Patent Application
20240263229
The present disclosure is generally directed to strategies for template capture and amplification during sequencing.
The detection of analytes such as nucleic acid sequences that are present in a biological sample has been used as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. A common technique for detecting analytes such as nucleic acid sequences in a biological sample is nucleic acid sequencing.
Advances in the study of biological molecules have been led, in part, by improvement in technologies used to characterise the molecules or their biological reactions. In particular, the study of the nucleic acids DNA and RNA has benefited from developing technologies used for sequence analysis.
Methods of nucleic acid amplification which allow amplification products to be immobilised on a solid support in order to form arrays comprised of clusters or “colonies” formed from a plurality of identical immobilised polynucleotide strands and a plurality of identical immobilised complementary strands are known. The nucleic acid molecules present in DNA colonies on the clustered arrays prepared according to these methods can provide templates for sequencing reactions.
One method for sequencing a polynucleotide template involves performing multiple extension reactions using a DNA polymerase to successively incorporate labelled nucleotides to a template strand. In such a “sequencing by synthesis” reaction a new nucleotide strand base-paired to the template strand is built up in the 5′ to 3′ direction by successive incorporation of individual nucleotides complementary to the template strand.
According to a first aspect of the disclosure, there is provided a method of amplifying a nucleic acid template, wherein the method comprises:
The method improves and/or addresses limitations of current amplification strategies, particularly those strategies that use bridging for amplification during cluster generation. Advantageously, it has been found that using both lawn and free solution primers for DNA amplification may result in less steric hindrance and a higher amplification flexibility. Furthermore, using primers that can only hybridize and extend, with no invasion capability, may minimise or prevent the formation of duplicates, which are detrimental amplification efficiency and downstream sequencing performance.
According to a further aspect of the disclosure, there is provided a method of sequencing a nucleic acid sequence, wherein the method comprises:
This method may significantly shorten paired-end read re-synthesis time.
According to a yet further aspect of the disclosure, there is provided a method of sequencing a target nucleic acid sequence, wherein the method comprises:
Again, this method may significantly shorten paired-end read re-synthesis timecycles. The methods of the present disclosure can be advantageously used in pairwise sequencing of target nucleic acid sequences.
According to a yet further aspect of the disclosure, there is provided a solution-phase primer comprising or consisting of a nucleic acid sequence as defined in SEQ ID NO: 5, 6 or 7 or a variant thereof.
The solution-phase primers of the present disclosure may be useful in the methods of the present disclosure and may advantageously minimise or prevent duplication.
According to a yet further aspect of the disclosure, there is provided a re-synthesis primer, the primer comprising a nucleic acid sequence selected from SEQ ID NO: 9, 10 or 11 or a variant thereof, wherein the primer is blocked at the 3′ end, wherein the block prevents extension of the primer until the block is removed.
According to a yet further aspect of the disclosure, there is provided a solid support for use in sequencing, wherein the support comprises a plurality of lawn primers immobilised thereon and a plurality of dormant lawn primers immobilised thereon, wherein the dormant lawn primers comprise a blocking 3′ group that prevents extension until removed.
The re-synthesis primers according to the present disclosure advantageously prevent bridged amplification during initial cluster generation which may minimise or avoid amplification propagating into adjacent wells. It may also advantageously provide pristine primers to be available during a second sequencing read, as the primers have not previously be used during bridge amplification.
According to a yet further aspect of the disclosure, there is provided a hybridisation buffer, wherein the hybridisation buffer comprises a denaturation agent and at least one solution-phase primer of the disclosure.
According to a yet further aspect of the disclosure, there is provided a buffer, wherein the buffer comprises at least one solution-phase primer of the disclosure.
The following features apply to all aspects of the present disclosure.
The present disclosure can be used in sequencing, for example pairwise sequencing. Methodology applicable to the present disclosure have been described in WO 08/041002, WO 07/052006, WO 98/44151, WO 00/18957, WO 02/06456, WO 07/107710, WO05/068656, U.S. Ser. No. 13/661,524 and US 2012/0316086, the contents of which are herein incorporated by reference. Further information can be found in US 20060024681, US 200602926U, WO 06110855, WO 06135342, WO 03074734, WO07010252, WO 07091077, WO 00179553 and WO 98/44152, the entire contents of each which are incorporated by reference herein.
Sequencing generally comprises four fundamental steps: 1) library preparation to form a plurality of template molecules available for sequencing; 2) cluster generation to form an array of amplified single template molecules on a solid support; 3) sequencing the cluster array; and 4) data analysis to determine the target sequence.
Library preparation is the first step in any high-throughput sequencing platform. During library preparation, nucleic acid sequences, for example genomic DNA sample, or cDNA or RNA sample, is converted into a sequencing library, which can then be sequenced. By way of example with a DNA sample, the first step in library preparation is random fragmentation of the DNA sample. Sample DNA is first fragmented and the fragments of a specific size (typically 200-500 bp, but can be larger) are ligated, sub-cloned or “inserted” in-between two oligo adapters (adapter sequences). This may be followed by amplification and sequencing. The original sample DNA fragments are referred to as “inserts.” Alternatively “tagmentation” can be used to attach the sample DNA to the adapters. In tagmentation, double-stranded DNA is simultaneously fragmented and tagged with adapter sequences and PCR primer binding sites. The combined reaction eliminates the need for a separate mechanical shearing step during library preparation. The target polynucleotides may advantageously also be size-fractionated prior to modification with the adaptor sequences.
As used herein an “adapter” sequence comprises a short sequence-specific oligonucleotide that is ligated to the 5′ and 3′ ends of each DNA (or RNA) fragment in a sequencing library as part of library preparation. The adaptor sequence may further comprise non-peptide linkers.
As will be understood by the skilled person, a double-stranded nucleic acid will typically be formed from two complementary polynucleotide strands comprised of deoxyribonucleotides joined by phosphodiester bonds, but may additionally include one or more ribonucleotides and/or non-nucleotide chemical moieties and/or non-naturally occurring nucleotides and/or non-naturally occurring backbone linkages. In particular, the double-stranded nucleic acid may include non-nucleotide chemical moieties, e.g. linkers or spacers, at the 5′ end of one or both strands. By way of non-limiting example, the double-stranded nucleic acid may include methylated nucleotides, uracil bases, phosphorothioate groups, also peptide conjugates etc. Such non-DNA or non-natural modifications may be included in order to confer some desirable property to the nucleic acid, for example to enable covalent, non-covalent or metal-coordination attachment to a solid support, or to act as spacers to position the site of cleavage an optimal distance from the solid support. A single stranded nucleic acid consists of one such polynucleotide strand. Where a polynucleotide strand is only partially hybridised to a complementary strand—for example, a long polynucleotide strand hybridised to a short nucleotide primer—it may still be referred to herein as a single stranded nucleic acid.
An example of a typical single-stranded nucleic acid template is shown in
In one embodiment, the P5′ and P7′ primer-binding sequences are complementary to short primer sequences (or lawn primers) present on the surface of the flow cells. Binding of P5′ and P7′ to their complements (P5 and P7) on—for example—the surface of the flow cell, permits nucleic acid amplification. As used herein “'” denotes the complementary strand.
The primer-binding sequences in the adaptor which permit hybridisation to amplification primers will typically be around 20-40 nucleotides in length, although, in embodiments, the disclosure is not limited to sequences of this length. The precise identity of the amplification primers, and hence the cognate sequences in the adaptors, are generally not material to the disclosure, as long as the primer-binding sequences are able to interact with the amplification primers in order to direct PCR amplification. The sequence of the amplification primers may be specific for a particular target nucleic acid that it is desired to amplify, but in other embodiments these sequences may be “universal” primer sequences which enable amplification of any target nucleic acid of known or unknown sequence which has been modified to enable amplification with the universal primers. The criteria for design of PCR primers are generally well known to those of ordinary skill in the art. “Primer-binding sequences” may also be referred to as “clustering sequences” “clustering primers” or “cluster primers” in the present disclosure, and such terms may be used interchangeably.
The index sequences (also known as a barcode or tag sequence) are unique short DNA sequences that are added to each DNA fragment during library preparation. The unique sequences allow many libraries to be pooled together and sequenced simultaneously. Sequencing reads from pooled libraries are identified and sorted computationally, based on their barcodes, before final data analysis. Library multiplexing is also a useful technique when working with small genomes or targeting genomic regions of interest. Multiplexing with barcodes can exponentially increase the number of samples analyzed in a single run, without drastically increasing run cost or run time. Examples of tag sequences are found in WO05068656, the entire contents of which are incorporated by reference herein. The tag can be read at the end of the first read, or equally at the end of the second read. The disclosure is not limited by the number of reads per cluster, for example two reads per cluster: three or more reads per cluster are obtainable simply by dehybridising a first extended sequencing primer, and rehybridising a second primer before or after a cluster repopulation/strand resynthesis step. Methods of preparing suitable samples for indexing are described in, for example U.S. 60/899,221, the entire contents of which are incorporated by reference herein. Single or dual indexing may also be used. With single indexing, up to 48 unique 6-base indexes can be used to generate up to 48 uniquely tagged libraries. With dual indexing, up to 24 unique 8-base Index 1 sequences and up to 16 unique 8-base Index 2 sequences can be used in combination to generate up to 384 uniquely tagged libraries. Pairs of indexes can also be used such that every i5 index and every i7 index are used only one time. With these unique dual indexes, it is possible to identify and filter indexed hopped reads, providing even higher confidence in multiplexed samples.
The sequencing binding sites are sequencing and/or index primer binding sites and indicates the starting point of the sequencing read. During the sequencing process, a sequencing primer anneals (i.e. hybridises) to a portion of the sequencing binding site on the template strand. The DNA polymerase enzyme binds to this site and incorporates complementary nucleotides base by base into the growing opposite strand. In one embodiment, the sequencing process comprises a first and second sequencing read. The first sequencing read may comprise the binding of a first sequencing primer (read 1 sequencing primer) to the first sequencing binding site (e.g. SBS3′) followed by synthesis and sequencing of the complementary strand. This leads to the sequencing of the insert. In a second step, an index sequencing primer (e.g. i7 sequencing primer) binds to a second sequencing binding site (e.g. SBS12) leading to synthesis and sequencing of the index sequence (e.g. sequencing of the i7 primer). The second sequencing read may comprise binding of an index sequencing primer (e.g. i5 sequencing primer) to the complement of the first sequencing binding site on the template (e.g. SBS3) and synthesis and sequencing of the index sequence (e.g. i5). In a second step, a second sequencing primer (read 2 sequencing primer) binds to the complement of the primer (e.g. i7 sequencing primer) binds to a second sequencing binding site (e.g. SBS12′) leading to synthesis and sequencing of the insert in the reverse direction.
Once a double stranded nucleic acid template library is formed, typically, the library has previously been subjected to denaturing conditions to provide single stranded nucleic acids. Suitable denaturing conditions will be apparent to the skilled reader with reference to standard molecular biology protocols (Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds Ausubel et al). In one embodiment, chemical denaturation, such as NaOH or formamide, is used. Suitable denaturation agents include: acidic nucleic acid denaturants such as acetic acid, HCl, or nitric acid; basic nucleic acid denaturants such as NaOH; or other nucleic acid denaturants such as DMSO, formamide, betaine, guanidine, sodium salicylate, propylene glycol or urea. Preferred denaturation agents are formamide and NaOH, preferably formamide.
Following denaturation, a single-stranded template library is in one embodiment contacted in free solution onto a solid support comprising surface capture moieties (for example P5 and/or P7 primers). This solid support is typically a flowcell, although in alternative embodiments, seeding and clustering can be conducted off-flowcell using, for example, microbeads or the like.
As used herein, the term “solid support” refers to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g. due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fibre bundles, and polymers. A particularly useful material is glass. Other suitable substrate materials may include polymeric materials, plastics, silicon, quartz (fused silica), boro float glass, silica, silica-based materials, carbon, metals including gold, an optical fibre or optical fibre bundles, sapphire, or plastic materials such as COCs and epoxies. The particular material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of the desired wavelength, such as one or more of the techniques set forth herein. Conversely, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g. being opaque, absorptive or reflective). This can be useful for formation of a mask to be used during manufacture of the structured substrate; or to be used for a chemical reaction or analytical detection carried out using the structured substrate. Other properties of a material that can be exploited are inertness or reactivity to certain reagents used in a downstream process; or ease of manipulation or low cost during a manufacturing process manufacture. Further examples of materials that can be used in the structured substrates or methods of the present disclosure are described in U.S. Ser. No. 13/661,524 and US Pat. App. Pub. No. 2012/0316086 A1, the entire contents of each are incorporated by reference herein.
The disclosure may make use of solid supports comprised of a substrate or matrix (e.g. glass slides, polymer beads etc) which has been “functionalised”, for example by application of a layer or coating of an intermediate material comprising reactive groups which permit covalent attachment to biomolecules, such as polynucleotides. Examples of such supports include, but are not limited to, a substrate such as glass. In such embodiments, the biomolecules (e.g. polynucleotides) may be directly covalently attached to the intermediate material but the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g. the glass substrate). The term “covalent attachment to a solid support” is to be interpreted accordingly as encompassing this type of arrangement. Alternatively, the substrate such as glass may be treated to permit direct covalent attachment of a biomolecule; for example, glass may be treated with hydrochloric acid, thus exposing the hydroxyl groups of the glass, and phosphite-triester chemistry used to directly attach a nucleotide to the glass via a covalent bond between the hydroxyl group of the glass and the phosphate group of the nucleotide.
In other embodiments, the solid support may be “functionalised” by application of a layer or coating of an intermediate material comprising groups that permit non-covalent attachment to biomolecules. In such embodiments, the groups on the solid support may form one or more of ionic bonds, hydrogen bonds, hydrophobic interactions, π-π interactions, van der Waals interactions and host-guest interactions, to a corresponding group on the biomolecules (e.g. polynucleotides). The interactions formed between the group on the solid support and the corresponding group on the biomolecules may be configured to cause immobilisation or attachment under the conditions in which it is intended to use the support, for example in applications requiring nucleic acid amplification and/or sequencing. For example, the interactions formed between the group on the solid support and the corresponding group on the biomolecules may be configured such that the biomolecules remain attached to the solid support during amplification and/or sequencing.
In other embodiments, the solid support may be “functionalised” by application of an intermediate material comprising groups that permit attachment via metal-coordination bonds to biomolecules. In such embodiments, the groups on the solid support may include ligands (e.g. metal-coordination groups), which are able to bind with a metal moiety on the biomolecule. Alternatively, or in addition, the groups on the solid support may include metal moieties, which are able to bind with a ligand on the biomolecule. The metal-coordination interactions formed between the ligand and the metal moiety may be configured to cause immobilisation or attachment of the biomolecule under the conditions in which it is intended to use the support, for example in applications requiring nucleic acid amplification and/or sequencing. For example, the interactions formed between the group on the solid support and the corresponding group on the biomolecules may be configured such that the biomolecules remain attached to the solid support during amplification and/or sequencing.
When referring to immobilisation or attachment of molecules (e.g. nucleic acids) to a solid support, the terms “immobilised” and “attached” are used interchangeably herein and both terms are intended to encompass direct or indirect, covalent or non-covalent attachment, unless indicated otherwise, either explicitly or by context. In certain embodiments of the disclosure, covalent attachment may be preferred; in other embodiments, attachment using non-covalent interactions may be preferred; in yet other embodiments, attachment using metal-coordination bonds may be preferred. However, in general the molecules (e.g. nucleic acids) remain immobilised or attached to the support under the conditions in which it is intended to use the support, for example in applications requiring nucleic acid amplification and/or sequencing. When referring to attachment of nucleic acids to other nucleic acids, then the terms “immobilised” and “hybridised” are used herein, and generally refer to hydrogen bonding between complementary nucleic acids.
If the amplification is performed on beads, either with a single or multiple extendable primers, the beads may be analysed in solution, in individual wells of a microtitre or picotitre plate, immobilised in individual wells, for example in a fibre optic type device, or immobilised as an array on a solid support. The solid support may be a planar surface, for example a microscope slide, wherein the beads are deposited randomly and held in place with a film of polymer, for example agarose or acrylamide.
As described above, once a library comprising template nucleotide strands has been prepared, the templates are seeded onto a solid support and then amplified to generate a cluster of single template molecules.
By way of brief example, following attachment of the P5 and P7 primers, the solid support may be contacted with the template to be amplified under conditions which permit hybridisation (or annealing—such terms may be used interchangeably) between the template and the immobilised primers (also referred to herein as “lawn primers”). The template is usually added in free solution under suitable hybridisation conditions, which will be apparent to the skilled reader. Typically, hybridisation conditions are, for example, 5×SSC at 40° C. Solid-phase amplification can then proceed. The first step of the amplification is a primer extension step in which nucleotides are added to the 3′ end of the immobilised primer using the template to produce a fully extended complementary strand. The template is then typically washed off the solid support. The complementary strand will include at its 3′ end a primer-binding sequence (i.e. either P5′ or P7′) which in some methods is capable of bridging to the second primer molecule immobilised on the solid support and binding. In this method, further rounds of amplification (analogous to a standard PCR reaction) lead to the formation of clusters or colonies of template molecules bound to the solid support. Thus, in this example, solid-phase amplification by either the method analogous to that of WO 98/44151 or that of WO 00/18957 (the contents of which are incorporated herein in their entirety by reference) will result in production of a clustered array comprised of colonies of “bridged” amplification products. Both strands of the amplification products will be immobilised on the solid support at or near the 5′ end, this attachment being derived from the original attachment of the amplification primers. Typically, the amplification products within each colony will be derived from amplification of a single template (target) molecule. Other amplification procedures may be used, and will be known to the skilled person. For example, amplification may be isothermal amplification using a strand displacement polymerase; or may be exclusion amplification as described in WO 2013/188582, the entire contents of which are incorporated by reference herein. The method may also involve a number of rounds of invasion by a competing immobilised primer (or lawn primer) and strand displacement of the template to the competing primer. Further information on amplification can be found in WO0206456 and WO07107710, the entire contents of each of which are incorporated by reference herein. Through such approaches, a cluster of single template molecules is formed.
To facilitate sequencing, it is preferable if one of the strands is removed from the surface to allow efficient hybridisation of a sequencing primer to the remaining immobilised strand. Suitable methods for linearisation are described in more detail in application number WO07010251, the entire contents of which are incorporated by reference herein.
Sequence data can be obtained from both ends of a template duplex by obtaining a sequence read from one strand of the template from a primer in solution, copying the strand using immobilised primers, releasing the first strand and sequencing the second, copied strand. For example, sequence data can be obtained from both ends of the immobilised duplex by a method wherein the duplex is treated to free a 3′-hydroxyl moiety that can be used an extension primer. The extension primer can then be used to read the first sequence from one strand of the template. After the first read, the strand can be extended to fully copy all the bases up to the end of the first strand. This second copy remains attached to the surface at the 5′-end. If the first strand is removed from the surface, the sequence of the second strand can be read. This gives a sequence read from both ends of the original fragment. The process whereby the strand is regenerated after the first read is known as “Paired-end resynthesis”. The typical steps of pairwise sequencing are known and have been described in WO 2008/041002, the entire contents of which are incorporated by reference herein.
Sequencing can be carried out using any suitable “sequencing-by-synthesis” technique, wherein nucleotides are added successively to the free 3′ hydroxyl group, resulting in synthesis of a polynucleotide chain in the 5′ to 3′ direction. The nature of the nucleotide added is preferably determined after each addition. One particular sequencing method relies on the use of modified nucleotides that can act as reversible chain terminators. Such reversible chain terminators comprise removable 3′ blocking groups. Once such a modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3′-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides. Once the nature of the base incorporated into the growing chain has been determined, the 3′ block may be removed to allow addition of the next successive nucleotide. By ordering the products derived using these modified nucleotides it is possible to deduce the DNA sequence of the DNA template. Such reactions can be done in a single experiment if each of the modified nucleotides has attached thereto a different label, known to correspond to the particular base, to facilitate discrimination between the bases added at each incorporation step. Suitable labels are described in PCT application PCT/GB/2007/001770, the entire contents of which are incorporated by reference herein. Alternatively, a separate reaction may be carried out containing each of the modified nucleotides added individually.
The modified nucleotides may carry a label to facilitate their detection. In a particular embodiment, the label is a fluorescent label. Each nucleotide type may carry a different fluorescent label. However the detectable label need not be a fluorescent label. Any label can be used which allows the detection of the incorporation of the nucleotide into the DNA sequence. One method for detecting the fluorescently labelled nucleotides comprises using laser light of a wavelength specific for the labelled nucleotides, or the use of other suitable sources of illumination. The fluorescence from the label on an incorporated nucleotide may be detected by a CCD camera or other suitable detection means. Suitable detection means are described in PCT/US2007/007991, the entire contents of which are incorporated by reference herein.
Alternative methods of sequencing include sequencing by ligation, for example as described in U.S. Pat. No. 6,306,597 or WO06084132, the entire contents of each of which are incorporated by reference herein.
However, current bridge-based clustering methods may limit the density of nanowells that can be used on any solid support. As shown in
The disclosure solves this problem by clustering without bridging. This may be referred to as “hybrid clustering”. Clustering without bridging is achieved in this disclosure by the use of free solution primers, in addition to immobilised (or lawn primers). In an embodiment, these are either free solution P5 or free solution P7 primers, and replace the use of the respective P5 and P7 lawn primers.
One embodiment of the hybrid clustering method of the disclosure is shown in
Accordingly, the disclosure provides a method of amplifying a nucleic acid template, wherein the method comprises the following steps:
In an embodiment, steps (d) to (f) are repeated through multiple cycles in the presence of an isothermal recombinase at 38ºC for about 1 hour.
In an embodiment, in step (e), the solution containing the plurality of primers may be the same solution from step (a). In a further embodiment, the solution containing the solution primers may be a different solution. Said another way, the solution primers may be added into the system at various stages depending on the methodology used. In some embodiment, the solution primers may be added during the process whereas in other embodiments the solution primers are present at the start of the process.
Following step (i) of the recited method, the template strands may be washed off the solid support.
By “nucleic acid template library” is meant a plurality of template nucleic acid strands comprising an insert, which is the samples nucleic acid flanked by 5′ and 3′ adaptor sequences that allow amplification and sequencing of the insert. Examples of adaptor sequences are described above. Preferably the adaptor sequences comprise 5′ and 3′ primer-binding sequences. The template nucleic acid strands may be initially double-stranded as shown in
The term “cluster” refers to a discrete site on a solid support comprised of a plurality of identical immobilised nucleic acid strands.
By “complementary” is meant that the primer has a sequence of nucleotides that can form a double-stranded structure by matching base-pairs with the adaptor or primer sequence or part thereof. By “substantially complementary” is meant that the primer has at least 85%, 90%, 95%, 98%, 99% or 100% overall sequence identical to the complementary sequence.
The terms “hybridise” and “anneal” can be used interchangeably. In one embodiment, hybridisation occurs under 5×SSC (saline sodium citrate) at 38° C.
An extension reaction, in which nucleotides are added to the 3′ end of a primer is performed using a polymerase, such as a DNA or RNA polymerase. In one embodiment, the polymerase is a non-thermal isothermal strand displacement polymerase. Suitable non-thermostable strand displacement polymerases according to the present disclosure can be found, for example, through New England BioLabs, Inc. and include phi29, Bsu, Klenow, DNA Polymerase I (E. coli), and Therminator. A particularly preferred polymerase is Bsu.
In an embodiment, the template strands comprise either a first 3′ primer-binding sequence or a second 3′ primer binding sequence, where the sequence of the first and second primer binding sequences are different. In this embodiment, the lawn primer is substantially complementary to either the first or second 3′ primer-binding sequence and the primer added in solution (referred to herein as the solution phase primer) is substantially complementary to the first or second 3′ primer binding sequence, wherein the immobilised and solution phase primer do not bind to the same 3′ primer binding sequence. In other words, only one type of lawn primer participates in the amplification/cluster generation step.
In a preferred embodiment, each single stranded template comprise a 5′ primer-binding sequence that is either a P5 or P7 primer-binding sequence and a 3′ primer-binding sequence that is either a P5′ or P7′ primer-binding sequence. In one embodiment, the lawn primer is a P5 or P7 primer. In another embodiment, the solution phase primer is a P5 or P7 primer.
In one embodiment, the lawn primer is a P7 primer and the solution phase primer is a P5 primer. In this embodiment, the lawn primer binds to P7′ on the 3′ end of the template strand, where P7′ is substantially complementary to P7. In this embodiment, the solution-phase primer binds to P5′ on the 5′ end of the immobilised strand, where P5′ is substantially complementary to P5.
In an alternative embodiment, the lawn primer is a P5 primer and the solution phase primer is a P7 primer. In this embodiment, the lawn primer binds to P5′ on the 3′ end of the template strand, where P5′ is substantially complementary to P5. In this embodiment, the solution-phase primer binds to P7′ on the 5′ end of the immobilised strand, where P7′ is substantially complementary to P7.
In one embodiment, the sequence of P5 comprises or consists of SEQ ID NO: 1 or a variant thereof, the sequence of P5′ comprises or consists of SEQ ID NO: 3 or a variant thereof, the sequence of P7 comprises or consist of SEQ ID NO: 2 or a variant thereof and the sequence of P7′ comprises or consists of SEQ ID NO: 4 or a variant thereof.
The term “variant” as used herein with reference to any of the sequences recited herein refers to a variant nucleic acid that is substantially identical, i.e. has only some sequence variations, for example to the non-variant sequence. In one embodiment, a variant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid sequence.
Of course, reference to P5 and P7 could refer to different primer sequences. Any suitable primer sequence combinations are encompassed by the present disclosure. P5′ and P7′ are complementary (as defined herein) to P5 and P7.
Evidence that the non-bridging method of the disclosure resulted in the formation of clusters is shown in
The use of only one type of lawn primers combined with the use of solution primers in step (f) allows amplification of the template strand without needing a bridging step. This in turn prevents propagation of amplification into adjacent wells, resulting in less steric hindrance, reducing the pitch possible between wells and consequently leads to faster clustering.
Accordingly, in one embodiment, the lawn primer is grafted at a concentration in the range of 0.2 μM to 5 μM or 0.4 μM to 3 μM or 0.5 μM to 2.5 μM. In a further embodiment, the lawn primer is grafted at 0.5, μM or 1.1 μM or 2.2 μM. In a preferred embodiment, the lawn immobilised primer is grafted at 2.2 μM. The lawn primer is either a P5 or P7 primer.
In another embodiment, the solution-phase primer is used at a concentration in the range of 1 μM to 100 μM or 3 μM to 75 μM or 5 to 50 M. In a further embodiment, the solution-phase primer is used at 0.5, μM or 1.1 μM or 2.2 μM. In a preferred embodiment, solution-phase primer is used at 1 μM 5 μM, 10 μM, 25 UM or 50 μM. The solution-phase primer is either a P5 or P7 primer.
Following the step of hybridisation and extension from the solution-phase primers, it is possible for another solution-phase primer to invade the newly formed duplex and extend again the same template strand, thereby creating duplicates. This is shown in
In one embodiment, the extension reaction is carried out by recombinase polymerase amplification (RPA). RPA comprises three core enzymes—a recombinase, a single-stranded DNA binding protein (SSB) and strand-displacing polymerase. As described in Daher et al. (Rana K Daher, Gale Stewart, Maurice Boissinot, Michel G Bergeron, Recombinase Polymerase Amplification for Diagnostic Applications, Clinical Chemistry, Volume 62, Issue 7, 1 Jul. 2016). The recombinase is responsible for strand invasion by forming filaments with the primers. It has been found that preventing the formation of recombinase-primer filaments reduces the formation of duplicates. In one embodiment, this can be achieved by reducing the length of the primers. In particular, without wishing to be bound by theory, shortening the length of the primers may avoid filament formation between the recombinase and the primers, thereby leading to reduced or no strand displacement. In this manner a solution primer is achieved that is capable of hybridisation and elongation but not invasion, thereby preventing or reducing the formation of duplicates. This is shown in
In one embodiment, the length of the solution-phase primers is between 5 and 25 bp or between 9 and 20 bp or between 5 and 15 bp or between 9 and 15 bp. In one embodiment, the length of the solution-phase primers is 10 bp, 13 bp or 15 bp. As above, the solution-phase primer may be a P5 or P7 primer. In one embodiment, the solution-phase primer is a P5 primer. In one embodiment, the solution phase primer is between 5 and 25 bp or between 10 and 20 bp or between 5 and 15 bp, preferably 10 bp, 13 bp or 15 bp of SEQ ID NO: 1 or 2. In other words, the solution-phase primer can be any—e.g. 13 bp of SEQ ID NO: 1 or 2. As shown in
In addition, the resulting sequence performance (P90 and % PF) is comparable whether the smart solution primers of the disclosure or longer-length amplification primers are used. This is shown in
In a further embodiment of the disclosure, the solution-phase primers comprise or consist of a nucleic acid sequence as defined in SEQ ID NO: 5, 6 or 7 or a variant thereof. In one embodiment, the solution-phase primers comprise or consist of SEQ ID NO: 6 or a variant thereof.
In another aspect of the disclosure, there is provided a solution-phase primer comprising or consisting of SEQ ID NO: 5, 6 or 7 or a variant thereof.
Following amplification of a template strand into a cluster, the next step in the process of sequencing the insert is sequencing of the forward strand and re-synthesis and sequencing of the reverse strand. In one embodiment this may be carried out by paired-end (PE) re-synthesis.
In one embodiment, PE re-synthesis is achieved using “blocked” or “dormant” lawn primers. These primers do not participate in cluster generation but only in re-synthesis prior to sequencing. In one embodiment, the lawn primer is blocked at the 3′ end, which is removed prior to re-synthesis—e.g. following generation of the cluster. In this way the lawn primer can be considered dormant until the sequencing step. The 3′ block may be a phosphate group or another reversible blocking group.
An exemplary method of sequencing according to the disclosure is shown in
Accordingly, in a further aspect, the disclosure provides a method of sequencing a nucleic acid sequence, wherein the method comprises the following steps, as described above:
In a further aspect, the disclosure provides a method of sequencing a target nucleic acid sequence, wherein the method comprises:
Again, in one embodiment, the lawn primer may be a P5 primer and the dormant lawn primer may be a P7 primer. In another embodiment, the lawn primer may be a P7 primer and the dormant lawn primer a P5 primer. In other words, the lawn and the dormant lawn primers are different.
In one embodiment, the dormant lawn primer is a P5 primer and comprises or consists of a sequence as defined in SEQ ID NO: 8 or a variant thereof. This primer has a polyT provides spacer to reduce steric hindrance during the paired end turn re-synthesis. Shexynyl is a non-limiting example of a linking group that allows attachment of the primer to the surface of the sold support. Other linking groups would be apparent to the skilled person.
Paired-end re-synthesis in particular requires numerous cycles (11 in a standard cycle) because of surface P5 damage in the first linearization, where some of the P5 primers are not able to be extended. The damage can come from a possible incomplete chemical reaction (CCL1) or inaccurate enzyme (Uracil) catalysed cleavage. In the present disclosure, as only one type of lawn primers participate in generation of the cluster, the first linearization is not required in order to carry out read one (R1). Accordingly, the present disclosure provides a method of sequencing (e.g. by paired-end re-synthesis) that avoids damage to surface (i.e. lawn) primers (e.g. P5 lawn primers) during template amplification (i.e. cluster generation). This leads to more efficient PE re-synthesis. This is demonstrated by an increase in intensity of the second sequencing read (i.e. read 2) as shown in
The increased efficiency of the present disclosure is further shown in
In one embodiment, the dormant lawn primer is grafted at a concentration in the range of 0.2 μM to 5 μM or 0.4 μM to 3 μM or 0.5 μM to 2.5 μM. In a further embodiment, the dormant lawn primer is grafted at 0.5, μM or 1.1 μM or 2.2 μM. In a preferred embodiment, the dormant lawn primer is grafted at 2.2 μM. The dormant lawn primer is either a P5 or P7 primer.
It has also been found that the ratio of lawn primers and dormant lawn primers affects read 1 and 2 intensity. As shown in
As the solution-phase primers are also shorter in length, in one embodiment, the dormant lawn primer may also be correspondingly shorter in length. In a further embodiment, the dormant lawn primer may also be between 5 and 25 bp or between 7 and 20 bp or between 9 and 13 bp. In one embodiment, the length of the dormant lawn primer is 9 bp, 10 bp or 13 bp. The use of shorter-length dormant primers, in addition to primers with a 3′ blocking group, not only prevents extension until following cluster generation but also prevents invasion (i.e. unwanted annealing), which would decrease amplification efficiency. As shown in
In a further embodiment, the dormant lawn primers may comprise or consist of a nucleic acid sequence selected from SEQ ID NO: 9, 10 or 11 or a variant thereof. The primers may also be blocked at the 3′ end (i.e. a 3′ blocking group), where the block prevents extension of the primer until the block is removed.
In a further aspect of the disclosure, there is provided a re-synthesis primer, the primer comprising a nucleic acid sequence selected from SEQ ID NO: 9, 10 or 11 or a variant thereof, and wherein the primer comprises a 3′ blocking group that prevents extension of the primer until the blocking group is removed. By “re-synthesis” is meant a primer that is capable of synthesising the reverse or complement strand after the first sequencing read (i.e. read 1). The re-synthesis primer is also referred to herein as a dormant lawn primer, and such terms may be used interchangeably.
In one embodiment the blocking group is a phosphate group. In one embodiment the surface of the solid support is treated with a phosphatase to remove the block.
In another aspect of the disclosure there is provided a solid support for use in sequencing, wherein the support comprises a plurality of lawn primers immobilised thereon and a plurality of dormant lawn primers immobilised thereon, wherein the dormant lawn primers comprise a blocking 3′ group that prevents extension until removed.
In one embodiment, the lawn primer is selected from a P7 or a P5 primer.
In another embodiment, the dormant lawn primer is selected from a P5 or a P7 primer. In a further embodiment, the dormant lawn primer comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 8, 9, 10 or 11 or a variant thereof.
In one embodiment, the ratio of lawn:dormant lawn primer ratio is selected from 5:1, 4:1, 3:1, 2:1, 1:1 and 1:2, 1:3, 1:4 and 1:5. In a preferred embodiment, the ratio of lawn:dormant lawn primer ratio is selected from 2:1, 1:1 and 1:2.
In further embodiments, the solid support does not require dormant lawn primers to achieve PE re-synthesis. Such a strategy is possible where bridge re-synthesis is not required to enable the second read to take place. An example is a system whereby two pads containing their own set of unique primers and complementary linearization chemistry (one set for read 1 and one set for read 2) are provided. An example of this strategy is using PAZAM pads as described in WO 2020/005503, the entire contents of which are incorporated by reference herein. In such an embodiment, the present disclosure can utilise the primer in solution approach of the present disclosure which avoids/minimises invasion and duplicate formation but does not require dormant lawn primers as described above since it is not necessary to undertake paired-end resynthesis.
The disclosure is now described in the following non-limiting examples:
The disclosure is a new hybrid clustering methodology (as shown in
To demonstrate the effectiveness of the present method, hybrid clustering performance has been evaluated through investigation of kinetics and cluster intensity. This experiment addresses the concern that flexible solution primers could generate primer dimers, which would influence the final sequencing intensity.
A wide range of concentrations titration on solution primers (P5) has been conducted with surface primers grafting at 1.1 μM. Real-time kinetics plot uses subtraction value of real-time intensity and initial intensity as the readout, as EvagreenR would vary the background signal corresponding to the amount of single strand DNA. However, hybrid clustering may not be accurately reflected if only relying on real-time EvaGreen intensity, as the significant background signal from the free solution primers. Thus, the investigation of clustering has been performed in combination of recording real-time intensity of EvaGreen and capturing final cluster intensity.
According to the result shown in
Percentage of duplicate reads is an important parameter in the evaluation of sequencing performance. Several factors can cause the generation of duplicate colonies as showing in
In one embodiment, the clustering method is based on recombinase polymerase amplification (RPA) and it has been reported that the optimized length for RPA primers should be 30-35 bases long for the optimal formation of recombinase/primer filaments, with longer primers not being recommended. A hypothesis comes out that shortening the length of the primers may avoid the filament formation between recombinase and primer, and consequently lower or prevent invasion capability of the solution primers. Solution-based invasion and hyb/extension assays have been employed to test this hypothesis. Sequences of the primer at 10 (TACGGCGACC) (SEQ ID NO: 5), 13 (GGCGACCACCGAG) (SEQ ID NO: 6) and 15 (ACGGCGACCACCGAG) (SEQ ID NO: 7) bp length have been selected from 29 bp sequence of P5 primer. The scheme and the corresponding results of the invasion and hyb/extension of primers with different length are shown in the
For further validation, the sequencing performance has been evaluated. According to the result shown in
Overall, to prevent invasion but maintain hyb/extension capability, the solution primers need to be designed to only form filament with polymerase, but not recombinase. Thus, besides tuning the length of the primers, a series of other possible approaches have been considered, such as modifying the backbone of the primers (decorating backbone with fluorine, incorporation of several PNA/LNA bases, internal mismatches sequence of the primers, or implementations with carbon spacers within primer sequence, etc), separately and in combination with modifications to the recombinase and or polymerase.
To obtain capability of PE sequencing, phosphate blocked P5 primers are grafted with surface clustering primer (P7) on the lawn. Surface-bounded blocked P5 is employed only for PE re-synthesis purpose, thus they are deprotected prior to PE turn. (scheme showing in the
ACCGAG*A/ideoxyU/CTACAC
In this sense, the P5 lawn primers are ‘smart’ as well since they are designed to not only be blocked (preventing extension) but also be short enough to prevent invasion (non-productive) which could slow the ExAmp reaction (decreasing amplification efficiency).
PE re-synthesis efficiency was evaluated using hybrid clustering according to the present disclosure to quantify the effect of no surface P5 damage caused from the first linearization. PE re-synthesis test is firstly conducted by comparing the intensity of read 2 after different re-synthesis cycles (1, 2, 5, 11), where the normal Illumina clustering is carried out in parallel as the control experiment. The result suggests the hybrid clustering can achieve much higher read 2 intensity, and similar intensity under different re-synthesis cycles (blue bars in
This application claims the benefit of U.S. Provisional Patent Application No. 63/290,183, filed Dec. 16, 2021 and entitled “Hybrid Clustering,” the entire contents of which are incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/053005 | 12/15/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63290183 | Dec 2021 | US |
