding region can be covalently or non-covalently attached at or near the 5' end of oligonucleotide a or oligonucleotide B (e.g., one nucleotide, two nucleotides, three nucleotides, or more from the 5' end) such that the 3' ends of oligonucleotide a and oligonucleotide B are available for polymerase extension. Attachment of the nucleic acid (e.g., oligonucleotide a or oligonucleotide B) to the binding region may be mediated by any of a variety of surface chemistries, such as reaction of a carboxylate moiety or succinimidyl ester moiety on the binding region with an amine modified nucleic acid, reaction of an alkylating agent (e.g., iodoacetamide or maleimide) on the binding region with a thiol modified nucleic acid, reaction of an epoxysilane or isothiocyanate modified binding region with an amine modified nucleic acid, reaction of an aminophenyl or aminopropyl modified binding region with a succinylated nucleic acid, reaction of an aldehyde or epoxide modified binding region with a hydrazide modified nucleic acid, or reaction of a thiol modified binding region with a thiol modified nucleic acid. The members of the aforementioned reactive pair may be switched with respect to the presence on the binding region or on the nucleic acid. Click chemistry can be used to attach nucleic acids to surface regions. Exemplary reagents and methods for click chemistry are described in U.S. patent No. 6,737,236; 7,375,234; 7,427,678 and 7,763,736, each of which is incorporated herein by reference.

Template nucleic acid

The template nucleic acid to be inoculated and amplified may comprise a primer binding sequence (e.g., first sequence, second sequence, third sequence) and a target region comprising a target sequence. The template nucleic acid may be double-stranded or single-stranded.

In some embodiments, the template nucleic acid is a circular nucleic acid (e.g., circular DNA template) comprising a first primer binding region (e.g., formed from a first sequence and a second sequence) and a second primer binding region (e.g., formed from a third sequence). The first and second sequences may be adjacent to each other with no spacer in between, or the 3 'end of one sequence may be one nucleotide, two nucleotides, three nucleotides or more from the 5' end of the other sequence. The first and second primer binding regions and the target region may be in any configuration that enables complementary binding between the first and second primer binding regions and the oligonucleotides a and B of the template nucleic acid.

For example, at least one of the first and second primer binding regions of the nucleic acid template or a portion thereof may be substantially complementary to the capture sequence in oligonucleotide a, while the other primer binding region of the nucleic acid template or a portion thereof may be substantially identical to the sequence in oligonucleotide B.

In some embodiments, the template nucleic acid may be a linear nucleic acid having a target sequence flanked by two primer binding regions (e.g., adapter a and adapter B shown in fig. 1) at opposite ends of the target sequence (e.g., the 3 'end and the 5' end of the target sequence). Each of the primer binding regions comprises at least one of a first sequence, a second sequence, and a third sequence. For example, one of the first, second and third sequences may be located at one end of the target sequence, while the other two sequences are located at the other end of the target sequence. The first sequence may be located at the primer binding region at the 3 'end of the target nucleic acid, while the second sequence and the third sequence are located at the primer binding region at the 5' end of the target nucleic acid. The third sequence may be located 3' to the second sequence. Similarly, the second sequence may be located at the primer binding region at the 3 'end of the target nucleic acid, while the first sequence and the third sequence are located at the primer binding region at the 5' end of the target nucleic acid. The third sequence may be located 3' to the first sequence. In some embodiments, the first sequence and the third sequence are located at the primer binding region at the 3 'end of the target nucleic acid, and the second sequence is located at the primer binding region at the 5' end of the target nucleic acid. The third sequence may be located at the 5' end of the first sequence. Similarly, the second sequence and the third sequence are located in the primer binding region at the 3 'end of the target nucleic acid, while the first sequence is located in the primer binding region at the 5' end of the target nucleic acid. The third sequence may be located at the 5' end of the second sequence.

The linear nucleic acid templates may then be circularized to form circular nucleic acid templates (e.g., FIG. 1). A circular template nucleic acid can be prepared from a linear nucleic acid template for RCA using a variety of methods. In some embodiments, circularization of the linear nucleic acid template may be produced by an enzymatic reaction, e.g., by incubation with a ligase (e.g., DNA ligase). The ends of the linear nucleic acid templates may hybridize to a nucleic acid sequence (e.g., oligonucleotide a) such that the ends are proximal (see, e.g., fig. 1). Incubation with a ligase then may result in circularization of the hybridized linear nucleic acid template to produce a circular nucleic acid template. Cyclization of the linear nucleic acid templates brings together the first, second, and third sequences such that, in some embodiments, the first, second, and third sequences 101, 102, 103 are adjacent to one another after cyclization, as shown in fig. 1. The first, second, and third sequences may be adjacent to each other with no intervening sequence, or the 3 'end of one sequence may be one nucleotide, two nucleotides, three nucleotides, or more from the 5' end of the other sequence.

In some embodiments, the linear nucleic acid template molecule may be a linear library component having distinct 5 'and 3' adapter regions flanking the target region (e.g., in fig. 1). The adapter region can have any of a variety of functions, including, for example, providing a binding site that is complementary to a capture primer (e.g., oligonucleotide a attached to the binding region), providing a primer binding site for replication of a circular template, providing a primer binding site for replication of a complement of a circular template, providing a tag associated with the target region (e.g., a tag that indicates the source of the target region such as a bar code, or a tag that is used to identify errors introduced during amplification of the target region, etc.). The adaptor region or portion thereof may be common to the population of circular templates or the population of concatemers. Whether or not the adapter regions have a common sequence, the target regions in the circular template population or the target regions in the concatemer population can have different sequences (e.g., different target sequences). Thus, when comparing sequence units between two or more concatemers or between two or more circular templates, the sequence units may have a common sequence region (e.g., a universal primer binding site) and/or the sequence units may have regions of different sequences (e.g., different target sequences).

The linear library component may be annealed to a surface-bound oligonucleotide a having regions complementary to the 5 'adaptors and 3' adaptors of the linear library component or a portion thereof, and oriented such that the 5 'and 3' ends are positioned in proximity. The 5 'end and the 3' end of the linear library elements are ligated to form a circular library element. In some embodiments, the nucleic acid template may be a circular library component (e.g., a DNA library loop).

The methods described herein can accommodate any length of library component. For example, library components as used herein may have a length of less than 50, 45, 40, 35, 30, 25, 20, or less than 20 bases, or alternatively at least 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, or more than 3000 bases.

The template nucleic acid used in the methods, compositions, and systems set forth herein may be DNA such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), and the like. The template nucleic acid as used herein can also be an RNA such as mRNA, ribosomal RNA, tRNA, etc. Template nucleic acids as used herein may also include nucleic acid analogs that comprise modifications to the phosphate moiety, sugar moiety, and/or nitrogen-containing base of the nucleotide analog.

The length of the target region in the template nucleic acid may be selected to suit the particular application of the methods described herein. For example, the length may be about, at least about, up to, or up to about 50, 100, 250, 500, 1000, 1 x 10 ⁴ 、1×10 ⁵ One or more nucleotides.

The target nucleic acid as used herein may be derived from biological sources, synthetic sources or amplified products. Exemplary organisms from which nucleic acids may be derived include, for example, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, primates, humans or non-human primates; plants such as arabidopsis thaliana (Arabidopsis thaliana), maize, sorghum, oat, wheat, rice, canola, or soybean; algae such as chlamydomonas reinhardtii (Chlamydomonas reinhardtii); nematodes such as caenorhabditis elegans (Caenorhabditis elegans); insects such as drosophila melanogaster (Drosophila melanogaster), mosquito, drosophila, bee or spider; fish such as zebra fish; a reptile; amphibians such as frog animals or Xenopus laevis (Xenopus laevis); the reticulum dish (dictyostelium discoideum); fungi such as pneumocystis californicus (pneumocystis carinii), fugu rubripes (Takifugu rubripes), yeast, saccharomyces cerevisiae (Saccharamoyces cerevisiae) or schizosaccharomyces pombe (Schizosaccharomyces pombe); or plasmodium falciparum (plasmodium falciparum). The nucleic acid may also be derived from a prokaryote such as bacteria, E.coli (Escherichia coli), staphylococci (staphylococci) or Mycoplasma pneumoniae (mycoplasma pneumoniae); archaea; viruses such as hepatitis c virus, influenza virus, coronavirus or human immunodeficiency virus; or a viroid. The nucleic acid may originate from a homogeneous culture or population of the above organisms, or from a collection of several different organisms in, for example, a community or an ecosystem. Nucleic acids may be isolated using methods known in the art, including, for example, those described in Sambrook et al, molecular Cloning: A Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory, new York (2001) or Ausubel et al, current Protocols in Molecular Biology, john Wiley and Sons, baltimore, md. (1998), each of which is incorporated herein by reference.

Surface and bonding area

In the methods, compositions, and systems described herein, a surface, such as a flow cell surface, is provided that comprises one or more binding regions. The binding regions can be used, for example, to seed and amplify nucleic acid templates according to the methods disclosed herein. Any of a variety of suitable methods may be used to create the flow cell surface. For example, the flow cell surface may be created using top-down particle lithography or bottom-up particle self-assembly as described in U.S. provisional application No. 63/137,064, entitled "Surface Structuring With Colloidal Assembly," filed on 1 month 13 of 2021, the contents of which are incorporated herein by reference in their entirety.

The surface as used herein may be created using colloidal self-assembly (bottom-up) of particles. For example, a surface as used herein may be prepared by providing a structure contained in a liquid, delivering more than one particle (e.g., bead) to the surface of the liquid. The surface of the liquid may be the top surface of the liquid in contact with another medium such as air. A percentage of more than one particle (e.g., 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or numbers or ranges between any two of these values, or more) may self-assemble into tightly packed, ordered particles. The method may include removing liquid between the particles and the planar structure such that more than one particle is in contact with the planar structure. More than one particle on the surface of the liquid and/or in contact with the planar structure may designate more than one binding area on the planar structure.

The surface as used herein may also be prepared, for example, by providing a planar structure having an active site layer (or substance layer or binding site layer) and a masking layer deposited thereon. The method may include depositing more than one particle (e.g., bead) onto the masking layer of the planar structure. The method may include an act of exposing the planar structure to an etchant to differentially remove the masking layer from areas not masked by the more than one bead. The method may include removing the masking layer and the active site layer from areas not masked by more than one bead from the etchant. The method may include removing the remaining masking layer from the region shielded by the more than one bead, leaving the active site layer in the bonding region. Thus, the method designates more than one binding region comprising the remaining active site layer.

The number of binding regions on the flow cell surface may be below, about below, at least about below, up to below, or up to about below: 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000 or a number or range between any two of these values. In some embodiments, the surface comprises about 10 ⁴ From a binding area to about 10 ⁸ And binding regions. In some embodiments, the surface comprises more than one binding region of at least 10,000 binding regions. Each of the more than one bonding regions may correspond to a variety of different forms and shapes, such as annular, circular, oval, rectangular, or square. Each of the more than one bonding regions may have a center point and a diameter. The binding regions on the flow cell surface may be ordered, well-packed and/or may have a high density. The location (or position) of the binding areas on the surface of the flow cell may be random and non-predetermined. In some cases, the flow cell surface comprises a surface defined by discontinuities (e.g., 5, 10, 15, 20, 25, 30, 35, 40Numbers or ranges between 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any two of these values, or more discontinuities) of at least 10,000 ordered, well-packed, and/or high density bonding regions. The configuration of the ordered bonding regions and/or discontinuities may be non-predetermined and/or may be randomly distributed.

The bonded regions may take into account the different spacing or pitch between any two adjacent bonded regions. In some embodiments, the spacing between any bonded region and any nearest neighbor bonded region measured from the center of the first bonded region to the center of the nearest neighbor bonded region may be at least twice the diameter of the first bonded region. For example, the spacing between any bonding region and any nearest neighbor bonding region may be about 1nm to about 100 μm. The spacing between any bonded region and any nearest neighbor bonded region measured from the edge of the first bonded region to the center of the nearest neighbor bonded region is at least twice the diameter of the first bonded region. The two edges are closer (or at least as close) than the distance between any other edge of the first bonding region and any edge of the second bonding region. The size (e.g., radius, diameter, width, or height) of more than one bonding region may be about 1nm to about 100 μm.

More than one bonding region or a percentage of bonding regions may share a common pattern (e.g., shape or size) or different patterns. In some embodiments, more than one binding region comprises an unpatterned binding region. More than one binding region or a percentage of binding regions may be randomly located on the flow cell surface or may be arranged (registered) in a predetermined set of positions on the flow cell. In some embodiments, more than one binding region is arranged to form a first region having regularly positioned (or ordered) binding regions and a second region having randomly (or irregularly) positioned binding regions.

The binding region on the surface may be functionalized to facilitate attachment of the primer. For example, the binding region may comprise a carboxylate moiety or a succinimidyl ester moiety to attach an amine modified primer, an alkylating agent (e.g., iodoacetamide or maleimide) to attach a thiol modified primer, an epoxysilane or isothiocyanate to attach an amine modified primer, an aminophenyl or aminopropyl moiety to attach a succinylated primer, an aldehyde or epoxide moiety to attach a hydrazide modified primer, or a thiol group to attach a thiol modified primer. The members of the aforementioned reactive pair may be switched with respect to the presence on the binding region or on the primer. Click chemistry can be used to attach nucleic acids to surface regions. Exemplary reagents and methods for click chemistry are described in U.S. patent No. 6,737,236; 7,375,234; 7,427,678 and 7,763,736, each of which is incorporated herein by reference. Methods for functionalizing the binding region of the flow-through cell surface are also described in U.S. provisional application No. 63/137,064, entitled "Surface Structuring With Colloidal Assembly," filed on 1 month 13 of 2021, the contents of which are incorporated herein by reference.

Polymerase enzyme

Any of a variety of polymerases can be used in the methods or compositions set forth herein, e.g., to form a polymerase-nucleic acid complex or to perform primer extension. Polymerases that can be used include naturally occurring polymerases and modified variants thereof, including but not limited to mutants, recombinants, fusions, genetic modifications, chemical modifications, syntheses, and the like. Naturally occurring polymerases and modified variants thereof are not limited to polymerases having the ability to catalyze polymerization reactions. Naturally occurring and/or modified variants thereof may, for example, have the ability to catalyze polymerization under conditions that are not used during formation or examination of the stabilized ternary complex. Naturally occurring and/or modified variants that participate in the polymerase-nucleic acid complex may, for example, have modified properties, e.g., enhanced binding affinity to nucleic acid, reduced binding affinity to nucleic acid, enhanced binding affinity to nucleotide, reduced binding affinity to nucleotide, enhanced specificity for the next correct nucleotide, reduced catalytic rate, no catalytic activity, etc. Mutant polymerases include, for example, polymerases in which one or more amino acids are replaced with other amino acids or one or more amino acids are inserted or deleted. Exemplary polymerase mutants that can be used to form the stabilized ternary complex include, for example, those set forth in the following: U.S. patent application publication 2020/0087637 and U.S. patent nos. 10,584,379 and 10,597,643, each of which is incorporated herein by reference. In some embodiments, the polymerase used herein has strand displacement activity alone or in combination with a strand displacement factor such as a helicase.

The polymerases used herein can be attached with an exogenous labeling moiety (e.g., an exogenous fluorophore) that can be used to detect the polymerase. For example, the labeling moiety may be attached after at least partially purifying the polymerase using protein separation techniques. For example, the exogenous labeling moiety can be covalently linked to the polymerase using a free thiol or free amine moiety of the polymerase. This may involve covalent attachment to the polymerase through the side chain of the cysteine residue or through the free amino moiety of the N-terminus. Exogenous tag moieties can also be attached to the polymerase via protein fusion. Exemplary labeling moieties that can be attached via protein fusion include, for example, green Fluorescent Protein (GFP), phycobiliprotein (e.g., phycocyanin and phycoerythrin), or GFP or wavelength shifted variants of phycobiliprotein. In some embodiments, the polymerase used herein need not be attached to an exogenous label.

The different activities of the polymerase can be utilized in the methods set forth herein. The polymerase can be used, for example, for RCA amplification (e.g., in a primer extension step) or nucleic acid sequencing. The polymerase may be obtained from a variety of known sources and used in accordance with the teachings set forth herein and the accepted activity of the polymerase. The polymerase may be a DNA polymerase, an RNA polymerase or other type of polymerase such as reverse transcriptase.

Exemplary DNA polymerases include, but are not limited to, bacterial DNA polymerase, eukaryotic DNA polymerase, archaeal DNA polymerase, viral DNA polymerase, and phage DNA polymerase. Bacterial DNA polymerases include E.coli (E.coli) DNA polymerase I, II and Klenow fragment of III, IV and V, E.coli DNA polymerase, clostridium faecium (Clostridium stercorarium) (Cst) DNA polymerase, clostridium thermocellum (Clostridium thermocellum) (Cth) DNA polymerase and sulfolobus solfataricus (Sulfolobus solfataricus) (Sso) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, fahrenheit, η, ζ, λ, σ, μ, and k, and Revl polymerase (terminal deoxycytidine transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29DNA polymerase, GA-l, phi-29 like DNA polymerase, PZA DNA polymerase, phi-15DNA polymerase, cpl DNA polymerase, cp 7DNA polymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNA polymerases include thermostable DNA polymerases and/or thermophilic DNA polymerases such as Thermus aquaticus (Thermus aquaticus) (Taq) DNA polymerase, thermus aquaticus (Thermus filiformis) (Tfi) DNA polymerase, rhodococcus tzeri (Thermococcus zilligi) (Tzi) DNA polymerase, thermus thermophilus (Thermus thermophilus) (Tth) DNA polymerase, yellow perch (thermomus flavus) (Tfl) DNA polymerase, volcanic fireball (Pyrococcus woesei) (Pwo) DNA polymerase, pyrococcus furiosus (Pyrococcus furiosus) (Pfu) DNA polymerase and turbopfu DNA polymerase, seathermophilic coccus (Thermococcus litoralis) (Tli) DNA polymerase, fireball species (Pyrococcus sp.) GB-D polymerase, maritime (Thermotoga maritima) (a) DNA polymerase, stearothermophilus (Bacillus stearothermophilus) (Bst) DNA polymerase, pyrococcus Kodakaraensis (KOD) DNA polymerase, pfx thermophilus (tmf) DNA polymerase, jd) DNA polymerase (jd) DNA polymerase, jd.25-jd.sp.sp.sp.sp.sp.sp.sp.dna polymerase; a sulfolobus acidocaldarius (Sulfolobus acidocaldarius) DNA polymerase; 9 ° (Thermococcus sp.9 °) N-7DNA polymerase; cryptic heat transfer bacteria (Pyrodictium occultum) DNA polymerase; a methanococcus vortioides (Methanococcus voltae) DNA polymerase; a methanobacterium thermoautotrophicum (Methanococcus thermoautotrophicum) DNA polymerase; methanococcus jannaschii (Methanococcus jannaschii) DNA polymerase; a thiococcus (Desulfococcus) strain TOK DNA polymerase (D.Tok Pol); a deep sea pyrococcus (Pyrococcus abyssi) DNA polymerase; a Horikoshi (Pyrococcus horikoshii) DNA polymerase; a rhodococcus island (Pyrococcus islandicum) DNA polymerase; a Thermococcus fumigatus (Thermococcus fumicolans) DNA polymerase; an aeropyretic (Aeropyrum pernix) DNA polymerase; and heterodimeric DNA polymerase DP1/DP2. Engineered and modified polymerases can also be used in the disclosed technology. For example, a modified variant of the extremely thermophilic marine archaea thermochromatic species 9°n (e.g., from New England BioLabs inc.; thermo DNA polymerase of Ipswich, MA) may be used.

Exemplary RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kll polymerase; eukaryotic RNA polymerases, such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV and RNA polymerase V; archaea RNA polymerase.

Exemplary reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1 HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastosis virus, and telomerase reverse transcriptase that maintains eukaryotic chromosome telomeres.

Application in sequencing

Amplified nucleic acids generated on the binding region of the flow cell surface can be sequenced. The methods disclosed herein can be used in a variety of sequencing platforms, including but not limited to sequencing by synthesis or sequencing by binding (sometimes collectively referred to as incorporating sequencing chemistry), pH-based sequencing, sequencing by polymerase monitoring, sequencing by hybridization, and other methods of large-scale parallel sequencing or next generation sequencing. In some embodiments, sequencing is performed as described in U.S. patent No. 10,077,470, which is incorporated herein by reference in its entirety. Suitable surfaces for sequencing include, but are not limited to, planar substrates, hydrogels, nanopore arrays, microparticles or nanoparticles.

Binding sequencing

The methods, compositions, and systems disclosed herein for performing RCA can be used in methods, compositions, and systems for sequencing-by-Sequencing (SBB).

For example, combined sequencing is described in U.S. patent nos. 10,443,098 and 10,246,744 and U.S. patent application publication No. 2018,0044727, the contents of each of which are incorporated herein by reference in their entirety. In SBB, the polymerase undergoes a conformational transition between an open conformation and a closed conformation during discrete steps of the reaction. In one step, the polymerase binds to the primed template nucleic acid to form a binary complex, also referred to herein as a pre-insertion conformation. In a subsequent step, the incoming nucleotides are bound and the polymerase is turned off, forming a chemical pre-conformation comprising the polymerase, the primed template nucleic acid and the nucleotides, wherein the bound nucleotides have not been incorporated. This step is also called an inspection step. Nucleotides may be labeled or unlabeled. Likewise, the polymerase may be labeled or unlabeled. The detecting step may involve providing an primed template nucleic acid and contacting the primed template nucleic acid with a polymerase (e.g., a DNA polymerase) and one or more test nucleotides that are investigated as possible next correct nucleotides. The polymerase configuration and/or interactions with the primed template nucleic acid and further with nucleotides can be monitored during the checking step to identify the next correct base in the template nucleic acid. Thus, in some embodiments, the SBB procedure includes a monitoring step that monitors or measures the interaction between the polymerase and the primed template nucleic acid in the presence of the test nucleotide. In some embodiments, the checking step determines the identity of the next correct nucleotide without the need to incorporate the nucleotide (e.g., without or prior to the nucleotide being chemically linked to the 3' -end of the primer by a covalent bond). For example, a primer for a primed template nucleic acid molecule may include a blocking group that prevents enzymatic incorporation of an incoming nucleotide into the primer. The reaction mixture used in the checking step may, for example, contain low or insufficient levels of catalytic metal ions to prevent nucleotide incorporation into the primer of the primed template nucleic acid. In some embodiments, the reaction mixture used in the checking step comprises a stabilizer that stabilizes the ternary complex while preventing any nucleotide incorporation into the primer, such as a non-catalytic metal ion that inhibits polymerization.

Typically, the step of examining involves binding a polymerase to the polymerization initiation site of the initiated template nucleic acid in a reaction mixture comprising one or more nucleotides, and monitoring the interaction. The checking step generally comprises the following sub-steps: (1) Providing a primed template nucleic acid (i.e., a template nucleic acid molecule that hybridizes to a primer that may optionally be blocked from extension at its 3' end); (2) Contacting the primed template nucleic acid with a reaction mixture comprising a polymerase and at least one nucleotide; (3) Monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of nucleotides and without any chemical incorporation of nucleotides into the primed template nucleic acid; and (4) determining the identity of the next base (i.e., the next correct nucleotide) in the template nucleic acid based on the monitored interactions. The assay typically involves detecting the interaction of the polymerase with the template nucleic acid. Detection may include optical, electrical, thermal, acoustic, chemical, and mechanical means. In some embodiments, the checking step of the sequencing reaction may be repeated 1, 2, 3, 4 or more times prior to the optional incorporation step.

In SBB, the reaction mixture used for the examination step may contain 1, 2, 3 or 4 types of nucleotide molecules. The nucleotide may be selected from dATP, dTTP (or dUTP), dCTP and dGTP. The assay reaction mixture may comprise one or more nucleotide triphosphates and one or more nucleotide triphosphates. Ternary complexes can be formed between the primed template nucleic acid, the polymerase and any of the four nucleotide molecules, such that four types of ternary complexes can be formed.

The incorporation step may be performed simultaneously with the inspection step or may be separate from the inspection step. In some embodiments of the SBB procedure, the checking step is followed by an incorporation step that adds one or more complementary nucleotides to the primer of the primed template nucleic acidThe 3' end of the component. The polymerase, primed template nucleic acid and newly incorporated nucleotide create a post-chemical conformation. Both the pre-chemical conformation and the post-chemical conformation may be referred to as ternary complexes, each comprising a polymerase, a primed template nucleic acid and a nucleotide, wherein the polymerase is in a blocked state and facilitates interaction between the next correct nucleotide and the primed template nucleic acid. During the incorporation step, divalent catalytic metal ions, such as Mg ²⁺ A chemical step involving nucleophilic displacement of the 3' -hydroxy-focused phosphate (PPi) at the primer terminus. The polymerase returns to the open state after PPi release.

The incorporation step may be facilitated by incorporation of the reaction mixture. The incorporation reaction mixture may have a composition different from the nucleotide of the inspection reaction. For example, the inspection reaction may include one type of nucleotide, and the incorporation reaction may include another type of nucleotide. By way of another example, the check reaction includes one type of nucleotide and the incorporation reaction includes four types of nucleotide, and vice versa. The check reaction mixture may be modified or replaced by incorporation into the reaction mixture.

In some embodiments of the SBB procedure, the checking step is followed by removal of unincorporated labeled nucleotides and then deblocking of the 3' end of the primer (or extended primer) of the primed template nucleic acid in order to render it suitable for extension. Unlabeled 3' blocked nucleotides are then added followed by a chemical incorporation step in which phosphodiester bonds are formed, with cleavage from the pyrophosphates of the nucleotides (nucleotide incorporation), to form an extended strand that has been extended by one base and cannot be extended further without modification. The unincorporated blocked extension bases are removed and labeled bases are added so that they can form a ternary complex at the site of base pairing with the template. The fluorescence or other output of these ternary complexes is assayed to determine the identity of the paired bases, and the process is then repeated by removing the labeled bases, chemically modifying the extension strand to expose the 3'oh, and contacting with a population of 3' blocked unlabeled nucleotides to obtain another single base extension.

Sequencing by synthesis

The methods, compositions and systems for performing RCA disclosed herein may be used in methods, compositions and systems for Sequencing By Synthesis (SBS).

SBS generally involves enzymatic extension of nascent primers by iterative addition of nucleotides to the template strand hybridized to the primer. SBS differs from SBB described above in that the labeled nucleotides are incorporated into the extended strand, measured, and then the label is removed or inactivated, and the 3' closure is removed to iteratively sequence the template. In SBB, the labeled bases are not incorporated into the extended strand. In contrast, ternary complex formation is determined, typically for the presence of a labeled base, but sometimes for the presence of a labeled polymerase or other feature, after which the complex is broken down and the 3' blocked unlabeled base is used to extend the primer strand. Briefly, SBS can be initiated by contacting a target nucleic acid attached to a site in a flow cell with one or more labeled nucleotides, DNA polymerase, or the like. Those sites that use the target nucleic acid as a template extension primer will incorporate labeled nucleotides that can be detected. Detection may include scanning using the apparatus or methods set forth herein. For example, the labeled nucleotide may also include reversible termination properties that terminate further primer extension after the nucleotide has been added to the primer. For example, a nucleotide analog with a reversible terminator moiety may be added to the primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for embodiments using reversible termination, the deblocking agent may be delivered to the container (either before or after detection occurs). Washing may be performed between the delivery steps. The cycle may be performed n times to extend the primer by n nucleotides to detect a sequence of length n. For example, in Bentley et al, nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; U.S. patent No. 7,057,026; 7,329,492; 7,211,414; exemplary SBS procedures, reagents, and detection components that may be readily adapted for use with the methods, systems, or apparatus of the present disclosure are described in 7,315,019 or 7,405,281 and U.S. patent application publication No. 2008/0108082 A1, each of which is incorporated herein by reference.

System and method for controlling a system

The systems for nucleic acid amplification, detection and/or sequencing disclosed herein may include a container, solid support, or other device for performing nucleic acid amplification, detection and/or sequencing. For example, the system may include an array, flow cell, multiwell plate, cuvette, collection of channels, droplets or vesicles in a substrate, tray, centrifuge tube, tubing, or other convenient device. The device may be removable, allowing it to be placed into or removed from the system. Thus, the system may be configured to process more than one device (e.g., a container or solid support) sequentially or in parallel. The system may include a fluidic component configured to deliver one or more reagents (e.g., one or more reagents in solution) to a container or solid support, for example, via a channel or droplet transfer device (e.g., an electrowetting device). Any of a variety of detection devices may be configured to detect a container or solid support in which a reagent interacts. An exemplary system has a fluidic component and a detection component, which are set forth in the following: U.S. patent application publication No. 2018/0280975 A1; U.S. patent No. 8,241,573; 7,329,860 or 8,039,817; or U.S. patent application publication No. 2009/0272914A1 or 2012/0270305A1, each of which is incorporated herein by reference.

In some embodiments, a system for nucleic acid amplification comprises a flow-through cell having more than one binding region distributed thereon, each binding region having attached thereto more than one oligonucleotide a and more than one oligonucleotide B described herein. Oligonucleotide a is designed to be complementary to a portion of the template nucleic acid to be amplified, while oligonucleotide B is designed to be substantially identical to a different portion of the template nucleic acid. In some embodiments, more than one binding region or population of binding regions may have universal oligonucleotide primers attached such that the same oligonucleotide a and oligonucleotide B are present on the binding region.

In at least some of the previously described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically not feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. For clarity, various singular/plural permutations may be explicitly set forth herein. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to encompass "and/or" unless otherwise specified.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims), are generally intended as "open" terms (e.g., the term "include" should be interpreted as "including but not limited to (including but not limited to)", the term "having" should be interpreted as "having at least (having at least)", the term "include" should be interpreted as "including but not limited to (includes but is not limited to)", etc.). Those skilled in the art will further understand that if a specific number is intended in the recited claim recitation, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles to introduce claim recitations. Furthermore, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B and C, etc." is used, such a syntactic structure is generally intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include, but not be limited to, a system having a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). In those instances where a convention analogous to "at least one of A, B or C, etc." is used, such a syntactic structure is generally intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B or C" would include, but not be limited to, a system having a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). Those skilled in the art will further appreciate that, in fact, any separating word and/or expression presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

Further, when features or aspects of the present disclosure are described in terms of Markush groups (Markush groups), those skilled in the art will recognize that the present disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by those of skill in the art, for any and all purposes, such as in providing a written description, all ranges disclosed herein also include any and all possible subranges and combinations of subranges of the range. Any listed range can be readily identified as sufficiently descriptive and that the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be readily broken down into a lower third, a middle third, an upper third, and the like. As will also be understood by those skilled in the art, all language such as "up to", "at least", "greater than", "less than" and the like include the stated numbers and refer to ranges that may be subsequently broken down into subranges as discussed above. Finally, as will be appreciated by those skilled in the art, a range includes each individual member. Thus, for example, a group of 1-3 items refers to a group of 1, 2, or 3 items. Similarly, a group of 1-5 items refers to a group of 1, 2, 3, 4, or 5 items, and so on.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method of nucleic acid amplification comprising:

(a) Providing a first circular DNA template comprising a first sequence, a second sequence, and a third sequence;

(b) Providing a surface comprising more than one binding region, wherein each of the more than one binding region has attached more than one oligonucleotide a and more than one oligonucleotide B, wherein the oligonucleotide a comprises a first capture sequence complementary to the first sequence and a second capture sequence complementary to the second sequence, and wherein the oligonucleotide B comprises the third sequence; and

(c) Contacting the first circular DNA template with the more than one oligonucleotide a attached to a first binding region of the more than one binding region in the presence of a DNA polymerase to produce an amplified single-stranded concatemer of the first DNA template via Rolling Circle Amplification (RCA), and contacting a single-stranded, oligonucleotide a-primed concatemer with the more than one oligonucleotide B to produce a complementary concatemer of the first DNA template.

2. The method of claim 1, further comprising providing a second circular DNA template comprising the first sequence, the second sequence, and the third sequence; and contacting the second circular DNA template with the more than one oligonucleotide a attached to a second binding region of the more than one binding region in the presence of the DNA polymerase to produce an amplified single-stranded concatemer of the second DNA template via RCA, and contacting a single-stranded, oligonucleotide a-primed concatemer of the second DNA template with the more than one oligonucleotide B to produce a complementary concatemer of the second DNA template.

3. The method of claim 1 or 2, wherein contacting the first circular DNA template with the more than one oligonucleotide a attached to a first binding region of the more than one binding region occurs simultaneously in the presence of a DNA polymerase and contacting the second circular DNA template with the more than one oligonucleotide a attached to a second binding region of the more than one binding region in the presence of the DNA polymerase.

4. The method of any one of claims 1-3, wherein contacting the first circular DNA template with the more than one oligonucleotide a attached to the first binding region of the more than one binding region in the presence of the DNA polymerase to produce an amplified single-stranded concatemer of the first DNA template via RCA comprises:

Hybridizing the first circular DNA template to oligonucleotide a attached to the first binding region of the more than one binding regions; and

extending the oligonucleotide a bound to the first circular DNA template along the first circular DNA template by the DNA polymerase, thereby producing an amplified single-stranded concatemer of the first DNA template.

5. The method of any one of claims 1-4, wherein contacting the single-stranded, oligonucleotide a-primed concatemer with the more than one oligonucleotide B to produce a complementary concatemer of the first DNA template comprises:

hybridizing a single-stranded, oligonucleotide a-primed concatemer with oligonucleotide B attached to the first binding region of the more than one binding regions; and

extending said oligonucleotide B bound to said single stranded, oligonucleotide a-initiated concatemer by said DNA polymerase, thereby generating a complementary concatemer of said first DNA template.

6. The method of claim 5 or 6, wherein extending oligonucleotide a bound to the first circular DNA template along the first circular DNA template by the DNA polymerase occurs simultaneously with extending the oligonucleotide B bound to the single-stranded, oligonucleotide a-initiated concatemer.

7. The method of any one of claims 1-6, comprising contacting a complementary concatamer of the first DNA template with one or more of the more than one oligonucleotides a attached to the first binding region to produce an additional concatamer of the first DNA template.

8. The method of any one of claims 2-7, comprising contacting a complementary concatamer of the second DNA template with one or more of the more than one oligonucleotides a attached to the second binding region to produce an additional concatamer of the second DNA template.

9. The method of any one of claims 1-8, wherein the complementary concatamer of the first DNA template is reverse-complementary to the single-stranded concatamer of the first DNA template.

10. The method of any one of claims 1-9, wherein the first circular DNA template and the second circular DNA template are provided in the same sample.

11. The method of any one of claims 1-10, wherein the first circular DNA template, the second circular DNA template, or both are single stranded DNA.

12. The method of any one of claims 1-11, wherein the first circular DNA template, the second circular DNA template, or both are circularized by a linear nucleic acid template.

13. The method of any one of claims 1-12, wherein the surface is a flow cell surface.

14. The method of claim 13, wherein the RCA reaction is performed within a flow-through cell.

15. The method of any one of claims 1-14, comprising terminating the RCA reaction by depleting oligonucleotide a, oligonucleotide B, or both.

16. The method of any one of claims 1-15, wherein the method does not comprise terminating the RCA reaction by denaturing the DNA polymerase.

17. The method of any one of claims 1-15, wherein the method does not comprise terminating the RCA reaction by removing the DNA polymerase.

18. The method of any one of claims 1-17, wherein the RCA is performed at about 37 ℃.

19. The method of any one of claims 1-18, wherein the DNA polymerase is Phi29 DNA polymerase.

20. The method of any one of claims 1-19, wherein on the oligonucleotide a the first capture sequence is 5' to the second capture sequence.

21. The method of any one of claims 1-19, wherein on the oligonucleotide a the second capture sequence is 5' to the first capture sequence.

22. The method of any one of claims 1-21, wherein the more than one oligonucleotide a, the more than one oligonucleotide B, or both are covalently conjugated to the first binding region, the second binding region, or both.

23. The method of any one of claims 1-21, wherein the more than one oligonucleotide a, the more than one oligonucleotide B, or both are non-covalently attached to the first binding region, the second binding region, or both.

24. The method of any one of claims 1-23, wherein the more than one oligonucleotide a and the more than one oligonucleotide B are each attached at or near the 5' end of the oligonucleotide a or oligonucleotide B.

25. The method of any one of claims 1-24, wherein the more than one oligonucleotide a and the more than one oligonucleotide B are not reverse complementary to each other.

26. The method of any one of claims 1-25, wherein a binding region of the more than one binding region is attached to a single oligonucleotide a, a single oligonucleotide B, or both.

27. The method of any one of claims 1-25, wherein a binding region of the more than one binding region has attached at least 10,000 oligonucleotides a, at least 10,000 oligonucleotides B, or both.

28. The method of any one of claims 1-27, wherein the ratio of the more than one oligonucleotide a and the more than one oligonucleotide B attached to the binding region of the more than one binding region is about 100:1 to about 1:100.

29. The method of any one of claims 1-28, wherein the first binding region comprises a clonal population of the first DNA templates.

30. The method of any one of claims 1-29, wherein the first binding region does not comprise the second DNA template.

31. The method of any one of claims 2-30, wherein the second binding region comprises a clonal population of the second DNA templates.

32. The method of any one of claims 2-31, wherein the second binding region does not comprise the first DNA template.

33. The method of any one of claims 1-32, wherein at least 90% of the binding regions comprise clonal populations of no more than one nucleic acid template.

34. The method of any one of claims 1-33, wherein at least 90% of the binding regions comprise template nucleic acids that are different from each other.

35. The method of any one of claims 1-34, wherein the first sequence and the second sequence are adjacent to each other.

36. The method of any one of claims 1-35, wherein the third sequence is adjacent to the first sequence or the second sequence.

37. The method of any one of claims 1-36, wherein the concatemer of the first DNA template and the complementary concatemer of the first DNA template are attached to the first binding region of the more than one binding region via the more than one oligonucleotide a and the more than one oligonucleotide B attached to the first binding region.

38. The method of any one of claims 1-37, wherein the concatemer of the second DNA template and the complementary concatemer of the second DNA template are attached to the second binding region of the more than one binding region via the more than one oligonucleotide a and the more than one oligonucleotide B attached to the second binding region.

39. The method of any one of claims 1-36, wherein the surface comprises about 10 ⁴ From a binding area to about 10 ⁸ And binding regions.

40. The method of any one of claims 1-37, wherein the surface comprises at least 10,000 ordered binding regions separated by non-predetermined and/or randomly distributed discontinuities.

41. The method of any one of claims 1-40, wherein one, one or more, or each of the more than one binding regions has a circular shape.

42. The method of any one of claims 1-41, wherein one, one or more, or each of the more than one binding region is about 10 in size ^-9 m to about 10 ^-4 m。

43. The method of claim 42, wherein the dimension of one, one or more, or each of the more than one bonding regions is a width or radius of the bonding region.

44. The method of any one of claims 1-43, wherein the surface is a planar surface.