Clonal amplification

Abstract

The present invention provides methods for the preparation of clonal clusters of polynucleotides on surfaces, and also provides surfaces containing such clusters. These methods and tethered clonal clusters are particularly useful in nucleotide sequencing methodologies, such as sequencing-by-synthesis.

Description

[Technical Field]

[0001] The present invention relates, inter alia, to methods for the clonal amplification of nucleotide sequences of interest (SOIs), to methods of nucleotide sequencing incorporating these approaches, and to surfaces containing clonally amplified nucleotide sequences. [Background technology]

[0002] In solid-phase sequencing workflows, individual nucleotide fragments of interest are typically captured by oligonucleotides immobilized on a surface, such as a slide, bead, or a feature in a microfluidic system. A sequencing reaction is then performed on the tethered molecule. This allows, among other things, the use of very small amounts of material, easy control of the necessary fluidics, and the ability to perform many similar reactions arranged within a small space. In a recent approach to nucleotide sequencing, a sequencing primer (sequencing primer) is annealed to the tethered sequence, and as the primer is extended, the incorporation of each base is detected sequentially (the incorporation signal) by, for example, fluorescence or detection of ions generated in the reaction. To increase the incorporation signal, a single tethered molecule can be amplified to generate a clonal cluster of tethered amplicons derived from a single polynucleotide. This "clonal amplification" (CA) provides many replicated sequencing targets, thus allowing multiple incorporation events to occur simultaneously within the cluster, thereby amplifying the signal many-fold. Numerous approaches to CA are known and have been developed and commercialized for use, for example, in sequencing workflows. These include bridge PCR (U.S. Patent No. 9,593,328), emulsion PCR (U.S. Patent Application Publication No. 20100261230), and kinetic exclusion amplification (U.S. Patent No. 9,169,513). Clonal amplification by recombinase polymerase amplification (RPA) has also been reported and is discussed further in co-pending application GB Patent Application Publication No. 2110479.9 and herein below.

[0003] While CA methodologies typically provide increased signal, the level of signal and / or its level above background can still be challenging in some cases. Optimizing a particular CA methodology can improve the density of anchored amplicons, but the density, and therefore the signal, can still be limited by factors such as the density of surface-immobilized capture oligos and steric factors that reduce polymerase access to locally crowded molecules during clonal amplification. Furthermore, approaches that increase the nucleotide or target density within an anchored cluster can also affect the spread of the cluster, increasing the incidence of contamination from neighboring amplification events, reducing clonality, interfering with read quality, increasing background, and negatively impacting the signal-to-noise ratio, especially near the edges of the cluster. Increasing the density of clonal clusters may also reduce the space occupied by each cluster, thus providing room for increasing the number of clusters in a given area without reducing the signal generated by each cluster.

[0004] In some situations, it may be desirable to provide clusters based on known or partially known sequences, for example, when polymorphisms within a sequence are to be determined by sequencing, or when the presence of a particular nucleotide sequence within a sample is to be determined. In some situations, it may be desirable to provide clonal clusters of unknown sequences, for example, when genomic fragments are to be sequenced for later assembly, or when other unknown nucleotide sequences are to be determined.

[0005] It would therefore be desirable to provide improved methods of clonal amplification. It would also be desirable to provide improved methods of clonal amplification without adversely affecting clonality, cluster spreading, or signal-to-noise ratio. It would also be desirable to provide clonal amplification methods that can be easily adapted for use with known, partially known, or unknown sequences. The present invention addresses one or more of the above problems. Summary of the Invention

[0006] In a first embodiment, the present invention provides a method for preparing a clonal cluster of a nucleotide sequence of interest, the method comprising the steps of: (i) providing a single-stranded polynucleotide comprising the nucleotide sequence of interest (SOI), wherein the polynucleotide is anchored to a surface via its 5' end; (ii) performing a first clonal amplification of the single-stranded polynucleotide to provide a first cluster of polynucleotides comprising a plurality of single-stranded polynucleotides anchored to the surface via their 5' ends and comprising the nucleotide sequence of interest; and (iii) extending the anchored single-stranded polynucleotides in the first cluster by addition of one or more further copies of the nucleotide sequence of interest to provide a cluster of anchored polynucleotides, wherein the polynucleotides of the cluster comprise multiple copies of the nucleotide sequence of interest.

[0007] A second embodiment provides a method for preparing a clonal cluster of single-stranded polynucleotides comprising a sequence of interest, the method comprising: (ii) hybridizing a polynucleotide fragment comprising a complementary copy of the sequence of interest to a capture oligo, wherein the capture oligo is tethered to a surface via its 5' end; (ii) extending the 3' end of the capture oligo using a polymerase to provide a single-stranded polynucleotide comprising the sequence of interest tethered to the surface via its 5' end; (iii) performing a first clonal amplification of the tethered single-stranded polynucleotide to provide a first cluster of polynucleotides comprising a plurality of single-stranded polynucleotides tethered to the surface via their 5' ends and comprising the nucleotide sequence of interest; and (iii) extending the 3' end of the tethered single-stranded polynucleotides in the first cluster by the addition of one or more further copies of the sequence of interest to provide a cluster of tethered polynucleotides, wherein the polynucleotides of the cluster comprise multiple copies of the nucleotide sequence of interest.

[0008] In one embodiment, the polynucleotide is hybridized to the capture oligo via its 5' end portion. The polynucleotide may be provided with a 3' adaptor and optionally a 5' adaptor, at least a portion of which is hybridized to the capture oligo.

[0009] In a third embodiment, a method for determining the nucleotide sequence of a sequence of interest is provided, comprising: (i) anchoring the sequence of interest to a surface via its 5' end; (ii) performing a first clonal amplification step to clonally amplify the SOI to provide a first clonal cluster of oligonucleotides, each comprising one copy of the SOI; (iii) extending single-stranded polynucleotides in the first cluster by adding one or more further copies of the SOI to provide a cluster of anchored polynucleotides, wherein the polynucleotides of the cluster comprise multiple copies of the nucleotide sequence of interest; and (iv) at least partially sequencing the polynucleotides of the cluster to determine the nucleotide sequence of the SOI.

[0010] The tethered SOI may be a complementary copy of a polynucleotide obtained by an earlier workflow step.

[0011] As referred to herein, a "sequence of interest" or SOI can be any nucleotide sequence that a user desires to clonally amplify. As referred to herein, the nucleotide sequence of an SOI is the nucleotide sequence of a polynucleotide that was anchored via its 5' end prior to the initial clonal amplification to provide the first clonal cluster. This sequence can be a complementary copy of a polynucleotide generated at an earlier stage in the workflow as a result of the anchoring process described further herein.

[0012] Typically, an SOI is a polynucleotide sequence about which a user desires information. This can be, for example, sequence information, including the sequence or subsequence of a polynucleotide, or the presence or absence of a particular sequence or polymorphism. Alternatively, it can be binding information, such as the ability of a sequence to bind a particular binding partner, such as a primer or other polynucleotide, a nucleotide-binding protein, or a drug.

[0013] The SOI can be any length required depending on the workflow parameters and the information required. Typically, the SOI is 1 to 100,000 bases long. For example, the SOI can be 1 to 10,000 bases long, preferably 5 to 1,000 bases long, but is typically about 10 to 500 bases or 50 to 250 bases long.

[0014] In some cases, an SOI can be a polynucleotide of known sequence, or an SOI can be a polynucleotide of unknown or partially unknown sequence. An SOI can be DNA or RNA. For example, without limitation, an SOI can be a fragment of genomic DNA to be sequenced, or an SOI can be a polynucleotide encompassing a polymorphic region, a cDNA derived from an RNA sequence (such as mRNA, rRNA, or tRNA) by reverse transcription, or a polynucleotide fragment whose sequence is diagnostic of or indicative of a particular disease, condition, or organism (such as an infectious agent). Those skilled in the art will know that sequence information obtained for the forward strand can also be obtained from the sequence of the complementary (reverse) strand.

[0015] In some embodiments, the polynucleotide or SOI is provided with an adapter. The adapter can be a single- or double-stranded nucleotide sequence added to the 3' or 5' end of the sequence of interest, but preferably to both ends. Adapters are typically of known sequence and are designed to contain distinct or overlapping functional sequences configured for a specific downstream purpose. Such sequences can, for example, hybridize to a primer for a capture oligo (e.g., a sequencing primer or a clonal amplification primer) or to a complementary sequence in an intramolecular inversion probe, or can be configured to facilitate attachment to a bead or other surface. Additional functional sequences include identification tags such as nucleic acid barcodes. In distinct functional sequences, none of the nucleotides of a sequence form part of another functional sequence. In overlapping functional sequences, the nucleotides of one sequence can also be part of a second functional sequence; for example, n 5' nucleotides of a first functional sequence can form n 3' nucleotides of a second functional sequence. A universal adapter is an adapter carried by all polynucleotides in a population.

[0016] Adapters can be provided as part of an upstream workflow (i.e., an earlier step) or can be provided separately for the purpose of clonal amplification. The workflow can include one or more steps such as (by way of non-limiting example) isolation and fragmentation of larger polynucleotides such as genomic DNA, amplification of the fragments by PCR or isothermal amplification, and addition of adapters to the 5' and / or 3' ends of the fragments. In some embodiments, adapters can be ligated to the 5' and / or 3' ends of double-stranded fragments, which can then be denatured to provide single-stranded polynucleotides. Alternatively, adapters can be incorporated via a tagmentation reaction in which a transposase such as Tn5 cleaves DNA and adds short tags that can include the adapter sequence. In a further approach, adapters can be incorporated during a step in which fragments are amplified or replicated using tailed primers.

[0017] Typically, the surface is the surface of a substrate to which the polynucleotide is tethered. In non-limiting examples, the substrate can be an ISFET, a glass or silica substrate, an insoluble particulate substrate such as a microsphere or nanosphere, or part of a microfluidic device adapted to contact the tethered polynucleotide with a liquid reagent. The surface can be planar, such as the surface of a slide or semiconductor chip, or can be in the form of a well or other 3D structure (features). The surface / substrate can comprise a variety of materials, including inorganic materials such as glass, silica, or Ta2O5, or organic materials, including various polymers.

[0018] In one embodiment, the surface is the surface of a semiconductor chip comprising a field-effect transistor (FET) array for sensing chemical and / or biological reactions, including sequencing reactions. Such devices are well known in the art (e.g., U.S. Pat. Nos. 7,686,929; 8,685,228; 8,986,525; U.S. Patent Application Publication No. 2010 / 0137143). A preferred embodiment includes a semiconductor chip comprising an ion-sensitive field-effect transistor (ISFET) array useful as a sensing device for various reactions, including nucleic acid sequencing reactions. In particularly preferred embodiments, the chip further comprises an array of wells disposed above and in fluid contact with the ISFET array. In these embodiments, sequencing reactions typically occur within the wells, and ion release is detected by the ISFET sensor. In particularly preferred embodiments, the chip comprises a flow cell mounted on the chip (with or without wells) for delivery and removal of fluids to and from the chip / ISFET array. The term surface also includes, but is not limited to, surfaces that have been modified to provide functional groups to which polynucleotides can be attached, such as by directly functionalizing the surface or by coating the surface with a polymer bearing appropriate functional groups.

[0019] Polynucleotides can be tethered to a surface using a number of approaches, for example, they can be chemically bound to the surface, such as by covalent bonding, or they can be captured by a capture moiety that is itself bound to the surface.

[0020] In some embodiments, tethering can occur via an affinity tag, such as the interaction between a protein or peptide and its cognate ligand. Typically, the ligand is attached to the polynucleotide, and the protein or peptide is attached to the surface as a capture moiety. Such pairs include, for example, biotin and avidin, biotin-binding partners such as NeutrAvidin™ (Thermo Fisher) or streptavidin, although many more are known, well-studied, and commercially available. Biotinylation of nucleic acids is well-known, and biotin is particularly useful because of the strong interaction between biotin and its binding partner. Furthermore, biotin-tagged oligos are available, for example, from numerous sources. In some cases, surfaces can be coated to provide appropriate functional groups to which capture moieties, such as polynucleotides or capture oligos, can be linked.

[0021] Polynucleotides or oligos can be tethered via their 5' or 3' termini. Tethering can occur via functionalized 5' or 3' nucleotides. Typically, in the present invention, polynucleotides and oligos are tethered to surfaces via their 5' termini. The exact chemistry of attachment of polynucleotides to surfaces depends on the surface involved, and many chemistries are commercially available for this purpose, including numerous so-called "click chemistry" approaches (e.g., Click Chemistry, a Powerful Tool for Pharmaceutical Sciences (2008) Hein, et al.; Pharm Res 25(10):2216-2230 and A Hitchhiker's Guide to Click-Chemistry with Nucleic Acids (2021) Fantoni et al., Chem Rev, 121:7122-7154).

[0022] In some embodiments, a tethered polynucleotide is provided by hybridizing a polynucleotide to a capture oligo and extending the capture oligo to provide a complementary copy of the polynucleotide tethered to the surface via its 5' end. Hybridization can occur between a complementary sequence in the 3' portion of the capture oligo and the 3' portion of the polynucleotide. The 3' portion of the polynucleotide can include an adapter that includes a sequence complementary to the 3' "capture" region of the capture oligo.

[0023] After hybridization, the 3' end of the capture oligo acts as a primer and is extended onto the captured polynucleotide using a polymerase to provide a double-stranded polynucleotide. The two strands are then denatured, leaving a single-stranded polynucleotide tethered to the surface via its 5' end. This tethered polynucleotide contains a complementary copy of the original captured polynucleotide (including any 5' and 3' adapters), which is now contiguous with the capture oligo and thus tethered to the surface via its 5' end.

[0024] In some embodiments, the surface is provided with a single population of capture oligos having identical 3' capture sequences. In other embodiments, the surface may be provided with a mixed population of two, three, four, or more capture oligos, each containing a different 3' capture sequence. For example, the surface may be provided with two mixed populations of capture oligos, where the first population contains a 3' sequence complementary to the 3' adapter (reverse primer) and the second population contains a 3' sequence complementary to the 3' end of the extended complementary polynucleotide and acts as a forward primer, as further described herein.

[0025] The capture oligo may contain nucleotide sequences or other mechanisms that provide additional functionality (other than the 3' capture moiety), such as, for example, spacer sequences or chemical spacers that distance the tethered moiety from surface interactions, and chemically modified nucleotides or linkages (such as phosphorothioate linkages) at the 3' end that protect the oligo from the 3' to 5' exonuclease activity of polymerase enzymes.

[0026] In some embodiments, a single-stranded polynucleotide may comprise, from 5' to 3', a 5' flanking region, a nucleotide sequence comprising or consisting of a nucleotide sequence of interest, and a 3' flanking region. The 5' and 3' flanking regions are directly linked to the 5' and 3' ends of the sequence of interest so as to be contiguous with the sequence of interest. Typically, the 5' and 3' flanking regions are adapters.

[0027] In a preferred configuration, the 5' and 3' flanking regions are of known and predetermined sequence. Typically, these are "universal adapters," i.e., adapters common to all oligonucleotides applied to the surface. The 5' and 3' flanking regions or adapters are typically of different sequences. Each flanking region can contain one or more sequences complementary to primers suitable for various functions. For example, a flanking region or adapter can contain one or more regions complementary to clonal amplification primers, and / or the 3' flanking region can contain a region complementary to a sequencing primer. As described further below, they can contain separate universal regions complementary to intramolecular inversion probe (MIP) 3' and / or 5' complementary regions. In some cases, these can be separated from other binding regions, allowing for independent design of highly specific binding regions for MIP complementary regions.

[0028] In the case of a 3'-flanking region, it is advantageous to link the flanking region directly to the 3'-end of the sequence of interest so that the 5'-most nucleotide of the flanking region is directly linked to the 3'-most nucleotide of the sequence of interest. A sequencing primer complementary to the adapter sequence and having its 3'-end coterminous with the 5'-end of the adapter can initiate sequencing at the 3'-most end of the sequence of interest. Alternatively, a short, known sequence of nucleotides in the flanking region between the 3'-end of the sequencing primer and the 5'-end of the sequence of interest can serve as an internal sequence calling standard. For example, a short sequence of one, two, three, four, or five nucleotides can be used. Other functional sequences within the flanking region or adapter include barcodes or other identification sequences.

[0029] In some embodiments, the 3' end of the 3' flanking region is located at the 3' end of the tethered polynucleotide. In some embodiments, the 3' end of the 3' flanking region is located at the 3' end of the tethered polynucleotide and is flush with the 3' end of the tethered polynucleotide.

[0030] In some embodiments, the 3' end of the sequence of interest is located at the 3' end of the tethered polynucleotide. In some embodiments, the 3' end of the sequence of interest is located at the 3' end of the tethered polynucleotide and ends at the same end as the 3' end of the tethered polynucleotide. This may be the case, for example, when the sequence of interest does not have a 3' flanking region.

[0031] In some embodiments, a single type of polynucleotide can be dotted on the surface in different regions. In another approach, polynucleotides can be applied to the surface by immersing the surface in a dilute solution of a population of polynucleotides that may contain different sequences of interest. The dilution of polynucleotides is calculated so that single polynucleotides are captured in a spatially separated manner, so that clonal amplification provides separate clusters. In some embodiments, the surface is equipped with features such as wells that are (statistically) intended to capture single polynucleotides.

[0032] Once anchored to the surface, the polynucleotides can be clonally amplified to provide a first clonal cluster comprising a plurality of single-stranded polynucleotides anchored to the surface via their 5' ends and comprising the nucleotide sequence of interest. The first clonal amplification can be performed using a number of methodologies well known in the art and selected according to the needs. A non-limiting selection of these is described below. Methods include (but are not limited to) bridge amplification (see U.S. Pat. No. 9,593,328), rolling circle amplification, exAMP (see U.S. Pat. No. 9,169,513), emulsion PCR (particularly suitable for use with microspheres, see U.S. Pat. App. Pub. No. 20100261230), and template walking (e.g., U.S. Pat. App. Pub. No. 2012 / 0156728). Clonal amplification by recombinase polymerase amplification (RPA) has also been reported and is discussed in co-pending application GB 2110479.9. Any method of clonal amplification is suitable as long as it provides a clonal cluster comprising a plurality of, preferably single-stranded, polynucleotides tethered to a surface via their 5' ends. In some embodiments, the selected clonal amplification method provides only a sense copy of the SOI or an antisense copy of the SOI, but not both, in the first cluster. In some embodiments, the method provides both a sense copy and an antisense copy of the SOI in the same first cluster. In some embodiments, the polynucleotides in the first cluster comprise only a single copy of the SOI. In some embodiments, the tethered polynucleotide preferably comprises a free 3' end. In one embodiment, the clonal amplification method provides a clonal cluster comprising a plurality of polynucleotides with free 3' ends tethered to a surface via their 5' ends, preferably comprising only one copy of the SOI.

[0033] In one approach for bridge amplification, fragments are provided with 5' and 3' adapters. A mixed population of both forward and reverse primers is tethered to a surface. The single-stranded template binds to the reverse primer on the surface via its 3' adapter, and a polymerase extends the primer onto the template to generate a tethered, complementary copy of the template with its 3' and 5' adapters. The duplex is then denatured (typically by exposing the duplex to a denaturing solution or by cleaving the duplex. m This duplex is denatured (by heating above 1000 kJ / min) to release the original template. The 5' adapter of the tethered complementary copy is then captured by the tethered forward primer, forming a bridge attached to the surface. Extension of the forward primer using the tethered, bridged complementary strand provides a copy of the original single-stranded fragment linked to the surface via the second capture oligo. The double-stranded bridge is then denatured, leaving copies of both the forward and reverse strands tethered to the surface via their 5' ends. After washing, the 3' ends of these strands are then captured by the tethered forward and reverse primers, and further cycles of extension and denaturation then occur, spreading the clonal clusters across the surface until clonal amplification is complete (see, e.g., U.S. Pat. Nos. 10,370,652 and 7,972,820).

[0034] Clonal amplification by recombinase polymerase amplification (RPA) utilizes two primers, one of which (the reverse primer) is a capture oligo tethered to a surface, and the second (the forward primer) is in solution. A 5' and 3' adapter is attached to the polynucleotide fragment. The 3' adapter hybridizes to the capture oligo, and a polymerase is then used to extend the 3' end of the capture oligo onto the polynucleotide template, providing a complementary copy of the polynucleotide that is tethered to the surface via its 5' end but free at its 3' end. A reaction mixture containing recombinase(s), a single-stranded DNA-binding protein, and a strand-displacing polymerase is then provided, and the concerted action of the recombinase(s) and the single-stranded DNA-binding protein separates the 3' ends of the duplex. This allows the forward (solution-phase) primer to access the 3' end of the tethered complementary strand, allowing the strand-displacing polymerase to extend the forward primer and ultimately expel the original fragment into solution. In some systems (e.g., T4), a recombinant mediator protein can be provided to mediate polymerase access to the single-stranded DNA. The released fragment is then free to hybridize to additional capture oligos to repeat the process. Increasing the viscosity of the reaction mixture can reduce local diffusion of the released template, thus reducing cluster spreading. This approach is further discussed in relation to Figure 1.

[0035] In template walking (U.S. Patent Application Publication No. 2012 / 0156728), the capture oligo has a low T m and a 3' end of, for example, a low T m The 3' end of the can be a portion with a high proportion of A or T (or U). mThe 3' end of the SOI may be, for example, a sequence of 20A. The SOI is provided with an adaptor, and the 3' adaptor contains a region (e.g., 20A or 30A) complementary to the 3' region of the capture oligo, with the remainder of the SOI having a higher Tm than the 3' end of the adaptor. The polynucleotide with the 5' and 3' adaptors is provided with a low Tm of the capture oligo. m The 3' end of the capture oligo is hybridized to the polynucleotide, and the 3' end of the capture oligo is extended using the polynucleotide as a template to provide a complementary copy that is tethered to the surface via its 5' end. The duplex has a relatively high T m and low T m The temperature is then increased to dehybridize the 3' end of the original polynucleotide from the capture oligo without dissociating the entire duplex across the SOI. As the temperature is decreased, the low T m The region can rehybridize to an adjacent capture oligo, allowing the capture oligo to be extended once more from there, thereby providing a template for displacing the original polynucleotide from the original extended capture oligo.

[0036] Binding equilibrium exclusion amplification (exAMP) was developed in response to the problem of clonal contamination, particularly in well-like setups in which the workflow attempts to capture and amplify a single polynucleotide fragment per well on a surface, with capture and amplification occurring simultaneously. While capture is rapid, if amplification is slow, there is a high probability that a second polynucleotide fragment of a different sequence will be captured nearby, resulting in clusters generated from two different sequences, resulting in overlap and reduced clonality. Binding equilibrium exclusion amplification attempts to maintain clonality by ensuring that clonal amplification exceeds the rate of capture of new polynucleotide species. This reduces the local supply of capture oligos and prevents new polynucleotide species from accessing the capture oligo lawn.

[0037] One way to do this is to provide the oligonucleotide fragments (in double-stranded form) along with a DNA polymerase, a single-stranded binding protein (ssBP), and a recombinase. The concentration of polynucleotides in solution is adjusted so that the rate of capture of single-stranded polynucleotides by capture oligos is much slower than the rate of clonal amplification and the resulting depletion of nearby capture oligos.

[0038] Clonal amplification methods can provide clusters of only one strand (such as RPA) or both forward and reverse strands (e.g., bridge amplification). The selected CA method preferably results in a cluster of linear polynucleotides containing the sequence of interest and having a free 3' end. In one embodiment, the linear polynucleotide contains a single copy of the SOI and has a free 3' end. Clonal amplification by RPA is typically the preferred approach used in the present invention.

[0039] Once the first clonal amplification is complete, the surface comprises clusters of polynucleotides arranged in a two-dimensional feature (or "lawn") on the surface, with each polynucleotide comprising a sequence of interest. Typically, at this point, each polynucleotide comprises only one copy of the SOI. However, by extending at least some of the tethered single-stranded polynucleotides in the first cluster by the addition of one or more additional copies of the SOI, it is possible to provide a cluster of tethered polynucleotides comprising multiple copies of the original nucleotide sequence of interest (or at least some of the tethered polynucleotides comprise multiple copies of the original nucleotide sequence of interest). In this way, the number of copies of the sequence of interest in the cluster can be increased several-fold without having to tether additional copies of the oligonucleotide to the surface, yet clonality is maintained. This approach overcomes the problem of increasing the number of SOIs per unit area in a manner that does not tend to spread the cluster. This tends to improve signal by providing what is effectively a three-dimensional cluster with multiple copies of the SOI, and may also provide multiple copies of sequencing adapters and other functional sequences useful in downstream processing of the SOI.

[0040] In one advantageous embodiment, the anchored single-stranded polynucleotides in the first cluster can be extended (at least of the SOI) by rolling circle amplification (RCA). RCA can be used to replicate only the sequence of interest (or at least a portion thereof), or can also replicate all or part of the 5' and / or 3' adapters. In this way, the method provides copies of the sequencing adapters, if present in the original polynucleotide, positioned to initiate sequencing from each SOI, as well as copies of any other functional sequences, if present in the original polynucleotide, that are useful in downstream processing of the SOI.

[0041] RCA extension can be achieved in many ways and can be applied whether the SOIs have known or partially known sequence or unknown sequence, making it a useful approach for increasing the density of SOIs within clonal clusters and adaptable to a variety of workflows.

[0042] In one embodiment, rolling circle amplification may be performed by a process comprising: (a) providing a single-stranded circular nucleotide probe comprising a nucleotide sequence complementary to the SOI in a first clonal cluster and, optionally, to 5' and 3' adapters or portions thereof; and (b) using the cyclic nucleotide probe as a template, preferably using a strand-displacing polymerase, to extend the 3' end of the tethered single-stranded polynucleotide, thereby extending the 3' end of the tethered single-stranded polynucleotide (or at least a portion thereof) by the addition of one or more additional copies of the sequence of interest.

[0043] In some embodiments, a single-stranded circular probe can be provided as a preformed circular probe containing a complementary copy of the target sequence.The complementary copy of the SOI is then hybridized to the anchored SOI (and optionally to any 5' or 3' adapter or part thereof), and the 3' end of the anchored oligonucleotide is then extended by repeatedly replicating the circular probe with a strand-displacing polymerase.This approach is particularly useful when the SOI is of a known sequence.Usefully, a circular probe can be prepared in solution, for example, by ligating the free ends of a linear probe containing a complementary copy of the SOI.

[0044] In a further modification of this approach, the single-stranded circular nucleotide probe contains a nucleotide sequence complementary to the SOI and, adjacent to the SOI, complementary copies of 5' and / or 3' adapters whose sequences are not present in the anchored polynucleotide. RCA of the anchored polynucleotide then incorporates functional adapters into the extending strand.

[0045] In either case, the circular probe may further comprise an optional linker region extending from the 3' end of the sequence complementary to the SOI and, optionally, the 3' adaptor, to the 5' end of the SOI and, optionally, any 5' adaptor. Such single-stranded circular probes may be constructed in a number of ways well known to those of skill in the art and discussed elsewhere herein.

[0046] In some embodiments, the circular probe is not provided as a preformed circular probe, but rather as a linear probe from which it can be synthesized in situ, i.e., on a surface, rather than as a preformed probe. In practice, this means that at least a portion of the linear probe is hybridized to the polynucleotide while the circular probe is being formed; preferably, the circularization process occurs while the probe is hybridized to the polynucleotide.

[0047] In one approach, the in situ formation of a circular probe is (a) providing a linear single-stranded nucleotide probe comprising, in a 5' to 3' direction, a 5' complementary region (5'CR), an optional linker region, and a 3' complementary region (3'CR), wherein the 5'CR and 3'CR are configured to hybridize to distinct sequences (hybridization sites) on an anchored single-stranded polynucleotide; (b) hybridizing the 5' CR of the probe and the 3' CR of the probe to the anchored single-stranded polynucleotide, wherein the 5' CR hybridizes to a sequence upstream of the sequence to which the 3' CR hybridizes (i.e., closer to the 5' end of the anchored polynucleotide than the sequence to which the 3' CR hybridizes); (c) optionally extending the 3' end of the linear probe using the sequence of the single-stranded polynucleotide as a template; and (d) circularizing the probe by ligating the optionally extended 3' end of the probe to the 5' end of the probe. Such linear probes are sometimes called "molecular inversion probes."

[0048] The sequences of the 5' CR and 3' CR of the probe are selected so that, after optional extension and ligation, a single-stranded circular probe containing a complementary copy of the sequence of interest is provided. The sequences of the 5' and 3' CR can be selected in a number of configurations.

[0049] In non-limiting examples, a 5' CR can hybridize to the 5' adapter only, to the 5' adapter and the 5'-most portion of the SOI, or to the 5'-most portion of the SOI only. Similarly, a 3' CR can hybridize to the 3' adapter only, to the 3' adapter and the 3'-most portion of the SOI, or to the 3'-most portion of the SOI only.

[0050] The 5'CR may hybridize to a sequence entirely upstream of the SOI (i.e., toward the 5' end of the anchored polynucleotide from the SOI) (e.g., so that its 5'-most nucleotide is complementary to a nucleotide upstream of the 5'-most end of the SOI), or the 5'CR may hybridize to a sequence overlapping the 5' end of the SOI (e.g., so that its 5'-most nucleotide is complementary to a nucleotide within the SOI), or the 5'CR may hybridize to a sequence entirely within the SOI (e.g., so that the 3'-most nucleotide of the 5'CR may hybridize to the 5'-most nucleotide or to a nucleotide closer to the 3' end of the SOI).

[0051] Similarly, the 3'CR may hybridize to a sequence completely downstream of the SOI (i.e., toward the 3' end of the anchored polynucleotide from the SOI) (e.g., so that its 3'-most nucleotide is complementary to a nucleotide upstream of the 3'-most end of the sequence of interest), or the 3'CR may hybridize to a sequence overlapping the 3' end of the SOI (e.g., so that its 3'-most nucleotide is complementary to a nucleotide within the SOI), or the 3'CR may hybridize completely to a sequence within the SOI (e.g., the 5'-most nucleotide of the 3'CR may hybridize to the 3'-most nucleotide of the SOI).

[0052] In some situations, the 3'CR may hybridize and extend before the 5'CR hybridizes to its target. In these cases, the extending 3'CR may block the hybridization of the 5'CR, causing amplification to fail. This may result in a lower yield of extended polynucleotides and a poorer signal in the sequencing reaction. To prevent this from happening, the 5'CR may be hybridized to the anchored polynucleotide before the 3'CR is hybridized. This ensures that the 3' end of the probe cannot be extended before the 5' end of the probe is hybridized (see Figure 4A). In one approach, the sequences of the 5' and 3'CR are aligned such that the T of the 5'CR is aligned with the T of the 5'CR. m T in 3'CR m A protocol including an appropriate temperature profile is then selected to ensure that 3' extension and ligation occur as needed. For some nucleotide sequences, an appropriate T m It may be possible to select a sequence in which the 5' and 3' CRs of the MIP hybridize in the manner described above. For other nucleotide sequences, it may be possible to select a sequence in which the appropriate T m It may be necessary to provide 5' and 3' adapters containing sequences having the following structure: Such adapters can be added to the polynucleotide or sequence of interest using standard methodologies at an appropriate point in any preparation protocol or workflow.

[0053] The Tms of the 5' and 3' CRs should be sufficiently different to ensure that the 5' CR hybridizes before the 3' CR when an appropriate temperature profile is used. The profile takes into account throughput speed, yield, and unwanted by-products. Typically, the Tms should differ by 2-20°C, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15°C or more, although a difference of about 10°C has been shown to provide good results.

[0054] Thus, in a further embodiment, the present invention provides an improved intramolecular inversion probe for use in preparing a clonal cluster of anchored polynucleotides, such as those further described herein, the improved intramolecular inversion probe comprising or consisting of, in a 3' to 5' direction: (a) a 3' CR, (b) an optional linker region, and (c) a 5' CR, wherein the sequence of the 3' CR is selected to hybridize to a first portion of the anchored polynucleotide, and the sequence of the 5' CR is selected to hybridize to a second portion of the anchored polynucleotide that is distinct from the first portion, and wherein the Tm of the 5' CR is higher than the Tm of the 3' CR. This approach is illustrated in Figure 4B.

[0055] As described above and in further detail elsewhere herein, the first portion of the anchored polynucleotide may be separated from the second portion of the anchored polynucleotide (i.e., the two hybridization sites for the CRs of the MIP) by the sequence of interest, the complement of the sequence of interest, or portions thereof, depending on the selection of the sequences to which the 5' and 3' CRs hybridize.

[0056] Step (b) of the above process for in situ formation of a circular probe then comprises the addition of a T m Higher but 5'CR T mhybridizing the 5'CR of the probe to the tethered single-stranded polynucleotide at a temperature below T m Lower temperatures, especially the T of the 3'CR m The method may include hybridizing the 3'CR of the probe to the polynucleotide at a temperature of:

[0057] An exemplary MIP for use in this protocol is shown below: This MIP may be used in the embodiment of Example 6, as further described below, with a temperature profile of 95°C for 2 minutes, 55°C for 10 minutes to allow hybridization of the 5' CR, followed by 45°C for 30 minutes to allow hybridization of the 3' CR prior to extension and ligation.

number

[0058] In a further aspect of the present invention, there is provided a method for preparing a clonal cluster of tethered polynucleotides, the method comprising the steps of: (i) providing single-stranded polynucleotides tethered to a surface via either of their 5' ends; (ii) performing a first clonal amplification of the single-stranded polynucleotides to provide a first cluster of polynucleotides tethered to the surface via their 5' ends; and (iii) extending the tethered single-stranded polynucleotides in the first cluster by rolling circle amplification using a MIP described herein, in particular the improved MIP described above. The tethered polynucleotides may be any of those further described elsewhere herein. In one approach, a method for rolling circle amplification may include the steps of: (a) contacting polynucleotides of a first cluster with a MIP, such as the improved MIP described above; (b) hybridizing the 5' CR of the MIP to the tethered single-stranded polynucleotide at a temperature above the Tm of the 3' CR but below the Tm of the 5' CR, and then hybridizing the 3' CR of the probe to the polynucleotide at a temperature below the Tm of the 3' CR; (c) optionally extending the 3' end of the MIP; (c) circularizing the probe by ligating the optionally extended 3' end of the probe to the 5' end of the probe; and (d) contacting the circularized probe with a strand-displacing polymerase to extend the single-stranded polynucleotide.

[0059] As already mentioned, the RCA approach to sequence extension can be applied whether the SOI has a known sequence, a partially known sequence, or a completely unknown sequence. The unknown sequence can be amplified if the 5' and 3' CRs hybridize only to sequences within the 5' and 3' adapters of the SOI. Other configurations in which the adapters hybridize at least partially to the SOI generally require knowledge of the sequence of the SOI, or at least a portion of it.

[0060] A linear probe may further comprise a linker region. If present, the linker region extends from the 3' end of the 5'CR to the 5' end of the 3'CR. Similarly, if the probe is provided as a preformed circular probe, the probe may also further comprise a linker region extending from the 3' end of the sequence complementary to the SOI to the 5' end of the sequence complementary to the SOI, or the probe may comprise a region complementary to any flanking region present in the anchored polynucleotide, if a flanking region is present, from the 3' end of the 3' flanking region to the 5' end of the 5' flanking region.

[0061] In either case, the linker may contain one or more functional sequences that are replicated in complementarity as the linker is extended. These may include, for example, sequences complementary to, for example, a sequencing primer or an amplification primer, or may include sequences complementary to additional capture oligos as described elsewhere herein.

[0062] In one embodiment, the linker comprises a sequence that, when replicated to its complement, hybridizes to a second species of surface-bound capture oligo. In some embodiments, this sequence is different from that of the 3' or 5' adapter, providing for the optional use of branching RCA in the second round of clonal amplification, as further described elsewhere herein.

[0063] The length of the linker region need only be sufficient to accommodate any required functional sequences and ensure efficient circularization of the probe. Therefore, the linker can be of any appropriate length. For example, the linker can be 40 to 160 nt long, with 40 to 70 nt being preferred. A shorter linker ensures a smaller circular probe, thus ensuring less steric hindrance and more efficient RCA requiring fewer nucleotides.

[0064] In some embodiments, when hybridized to a tethered polynucleotide, the 3' end of the linear probe can be separated from the 5' end by one or more nucleotides. Typically, the 3' and 5' ends are separated by all or part of the SOI, depending on the position of the sequence to which the 5' and 3' CR hybridize. The 3' end of the probe can be extended using a polymerase, preferably a polymerase lacking 5' to 3' exonuclease activity. Examples of polymerases include T4 polymerase or Taq polymerase. Once the 3' end is extended, the extended 3' end can be ligated to the 5' phosphorylated end using a ligase such as T4 ligase or Taq ligase to circularize the probe. In some embodiments, the 5' and 3' CRs of the linear probe are together extended to cover the entire SOI, in which case no extension is required and the 3' and 5' ends are simply ligated to circularize the probe.

[0065] Whether the circular probe is provided preformed or formed in situ, once the probe is in place, the 3' end of the tethered polynucleotide can be extended using the circular probe as a template. Adding one or more additional copies of the SOI to the 3' end of the tethered polynucleotide provides a cluster of tethered polynucleotides in which the polynucleotides of the cluster (or at least some of them) contain multiple copies of the nucleotide sequence of interest. Extending the tethered single-stranded polynucleotides in the first cluster provides additional copies of the SOI without requiring an increase in the density of capture oligos and without expanding the area occupied by the original cluster.

[0066] In some embodiments, for example, if the circular probe does not hybridize to the entire 3' adapter of the SOI, there may be a short single-stranded 3' overhang, which can be removed using a 3'-5' exonuclease or the 3'-5' exonuclease activity of a polymerase such as Phi29 polymerase can be utilized.

[0067] In some cases, the 3' end of the tethered polynucleotide may be extended around the probe before circularization and ligation are complete. If that occurs, the 5' end of the probe may be removed from the polynucleotide, and circularization and ligation cannot occur. Thus, in some embodiments, extension of the 3' end of the polynucleotide is prevented (or at least delayed) while gap-filling extension and ligation are completed. In some embodiments, this is achieved by hybridizing a single-stranded circular polynucleotide probe to the tethered polynucleotide, leaving a 3' single-stranded overhang. Advantageously, the overhang is not complementary to the probe, thus preventing the formation of a double-stranded feature suitable for extension by a polymerase. The 3' extension may then be removed by exonuclease activity prior to replication of the circular probe.

[0068] In a further embodiment, a single-stranded circular polynucleotide probe is hybridized to a 3' adapter, leaving a 3' overhang sufficient to hybridize a protection oligonucleotide whose 5' end coincides with the 3' end of the overhang. The oligoduplex has a Tm lower than that of the probe, preventing chain extension until the oligo is removed by raising the temperature above the Tm of the oligoduplex. The overhang can then be removed as described above.

[0069] The 3' end of the anchored polynucleotide around the probe is extended using a polymerase, preferably a strand-displacing polymerase. In this way, as synthesis proceeds and multiple copies of the SOI are synthesized, the probe is constantly separated from the extending amplicon. In the extended amplicon, the SOI copies are interspersed with sequences complementary to the remainder of the circular probe. Depending on the sequences to which the 5' and 3' CR of the linear probe hybridize, the SOI can also be flanked by full-length or shortened forms of adapters.

[0070] If the CR incorporates a complementary copy of the various functional sequences in the adapter, the functional sequences are replicated in the extending amplicon, and the functional sequences remain associated with the SOI. Thus, in some embodiments, the sequences of the 5' CR of the probe and the 3' CR of the probe are selected to provide, after optional extension and ligation, a single-stranded circular probe containing a complementary copy of the sequence of interest and copies of either the 5' adapter, the 3' adapter, or both; or copies of any adapter sufficient to include one or more functional sequences, such as any of the functional sequences further discussed herein. When the 3' adapter contains a region complementary to a sequencing primer, it is particularly preferred that the sequence of the 3' CR be selected to preserve the functional relationship between the sequencing primer and the SOI, as further described herein.

[0071] The combination of MIP hybridization, circularization and rolling circle amplification is sometimes referred to herein as Linear Cluster Concatemerization or LCC.

[0072] In one embodiment, a second species of capture oligo can be provided on the surface as a mixed population with the first capture oligo. The second capture oligo hybridizes to the complement of the sequence within the linker region of the circular probe, but does not hybridize to the SOI or its 5' or 3' adapter. As the circular probe is repeatedly replicated, it provides complementary copies of the linker interspersed with copies of the SOI and adapter. The complementary copies of the linker expose multiple sequences that hybridize to the second species of capture oligo, so that the extending strand is captured at multiple points by the second capture oligo. The 3' end of the capture oligo is then extended by a polymerase to form a complementary copy of the SOI and a sense copy of the linker. As the new strand extends, it displaces the next duplex from the next copy of the second capture oligo, generating multiple tethered polynucleotides containing multiple complementary copies of the SOI alternating with copies of the linker region. Using sequencing primers complementary to the appropriate adapters, it is then possible to sequence both the forward and reverse strands of the SOI from the same cluster.

[0073] Once the expanded clonal clusters are provided, the expanded clonal clusters can be used for various downstream processes, as further described herein. In one embodiment, the sequence or subsequence of the SOI within the 3-D cluster can be determined by nucleotide sequencing methods.

[0074] Sequencing can be by any method suitable for surface-immobilized or tethered polynucleotides, particularly the approach known as "sequencing by synthesis (SBS)." In this approach, after preparation of a clonal cluster, a sequencing primer is annealed to the 3' adapter, and incorporation of successive nucleotides into the growing strand complementary to the SOI is detected. In some approaches, the nucleotides are labeled with fluorescent dyes. The dyes can act as chain synthesis terminators. After each dNTP incorporation, the dye is imaged to identify the base and cleaved to allow incorporation of the next nucleotide. In other approaches, clonally amplified polynucleotides are tethered to the surface of a semiconductor chip, such as an ISFET, and incorporation of each nucleotide in the SOI is detected by detecting the protons released during incorporation of the dNTP into the sequence (one per incorporated nucleotide). In this case, fluorescently labeled dNTPs are not required.

[0075] While many variations of next-generation sequencing exist, including those that rely on SBS, the clonal amplification approach described herein is suitable for use with any that can be performed on a fixed population of polynucleotides.

[0076] In a further embodiment, the present invention also provides a substrate having attached to its surface a plurality of single-stranded polynucleotides arranged in a plurality of clonal clusters, wherein the polynucleotides within each clonal cluster comprise multiple alternating copies of a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is common to the single-stranded polynucleotides of each cluster and the first sequence is not common to the single-stranded polynucleotides of each cluster. In a further embodiment, the present invention also provides a clonal cluster of single-stranded polynucleotides as described herein, wherein the polynucleotides of the cluster are tethered to a surface via their 5' ends.

[0077] In some embodiments, the first sequence, which is identical to the sequence of interest discussed elsewhere herein, can be present in the cluster in either sense or antisense form, but not both. In some embodiments, both the sense and antisense versions of the first sequence are present in the cluster. In this case, the polynucleotides of each cluster comprise sense and antisense copies of the first sequence and sense and antisense copies of the second sequence, and the sense and antisense copies of the second sequence are common to the single-stranded nucleotides of each cluster, but the sense and antisense copies of the first sequence are not common to the single-stranded nucleotides of each cluster.

[0078] Typically, the polynucleotides in each cluster comprise multiple copies of a first sequence (or its antisense). In a preferred embodiment, the polynucleotides in each cluster comprise at least two copies, preferably at least three copies, more preferably at least four copies, and even more preferably at least five copies of the first sequence or its antisense. Similarly, the polynucleotides in each cluster comprise multiple copies of a second sequence (or its antisense). In a preferred embodiment, the polynucleotides in each cluster comprise at least two copies, preferably at least three copies, more preferably at least four copies, and even more preferably at least five copies of the second sequence or its antisense.

[0079] Typically, the polynucleotides within each cluster have a free 3' end. Typically, the surface initially contains one, two, three, four, five, or more species of capture oligos, so in some instances, regions that do not contain clustered polynucleotides contain unused capture oligos and therefore may also contain one, two, three, four, five, or more species of capture oligos that typically remain unhybridized to polynucleotides. However, unused capture oligos are typically locally depleted during the first round of clonal amplification, so that unused capture oligos are rare within a cluster. In some embodiments, the surface contains a single species of capture oligo, and in some embodiments, the surface contains two species of capture oligos.

[0080] The number of clusters containing the same first sequence depends on the number of unique SOIs present, so for example, if there are only two SOIs, 50% of the clusters will contain the same first sequence.

[0081] As used herein, a free 3' end means that the terminal 3' nucleotide has a free 3'-OH group that can be extended by a polymerase, such as a strand-displacing polymerase. The free 3' end is not part of a hairpin or other secondary structure.

[0082] As used herein, clonal amplification refers to the expansion of a single polynucleotide to provide multiple copies (clones) with the same sequence. Single polynucleotides and their clones are typically anchored to the surface of a substrate via their 5'- or 3'-terminal nucleotide, preferably via the 5'-terminal nucleotide. A clonal cluster is a plurality of polynucleotide clones grouped in a particularly tightly packed manner in a discrete region of a surface. Clonal clusters are typically derived from the clonal amplification of a single, original anchored polynucleotide, but may alternatively be derived by dotting a surface with a composition comprising a single type of polynucleotide. In some embodiments, clonal clusters may intentionally contain more than one type of polynucleotide. For example, a clonal cluster may contain a copy of a single-stranded polynucleotide and a complementary copy of the single-stranded polynucleotide. Clonal clusters may overlap with other clonal clusters, but preferably do not overlap.

[0083] As used herein, two sequences are considered complementary if every nucleotide in one strand undergoes continuous Watson-Crick base pairing with a nucleotide in the other strand; these two strands are complementary copies of each other.

[0084] As used herein, when two sequences are said to hybridize to one another, the two sequences hybridize under the conditions of the process in question; thus, when a primer is said to hybridize to a nucleotide sequence, the primer hybridizes under the conditions (e.g., ionic strength, pH, temperature) of primer extension, sequencing, etc.

[0085] As used herein, the term "polynucleotide" refers to a polymer of two or more nucleotides linked by covalent bonds, typically phosphodiester bonds. In some cases, polynucleotides may contain "non-natural" bonds, such as phosphorothioate bonds, where it is advantageous to reduce or prevent exonuclease activity, for example. The terms polynucleotide fragment, oligonucleotide, or oligo have the same meaning as polynucleotide, and these terms are used interchangeably. Polynucleotides can be single-stranded or double-stranded, and can be DNA, RNA, or a hybrid DNA / RNA duplex.

[0086] As used herein, the term "tethered" means that the referenced polynucleotide is attached via a chemical bond, preferably a covalent bond, to the surface or to any coating applied to the surface. [Brief explanation of the drawings]

[0087] [Figure 1A]

[0023] Figure 1A provides an exemplary schematic diagram of clonal amplification using a recombinase polymerase approach. Figure 1A is a double-stranded DNA fragment. [Figure 1B] An exemplary schematic diagram of clonal amplification using a recombinase polymerase approach is provided. Figure 1B is a fragment with 5' and 3' adapters. [Figure 1C] An exemplary schematic diagram of clonal amplification using a recombinase polymerase approach is provided. Figure 1C shows the melted duplex. [Figure 1D]

[0023] Figure ID provides an exemplary schematic diagram of clonal amplification using a recombinase polymerase approach. Figure ID illustrates the process of clonal amplification using recombinase polymerase amplification. [Figure 1E]

[0023] Figure 1E provides an exemplary schematic diagram of clonal amplification using a recombinase polymerase approach. Figure 1E is a substrate with tethered clusters of identical sequences. [Figure 2] FIG. 2 provides an exemplary schematic illustrating one approach to rolling circle amplification of a sequence of interest, which is particularly applicable when the sequence is known. [Figure 3] FIG. 3 provides a further exemplary schematic illustrating one approach to rolling circle amplification of a sequence of interest using intramolecular inversion probes (MIPs). [Figure 4A] Figure 4A shows the effect of 3'CR ligation prior to 5'CR, (i) indicates successful circularization and (ii) indicates unsuccessful circularization. [Figure 4B] Figure 4B shows a modified MIP in which the 5' CR has a Tm of 60°C and the 3' CR has a Tm of 55°C. An exemplary circularization protocol to be used would then be denaturation at -95°C for 2 minutes, 5' annealing at -60°C for 5 minutes, 3' annealing, extension and ligation at -50°C for 10 minutes. [Figure 5A] Figure 5A is a fluorescent micrograph composite image showing the results of a spotting trial of clonal amplification with 50 uM capture oligo (optimal for RPA) and 2 uM capture oligo (optimal for RPA + LCC) on both YdfU and ddl templates. Test templates were clonally amplified by either RCA alone or RCA followed by LCC. Templates were tethered to the surface of an ISFET. At this magnification, individual clonal clusters are visible in only a few spots. [Figure 5B] FIG. 5B shows SensoSpot® scan data of the spot in 5A. [Figure 6]Figure 6 shows data from sequencing reactions performed on the RPA clonal cluster (YdfU) and the double-template RPA+LCC clonal cluster (YdfU and ddl). Graph A provides read length data for the RPA and RPA+LCC clusters. Graph B gives the distribution of ARL-e. In Graph A, individual reads are plotted as aligned read length (ARL; total length from start to end) on the x-axis versus aligned read length minus error (ARL-e; total alignment length from start to end minus the number of errors) on the y-axis. Portions of the histogram show the distribution of reads on the opposite axis. The histogram across the top shows the distribution of ARL (bp), and the histogram to the right of the plot shows the distribution of ARL-e (bp). Graph B gives the distribution of ARL-e. Graph C shows the signal resulting from the incorporation of a single nucleotide into the growing sequencing strand during sequencing by synthesis (RPA and LCC 1-mers distinguished from each other). [Figure 7] Figure 7 shows fluorescence micrographs illustrating clonal clusters derived from single captured polynucleotides on a flood-filled ISFET chip. Clusters induced by RPA (A) can be seen as faint cloud-like features (arrows). White dots are control spots. Micrograph B shows clonal clusters resulting from LCC amplification after RPA (arrows). RPA was performed with capture oligo applied at 25 uM, whereas RPA + LCC was performed with oligo applied at 1 uM. The capture oligo was extHDA72R and the template was YdfU. Graph (C) shows the intensity of individual pixels across each image for five chip images. For each chip, intensity (X-axis) is plotted against the % of pixels (Y) of at least that intensity. [Figure 8]Figure 8 shows data from an experiment similar to Figure 5, but in which an improved MIP was used. The graph, as in Figure 7, shows the intensity of individual pixels across each image for six spot images. For each chip, intensity (X axis) is plotted against the % of pixels (Y) that are at least that intensity. [Figure 9] Figure 9 provides a grayscale image of a single spot resulting from tethering of the capture oligo onto a glass slide, followed by hybridization of an equimolar mixture of YdfU and ddl templates and subsequent clonal amplification. After the RPA step and then after LCC, the spot was probed with both the YdfU and ddl fluorescent probes. In the image on the left, arrows indicate individual ddl clonal clusters. The image on the right highlights the same clones as dark spots, indicating that these clusters are not detected by the YdfU probe even after LCC and that clonality is maintained. [Figure 10] Figure 10 shows 1-mer intensity data from sequencing reactions performed on YdfU clonal clusters derived from LCCs using RPA-only or RPA+ solutions to generate circles. Graph A provides read length data for clusters generated by RPA and by LCCs using circles generated in RPA+ solutions. Graph B provides the distribution of ARL-e as in Figure 6. Graph C shows the signal derived from the incorporation of a single nucleotide into the growing sequencing strand. [Figure 11] FIG. 11 illustrates a custom flow cell attached to the ISFET chip to assist in the delivery and flow of reagents onto the ISFET chip surface.

[0088] DESCRIPTION OF THE DRAWINGS Referring to Figure 1 and the description elsewhere herein, clonal amplification using surface-phase recombinase polymerase amplification (RPA) uses a reverse primer (capture oligo) (1) attached to a surface (2) to capture a single-stranded polynucleotide (3). Methods for tethering the capture oligo are described elsewhere herein, and the generality of this method is well known.

[0089] The single-stranded polynucleotide (3) can be derived from exemplary upstream workflows (A, B, C), which can include providing a polynucleotide fragment (4) and providing adapters (5, 6) at the 5' and / or 3' ends. Separation of the duplex (if the fragment is double-stranded) provides the single-stranded polynucleotide (3).

[0090] The capture oligo (1) has a sequence in its 3' region that is complementary to the 5' region of the polynucleotide, such as the 5' adapter (7), facilitating capture of the polynucleotide. The 3' end of the capture oligo is extended by a polymerase along the captured polynucleotide to provide a complementary copy of the polynucleotide. This complementary copy (8) is tethered to the substrate via its 5' end, which now serves as the extended portion of the original capture oligo (1). The coordinated action of a recombinase and an ssDNA-binding protein allows access to the duplex for a solution-phase forward primer (10) that is complementary to the 3' end of the tethered complementary copy. A strand-displacing polymerase then extends the hybridized forward primer onto the complementary strand, displacing the original oligonucleotide while providing a duplicate copy of the original oligonucleotide. The original polynucleotide (3) is then free to be captured by an additional local capture oligo (12). Repeated rounds of primer hybridization and extension provide multiple copies of the original polynucleotide, which are themselves released into solution and captured by localized capture oligos to provide templates for further rounds of amplification. After denaturing the remaining duplex molecules, the tethered oligonucleotides in the clonal clusters are clonal copies of, but complementary to, the original polynucleotide (14).

[0091] Referring to Figure 2, in some approaches, particularly when the SOI is known, a circular probe can be provided as a preformed circular probe (21) containing a complementary copy (22) of the sequence of interest. The complementary copy of the SOI can then be hybridized to an anchored SOI (23) (and optionally to any 5' or 3' adapters (24, 25)), and the 3' end (26) of the anchored oligonucleotide is then extended by complementary repeated replication of the circular probe using a strand-displacing polymerase (P). The circular probe can be prepared from a linear probe (27) containing a complementary copy (22) of the sequence of interest. One or more flanking regions (28, 29) can be included. The 5' and 3' ends of the linear probe are then hybridized to a complementary splice at the two ends. The circular probe is then joined and ligated (L) by a linto-oligo (30). In the illustrated approach, optional flanking regions are joined to form a linker (31). If necessary, separation of the duplex and digestion of the splint completes the circular probe. If the flanking regions contain complementary copies of adapters, these will be incorporated into the extending amplicon even if they are not present on the tethered polynucleotide. In practice, the link between the free ends of the circle can occur at any point on the circle and does not have to be between two flanking regions; see Example 8.

[0092] Referring to Figure 3, in one approach, a circular probe is provided as a linear, single-stranded polynucleotide (40) comprising, from 5' to 3', a 5' CR (41), a linker region (42), and a 3' CR (43). These CRs hybridize to separate sequences on an tethered polynucleotide (44). In this case, these CRs hybridize to complementary sequences within the 5' and 3' adapters (45, 46). The 3' end of the linear probe is extended using a DNA polymerase that uses the tethered polynucleotide as a template to provide a complementary copy of the SOI (47). The probe is then circularized by ligating the extended 3' end of the probe to the 5' end of the probe. The 3' end of the tethered polynucleotide template is extended around the probe using a strand-displacing polymerase, which ultimately displaces the 3' end of the duplex (48) as it extends the amplicon.

[0093] Extension of the amplicon using RCA provides multiple copies of the tethered polynucleotide template (49), separated from each other by complementary copies of the linker (50). The 3' end of the original tethered polynucleotide is extended by repeated copies of the complementary circular probe.

[0094] A and B illustrate approaches to prevent extension of the 3' end of a tethered polynucleotide beyond that of the MIP, thus displacing the MIP from the template. In A, the 3' CR of the MIP hybridizes to the tethered polynucleotide (44), leaving a 3' overhang (51) that is not complementary to the MIP. This prevents the formation of a double-stranded form with a free 3' end suitable for extension by a polymerase. The 3' extension can then be removed by 3'-5' exonuclease activity prior to replication of the circular probe. In B, the 3' overhang (52) is sufficient to hybridize a short protective oligonucleotide (53) whose 5' end coincides with the 3' end of the overhang. The short duplex is then removed by the T of the probe. m Lower T m and the temperature is Tm Raising higher prevents chain extension until the oligo is removed, and the overhang can then be removed as described above.

[0095] Referring to Figure 11, a circuit board (91) includes an ISFET chip (92) having a surface (93). A gasket (94) provides a seal between the chip (92) and a flow cell (95) that is secured onto the ISFET chip (92) using screws (96) and provides a flow path on the chip between an inlet (97) and an outlet (98). The inlet and outlet may be sealed by threaded plugs (99) sealed with "O" rings (100) to prevent evaporation.

[0096] Other figures are further discussed in appropriate examples. The invention will now be illustrated by non-limiting examples and drawings. Further embodiments of the invention will be apparent to those skilled in the art in the light of these. [Example]

[0097] Unless otherwise stated, the following buffers and reaction mixtures were used in the examples below:

[0098] [Table 1]

[0099] [Table 2]

[0100] [Table 3]

[0101] [Table 4]

[0102] [Table 5]

[0103] Example 1: Tethering capture oligos to a surface The semiconductor chip used in this example was fabricated using standard CMOS techniques and contained an array of ion-sensitive field-effect transistor (ISFET) sensors whose voltage output responded to changes in pH in the fluid solutions present in the wells on the IC. The wells were micron-sized and fabricated by a standard photoetching process. To facilitate fluid delivery over the surface of the chip, a custom-made flow cell device (see Figure 9) was attached above the IC and well assembly. Oligonucleotides were tethered to the well surface by first activating the well surface with an acrylamide-based polymer coating (MCP-Click™ - Lucident Polymers, Sunnyvale, California, USA) containing free azide groups, followed by immersing the surface in a solution of the appropriate 5' DBCO capture oligo at a concentration optimized for either the 2-D RPA approach (50 μM) or the 3-D (RPA+LCC) approach (2 μM). Standard dibenzocyclooctyl (DBCO) / azide click chemistry was used to covalently attach the modified oligos. The sequence of the extHDA72R capture oligo is shown below:

number

[0104] In another approach, rather than immersing the chip in liquid, capture oligos were dotted onto the pre-washed chip surface in a volume of 280–300 picoliters. The oligos were then ligated onto the surface in the same manner.

[0105] In some experiments, glass slides were used instead of ISFET chips. When glass slides were used as substrates, they were first coated with Ta2O5 using electron beam evaporation according to standard methods, and then coated with the acrylamide-based polymer coating with free azide groups described above before conjugating the capture oligos.

[0106] Example 2: Hybridization of test template to capture oligo Synthetic DNA oligos (purchased from IDT Technologies) representing the D-alanine-D-alanine ligase gene (ddl) from Enterococcus faecalis and the Qin prophage protein YdfU gene (YdfU) from Escherichia coli were used as test templates. Their sequences are shown below: ddl template (SEQ ID NO: 3)

number

number

[0107] The underlined sequences are the universal forward and reverse primer binding sites; the bolded sequences are the universal binding sites for MIP 5' and 3' CR.

[0108] An equimolar mix of YdfU and ddl (2.5e4 copies / ul)—40 ul in 1x annealing buffer (20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 5 mM magnesium acetate, 0.01% v / v Tween 20, and 5% v / v DMSO)—was hybridized to the surface-conjugated extHDA72R oligo (conjugated as a spot or flood) via the universal adapter region by incubation at 95°C for 2 min, 50°C for 5 min, and 20°C for 10 min. After hybridization, the surface was washed twice with 60 ul of 1x RPA wash buffer (0.06% SSC pH 7, 0.06% v / v Tween 20).

[0109] Example 3: Clonal amplification of captured test templates by RPA Clonal amplification was performed by surface-phase recombinase polymerase amplification (RPA). Captured test templates were incubated with 40 μl of RPA final mix (Table 3) at 43°C for 1 hour, followed by a 10-minute incubation at 75°C and two 60 μl water washes. After amplification, oligoduplexes were melted by incubation with 40 μl of 40 mM NaOH at 20°C for 10 minutes and washed twice with 1× RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20).

[0110] Example 4: Visualizing clonal clusters The initial clonal clusters amplified in the plane of the surface (2D clusters) were confirmed by annealing fluorescent oligos directed to specific sequences within the surface-tethered templates.

[0111] Clonally amplified, surface-conjugated templates were incubated with an equimolar mix of 5 μM YdfU (Cy5)- and ddl (Cy3)-specific probes in 1× annealing buffer (20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 5 mM magnesium acetate, 0.01% v / v Tween 20, and 5% v / v DMSO) at 95°C for 2 minutes, 50°C for 5 minutes, and 20°C for 10 minutes. After hybridization, the surface was washed twice with 1× RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20). The sequences of these probes are shown below.

number

number

[0112] Annealed fluorescent probes were visualized by imaging with a Sensospot® microarray scanner (Sensovation AG) using a red excitation filter for the YdfU Cy5 probe and a green excitation filter for the ddl Cy3 probe, both with an exposure time of 10 ms.

[0113] Example 5: Sequencing (a) Preparation of buffers / reagents Sequencing solution: (7.5 mM MgCl, 200 mM NaCl, 0.02% Tergitol NP-9) was prepared in a glass bottle using deionized water (18 MΩ; Merck Millipore), 1 M magnesium chloride solution, 5 M NaCl, and Tergitol™ NP-9 (solvent-free; Merck). MgCl and NaCl stock solutions were added to deionized water to give final concentrations of 7.5 and 200 mM, respectively. The solution was mixed using a magnetic stir plate until homogeneous. The solution was then stirred with nitrogen sparging and maintained under nitrogen to ensure removal of dissolved carbon dioxide. Tergitol NP-9 (250 μl) was added to a final concentration of 0.02%.

[0114] Nucleotides: 10 μM solutions of each of dGTP, dCTP, dATP, and dTTP were prepared by adding 12.5 μL of a 100 mM stock solution of the selected nucleotide (Fisher Scientific, 11843933) to 125 mL of sequencing solution and adjusting the pH to 8.05 ± 0.01 using 10 mM NaOH. All dNTP solutions were prepared in a CO2-free, nitrogen-controlled environment.

[0115] The wash solution was prepared by adjusting the pH of the sequencing solution to 8.05±0.01 with 10 mM NaOH in a CO2-free, nitrogen-controlled environment.

[0116] Annealing buffer (1x) was prepared by diluting a 20x stock saline sodium citrate buffer (Life Technologies) with Molecular Grade Water (Sigma) to a final 1x concentration of 150 mM NaCl and 15 mM sodium citrate.

[0117] Sequencing Primer: A 5 μM working solution of sequencing primer (the fluorescent probe from Example 4 was used as the sequencing primer) was prepared by adding 1.25 μL of sequencing primer (100 μM stock solution) to 5 μL of 1× annealing buffer to give a final composition of 5 μM sequencing primer in 0.8× annealing buffer.

[0118] Polymerase: A 25.4 U / μL working stock of sequencing polymerase was prepared by diluting 1 μL of 2 kU / μL IsoPol BST+ DNA Polymerase (ArcticZymes) in 79 μL of sequencing solution and mixing thoroughly.

[0119] (b) General sequencing protocol The ISFET chip was equipped with a custom-built flow cell device (Figure 11), in which the ISFET surface was exposed to a solution flowing through the cell. This allowed for easy delivery and control of reagent flow over the surface. The flow cell was flushed twice with 1x ThermoPol Buffer [4.5 mL of 10x ThermoPol Buffer (New England Biolabs), 27 μL of Tween 20 (100% stock solution, Merck Life Science, P9416), 40.5 mL of Molecular Grade Water (Sigma)].

[0120] The fluorescent probe applied as part of the workflow according to Example 4 served as a sequencing primer. Sequencing polymerase (25 U / µL) was loaded into the flow cell and incubated at ambient temperature (approximately 20-26 °C) for 10 minutes. 200 µL of 1x ThermoPol Buffer was flushed through the flow cell.

[0121] A priming step was performed in which wash solution was flowed across the chip at 5 mL / min. Electrical response testing was performed by biasing the reference electrode with increasing voltage steps. The resulting change in mV output measured by the ISFET on the IC was used to determine the relationship between the reference electrode potential and the corresponding potential seen on the ISFET, which was then used to determine the optimal reference electrode potential for the experiment.

[0122] Sequencing was then performed cycle by cycle. During each sequencing cycle, each of the four individual dNTP solutions (10 μM each; see preparation details above) was flowed sequentially across the chip (5 ml / min for 15 seconds for each nucleotide), with each nucleotide flow separated by a wash step. The wash step was performed in two stages: 1) a "through wash" to flush the nucleotide solution from the fluidic channels and flow cell, and 2) a purge wash, in which the wash channel bypassed the nucleotide valving device to clean the chip while simultaneously directing the next nucleotide solution through the purge channel to a waste container. Upon nucleotide incorporation, the subsequent proton release was detected by the integrated circuit as a voltage change.

[0123] Example 6: Clonal amplification by RPA compared with clonal amplification by RPA followed by LCC. The density of capture oligos on the surface is a factor in determining optimal capture and amplification. Optimal density for RPA was previously found to be obtained using 50 μM capture oligos in the ligation reaction. For RPA followed by LCC, the optimal concentration was 2 μM. Data not shown.

[0124] Glass slides were prepared using the optimal density of capture oligos (extHDA72R) arranged as spots according to Example 1. YdfU and ddl test templates were then hybridized to the capture oligos according to Example 2. All spots were subjected to clonal amplification by optimized surface-phase RPA as in Example 3. Fluorescent probes were annealed and the slides were imaged according to Example 4. The fluorescent probes were dehybridized with 40 mM NaOH, washed, and subjected to LCC as described below.

[0125] (a) Hybridization of intramolecular inversion probes. A schematic of this approach is shown in Figure 3 and described in detail above. The intramolecular inversion probe (MIP) was a single-stranded polynucleotide that had at its 5' end a 20-nucleotide sequence complementary to the 5' universal adapter sequence of the tethered polynucleotide and at its 3' end a 19-nucleotide sequence complementary to the 3' universal adapter sequence. Between these sequences was a 64-nucleotide linker sequence.

[0126] Forty μl of MIPs (6.25e11 copies / μl) in 1× annealing buffer (20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 5 mM magnesium acetate, 0.01% v / v Tween 20, and 5% v / v DMSO) were hybridized through the universal adapter regions of the clonally amplified oligonucleotides anchored to the 2-D clusters by incubation at 95°C for 2 minutes, ramping from 60°C to 50°C at a rate of 0.2°C / sec, followed by a final incubation at 20°C for 10 minutes. After hybridization, the surface was washed twice with 1× RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20). The MIP sequence is shown below. The 5' and 3' complementary regions are underlined:

number

[0127] (b) Extension and circularization of the 3' end of the MIP The MIPs were extended downstream of the 3' end to include the sequence of interest from the ddl or ydfU fragment of each surface-conjugated oligo, up to the phosphorylated 5' of the MIP. The MIPs were then ligated to form annealed circles to the surface-tethered oligos. This process was carried out by incubating the surface-tethered oligos with 40 μl of 1× circularization mix (Table 4) at 20°C for 30 minutes, followed by 60°C for 10 minutes. After circularization, the surface was washed twice with 1× RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20).

[0128] (c) Rolling circle amplification of the sequence of interest Oligonucleotides tethered to the surface of the first (2-D) clonal clusters were replicated by incubation with 40 μl of 1× RPA mix (Table 5) at 45°C for 15 minutes, followed by 10 minutes at 75°C. After replication, the surface was washed twice with 60 μl of 1× RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20). After amplification, the oligoduplexes were melted by incubation with 40 μl of 40 mM NaOH at 20°C for 10 minutes and washed twice with 60 μl of 1× RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20).

[0129] Clonal clusters were visualized using a fluorescent probe (Figure 5A), and fluorescence was quantified according to Example 4 (Figure 5B). After rolling circle amplification of the RPA clusters to form 3-D clusters, there were some regions of increased intensity around the edges of the spots, but there was little difference in the fluorescence intensity of the 50 μM spots. In contrast, there was significantly increased fluorescence intensity of the 2 μM primer spots. A comparison of the spot fluorescence data is shown in Figure 5B. For both YdfU and ddl, RPA + LCC resulted in a 2- to 3-fold increase in fluorescence intensity.

[0130] This example was repeated using the improved MIP described above, whose sequence was designed to prevent premature 3'CR binding and extension. The sequence of the MIP is shown below. Simultaneous MIP hybridization and circularization was achieved by adding the MIP to the circularization mix. A thermostable polymerase (Titanium Taq) and ligase (HiFi Ligase) were used to allow hybridization of the MIP at higher temperatures using the following heating profile: 95°C for 2 minutes, 55°C for 10 minutes, and 45°C for 30 minutes.

[0131] After circularization, the surface was washed twice with 1x RPA wash buffer (0.06% SSC, 0.06% v / v Tween 20). The results are shown in Figure 8. Fluorescence was again quantified according to Example 4, and the distribution of fluorescence across all pixels in the image was plotted. The Y axis is the percentage of pixels with an intensity of at least the X value.

number

[0132] Example 7: Sequencing from clonal clusters For either RPA or RPA+LCC, ISFET chips were coated with ExtHD72R capture oligos according to Example 1. YdfU template was captured on chips prepared for RPA according to Example 2, and an equimolar mixture of YdfU and dll templates was captured on chips prepared for RPA+LCC. Clonal amplification was performed according to Example 3 for RPA chips and Examples 3 and 6 for RPA+LCC chips. Sequencing from clonal clusters was performed according to Example 5, and the results are shown in Figure 6. Graph C shows the signal from incorporation of a single nucleotide into the growing sequencing strand during SBS according to Example 5. The voltages generated by the ISFET are shown in Table 6.

[0133] Figure 6A provides read length data for the RPA (YdfU) and RPA+LCC (YdfU and ddl) clusters. Read data is also shown in Table 6. [Table 6]

[0134] The signal generated from the RPA+LCC cluster was superior to the signal generated from RPA alone, which is reflected in improved read length and read quality.

[0135] Figure 7 illustrates a further comparison between RPA and RPA+LCC clonal amplification. RPA was performed with capture oligo applied at 25 uM, whereas RPA+LCC was performed with oligo applied at 1 uM. Amplification was performed on a submerged ISFET chip according to Examples 1-4 and 6. The capture oligo was extHDA72R and the template was YdfU. An unmodified MIP was used (SEQ ID NO: 7).

[0136] Fluorescence micrographs show: A - RPA clonally amplified clusters, visible as pale cloud-like features (arrows). White dots are control spots; B - Clonal clusters resulting from RPA followed by LCC amplification. The fluorescence (arrows) of the RPA+LCC clusters is significantly brighter, indicating a higher density of oligonucleotide clones.

[0137] Graph C illustrates the distribution of fluorescence across all pixels in the image of the chip. The Y axis is the percentage of pixels that have an intensity of at least X value.

[0138] Example 8: Visualizing clonality As outlined in Example 1, ExtHD72R capture oligos were spotted onto an ISFET chip at 10 μM. Template (an equimolar mixture of YdfU and ddl) was applied to the capture oligos according to Example 2 and subjected to RPA clonal amplification according to Example 3. At this point, the YdfU and ddl clusters were visualized and imaged using a fluorescent probe according to Example 4. The chip was washed with 40 mM NaOH to remove the fluorescent probe and subjected to LCC according to Example 6. The spots were relabeled with the fluorescent probe according to Example 4 and imaged again. Figure 9 shows grayscale images of clonally amplified templates after RPA and RPA + LCC. Arrows indicate individual ddl clonal clusters, the intensity of which is improved in RPA + LCC compared to RPA alone. The image on the right shows that these clusters are not detected by the YdfU probe, even after LCC.

[0139] Example 9: Formation of YdfU rings in solution In this example, the following reagent mix was used:

[0140] [Table 7]

[0141] [Table 8]

[0142] (a) Linear MIP / sprint hybridization 6×10 annealing buffer (20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 5 mM magnesium acetate, 0.01% v / v Tween 20, and 5% v / v DMSO) according to Table 7. 13 1.8 x 10 copies of the linear YdfU MIP 1450 μl of YdfU linear MIP / sprint hybridization mix was prepared by mixing 1:3 YdfU MIP splint with 1:1 YdfU MIP splint. This mix was heated to 95°C for 3 minutes and then incubated at 60°C for 1 hour.

[0143] Linear YdfU MIP (SEQ ID NO: 8)

number

[0144] YdfU MIP splint (SEQ ID NO: 9)

number

[0145] (b) YdfU ring connection 125 ul of YdfU linear MIP / splint ligation mix was prepared by adding T4 DNA ligase to the hybridization mix (Table 7) in 1x T4 DNA ligase buffer. The mix was then incubated at 20°C for 1 hour, followed by heat kill at 75°C for 15 minutes.

[0146] (c) Extension of the tethered polynucleotide RPA-amplified YdfU clusters on ISFET chips were prepared according to Examples 1-3. A circular probe was hybridized to the tethered polynucleotide according to Example 6(a), and the polynucleotide was extended according to Example 6(c). Sequencing was then performed according to Example 5. The results are shown in Figure 10. The signal generated from RPA+LCC clusters using LCC, where the rings are generated in solution rather than on a surface using MIPs, was again superior to the signal generated from RPA alone, as reflected in improved read length and read quality.

Claims

1. 1. A method for preparing a clonal cluster of nucleotide sequences of interest, comprising: (i) providing a single-stranded polynucleotide comprising the nucleotide sequence of interest, wherein the polynucleotide is tethered to a surface via its 5' end; (ii) performing a first clonal amplification of the single-stranded polynucleotides to provide a first cluster of polynucleotides comprising a plurality of single-stranded polynucleotides tethered to the surface via their 5' ends and comprising the nucleotide sequence of interest; (iii) extending the anchored single-stranded polynucleotides in the first cluster by the addition of one or more additional copies of the nucleotide sequence of interest to provide a cluster of anchored polynucleotides, wherein the polynucleotides of the cluster comprise multiple copies of the nucleotide sequence of interest; A method comprising:

2. The method of claim 1 , wherein the single-stranded polynucleotide comprises a 5′ and / or 3′ adaptor.

3. 3. The method of claim 2, wherein the 5' adapter comprises a nucleotide sequence configured to hybridize to a sequencing primer.

4. 4. The method of claim 2 or 3, wherein the 3' adapter of the template polynucleotide comprises a nucleotide sequence configured to hybridize to a clonal amplification primer.

5. 5. The method of any one of claims 1 to 4, wherein the 3' adaptor and / or the sequence of interest is at the 3' end of the tethered polynucleotide, preferably coterminous with the 3' end of the tethered polynucleotide.

6. 6. The method of any one of claims 1 to 5, wherein the anchored single-stranded polynucleotides in the first cluster are extended by rolling circle amplification of the sequence of interest.

7. The rolling circle amplification (a) providing a single-stranded circular polynucleotide probe comprising a nucleotide sequence that is complementary to the sequence of interest in the first clonal cluster; (b) using the cyclic nucleotide probe as a template, extending the 3' ends of the anchored single-stranded polynucleotides in the first clonal cluster using a strand-displacing polymerase, thereby extending the 3' ends of the anchored single-stranded polynucleotides by the addition of one or more additional copies of the sequence of interest; The method of claim 6 , performed by a process comprising:

8. 8. The process of claim 7, wherein the single-stranded circular polynucleotide probe is synthesized in solution from a linear polynucleotide containing a complementary copy of the sequence of interest.

9. 8. The process of claim 7, wherein the single-stranded circular polynucleotide probe is synthesized in situ, preferably from an intramolecular inversion probe whose 5' and 3' complementary regions hybridize to distinct nucleotide sequences of the anchored polynucleotide.

10. the single-stranded cyclic nucleotide probe (a) providing a linear single-stranded nucleotide probe comprising, in a 5' to 3' direction, a 5' complementary region, an optional linker region, and a 3' complementary region, wherein the 5' complementary region and the 3' complementary region are configured to hybridize to distinct sequences on the anchored single-stranded polynucleotide; (b) hybridizing the 5' complementary region of the probe and the 3' complementary region of the probe to the anchored single-stranded polynucleotide, wherein the 5' complementary region hybridizes to a sequence upstream of the sequence to which the 3' complementary region hybridizes; (c) optionally extending the 3' end of the probe using the sequence of the single-stranded polynucleotide as a template; (d) circularizing the probe by ligating the optionally extended 3' end of the probe to the 5' end of the probe; is formed by 10. The process of claim 9, wherein the sequences of the 5' complementary region of the probe and the 3' complementary region of the probe are selected such that any extension and ligation provides a single-stranded circular probe containing a complementary copy of the sequence of interest.

11. 11. The method of claim 10, wherein the sequence of the 5' complementary region is selected to hybridize to the 5' adapter only, to the 5' adapter and the 5'-most portion of the SOI, or to the 5'-most portion of the SOI only.

12. 12. The method of claim 10 or 11, wherein the sequence of the 3' complementary region is selected to hybridize to the 3' adapter only, to the 3' adapter and the 3'-most part of the SOI, or to the 3'-most part of the SOI only.

13. 11. The method of claim 10, wherein the 5' complementary region is selected to hybridize only to sequences in the 5' adapter, and the 3' complementary region is selected to hybridize only to sequences in the 3' adapter.

14. The method of any one of claims 9 to 13, wherein the 3' end of the probe is extended using a polymerase that lacks 5' to 3' exonuclease activity.

15. The method of any one of claims 7 to 13, wherein the single-stranded circular polynucleotide probe binds to the tethered polynucleotide leaving a 3' single-stranded overhang.

16. 16. The method of claim 15, wherein a protective oligonucleotide hybridizes to the 3' overhang.

17. 16. The method of claim 15, wherein the 3' single-stranded overhang is removed by 3' to 5' exonuclease activity, removing the protective oligonucleotide, if present.

18. The method of any one of claims 10 to 17, wherein a linking region is present.

19. 19. The method of claim 18, wherein the linking region comprises one or more functional sequences, preferably functional sequences selected such that when complementarily replicated, they hybridize to a sequencing primer or other primer or to a capture oligo.

20. 1. A method for preparing a clonal cluster of single-stranded polynucleotides comprising a sequence of interest, comprising: (i) hybridizing a polynucleotide fragment containing a complementary copy of the sequence of interest to a capture oligo that is tethered to a surface via its 5' end; (ii) extending the 3' end of the capture oligo using a polymerase to provide a single-stranded polynucleotide comprising the sequence of interest tethered to the surface via its 5' end; (iii) clonally amplifying the sequence of interest using the method of any one of claims 1 to 19 to provide a cluster of anchored polynucleotides, wherein the polynucleotides of the cluster comprise multiple copies of the nucleotide sequence of interest; A method comprising:

21. 1. A method for determining the nucleotide sequence of a single-stranded nucleotide fragment, comprising: (i) tethering the single-stranded fragment to a surface to provide a single-stranded polynucleotide comprising a nucleotide sequence of interest tethered to the surface via its 5' end; (ii) clonally amplifying the sequence of interest using the method of any one of claims 1 to 19 to provide a cluster of anchored polynucleotides, wherein the polynucleotides of the cluster comprise multiple copies of the nucleotide sequence of interest; (ii) at least partially sequencing the SOIs of the cluster to determine the nucleotide sequences of the SOIs; A method comprising:

22. A substrate having attached to its surface a plurality of single-stranded polynucleotides arranged as a plurality of clonal clusters, the polynucleotides within each cluster comprising alternating copies of a first nucleotide sequence and a second nucleotide sequence, the second nucleotide sequence being common to the single-stranded nucleotides of each cluster, and the first sequence being not common to the single-stranded nucleotides of each cluster.

23. 23. The substrate of claim 22, wherein the first sequence is present within the cluster in the sense or antisense orientation, but not both.

24. 24. The substrate of claim 22 or 23, wherein the polynucleotides in each cluster comprise at least two copies, preferably at least three copies, more preferably at least four copies, even more preferably at least five copies of the first sequence.

25. The substrate according to claims 22 to 24, wherein the polynucleotides in each cluster have a free 3' end.

26. The substrate of claims 22-25, wherein a single species of capture oligo is tethered to the surface via its 5' end.

27. The substrate according to claims 22 to 26, wherein the polynucleotides in each cluster are covalently attached to the surface via their 5' ends.

28. 28. The substrate of any of claims 22-27, which is part of an ISFET, a glass or silica substrate, an insoluble particulate substrate, or a microfluidic device adapted to contact the tethered polynucleotide with a liquid reagent.

29. 1. An intramolecular inversion probe for use in preparing a clonal cluster of anchored polynucleotides, said intramolecular inversion probe comprising: (a) 3′CR, (b) an optional linker region, and (c) 5′CR Including, An intramolecular inversion probe, wherein the sequence of the 3'CR is selected to hybridize to a first portion of an anchored polynucleotide and the sequence of the 5'CR is selected to hybridize to a second portion of the anchored polynucleotide that is distinct from the first portion, and wherein the Tm of the 5'CR is higher than the Tm of the 3'CR.

30. 30. The intramolecular inversion probe of claim 29, wherein the first portion of the tethered polynucleotide is separated from the second portion of the tethered polynucleotide by a sequence of interest, or a complement of a sequence of interest, or a portion thereof.

31. 1. A method for preparing a clonal cluster of anchored polynucleotides, comprising: (i) providing a single-stranded polynucleotide tethered to a surface via its 5' end; (ii) performing a first clonal amplification of the single-stranded polynucleotides to provide a first cluster of polynucleotides anchored via their 5' ends to the surface; (iii) extending the anchored single-stranded polynucleotides in the first cluster by rolling circle amplification using an intramolecular inversion probe, preferably an intramolecular inversion probe according to any of claims 29 or 30; A method comprising:

32. The rolling circle amplification (a) contacting the polynucleotides of a first cluster with the intramolecular inversion probe; (b) hybridizing the 5'CR of the intramolecular inversion probe to the tethered single-stranded polynucleotide at a temperature above the Tm of the 3'CR but below the Tm of the 5'CR, and then hybridizing the 3'CR of the probe to the polynucleotide at a temperature below the Tm of the 3'CR; (c) optionally extending the 3' end of the intramolecular inversion probe; (c) circularizing the intramolecular inversion probe by ligating the optionally extended 3' end of the probe to the 5' end of the probe; (d) contacting the circularized intramolecular inversion probe with a strand displacement polymerase to extend the single-stranded polynucleotide; The method of claim 10, comprising:

Images