Nickase-mediated linear amplification of library constructs for duplex sequencing

Abstract

The present invention relates to methods, compositions, and kits used for library preparation for duplex Sequencing by Expansion that include linear amplification of a duplex nucleic acid template construct and further methods that include a target enrichment step in the library preparation workflow. Also provided are methods for the amplification of a duplex template construct that combine a first linear amplification step and a second linear amplification step in which the template strands are switched between the first and second amplification steps. Further template amplification strategies are also provided in the present disclosure.

Description

NICKASE-MEDIATED LINEAR AMPLIFICATION OF LIBRARY CONSTRUCTS FOR DUPLEX SEQUENCINGBACKGROUND OF THE INVENTION

[0001] Commonly used NGS platforms often rely on PCR amplification during library construction to increase the copy number of library fragments to meet the needs of the sequencing platforms. However, the most problematic step in sample preparation procedures is amplification. This is because PCR amplification stochastically introduces biases, which can propagate to later cycles. In addition, PCR also amplifies different molecules with unequal probabilities, leading to uneven amplification of DNA molecules. For example, fragments with high AT or GC content may become underrepresented or completely lost during library preparation. Another challenge in relying on PCR for sequencing library preparation is the mandatory requirement for thermal cycling conditions and miniaturization of PCR instrumentation. These PCR-mediated changes introduced during sample preparation severely compromise the quality of the data, complicating data quantitation and representation (coverage), which can only be partially compensated for by deeper sequencing.

[0002] Recently, different methods have been proposed to reduce reliance on PCR amplification, such as PCR-free protocols and isothermal amplification. However, such methods possess shortcomings when sequencing duplexed, or paired-end, library constructs. Therefore, there is a need in the art for the development of improved methods resulting in linear amplification of sequencing libraries, particularly for duplex sequencing.

[0003] Provided herein are novel and useful compositions and methods for carrying out linear amplification of paired-end, duplexed DNA template constructs. These compositions and methods provide advantages to several sequencing methods, e.g., nanopore-based, single molecule sequencing methods.

[0004] All of the subject matter discussed in the Background section is not necessarily prior art and should not be assumed to be prior art merely as a result of its discussion in the Background section. Along these lines, any recognition of problems in the prior art discussed in the Background section or associated with such subject matter should not be treated as prior art unless expressly stated to be prior art. Instead, the discussion of any subject matter in the Background section should be treated as part of the inventor’s approach to the particular problem, which in and of itself may also be inventive.BRIEF SUMMARY OF THE INVENTION

[0005] The present disclosure provides improved methods, compositions, and kits for linear amplification of duplex nucleic acid template constructs and their use in duplex sequencing methods, including, e.g., Sequencing by Expansion.

[0006] In one aspect, the invention provides a method of amplifying a duplex nucleic acid template, the method including the steps of: (a) providing a first double stranded nucleic acid product, in which a first strand of the first double stranded nucleic acid product includes the duplex nucleic acid template and a second strand of the first double stranded nucleic acid product includes a copy of the duplex nucleic acid template, and in which the copy of the duplex nucleic acid product includes a nickase endonuclease cleavage site; (b) contacting the double stranded nucleic acid product with a nickase endonuclease under endonuclease conditions, in which the nickase endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the copy of the duplex nucleic acid template; (c) contacting the free 3’ end in the copy of the duplex nucleic acid product with a strand displacing nucleic acid polymerase under nucleic acid synthesis conditions, in which the strand displacing nucleic acid polymerase synthesizes a new copy of the duplex nucleic acid template, in which the new copy displaces the copy of step (a) from the first double stranded nucleic acid product; and (d) repeating steps (a) through (c) to provide an amplified population of copies of the duplex nucleic acid template.

[0007] In some embodiments, the method further comprises the steps of: (e) contacting the amplified population of copies of the duplex nucleic acid template described above with an oligonucleotide probe under nucleic acid hybridization conditions, in which the oligonucleotide probe includes the nucleic acid target sequence, and in which the oligonucleotide probe is bound to a solid support; (f) washing the solid support to selectively retain an enriched copy of the duplex nucleic acid template, in which the enriched copy includes the nucleic acid target sequence, and in which the enriched copy is specifically hybridized to the oligonucleotide probe; (g) releasing the enriched copy of the duplex nucleic acid template from the solid support; (h) providing a second double stranded nucleic acid product in which a first strand of the second double stranded nucleic acid product includes the enriched copy of the duplex nucleic acid template and a second strand of the second double stranded nucleic acid product includes a copy of the enriched copy, in which the copy of the enriched copy of the duplex nucleic acid template includes a nickase endonuclease cleavage site; (i) contacting the second double stranded nucleic acid product with a nickase endonuclease under endonuclease conditions, in which the nickase endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the copy of the enriched copy; (j) contacting the free 3’ end in the copy of the enriched copy of the duplex nucleic acid template with a strand displacing nucleic acid polymerase under nucleic acidsynthesis conditions, in which the strand displacing DNA polymerase synthesizes a new copy of the enriched copy of the duplex nucleic acid template, and in which the new copy displaces the copy of step (h); and (k) repeating steps (h) through (j) to provide an amplified population of copies of the enriched copy of the duplex template construct comprising the nucleic acid target sequence.

[0008] In some embodiments, the method further comprises the steps of (e) contacting the amplified population of copies of the duplex nucleic acid template described above with an oligonucleotide primer under nucleic acid hybridization conditions, in which the nucleic acid sequence of the oligonucleotide primer is complementary to a nucleic acid sequence in the amplified copies of the duplex nucleic acid template, and in which the oligonucleotide primer is bound to a solid support, and in which the amplified copies of the duplex nucleic acid template hybridize to the oligonucleotide primer bound to the solid support; (f) contacting the oligonucleotide primer with a nucleic acid polymerase under nucleic acid synthesis conditions, in which the nucleic acid polymerase extends the oligonucleotide primer to form a second double stranded nucleic acid product, in which the second double stranded nucleic acid product includes a copy of the amplified copy of the duplex nucleic acid template bound to the solid support; (g) contacting the second double stranded nucleic acid product with denaturing conditions, in which the denaturing conditions produce a single stranded nucleic acid product bound to the solid support, in which the single stranded nucleic acid product includes the copy of the amplified copy of the duplex nucleic acid template; (h) contacting the single stranded nucleic acid product with an oligonucleotide primer under nucleic acid hybridization conditions, in which the oligonucleotide primer includes a nucleic acid sequence complementary to a nucleic acid sequence in the single stranded nucleic acid product and a nucleic acid polymerase under nucleic acid synthesis conditions, to produce a third double stranded nucleic acid product, in which the third double stranded nucleic acid product includes a copy of the single stranded nucleic acid product; and (i) contacting the third double stranded nucleic acid product with denaturing conditions to produce an amplified population of copies of the duplex nucleic acid template released from the solid support.

[0009] In another aspect, the invention provides a method of amplifying a duplex nucleic acid template including a nucleic acid target sequence, the method including the steps of: (a) contacting the amplified population of copies of the duplex nucleic acid template described above with an oligonucleotide probe under nucleic acid hybridization conditions, in which the oligonucleotide probe includes the nucleic acid target sequence, and in which the oligonucleotide probe is bound to a solid support; (b) washing the solid support to selectively retain an enriched copy of the duplex nucleic acid template, in which the enriched copy includes the nucleic acidtarget sequence, and in which the enriched copy is specifically hybridized to the oligonucleotide probe; (c) releasing the enriched copy of the duplex nucleic acid template from the solid support; (d) providing a second double stranded nucleic acid product in which a first strand of the second double stranded nucleic acid product includes the enriched copy of the duplex nucleic acid template and a second strand of the second double stranded nucleic acid product includes a copy of the enriched copy, in which the copy of the enriched copy of the duplex nucleic acid template includes a nickase endonuclease cleavage site; (e) contacting the second double stranded nucleic acid product with a nickase endonuclease under endonuclease conditions, in which the nickase endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the copy of the enriched copy; (f) contacting the free 3’ end in the copy of the enriched copy of the duplex nucleic acid template with a strand displacing nucleic acid polymerase under nucleic acid synthesis conditions, in which the strand displacing DNA polymerase synthesizes a new copy of the enriched copy of the duplex nucleic acid template, and in which the new copy displaces the copy of step (d); and (g) repeating steps (d) through (f) to provide an amplified population of copies of the enriched copy of the duplex template construct comprising the nucleic acid target sequence.

[0010] In another aspect, the invention provides a method of amplifying a duplex nucleic acid template, the method including the steps of (a) contacting the amplified population of copies of the duplex nucleic acid template described above with an oligonucleotide primer under nucleic acid hybridization conditions, in which the nucleic acid sequence of the oligonucleotide primer is complementary to a nucleic acid sequence in the amplified copies of the duplex nucleic acid template, and in which the oligonucleotide primer is bound to a solid support, and in which the amplified copies of the duplex nucleic acid template hybridize to the oligonucleotide primer bound to the solid support; (b) contacting the oligonucleotide primer with a nucleic acid polymerase under nucleic acid synthesis conditions, in which the nucleic acid polymerase extends the oligonucleotide primer to form a second double stranded nucleic acid product, in which the second double stranded nucleic acid product includes a copy of the amplified copy of the duplex nucleic acid template bound to the solid support; (c) contacting the second double stranded nucleic acid product with denaturing conditions, in which the denaturing conditions produce a single stranded nucleic acid product bound to the solid support, in which the single stranded nucleic acid product includes the copy of the amplified copy of the duplex nucleic acid template; (d) contacting the single stranded nucleic acid product with an oligonucleotide primer under nucleic acid hybridization conditions, in which the oligonucleotide primer includes a nucleic acid sequence complementary to a nucleic acid sequence in the single stranded nucleic acid product and a nucleic acid polymerase under nucleic acid synthesis conditions, to produce athird double stranded nucleic acid product, in which the third double stranded nucleic acid product includes a copy of the single stranded nucleic acid product; and (e) contacting the third double stranded nucleic acid product with denaturing conditions to produce an amplified population of copies of the duplex nucleic acid template released from the solid support.

[0011] In some embodiments, the nickase endonuclease is selected from the group consisting of Nb. BbvCl, Nb. Bsml, Nt. BstNBI, Nt. BspQI, Nt. BspD61, Nt. Bst9I, Nt. BstSEI, Nt. BsmAI, Nt. AIwI, Nb. BsrDl, and Nt. CviPII, or variants thereof. In some embodiments, the strand displacing nucleic acid polymerase is a strand displacing DNA polymerase selected from the group consisting of Bst wildtype, Bst 2.0, Bst 3.0, Bsu, and Klenow fragment, or variants thereof. In some embodiments, the nickase endonuclease conditions and the DNA synthesis conditions are provided in the same reaction mixture. In other embodiments, the reaction mixture includes a nucleotide analog, in which the nucleotide analog includes N4-Me dCTP or 7-deaza dGTP. In some embodiments, the reaction mixture imcludes one or more of a single stranded binding protein, a pyrophosphatase, a DPO4 polymerase or a variant thereof, and a translesion repair enzyme. In some embodiments, the nickase endonuclease conditions and the DNA synthesis conditions include isothermal conditions, in which the isothermal conditions include incubation at around 50 degrees Celsius to around 55 degrees Celsius for around 5 hours to around 24 hours. In some embodiments, the nucleic acid synthesis conditions of step (d) of the third method described above include native nucleotides. In some embodiments, the duplex nucleic acid template includes a double stranded nucleic acid fragment joined on a first end to a Y adapter and joined on a second end to a hairpin adapter, in which the hairpin adapter covalently joins the two strands of the double stranded nucleic acid fragment. In yet other embodiments, the Y adapter includes a nickase endonuclease recognition site. In some embodiments, the double stranded nucleic acid fragment is provided by a formalin-fixed, paraffin embedded (FFPE) sample. In yet other embodiments, the double stranded nucleic acid fragment is treated with one or more of an FFPE repair kit, an end-repair A-tail (ERAT) kit, an exonuclease enzyme, or an enzymatic fragmentation (FragTail) kit. In yet other embodiments, the FFPE repair kit includes a thermolabile exonuclease enzyme or a RecJf enzyme. In yet other embodiments, the double stranded nucleic acid fragment is treated with a fragmentation kit, a SPRI purification kit, a FFPE repair kit, a RecJf enzyme, a thermolabile proteinase K, and an ERAT kit. In other embodiments, the methods further includes the step of purifying the amplified population of copies of the duplex template construct with SPRI beads. In yet other embodiments, the purified copies of the duplex template construct are eluted from the SPRI beads with an elution buffer including one or more of a Tris buffer, a sodium salt, EDTA, and PEG8k. In some embodiments, the method further includes the step of real-time quantificationof the amplified population of copies of the duplex nucleic acid template using a molecular beacon probe. In some embodiments, the step of providing a first double stranded nucleic acid product includes contacting the duplex nucleic acid template with an oligonucleotide primer under nucleic acid hybridization conditions, in which the oligonucleotide primer includes a nucleic acid sequence complementary to a sequence a single stranded arm region of the Y adapter and a nucleic acid polymerase under nucleic acid synthesis conditions.

[0012] In some embodiments, the methods of the present invention further comprise contacting the amplified population of duplex nucleic acid templates of any of the above methods with an extension oligonucleotide under nucleic acid hybridization conditions, in which the extension oligonucleotide includes a nucleic acid sequence complementary to the duplex nucleic acid template, and with a variant of DPO4 polymerase under Xpandomer synthesis conditions. In some embodiments, the Xpandomer synthesis conditions include a buffer / salt system, polymerase cofactors, polymerase enhancing moieties (PEMs), XNTP substrates, a phosphate shield molecule, a solvent, a crowding agent, and a single stranded binding protein. In other embodiments, the extension oligonucleotide is bound to a solid support.

[0013] In another aspect, the invention provides a method of synthesizing an Xpandomer copy of a duplex nucleic acid template including contacting the amplified population of duplex nucleic acid templates of any of the above methods with an extension oligonucleotide under nucleic acid hybridization conditions, in which the extension oligonucleotide includes a nucleic acid sequence complementary to the duplex nucleic acid template, and with a variant of DPO4 polymerase under Xpandomer synthesis conditions. In some embodiments, the Xpandomer synthesis conditions include a buffer / salt system, polymerase cofactors, polymerase enhancing moieties (PEMs), XNTP substrates, a phosphate shield molecule, a solvent, a crowding agent, and a single stranded binding protein. In other embodiments, the extension oligonucleotide is bound to a solid support.

[0014] In another aspect, the invention provides a kit for linear amplification of a duplex template construct including one or more of an oligonucleotide primer, an isothermal amplification buffer, dNTPs, a pyrophosphatase, a single stranded binding protein, a strand displacing DNA polymerase, and a nicking endonuclease. In some embodiments, the isothermal amplification buffer includes one or more of Tris-HCl, (NEL^SCU, KC1, MgSCU, and Tween 20, the dNTPs include 7-deaza dGTP, the single stranded binding protein is selected from the group consisting of gp32, TTH, KOD, RPA, NCp7, RecA, and UvrD, the strand displacing DNA polymerase is a Bst polymerase or a variant thereof, and the nicking endonuclease is selected from the group consisting of Nb. BbvCl, Nb. Bsml, Nt. BstNBI, Nt. BspQI, Nt. BspD61, Nt. Bst9I, Nt. BstSEI, Nt. BsmAI, Nt. AIwI, Nb. BsrDl, and Nt. CviPII, or variants thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1 A and IB are condensed schematics summarizing one embodiment of the methods of nickase-mediated linear amplification of a duplex nucleic acid template construct of the present invention.

[0016] FIG. 2 is a condensed schematic summarizing one embodiment of the methods of dual round nickase-mediated linear amplification of a duplex nucleic acid template construct of the present invention integrating a target enrichment step.

[0017] FIGS. 3A and 3B are condensed schematics summarizing one embodiment of dual round nickase-mediated linear amplification of a nucleic acid template construct of the present invention in which the first round amplifies a non-duplexed template construct and the second round amplifies a duplexed template construct and in which the first and second round are separated by a target enrichment step and a hairpin adapter ligation step.

[0018] FIGS. 4 A and 4B are condensed schematics summarizing one embodiment of dual round amplification of a nucleic acid template construct of the present invention in which the first round amplifies a non-duplexed template construct by conventional PCR and the second round amplifies a duplexed template construct by linear amplification and in which the first and second round are separated by a target enrichment step and a hairpin adapter ligation step.

[0019] FIG. 5 is a condensed schematic summarizing one embodiment of the methods of generating a mate paired template construct of the present invention.

[0020] FIG. 6 is a condensed schematic summarizing another embodiment of the methods of generating a mate paired template construct of the present invention integrating a linear amplification step.

[0021] FIGS. 7A, 7B, 7C and 7D are condensed schematics summarizing one embodiment of the methods of generating a duplex nucleic template construct of the present invention and use in duplexed Xpandomer synthesis.

[0022] FIGS. 8 A and 8B are condensed schematics summarizing another embodiment of the methods of generating a duplex nucleic template construct of the present invention utilizing prenicked hairpin adapters.

[0023] FIGS. 9 A, 9B and 9C are condensed schematics summarizing another embodiment of the methods of generating a duplex nucleic template construct of the present invention employing solid-state synthesis and use in duplexed Xpandomer synthesis.

[0024] FIG. 10 is a condensed schematic summarizing one embodiment of the methods of generating a duplex parent-parent nucleic template construct of the present invention.

[0025] FIG. 11 is a condensed schematic summarizing another embodiment of the methods of generating a duplex parent-parent nucleic template construct of the present invention.

[0026] FIG. 12 is a simplified flow chart illustrating one embodiment of a template amplification workflow that incorporates a template switch step that is carried out on a solid support.

[0027] FIG. 13 is a simplified flow chart illustrating another embodiment of a template amplification workflow that incorporates a template switch step that is carried out on a solid support.

[0028] FIG. 14 is a simplified illustration of one embodiment of a generic xNTP substrate for Xpandomer synthesis.DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included herein. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0030] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and so forth which are within the skill of the art. Such techniques are explained fully in the literature. See e g., Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition (1989), OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984), the series METHODS IN ENZYMOLOGY (Academic Press, Inc ), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Siedman, J. A. Smith, and K. Struhl, eds., 1987).

[0031] Reference throughout this specification to “one embodiment” or “an embodiment” and variations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, i.e., one or more, unless the content and context clearlydictates otherwise. It should also be noted that the conjunctive terms, “and” and “or” are generally employed in the broadest sense to include “and / or” unless the content and context clearly dictates inclusivity or exclusivity as the case may be. Thus, the use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. In addition, the composition of “and” and “or” when recited herein as “and / or” is intended to encompass an embodiment that includes all the associated items or ideas and one or more other alternative embodiments that include fewer than all the associated items or ideas.

[0033] Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” and “include”, as well as variations thereof such as “comprises” and “comprising” are to be construed in an open, inclusive sense, e.g., “including, but not limited to.” The term "consisting essentially of' limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic and novel characteristics of the claimed invention.

[0034] The abbreviation, "e.g.," is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g.," is synonymous with the term "for example." It is also to be understood that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise, the term “X and / or Y” means “X” or “Y” or both “X” and “Y”, and the letter “s” following a noun designates both the plural and singular forms of that noun. In addition, where features or aspects of the invention are described in terms of Markush groups, it is intended, and those skilled in the art will recognize, that the invention embraces and is also thereby described in terms of any individual member and any subgroup of members of the Markush group, and Applicants reserve the right to revise the application or claims to refer specifically to any individual member or any subgroup of members of the Markush group.

[0035] Any headings used within this document are only being utilized to expedite its review by the reader, and should not be construed as limiting the invention or claims in any manner. Thus, the headings and Abstract of the Disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0036] Where a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated rangeincludes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0037] For example, any concentration range, percentage range, ratio range, or integer range provided herein is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated.Methods and Compositions for Nickase-Mediated Linear Amplification of Duplex Template Constructs

[0038] The methods of the present invention may be used for the linear amplification of any suitable nucleic acid template, such as a duplex template construct that, e.g., may be used as a template for Xpandomer synthesis. Linear amplification, also be referred to herein as isothermal amplification, is well known in the art (see, e.g., Joneja and Huang. 2011. Anal. Biochem. July 1; 414(l):58-69, the entire contents of which is herein incorporated by reference in its entirety). Linear amplification replicates a nucleic acid template in a non-exponential manner.Accordingly, the original template strand (i.e. the parental strand) serves as a template for all subsequent replication events, while the newly synthesized complementary copy strands (i.e. the daughter strands) are not replicated as part of the template amplification process.Advantageously, linear amplification prevents errors in replication from being exponentially propagated into the progeny population of complementary copy strands. Linear amplification is also advantageous in that it is not limited by constraints of thermal cycling well known in the art, e.g., dependence on heat-resistant DNA polymerases. In particular, many of the heat-resistant DNA polymerase used for conventional PCR do not possess robust strand displacement activity. As such, conventional PCR has limited use in the amplification of duplex template constructs (e.g., double stranded DNA fragments in which the two complementary strands are linked by an intervening hairpin adapter structure).

[0039] In certain embodiments, the methods of the present invention may be used for the linear amplification of duplex template constructs, such as those disclosed in Applicant’s published PCT application no.s WO / 2025 / 132779, entitled, “Methods and Compositions for Nucleic Acid Library and Template Preparation for Duplexed Sequencing by Expansion”, and WO / 2025 / 087393, entitled “Methods and Compositions for Nucleic Acid Library and TemplatePreparation using Extendable Adapters for Duplexed Sequencing by Expansion”, the contents of which are herein incorporated by references in their entireties. Certain exemplary duplex template constructs, and methods of making and using the same for Sequencing by Expansion are described further herein.

[0040] In certain embodiments, the methods of the present invention for linear amplification of duplex template constructs employ nicking endonuclease-enabled strand displacement by a DNA polymerase. In certain embodiments, the methods include the following steps: (1) a nicking enzyme recognizes and cleaves a specific site in a single strand of a double stranded region of DNA, creating a free 3’ end; (2) a strand displacing DNA polymerase extends a new strand from the newly created free 3 ’ end, the new strand including the recognition site for the nicking enzyme; (3) the non-template strand is displaced from the template strand by the nascent strand, e.g., the non-template strand is displaced as a free single strand in solution; (4) the newly synthesized strand includes the renewed nickase site for subsequent rounds of amplification using the original template strand as the template for the repeated cycles of nicking and extension.

[0041] As used herein, “nicking” refers to the cleavage of only one strand of a doublestranded portion of a fully or partially double-stranded nucleic acid. The position where the nucleic acid is nicked is referred to as the nicking site or nickase cleavage site. The recognition sequence that the nicking enzyme recognizes is referred to as the nicking enzyme binding or recognition site. “Capable of nicking” refers to an enzymatic capability of a nicking enzyme.

[0042] As used interchangeably herein, a nicking enzyme, a nickase, or a nicking endonuclease is a protein that binds to double-stranded DNA and cleaves one strand of a doublestranded duplex. The nicking enzyme may cleave either upstream or downstream of the binding site, or nicking enzyme recognition site. When used in connection with a nicking endonuclease, the letters “Nt” or “Nb” indicate whether it is the top (Nt) or bottom (Nb) strand that is cut by the enzyme. In certain embodiments, a suitable nickase will be one that has a relatively long recognition site in order to minimize the occurrence of cleavage at undesired sites in the library fragment. In other embodiments, single stranded cleavage may be achieved by use of a CRISPR- cas enzymatic system.

[0043] In certain embodiments, the nicking enzyme may be selected from the group consisting of one or more of the nicking enzymes listed in Table 1. Those of ordinary skill in the art will recognize that various nicking enzymes other than those mentioned specifically herein may be used in the present methods. Nicking enzymes are available from many commercial sources, for example, New England Biolabs (NEB). In certain embodiments, a suitable nicking enzyme is Nt.BspQl, commercially available from NEB.

[0044] In other embodiments, a suitable nicking enzyme may be a variant of any of the enzymes set forth in Table 1. In certain embodiments, the suitable variant may have from around 95% to around 99% identity, from around 90% to around 95%, from around 85% to around 90%, from around 80% to around 85% or less than around 80% identity to an enzyme set forth in Table 1.Table 1Exemplary Nicking Enzymes(note: D=A or G or T; N=A or G or C or T; “X” indicates that the complementary strand of the sequence is recognized and cut; “V” indicates that the sequence itself is recognized and cut).

[0045] According to the methods of the present invention, DNA polymerases with strand displacement activity are particularly advantageous. In certain embodiments, a suitable standdisplacing DNA polymerase is a Bst DNA polymerase. In other embodiments suitable strand displacing DNA polymerases may include Bsu, Klenow Fragment, Phi29, Phi29-XT, and vent DNA polymerase.

[0046] In certain embodiments, a variant of any suitable nucleic acid polymerase may be used according to the present invention. For example, a suitable variant may be a commercially available variant of wildtype Bst or Bst large fragment DNA polymerase, such as “Neobolt Bst” (available from Varizymes, Middleton, WI). In other embodiments, a suitable commercially available variant of Bst or Bst large fragment DNA polymerase may be Bst 2.0 or Bst 3.0, (available from NEB) or Thermo Lyo Ready Bst DNA polymerase, commercially available from ThermoFisher Scientific.

[0047] In other embodiments, a suitable variant polymerase may be a variant of any of the DNA polymerase disclosed herein. In certain embodiments, the suitable variant may have from around 95% to around 99% identity, from around 90% to around 95% identity, from around 85% to around 90% identity, from around 80% to around 85% or less than around 80% identity to any of the DNA polymerases set forth herein. Of course, a suitable variant will retain the ability to synthesize a complementary copy of a duplex template construct during the linear amplification methods disclosed herein.

[0048] Other reactive materials are needed in the linear amplification reaction, such as a suitable buffer, dNTPs or analogs thereof, and other application-dependent additives. In certain embodiments, the nicking enzyme is functional in the same reaction conditions as the polymerase, so balancing the conditions to favor the appropriate activities for each enzyme is necessary. In certain embodiments, for example, it may be desirable to optimize the activity of the DNA polymerase while minimizing the activity of the nickase enzyme. In other embodiments, it may be desirable to optimize the activity of the nickase enzyme while minimizing the activity of the DNA polymerase.

[0049] In certain non-limiting embodiments, a linear amplification protocol may include an extension reaction including template DNA, a primer that hybridizes to an end region of the template DNA, a mixture of dNTPs, a pyrophosphatase, a single stranded binding protein and a Bst DNA polymerase and an amplification reaction that further includes a nicking endonuclease added to the extension reaction. In some embodiments, the mixture of dNTPs may include 7- deaza dGTP and / or N4-methyl dCTP. In certain embodiments, the mixture of dNTPs may include from around 25% to around 100% 7-deaza dGTP and / or N4-methyl dCTP in place of native dGTP and dCTP.

[0050] In other embodiments, an extension reaction may include certain DNA polymerases and / or other additives to optimize the replication and amplification of templates that havesustained certain forms of DNA damage or other alterations that could challenge a strand displacing DNA, or other, polymerase. Examples of common forms of DNA damage are known in the art and include damaged bases in FFPE samples or other poorly stored samples of DNA, abasic or other modified sites that result from methylation conversion protocols, or other chemical or enzymatic conversion protocols employed during parent-parent or parent-daughter duplex library preparation protocols described herein.

[0051] In certain embodiments, an extension reaction may be optimized by including a DPO4 DNA polymerase or a DPO4 polymerase variant that is described further herein with reference to the SBX® reaction. In other embodiments, the extension reaction may include a translesion repair enzyme. Exemplary translesion repair enzymes include, but are not limited to, Pol r| (Pol eta), Pol r (Pol iota), Pol K (Pol kappa), and Revl. In other embodiments, the extension reaction may include modified conditions, such as those disclosed herein with reference to the SBX® reaction and Table 2. For example, an extension reaction may include a single stranded DNA binding protein (SSB). In other embodiments, the extension reaction may include one or more suitable modified dNTPs, e.g., 7-deaza dGTP and others disclosed herein.

[0052] As used herein, the terms “isothermal conditions” and “constant temperature” may be used interchangeably and refer to a set of reaction conditions where the temperature of the reaction is kept essentially or substantially constant during the course of the amplification reaction. An advantage of isothermal amplification is that the temperature does not need to be cycled between an upper temperature and a lower temperature. The nicking and extension reaction will work at the same temperature or within the same narrow temperature range. However, it is not necessary that the temperature be maintained at precisely one temperature.

[0053] In certain embodiments, the isothermal conditions may include incubation of an amplification reaction at a temperature from around 25 degrees Celsius to around 80 degrees Celsius, from around 35 degrees Celsius to around 70 degrees Celsius, from around 40 degrees Celsius to around 65 degrees Celsius, or around 45 degrees Celsius to around 60 degrees Celsius. In some embodiments, the isothermal conditions may include incubation of an amplification reaction at a temperature from around 50 degrees Celsius to around 55 degrees Celsius. In one embodiment, the isothermal conditions may include incubation of an amplification reaction at a temperature around 52 degrees Celsius.

[0054] In certain embodiments, the isothermal conditions may include incubation for around 5 hours to around 24 hours.

[0055] One embodiment of the methods of the present invention is depicted in FIGS. 1A-1B. In this embodiment, as shown in FIG. 1A, duplex template construct 100 is provided, whichincludes a target nucleic acid (e.g., a library) fragment with parent (+) strand 101a (i.e., a sense strand) and parent (-) strand 101b (i.e., an antisense strand). The library fragment is covalently joined on a first end to hairpin adapter 110, which includes a double stranded stem region and a single stranded loop region. The complementary strands of the double stranded stem portion of the hairpin adapter are each ligated to one of the strands of the library fragment. The library fragment is joined on a second end to Y adapter 120, which includes a double stranded stem region, a 5’ single stranded arm region, and a 3’ single stranded arm region. Likewise, the complementary strands of the double stranded stem region of the Y adapter are each ligated to one of the strands of the library fragment.

[0056] In step 1, a first double stranded DNA product is provided. In this embodiment, cleavable extension oligonucleotide 125 is provided with a nucleic acid sequence that is complementary to a nucleic acid sequence in the 3’ single stranded arm of the Y adapter. The cleavable extension oligonucleotide 125 includes an internal restriction site 127 (denoted in FIG. 1A by the arrow internal to the extension oligonucleotide) that can be specifically recognized and cleaved by a nicking endonuclease (e.g., a nickase). As used herein, the terms “restriction site”, “cleavage site” and “recognition site” may be used interchangeably and refer to either strand of a double stranded nucleic acid. The cleavable extension oligonucleotide is contacted with duplex template construct 100 under nucleic acid hybridization conditions. The cleavable extension oligonucleotide provides a free 3’ end for initiation of a DNA synthesis reaction, here mediated by strand displacing DNA polymerase 130. In other embodiments, the internal restriction site 127 may be included in the sequence of the 3’ single stranded arm region of the Y adapter, rather than in the extension oligonucleotide. The skilled artisan will recognize that the cleavage sites in the extension oligonucleotide and the Y adapter will be in opposite orientations (i.e., sense and antisense). In yet other embodiments, the Y adapter may include more than one internal restriction site, e.g., a first recognition site for a first nickase and a second recognition site for a second nickase.

[0057] In step 2, DNA synthesis and nicking endonuclease conditions are provided. Strand displacing DNA polymerase 130 initiates synthesis of a complementary copy of duplex template construct 100. In certain embodiments, the DNA synthesis conditions (i.e., the extension reaction) may include modified nucleotides that, e.g., weaken the strength of the hydrogen bonding between the template strand and the newly synthesized complementary copy strand. Exemplary, non-limiting, modified nucleotides include N4-Me dCTP and 7-deaza dGTP. Strand displacing DNA polymerase 130 synthesizes a contiguous complementary copy of duplex template construct 100 that includes, from the 3’ end of the cleavable extension oligonucleotide 125: daughter (+) strand copy 102a; hairpin strand copy 112; daughter (-) strand copy 102b, andY adapter 5’ single strand arm copy 122. Polymerase 130 continues synthesis of the complementary copy of the duplex template construct through the entire length of the construct, eventually terminating at the 5’ end of the Y adapter 120. While synthesis of the complementary copy of the duplex template construct proceeds, nickase endonuclease 135, also provided in the extension reaction mixture, is capable of recognizing and cleaving internal restriction site 127 in the extension oligonucleotide sequence (or, alternatively, in the Y adapter-derived sequence) . The nicked extension oligonucleotide produces a free 3’ end internal to the extension oligonucleotide that provides an initiation site for the strand displacing polymerase to reinitiate DNA synthesis. Advantageously, in certain embodiments, the modified nucleotides incorporated into the complementary copy of the duplex template construct protect the newly synthesized strand from endonuclease cleavage, as the nickase endonuclease is incapable of cleaving restriction sites that include the non-natural nucleotides.

[0058] FIG. IB illustrates the product of the first extension reaction (i.e. the first double stranded nucleic acid product), in which original duplex template construct 100 is now hybridized to newly synthesized complementary copy 200. The newly synthesized complementary copy includes a sense copy (i.e., “daughter +”) and an antisense copy (i.e., “daughter -“) of the original library fragment covalently linked by an intervening adapter sequence copy of the original hairpin adapter. As shown here, nickase endonuclease 135 has cleaved at restriction site 127 and created a single stranded break in the nucleic acid sequence derived from the extension oligonucleotide. This provides an initiation site for strand displacing DNA polymerase 130, shown here commencing extension of a second complementary copy of the original duplex template construct 100.

[0059] In step 3, the process of strand nicking, to provide a free 3’ end, and initiation of DNA synthesis from the free 3’ end continues. As DNA synthesis proceeds, the downstream, non-template strand is displaced while the nickase restriction site is regenerated. Displacement of the non-template strand is facilitated by the “weakening” non-natural nucleotides incorporated into a daughter strand during the previous cycle of extension. The continuous combined action of endonuclease-mediated strand nicking and polymerase-mediated strand displacement and DNA synthesis results in linear amplification of the duplex template construct to generate an amplified population of daughter amplicons (i.e., “copies”) 250. Each daughter copy in the population is a complementary copy of the original parental duplex template construct 100. Because each complementary copy daughter strand includes a sense and an antisense copy of the original library target fragment, these strands will self-hybridize to form the original duplex configuration, in which complementary regions of the strand are covalently joined on a first end by a hairpin adapter structure and joined on a second end to a Y adapter structure.

[0060] In certain embodiments, a sample of amplified copies of the duplex template construct may be subjected to additional treatments steps or purification steps prior to use as templates for, e.g., Xpandomer synthesis. For example, the sample may be treated with a proteinase K enzyme to digest and inactivate certain protein or other components of the linear amplification reaction. A typical proteinase K digest reaction may be incubated at around 55 degrees Celsius for around 10 minutes followed by incubation at 95 degrees Celsius for around 4 minutes. In other embodiments, the sample of amplified copies may be subjected to a purification step using, e.g., SPRI beads, as discussed herein. In one embodiment, the purified sample of amplified copies may be eluted from SPRI beads in an elution buffer including one or more of a buffer / salt system and one or more stabilizing agents. In certain embodiments, an elution buffer may include one or more of a Tris buffer, a sodium salt, EDTA, and PEG8K. In more specific embodiments, an elution buffer may include from around 50mM TrisCi to around 750mM TrisCi, from around 50mM to around 750mM NaCl, from around 0.1% to around 1.0% PEG8K, and from around 0.01 to around ImM EDTA. In one embodiment, an elution buffer may include one or more of around 50mM TrisCi, around 500mM NaCl, around 0.5% PEG, and around 0.05mM EDTA. In other embodiments, an elution buffer may include any of the additional additives disclosed herein, e.g., a single stranded binding protein or a PEM.

[0061] In certain embodiments, the linear amplification reaction may be monitored, or quantified, in real-time using molecular beacon-based technologies. For example, a hairpin probe can be designed with a molecular beacon joined to the end of one strand in the stem portion of the adapter that is quenched by proximity to a quenching moiety joined to the end of the other strand of the hairpin stem. The sequence of a region of the quenching strand of the hairpin stem can also be designed to hybridize to a complementary sequence in a single stranded arm region of the Y adapter component of a duplex template construct. When the hairpin probe hybridizes to the duplex template construct, the hairpin adapter linearizes and the molecular beacon signal at one end is liberated from the quenching effect of the quenching moiety at the other end and is thus capable of being detected.

[0062] In one embodiment, the molecular beacon probe is designed to hybridize to the 3’ single stranded arm of the Y adapter component of the amplified duplex copies of a duplex template construct. As the duplex copies are produced and displaced from the template during the next round of replication, the molecular beacon probe hybridizes to the complementary sequence in the 3’ single stranded arm and produces a fluorescent single that can be detected by, e.g., a conventional light cycler instrument. As the amplification reaction proceeds, the fluorescent signal will increase and can be monitored in real-time. Advantageously, this enables termination of the amplification reaction when a sufficient copy number of duplex copies hasbeen reached. The probe can then be displaced from the duplex copies and removed from the sample using art-recognized techniques.

[0063] In other embodiments, the present invention provides methods that include more than one round of nickase-mediated linear amplification. In one embodiment, an exemplary method includes a first round of nickase-mediated linear amplification, for example, as discussed with reference to FIGS. 1A and IB, and a second round of nickase-mediated linear amplification in which one or more of the daughter strand products of the first round of linear amplification function as a template strand for the second round of amplification. In certain embodiments, the second round of linear amplification may utilize a second nickase enzyme that is different than the nickase enzyme utilized in the first round of amplification. In certain embodiments, a target enrichment step may be performed on the products of the first round of linear amplification, prior to the second round of linear amplification. This may be desirable when a complex library of duplex template constructs is amplified during the first round of linear amplification. The enriched sequences of interest from the first round of amplification can then be used as templates in the second round of linear amplification to selectively amplify specific target sequences of interest.

[0064] One example illustrating a simplified workflow for double rounds of nickase- mediated linear amplification with target enrichment is depicted in FIG. 2. Here, the depiction includes certain upstream library preparation steps that produce duplex template construct 290 (similar upstream library prep steps were omitted from FIGS. 1 A and IB for the sake of simplicity). As shown in FIG. 2, nucleic acid target fragment (i.e., library fragment) 275 is provided that has been end-repaired and A-tailed, as discussed herein. In step 1, the target fragment is contacted with hairpin adapter 277, under DNA ligation conditions to produce asymmetric ligation product 280. Alignment and ligation of hairpin adapter 277 to library fragment 275 is facilitated by the single T overhang on the hairpin adapter. In step 2, Y adapter 287 is provided that, in certain embodiments, may be joined to solid support 285. Joining of the Y adapter to the solid support may be mediated by any suitable coupling chemistry. For example, the Y adapter may include terminal biotin moiety 281 joined to the end of the 5’ single stranded arm regions of the adapter by a flexible linker. Likewise, the solid support may include streptavidin moiety 283 joined to the support by a flexible linker. In certain embodiments, the 3’ end of the double stranded region of the Y adapter may include a single T overhang to facilitate ligation to the library fragment.

[0065] In step 3, the Y adapter is contacted with asymmetric ligation product 280 under DNA ligation conditions to produce duplex template construct 290. In certain embodiments, theduplex template construct may be released from the solid support by cleavage of a selectively cleavable bond in the 5’ single stranded arm of the Y adapter.

[0066] Next, a first round of nickase-mediated linear amplification of duplex template construct 290 is conducted by first replicating the duplex template construct using extension oligonucleotide 295, as discussed with reference to FIGS. 1A and IB. In certain embodiments, the nickase site may be provided either in the extension oligonucleotide or in a single stranded arm region of the Y adapter. As described previously, the alternating nicking of one strand of the double stranded duplex template construct by a nicking endonuclease produces a primer for a strand displacing polymerase to initiate DNA synthesis. As the extension proceeds, the downstream, non-template strand, is displaced, while the nicking site is regenerated. In certain embodiments, displacement of the non-template strand may be facilitated by the “weakening” non-natural nucleotides incorporated into the complementary copy strand during the previous cycle of extension. The continuous combination of nicking, by the endonuclease, and strand displacement synthesis, by the DNA polymerase, results in linear amplification of the duplex template construct.

[0067] In certain embodiments, when the methods of the present invention are applied to a plurality of duplex template constructs (e.g., a library), the population of amplified products can be subjected to a target enrichment step. In general terms, target enrichment enables, e.g., targeted sequencing of just the coding regions of specific genes or segments of chromosomes that are relevant to a particular disease. With this approach, the rest of the whole genome can be disregarded, simplifying downstream bioinformatic analysis and making it more efficient and affordable. Various target enrichment methods are known in the art, including hybridization capture based target enrichment and primer extension-based target enrichment (see, e.g., the KAPA HyperCap workflow and the KAPA HyperPETE workflow, both included with kits commercially available from Roche Sequencing Solutions). Dual primer extension-based target enrichment (PETE) is described in greater detail in Applicant’s U.S. Patent No. 11,773,388, the contents of which are herein incorporated by reference in its entirety.

[0068] One embodiment of a target enrichment step is shown in simplified form in FIG. 2, step 4. Here, population 300 of amplified duplex template constructs includes non-target constructs 300b, and construct 300a that includes the sequence of interest for enrichment. In this embodiment, target hybridization probe 310 is joined to solid support 305 via a biotinstreptavidin linkage, as discussed herein. The population of amplified duplex template constructs is contacted with the target hybridization probe under nucleic acid hybridization conditions. In certain embodiments, the hybridization conditions may include a single strandedbinding protein (SSB) and / or other reaction additives that facilitate opening of the duplex construct structure to enable hybridization of the target probe. In other embodiments, the target hybridization probe may include modified nucleotides that increase the melting temperature of probe-target hybrids so as to energetically favor hybridization of the target fragment to the probe, rather than to its complementary strand within the template construct. In step 5, the unbound, non-target duplex template constructs are removed and the enriched, target duplex template construct 300a is released from the support.

[0069] In step 6, a second round of nickase-mediated linear amplification is performed, initiated by replication of the enriched duplex template construct using extension nucleotide 305. In certain embodiments, the extension oligonucleotide may include an internal cleavage site for a second nicking endonuclease (in other embodiments, a second nickase site may be designed into the sequence of the Y adapter of the duplex template construct). In certain embodiments, the first nicking endonuclease used in the first round of linear amplification may be Nt. BSTNB1 and the second nicking endonuclease is other than Nt. BSTNB1, for example, Nt. BSPQ1. Otherwise, the second round of nickase-mediated linear amplification may be carried out in a similar manner to the first round. However, it will be readily understood that in the second round of amplification, the template is a daughter copy of the original duplex template construct used in the first round of amplification. In step 6, amplified product 310 of the second round of linear amplification may be used, e.g., as a duplex template construct for Xpandomer synthesis using extension oligonucleotide 315.

[0070] According to the methods of the present invention, the cycle of: first linear amplification - target enrichment - second linear amplification can be integrated at any suitable stage during library preparation for duplex sequencing. In certain embodiments, the cycle of first linear amplification - target enrichment - second linear amplification may be performed prior to ligation of the library fragment to the hairpin adapter. One such example is illustrated, in simplified form, in FIGS. 3A and 3B. In this embodiment, a double stranded nucleic acid target (i.e., library) fragment is provided that includes parent (+) strand (i.e., a sense strand) 300a and parent (-) strand (i.e., an antisense strand) 300b. The library fragment is end-repaired and A- tailed, as described herein. Also provided are Y adapters 305, which, in certain embodiments, may have the same overall structure and sequence. The double stranded region of the Y adapters may include any suitable feature for a particular sequencing application, such as, in certain embodiments, an SID. The terminal 3’ end of the double stranded region of the Y adapters may also include a single T overhang to facilitate alignment and ligation to the library fragment. The Y adapters include a 3’ single stranded arm region that include first nickase (antisense) cleavage site, 309. The Y adapters also include 5’ single stranded regions 311 that include a secondnickase (sense) cleavage site, 313. In step 1, the library fragment is contacted with the Y adapters under DNA ligation conditions, such that each end of the library fragment is ligated to a Y adapter to generate adapter-ligated library fragment 325.

[0071] In step 2, adapter-ligated library fragment 325 is subjected to a first round of nickase- mediated linear amplification. As described herein, first, an extension oligonucleotide is provided that includes a nucleic acid sequence complementary to a sequence in 3’ single stranded arm region 307 of Y adapter 305 (not shown). The 3’ end of the extension oligonucleotide is designed to abut, but not to overlap with, the first nickase endonuclease (antisense) cleavage site 309. The extension oligonucleotide is contacted with the adapter- ligated library fragment under nucleic acid hybridization conditions such that it hybridizes to the 3’ single stranded arm region of the Y adapter and provides an initiation site for DNA synthesis. The adapter-ligated library fragment is then contacted with a strand displacing DNA polymerase under DNA synthesis conditions. The DNA polymerase initiates DNA synthesis from the 3’ end of the extension oligonucleotide to produce complementary copy (sense strand) 330a of the adapter-ligated library fragment that includes parent (antisense) strand 300b. (For the sake of simplicity, only one of the extension products is depicted in this illustration; the skilled artisan will recognize that a second extension product will include a complementary copy of parent strand 300a). From the 5’ to the 3’ end, complementary copy (sense) strand 330a includes the following key features (other features are omitted for the sake of clarity): a first nickase endonuclease (sense) cleavage site, a daughter (sense) strand, and a complementary copy of the 5’ single stranded arm region of the Y adapter. Of note, newly formed cleavage site 329 for the first nickase endonuclease is produced in the proper (sense) orientation in the complementary copy so as to enable nickase-mediated cleavage of the strand, as depicted in FIG. 3A.

[0072] Linear amplification of parental strand 300b proceeds as discussed with reference to FIG. 1. Briefly, the first nickase endonuclease is provided along with a strand displacing DNA polymerase under endonuclease and DNA synthesis conditions. As depicted in step 2 of FIG. 3 A, the first nickase endonuclease produces a single stranded nick at site 329 to provide an initiation site for DNA synthesis. Repeated rounds of strand nicking, DNA synthesis and renewal of the nickase cleavage site, and displacement of the non-template strand by the newly synthesized complementary copy strand produces a population of copies 330a of the original parental strand 300b, derived from the library fragment.

[0073] In step 3, a pool of amplified copies of a library of target fragments is subjected to a target enrichment step. A skilled artisan will appreciate that the pool will include amplified copies of a plurality of target fragments, depicted in simplified form here as copies 330a, 331a, and 332a. In this example, copy 330a includes the target sequence of interest, while copies 331aand 33 lb include “off-target” sequences, which are not of interest. Blocker oligonucleotides 333 and 335 are designed with sequences that are complementary and common to sequences at the 5’ end and 3’ end of the amplified copies (corresponding to the known sequences of the 3’ and 5’ single stranded arm regions of Y adapters 305). The blocker oligonucleotides are contacted with the pool of amplified copies under nucleic acid hybridization conditions. The hybridized blocking oligonucleotides advantageously prevent non-specific carry-over of adapter derived and / or non-target sequences during target enrichment. Target hybridization probe 340 is then contacted with the pool of amplified copies under suitable nucleic acid hybridization conditions. In this example, the target hybridization probe hybridizes to the sequence of interest present in copy 330a, while the probe does not interact with off-target sequences in copies 331a and 332a. As discussed herein, the target hybridization probe may be joined to a moiety, such as biotin, to enable isolation of double stranded complexes including the target sequence of interest from a pool of unhybridized non-target sequences.

[0074] In step 4, enriched copy 330a is released from the target hybridization probe and the blocking oligonucleotides by subjecting the nucleic acid complex to denaturing (e.g., basic) conditions. Following suitable neutralizing and oligonucleotide purification steps, the enriched copy is then contacted under nucleic acid hybridization conditions with extension oligonucleotide 340, which includes a sequence complementary to a sequence at the 3’ end of the copy, which is derived from the known sequence of the 5’ single stranded arm region of Y adapter 305. The sequence of extension oligonucleotide 340 includes a modified cleavage site for the second nickase endonuclease that is refractory to nickase cleavage, e.g., by including a single base mismatch or an inosine residue. The hybridized extension oligonucleotide is contacted with a DNA polymerase under DNA synthesis conditions to produce complementary copy strand 345 that include a daughter (antisense) strand copy of the original library fragment. The resulting double stranded DNA product includes a first double stranded end and a second end that includes a 3’ single stranded tail. The double stranded end of the DNA product is end- repaired and A-tailed, as described herein.

[0075] In step 5, hairpin adapter 350 is ligated to the first double stranded end of the DNA fragment to produce duplex template construct 360, in which the daughter (sense) strand and the daughter (antisense) strand are covalently joined by the intervening hairpin adapter.

[0076] In step 6, extension oligonucleotide 365 is provided with a sequence complementary to a sequence in the 3’ single stranded tail region of duplex template construct 360 and contacted with the duplex template construct under nucleic acid hybridization conditions. The hybridized extension oligonucleotide is then contacted with a strand displacing DNA polymerase under DNA synthesis conditions. The DNA polymerase proceeds to synthesize a complementary copyof the duplex template construct, resulting in double stranded duplex template construct 370. Significantly, the newly synthesized complementary copy of double stranded duplex template construct 370 includes second nickase endonuclease (sense) cleavage site 313.

[0077] In step 7, a second round of linear amplification is performed, in which double stranded duplex template construct 370 is contacted with the second nickase endonuclease and a strand displacing DNA polymerase under nickase endonuclease and DNA synthesis conditions. As discussed herein, repetitive cycles of strand nicking, DNA synthesis and strand displacement are carried out to linearly amplify the enriched complementary copy strand of the original parental (antisense) strand of the library fragment, providing a population of amplified enriched copies 380 of the original library target fragment.

[0078] According to the methods of the present invention, when the method includes two rounds of library amplification, with an intermediary round of target enrichment, one round of amplification may be conventional (exponential) PCR, while the other round may be linear (isothermal) amplification. One example of a method of duplex library preparation and target enrichment integrating one round of PCR and one round of linear amplification is illustrated in simplified form in FIGS. 4A and 4B. Here, a double stranded nucleic acid target (i.e., library) fragment is provided that includes parent (+) strand (i.e., a sense strand) 400a and parent (-) strand (i.e., an antisense strand) 400b. The nucleic acid target fragment is end-repaired and A- tailed, as described herein. Also provided are Y adapters 405, which, in this embodiment, have the same structure and sequence. The double stranded regions of the Y adapters may include any suitable sequence for a particular sequencing application, such as, in certain embodiments, an SID. In certain embodiments, the terminal 3’ end of the double stranded region of the Y adapters may also include a single T overhang to facilitate alignment and ligation of the library fragment. The Y adapters include 3’ single stranded arm regions 407 and 5’ single stranded arm regions 411, that include first nickase endonuclease (sense) cleavage site, 413. In step 1, each end of the double stranded nucleic acid target fragment is ligated to a Y adapter to generate adapter-ligated library fragment 425.

[0079] In step 2, adapter-ligated library fragment 325 is subjected to a first round of PCR amplification. Forward primers are provided with a sequence complementary to a sequence in the 3’ end (407) of the Y adapters and reverse primers are provided with a sequence that is identical to a sequence in the free 5’ end (411) of the Y adapters. PCR amplification produces a population 430 of amplified copies of the adapter-ligated library fragment. In step 2 of FIG. 4A, one of the resulting double stranded PCR products is depicted (based on using the antisense strand, including parental strand 400b, as a template). This product includes complementarycopy strand 430a that includes a daughter (sense) strand copy of the original library fragment. Strand 430b includes the sequence of parental (antisense) strand 400b.

[0080] In step 3, a pool of amplified copies of a library of target fragments is subjected to a target enrichment step. The skilled artisan will appreciate that the pool will include amplified copies of a plurality of target fragments, depicted in simplified form here as copies 430a, 431a, and 432a. In this example, copy 430a includes the target sequence of interest, whiles copies 4321a and 432a include “off -target” sequences, which are not of interest. As discussed with reference to FIG. 3A, blocker oligonucleotides 433 and 435 are designed with sequences that are complementary and common to sequences in the 3’ and 5’ ends of the amplified copies (corresponding to the known sequences of the 3’ and 5’ single stranded arm regions of Y adapters 405). The blocker oligonucleotides are contacted with the pool of amplified copies under nucleic acid hybridization conditions. The hybridized blocking oligonucleotides advantageously prevent non-specific carry-over of adapter-derived and / or non-target sequences during target enrichment. Target hybridization probe 440 is then contacted with the pool of amplified copies under suitable nucleic acid hybridization conditions. In this example, the target hybridization probe hybridizes to the sequence of interest in strand 430a, while the probe does not interact with the other strands that include off-target sequences. As discussed herein, the target hybridization probe may be joined to a moiety, such as biotin, to enable isolation of double stranded complexes including the target sequence of interest from a pool of unhybridized nontarget sequences.

[0081] As shown in FIG. 4B, in step 4, enriched amplified copy 430a is released from the target hybridization probe and the blocking oligonucleotides by subjecting the complex to denaturing conditions. After suitable steps to, e.g., neutralize the conditions and remove undesired oligonucleotides, the enriched amplified copy is then contacted under nucleic acid hybridization conditions with extension oligonucleotide 440 that includes a sequence complementary to a sequence at the 3’ end of the amplified copy, which is derived from the known sequence of the 5’ single stranded end of the Y adapter 405. The sequence of extension oligonucleotide 440 includes a modified cleavage site for the nickase endonuclease that is refractory to nickase cleavage, e.g., by including a single base mismatch or an inosine residue. The hybridized extension oligonucleotide 440 is contacted with a DNA polymerase under DNA synthesis conditions to produce complementary copy strand 445 that include a daughter (antisense) strand copy of the original library fragment. The resulting double stranded DNA product includes a first double stranded end and a second end that includes a 3’ single stranded tail region. The double stranded end of the DNA product is end-repaired and A-tailed, as described herein.

[0082] In step 5, hairpin adapter 450 contacted with the DNA product under DNA ligation conditions to produce duplex template construct 460, in which the daughter (sense) strand and the daughter (antisense) strands are covalently joined by ligation to the intervening hairpin adapter.

[0083] In step 6, extension oligonucleotide 465 is provided with a sequence complementary to a sequence in the 3’ single stranded tail region of duplex template construct 460. The extension oligonucleotide is contacted with the duplex template construct under nucleic acid hybridization conditions. The hybridized extension oligonucleotide is then contacted with a strand displacing DNA polymerase under DNA synthesis conditions. The DNA polymerase proceeds to synthesize a complementary copy of the duplex template construct, resulting in double stranded duplex template construct 470. Significantly, the newly synthesized complementary copy of double stranded duplex template construct 470 includes nickase endonuclease (sense) cleavage site 413a.

[0084] In step 7, a round of linear amplification is performed in which double stranded duplex template construct 470 is contacted with a nickase endonuclease and a strand displacing DNA polymerase under nickase and DNA synthesis conditions. As discussed herein, repetitive cycles of strand nicking, DNA polymerization, and strand displacement are carried out to linearly amplify the enriched complementary copy strand of the original parental (antisense) strand of the library fragment and provide a population of amplified enriched copies 480 of the original library target fragment.

[0085] According to the methods of the present invention, in certain embodiments, duplex template construct preparation and amplification workflows can be adapted to facilitate sequence determination of large and diverse genomic regions. One example of this application, termed “Mate Pair using Circularization,” is illustrated in a simplified manner in FIG. 5. In this embodiment, genomic fragment 500 is provided that includes parent (sense) strand 500a and parent (antisense) strand 500b. The genomic fragment is end-repaired and tailed to generate single 3’ T overhangs in each strand, as discussed herein. The genomic fragments may be of any suitable length; however, the length of the fragment should be of sufficient length to enable circularization of the fragment. In certain embodiments, the length of the fragment may be from around 3kB to around 20kB. Also provided is double stranded biotin cassette 510. The cassette functions as an adapter and includes a simple recognition site and cleavable linker 515 covalently joined at its terminal end to biotin moiety 517. In this embodiment, the biotin cassette adapter also includes single 3’ A overhangs to facilitate ligation to genomic fragment 600. In certain embodiments, the biotin cassette adapter may include additional features that facilitatedownstream steps of the workflow, e.g., bioinformatic analysis of sequence data, such as UMI and / or SID sequences.

[0086] In step 1, genomic fragment 500 is contacted with biotin cassette adapter 510 and a DNA ligase enzyme under ligation conditions. The DNA ligase joins a first end of the genomic fragment to a first end of the biotin cassette adapter and a second end of the genomic fragment to a second end of the biotin cassette adapter to product circular genomic fragment construct 525. In certain embodiments, the ligation conditions may be “dilute”, meaning that the concentrations of the genomic fragment and the biotin cassette adapter are low enough to favor circularization of the genomic fragment, mediated by the biotin cassette, into the circular construct configuration. In certain embodiments, the ligation reaction is treated with an exonuclease enzyme to “clean up” the reaction by removing un-ligated linear by-products via exonuclease digestion.

[0087] In step 2, circular genomic fragment construct 525 is “captured” on streptavidin- bound solid support 527, via binding of biotin moiety 517 to the support bound streptavidin moiety. The captured circular genomic fragment construct is contacted with a restriction enzyme under restriction digestion conditions. The restriction enzyme may be any suitable enzyme that produces random double stranded breaks in the genomic fragment (e.g., at suitable predicted intervals) to yield support-bound digestion fragment product 530. Digestion fragment product 530 includes a first genomic region and a second genomic region joined by the intervening biotin cassette adapter 510. In certain embodiments, the restriction enzyme is selected to produce fragment product 530 with a length of around 600bp to around 800bp. In certain embodiments, step 2 may also include ligation of a Y adapter to an end of fragment product 530.

[0088] In step 3, digestion fragment product 530 is end-repaired and A-tailed, as described herein. The digestion fragment product is then contacted with pre-nicked hairpin adapters 535 and a DNA ligase enzyme under DNA ligation conditions. Here, pre-nicked hairpin adapter 535 includes a double stranded stem region and a single stranded loop region. The double stranded region includes a single stranded nick 537 in one of the strands. In certain embodiments, the sequence of the double stranded region of the adapter may include a single stranded nickase site. Following synthesis of the adapter, the hairpin may be contacted with a nickase enzyme, followed by a phosphatase enzyme, to generate a single stranded nick in one strand that is refractory to ligation due to the absence of a 5’ phosphate. In other embodiments, the nick in the pre-nicked hairpin adapter 535 may be introduced into the hairpin adapter by using chemical methods known in the art. The single stranded nick provides a free 3’ end capable of being extended by a DNA polymerase. However, the free 3’ end is not capable of being re-ligated to the free 5’ end by a DNA ligase. In certain embodiments, the hairpin adapters 535 may includesequence features that facilitate bioinformation analysis of sequence data, such as UMI and / or SDI sequences. Of note, the sequence features provided in the hairpin adapters 535 will be different than those provided in the biotin cassette adapter 510.

[0089] In other embodiments, as mentioned, as an alternative to ligating pre-nicked hairpin adapters to the digestion fragment product 530, standard Y adapters may be ligated to each end of the digestion product fragment. For a simplified workflow, Y adapter-ligated digestion fragment products can be directly amplified, either by conventional PCR or by linear / isothermal amplification prior to Xpandomer synthesis for nanopore-based sequence determination.

[0090] In step 4, the biotin linker feature of the biotin cassette adapter is cleaved to release the hairpin adapter-ligated digestion fragment product from the solid support and into solution. The released hairpin adapter-ligated digestion product fragment is then contacted with a stranddisplacing DNA polymerase under DNA synthesis conditions. As discussed herein, the DNA polymerase begins DNA synthesis from the free 3' end at sites 537 using the opposite strand of the fragment as a template, while simultaneously displacing the non-template strand of the fragment in the 5' to 3' direction. As the polymerases proceed along the fragment strands in opposite directions (and on different strands of the fragment), the two strands of the digestion product fragment are separated, resulting in extension products 540a and 540b. Each of the extension products includes a strand of the original restriction fragment product and a newly synthesized complementary copy stand. Each of extension products 540a and 540b includes a fully ligated hairpin adapter at one end (i.e., there are no nicks or gaps between the strands of the target fragment and strands of the adapter) and either a blunt or overhang terminus at the opposite end.

[0091] In step 5, hairpin-ligated extension products 540a and 540b are end-repaired and A- tailed, as discussed herein and contacted with Y adapter 545 and a DNA ligase under DNA ligation conditions. The Y adapter is ligated to the double stranded end of hairpin-ligated extension products 540a and 540b to produce mate-paired template construct 550. In certain embodiments, mate-paired template construct 550 may be used directly as a template for Xpandomer synthesis. In other applications, the mate-paired template constructs may be amplified prior to Xpandomer synthesis, either via conventional PCR or via linear / isothermal amplification, as discussed in further detail herein.

[0092] Another example of a “Mate Pair using Circularization” workflow is illustrated in a simplified manner in FIG. 6. In this embodiment, genomic fragment 600 is provided that includes parental (sense) strand 600a and parental (antisense) strand 600b. The genomic fragment is end repaired and tailed to generate single 3’ T overhangs in each strand, as discussed herein. The genomic fragment may be any suitable length; however, the fragment should be of asuitable length to enable circularization of the fragment. In certain embodiments, the length of the fragment may be from around 3kB to around 20kB. Also provided is double stranded biotin cassette adapter 610. The biotin cassette adapter includes two double stranded regions, 621a and 621b, that flank two single stranded regions, 623a and 623b. Each single stranded region may include a sequence, 625a and 625b, that provides a hybridization site for an extension oligonucleotide. The biotin cassette adapter includes cleavable linker 615 covalently joined at its terminal end to biotin moiety 617. In this embodiment, the biotin cassette adapter also includes single 3’ A overhangs to facilitate ligation of the genomic fragment. In certain embodiments, the biotin cassette may also include additional sequences that facilitate, e.g., bioinformatic analysis of sequences data, such as UMI and / or SID sequences. In one embodiment, double stranded region, 621a or 621b, includes a UMI. In certain embodiments, the cassette adapter sequence further includes an engineered polymerase termination site in each of the single stranded regions (represented in this depiction by the black circles).

[0093] In step 1, genomic fragment 600 is contacted with biotin cassette adapter 610 and a DNA ligase enzyme under ligation conditions. The DNA ligase joins a first end of the genomic fragment to a first end of the biotin cassette adapter and a second end of the genomic fragment to a second end of the biotin cassette adapter to produce circular genomic fragment construct 625. In certain embodiments, the ligation conditions are “dilute”, in that the concentrations of the genomic fragment and the biotin cassette adapter are low enough to favor circularization of the genomic fragment mediated by the biotin cassette adapter 610. The ligation reaction may be subsequently treated with an exonuclease enzyme to “clean up” the reaction by removing unligated linear by-products via exonuclease digestion.

[0094] In step 2, circular genomic fragment construct 625 is “captured” on streptavidin- bound solid support 627 via binding of biotin moiety 617 to the support bound streptavidin moiety. The captured circular genomic fragment construct is then contacted with a restriction enzyme under restriction digestion conditions. Restriction enzymes are well known in the art. The restriction enzyme may be any suitable enzyme that produces random double stranded breaks in the genomic fragment at a desired interval. Restriction digestion yields support-bound digestion fragment product 630 in which each double stranded restriction fragment joined to the biotin cassette adapter is of the approximate length desired. In certain embodiments, the restriction enzyme is selected to produce digestion fragment product 630 with a length of around 600bp to around 800bp.

[0095] In step 3, digestion fragment product 630 is end-repaired and A-tailed, as described herein. The digestion fragment product is then contacted with hairpin adapters 635 and a DNA ligase enzyme under DNA ligation conditions. In certain embodiments, the hairpin adapters 635may include sequence features that facilitate, e.g., bioinformation analysis of sequence data, such as UMI and / or SDI sequences. Of note, the sequence features provided in the hairpin adapters 635 will be different than those provided in the biotin cassette adapter 610.

[0096] In step 4, the biotin linker feature of the biotin cassette adapter is cleaved to release the hairpin adapter-ligated digestion fragment product from the solid support and into solution. The released hairpin adapter-ligated digestion product fragment is then subjected to linear nickase-mediated amplification. First, cleavable extension oligonucleotides 637a and 637b are contacted with the adapter-ligated digestion product fragment under nucleic acid hybridization conditions. The cleavable extension oligonucleotides 635a and 637b hybridize to their respective binding sites, 625a and 625b in the cassette adapter sequence. Then, the adapter- ligated digestion product fragment is contacted with a strand-displacing DNA polymerase under DNA synthesis conditions. As discussed herein, the DNA polymerase begins DNA synthesis from the free 3' end of the cleavable extension oligonucleotides using the opposite strand of the fragment as a template, while simultaneously displacing the non-template strand of the fragment in the 5' to 3' direction. DNA synthesis terminates at engineered termination sites in the cassette adapter sequence (represented in the figure by the black dots). Thus, each extension product 640a and 640b will include one half of the original adapter-ligated digestion product fragment. The adapter-ligated digestion product is concurrently contacted with a nickase endonuclease under endonuclease conditions and the nickase cleaves its cleavage site in the cleavable extension oligonucleotides 637a and 637b to produce a single stranded nick in the extension oligonucleotides, which provides an initiation site for the next round of DNA synthesis by the strand displacing DNA polymerase, which renews the cleavage site in the cleavable extension oligonucleotides. Repetitive cycles of nickase endonuclease-mediated cleavage and DNA synthesis of a new complementary copy of the digestion product fragment template halves 640a and 640b generates a population of mate-paired duplex template constructs 650.

[0097] According to the methods of the present invention, in certain embodiments, the methods include template amplification workflows that combines a first linear amplification protocol with a second template replication protocol. The ability to combine different template amplification and replication protocols provides, in certain embodiments, advantages, such as optimizing the copy number and the nucleotide composition of the final pool(s) of amplified library constructs. A simplified outline of one embodiment of such a combination template amplification and replication workflow is depicted in FIG. 12.

[0098] Here, in step 1210, a duplex template construct (referred to herein as “(Pl) duplexed template” is first copied with a DNA polymerase using an extension oligonucleotide with a sequence complementary to a sequence in the 3’ single stranded arm region of the Y adaptercomponent of the duplex template construct. In certain embodiments, either the extension oligonucleotide or the 3’ single stranded arm region of the Y adapter may include a nickase recognition site.

[0099] In step 1220, the double stranded (Pl) duplexed template is subjected to a first round of nickase-mediated linear amplification, as discussed with reference to FIGS. 1A and IB. The amplification reaction mixture may include, in certain embodiments, non-natural nucleotides (i.e., nucleotide analogs), as discussed herein. The resulting amplicon pool includes daughter strand (Di) copies with sequences that are the reverse complement of the Pi parental duplexed template and may include non-natural nucleotides, such as N4Me dCTP and / or others as disclosed herein. Advantageously, the make-up of the pool of nucleotides included in the linear amplification reaction can be tailored to achieve a specific outcome of interest. In certain embodiments, the sample of daughter (Di) copies may be subjected to one or more nucleic acid purification or quantification steps, if desired.

[0100] In step 1230, the sample of daughter (Di) copies is optionally treated with proteinase K to digest and inactivate the remaining enzymes and other proteins carried over from the linear amplification reaction. A biotinylated primer (i.e., extension oligonucleotide) is provided and contacted with the daughter (DI) copies under nucleic acid hybridization conditions. The sequence of the primer is designed to be complementary to a sequence in the 3’ single stranded arm region of the Y adapter component of the daughter Di copies. The 5’ end of the primer is joined to a biotin moiety via a flexible linker, as described herein. The 3’ end of the hybridized primers provide an initiation site for DNA synthesis, while the biotinylated 5’ end of the primers is capable of being captured via a streptavidin moiety (e.g., using streptavidin-coated beads). In certain embodiments, step 1230 may be followed by a wash step using, e.g., a conventional bead washing buffer.

[0101] In step 1240, the complexes of oligonucleotide primer-daughter (Di) copy hybrids are bound to streptavidin-coated beads and the beads may then be washed with, e.g., a bead washing buffer to remove unbound nucleic acids and other reaction components from step 1230.

[0102] In step 1250, DNA synthesis conditions are provided that include a suitable DNA polymerase, buffer and dNTPs. The DNA polymerase synthesizes (i.e., “prints”) (P2) copies of the daughter (Di) templates by extending the bead-bound extension oligonucleotide. Thus, the newly printed (P2) copies are bound to the streptavidin beads, while the daughter (Di) templates are associated with the beads by virtue of being hybridized to the printed (P2) copies. In certain embodiments, the nucleic acid synthesis conditions include native dNTPs, such that the newly printed (P2) copies are comprised of natural nucleotides. In some embodiments, step 1250 may be followed by a wash step using, e.g., a bead washing buffer.

[0103] In step 1260, the daughter (Di) templates are melted-off the bead-bound printed (P2) copies. For example, the double stranded nucleic acid hybrids may be treated with a water melt solution that breaks the hydrogen bonds between the two strands and releases the daughter (Di) templates into solution. In other embodiments, alternative melt solutions containing, e.g., a suitable concentration of NaOH, may be used to release the daughter (Di) templates from the bead-bound printed (P2) copies.

[0104] In step 1270, template “switching” is implemented in a template replication reaction. In this step, the bead-bound printed (P2) copies serve as the replication templates. The printed copies are contacted with an extension oligonucleotide and DNA polymerase under nucleic acid hybridization and synthesis conditions. The extension oligonucleotide has a sequence that is designed to be complementary to a sequence in the free (i.e., unbound) 5’ single strand arm region of the Y adapter component of the printed (P2) templates. The DNA polymerase synthesizes complementary (D2) copies of the printed templates. In some embodiments, the nucleic acid synthesis conditions include native nucleotides such that the newly synthesized (D2) copies comprise natural nucleotides. In some embodiments, step 1270 may be followed by a wash step using, e.g., a bead washing buffer.

[0105] In step 1280, the newly synthesized (D2) copies are released from the printed (P2) templates using a suitable melt solution, e.g., water or a suitable concentration of NaOH. The pool of eluted (D2) copies and / or (DI) copies provide an amplified sample of the original duplex template construct, which can find applications in downstream consensus sequencing protocols, such as synthesis of Xpandomer copies for nanopore sequencing.

[0106] A simplified outline of another embodiment of a combination template amplification and replication workflow is depicted in FIG. 13. Here, in step 1310, the duplex template construct (referred to herein as “(Pl) duplexed template” is first copied with a DNA polymerase using an extension oligonucleotide with a sequence complementary to a sequence in the 3’ single stranded arm of the Y adapter component of the duplex template construct. In certain embodiments, either the extension oligonucleotide or the 3’ single stranded arm of the Y adapter may include a nickase recognition site.

[0107] In step 1320, the double stranded (Pl) duplexed template is subjected to a first round of nickase-mediated linear amplification, as discussed with reference to FIGS. 1A and IB. The amplification reaction mixture may include, in certain embodiments, non-natural nucleotides (i.e., nucleotide analogs), as discussed herein. The resulting amplicon pool includes daughter strand (Di) copies with sequences that are the reverse complement of the Pi parental duplexed template and may include non-natural nucleotides, such as N4-Me dCTP and / or others as disclosed herein. Advantageously, the make-up of the pool of nucleotides included in the firstlinear amplification step can be tailored for specific applications. In certain embodiments, the sample of daughter (Di) copies can be treated to one or more purification steps or nucleic acid quantification steps, if desired.

[0108] In step 1330, a biotinylated primer (i.e., extension oligonucleotide) is provided and contacted with the (DI) copies under nucleic acid hybridization conditions. The sequence of the primer is designed to be complementary to a sequence in the 3’ single stranded arm regions of the Y adapter component of the daughter Di copies. The 5’ end of the primer is joined to a biotin moiety via a flexible linker, as described herein. The primers hybridize to the daughter copies and provide a free 3’ end for extension, while the biotinylated 5’ end of the primers is available for capture via a streptavidin moiety. The free 3’ end of the primer is extended by the DNA polymerase provided in the nucleic acid synthesis conditions to produce complementary (P2) copies of the daughter Di templates. As used herein, this step may be referred to as “template printing”. In certain embodiments, step 1330 may be followed by a wash step using, e.g., a bead washing buffer.

[0109] In step 1340, the complexes of (P2) copy- (Di) template hybrids are bound to streptavidin beads and the beads are then washed with, e.g., a bead washing buffer to remove unbound nucleic acids and other reaction components from step 1330.

[0110] In step 1350, the daughter (Di) templates are melted-off the bead-bound printed (P2) copies. For example, the double stranded hybrids may be treated with a water melt solution that breaks the hydrogen bonds between the two strands and releases the daughter (Di) templates from the beads into solution. In other embodiments, alternative melt solutions containing, e.g., a suitable concentration of NaOH, may be used to release the daughter (Di) templates from the bead-bound printed (P2) copies.

[0111] In step 1360, template “switching” is implemented in a template replication reaction. In this step, the bead-bound printed (P2) copies serve as the replication templates. The printed copies are contacted with an extension oligonucleotide under nucleic acid hybridization and synthesis conditions. The extension oligonucleotide has a sequence that is designed to be complementary to a sequence in the free (i.e., unbound) 5’ single strand arm of the Y adapter component of the printed (P2) templates. The DNA polymerase synthesizes complementary (D2) copies of the printed templates. In some embodiments, the nucleic acid synthesis conditions include native nucleotides such that the newly synthesized (D2) copies comprise natural nucleotides. In some embodiments, step 1270 may be followed by a wash step using, e.g., a bead washing buffer.

[0112] In step 1370, the newly synthesized (D2) copies are released from the printed (P2) templates using a suitable melt solution, e.g., water or NaOH. The pool of eluted (D2) copiesand / or (DI) copies can be used in downstream consensus sequencing protocols, such as synthesis of Xpandomers for nanopore sequence determination.Construct Libraries

[0113] Described herein are methods and compositions for producing duplex template constructs that may be subjected to the linear and / or other amplification protocols described in further detailed herein. In general terms, embodiments described herein may be applied any suitable sequencing platform, including next generation sequencing (NGS) and nanopore sequencing, but are particularly useful for Sequencing by Expansion (SBX®). Sequencing by Expansion is described in Applicant’s published PCT application, WO 2020 / 236526 Al, “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing,” filed May 14, 2020, and issued patent, US 7,939,259 B2, “High throughput nucleic acid sequencing by expansion,” filed June 19, 2008, the entire contents of which are both incorporated herein by reference for all purposes.

[0114] In general terms, Sequencing by Expansion (SBX® ) uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an “Xpandomer” using highly modified, non-natural nucleotide analog substrates referred to as “XNTPs”. The transcribed sequence is encoded along the Xpandomer backbone in high signal- to-noise reporter codes that are separated by ~10 nm and are designed for high-signal-to-noise, well-differentiated responses. The Xpandomer polymer thus preserves the original genetic information of the target nucleic acid template, while also increasing linear separation of the individual elements of the sequence data. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to natural DNA.

[0115] Sequencing by Expansion is a single molecule sequencing method in that the nanopore detector reads electronic signals from a single Xpandomer. As such, SBX® sequencing reads provide information from a single, contiguous DNA template. Of note, Sequencing by Expansion is not based on sequencing by synthesis in that the ultimate electronic measurement of the Xpandomer to produce sequence information is entirely uncoupled from the earlier step of synthesis of an Xpandomer copy of a duplex template, or other, library construct.

[0116] Sequencing by Expansion is discussed in greater detail further herein.

[0117] The present technology is based on duplex (i.e., paired-end) sequencing, which can be applied to both genomic and epigenomic (e.g., methylomic) sequence analysis. For Sequencing by Expansion applications, the methods and compositions of the present inventionallow for linear amplification of duplex library constructs prior to synthesis of duplexed Xpandomers for nanopore sequence determination.

[0118] The general method typically begins with a sample of double stranded nucleic acid fragments having defined ends, which could be blunt ends or ends with known overhang sequences (5' or 3' overhangs). These nucleic acid fragments can be of any size or size range and can include DNA, RNA, DNA-RNA hybrids (e.g., molecules produced by first-strand synthesis during cDNA preparation have one mRNA strand and one complementary DNA strand), genomic DNA, cDNA, mRNA, tRNA, etc. In some embodiments, the nucleotide sequence of the fragments is not known.

[0119] In some aspects, the invention provides for producing a library of paired-end nucleic acid template constructs from the sample of double stranded nucleic acid fragments for synthesis of Xpandomer copies for nanopore sequence determination. As discussed herein, the terms “paired-end” and “duplex” (or “duplexed”) may be used interchangeably as they relate to template construct for Xpandomer synthesis. Sequencing of the single, contiguous Xpandomer copies of the paired-end template provides duplexed reads of the original nucleic acid target fragments. The paired-end Xpandomer template constructs can be single nucleic acid chains that each have the following structure: adapter region 1, sense (i.e., forward) nucleic acid strand of the target fragment, adapter region 2, anti-sense (i.e., reverse) nucleic acid strand of the target fragment, adapter regions 3. In some embodiments, adapter region 2 forms a classic “hairpin” structure in which the stem portion of the hairpin adapter is double stranded and is ligated to one end of the double stranded nucleic acid target fragment. The loop portion of the hairpin adapter is single-stranded and operable joins (i.e., covalently links or couples) the sense and antisense strands of the double stranded nucleic acid target fragment. In some embodiments, adapter region 1 and adapter region 3 are derived from a classic “Y adapter” structure, in which the stem portion of the Y adapter is double stranded and is ligated to the opposite end of the double stranded nucleic acid target fragment. The arms of the Y adapter may be single stranded and provide a free 3’ end and a free 5’ end to the paired-end template construct. As described in further detail herein, in several embodiments, the hairpin and Y adapters structures of the present invention may include several novel features that enable synthesis of “daughter strand” copies of the paired-end template constructs useful for, e.g., epigenomic analyses. Certain, non-limiting, examples of alternative library formats and workflows to enable high accuracy duplex and methylome Sequencing by Expansion are discussed below.

[0120] In one aspect, DNA from a biological sample is obtained or provided. The DNA obtained or provided from the biological sample may be genomic DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or a combination thereof.

[0121] DNA samples may be obtained from a patient or subject, from an environmental sample, or from an organism of interest. In embodiments, the DNA sample is extracted, purified, or derived from a cell or collection of cells, a body fluid, a tissue sample, an organ, and / or an organelle. In some embodiments, the sample DNA is whole genomic DNA.

[0122] In some instances, genomic DNA and mitochondrial DNA may be obtained separately from the same biological sample or source. Many different methods and technologies are available for the isolation of genomic DNA and mitochondrial DNA. In general, such methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA. Removal of proteins can be achieved, for example, by digestion with proteinase K, followed by salting-out, organic extraction, gradient separation, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology). Mitochondrial DNA may be isolated similarly following initial isolation of mitochondria. DNA may be recovered by precipitation using ethanol or isopropanol. There are also commercial kits available for the isolation of nuclear or mitochondrial DNA. The choice of a method depends on many factors including, for example, the amount of sample, the required quantity and molecular weight of the DNA, the purity required for downstream applications, and the time and expense.

[0123] The methods of the present disclosure, in certain embodiments, utilize mild enzymatic and chemical reactions that avoid the substantial degradation associated with methods like bisulfite sequencing. Thus, the methods are useful in analysis of low-input samples, such as circulating cell-free DNA, circulating tumor DNA, and in single-cell analysis.

[0124] In some embodiments, the DNA sample is circulating cell-free DNA (cfDNA), which is DNA found in the blood and is not present within a cell. cfDNA can be isolated from blood or plasma using methods known in the art. Commercial kits are available for isolation of cfDNA including, for example, the Circulating DNA Kit (Qiagen). The DNA sample may result from an enrichment step, including, but is not limited to antibody immunoprecipitation, chromatin immunoprecipitation, restriction enzyme digestion-based enrichment, hybridization-based enrichment, or chemical labeling-based enrichment.

[0125] In some instances, the isolated DNA is fragmented into a plurality of shorter double stranded DNA target fragments. In general, fragmentation of DNA may be performed physically, or enzymatically.

[0126] For example, physical fragmentation may be performed by acoustic shearing, sonication, microwave irradiation, or hydrodynamic shear. Acoustic shearing and sonication are the main physical methods used to shear DNA. For example, the Covaris® instrument (Woburn, MA) is an acoustic device for breaking DNA into 100 bp - 5 kb. Covaris also manufactures tubes(gTubes) which will process samples in the 6-20 kb for Mate-Pair libraries. Another example is the Bioruptor® (Denville, NJ), a sonication device utilized for shearing chromatin, DNA and disrupting tissues. Small volumes of DNA can be sheared to 150 bp - 1 kb in length. The Hydroshear® from Digilab (Marlborough, MA) is another example and utilizes hydrodynamic forces to shear DNA. Nebulizers, such as those manufactured by Life Technologies (Grand Island, NY) can also be used to atomize liquid using compressed air, shearing DNA into 100 bp - 3 kb fragments in seconds. As nebulization may result in loss of sample, in some instances, it may not be a desirable fragmentation method for limited quantities samples. Sonication and acoustic shearing may be better fragmentation methods for smaller sample volumes because the entire amount of DNA from a sample may be retained more efficiently. Other physical fragmentation devices and methods that are known or developed can also be used.

[0127] Various enzymatic methods may also be used to fragment DNA. For example, DNA may be treated with DNase I, or a combination of maltose binding protein (MBP)-T7 Endo I and a non-specific nuclease such as Vibrio vulnificus nuclease (Vvn). The combination of nonspecific nuclease and T7 Endo synergistically work to produce non-specific nicks and counter nicks, generating fragments that disassociate 8 nucleotides or less from the nick site. In another example, DNA may be treated with NEBNext® dsDNA Fragmentase® (NEB, Ipswich, MA). NEBNext® dsDNA Fragmentase generates dsDNA breaks in a time-dependent manner to yield 50-1,000 bp DNA fragments depending on reaction time. NEBNext dsDNA Fragmentase contains two enzymes, one randomly generates nicks on dsDNA and the other recognizes the nicked site and cuts the opposite DNA strand across from the nick, producing dsDNA breaks. The resulting DNA fragments contain short overhangs, 5 '-phosphates, and 3 '-hydroxyl groups.

[0128] In some instances, the DNA sample is fragmented into specific size ranges of target fragments. For example, the DNA sample may be fragmented into fragments in the range of about 25-100 bp, about 25-150 bp, about 50-200 bp, about 25-200 bp, about 50-250 bp, about 25-250 bp, about 50-300 bp, about 25-300 bp, about 50-500 bp, about 25-500 bp, about 150-250 bp, about 100- 500 bp, about 200-800 bp, about 500-1300 bp, about 750-2500 bp, about 1000- 2800 bp, about 500-3000 bp, about 800-5000 bp, or any other size range within these ranges. For example, the DNA sample may be fragmented into fragments of about 50-250 bp. In some instances, the fragments may be larger or smaller by about 25 bp.

[0129] In certain embodiments, the fragments are treated to produce blunt ends that are compatible with ligation to a first adapter having a compatible blunt end. Any convenient method for producing blunt ends may be employed, including treatment with one or more enzyme having 5' and / or 3' single strand exonuclease activity (e.g., E. coli Exonuclease III)and / or performing a fill-in reaction to extend 3' recessed ends (e.g., with T4 DNA polymerase). No limitation in this regard is intended.

[0130] In certain embodiments, the methods of the present invention may be used for sequence analysis of nucleic acids extracted from formalin-fixed paraffin-embedded (FFPE) tissue samples. FFPE tissue plays a critical role in histopathological diagnosis, as it allows for the examination of tissue samples under a microscope. It is particularly valuable in the diagnosis of a wide variety of diseases like cancer. Pathology departments routinely archive large numbers of FFPE tissue blocks, making them a valuable resource for translational clinical research. While FFPE tissue is ideal for preserving tissue morphology, it presents challenges for molecular analysis. For example, the formalin fixation process induces cross-linking of proteins, resulting in the degradation and fragmentation of nucleic acids. This can make it difficult to extract high- quality DNA, RNA, and proteins from FFPE tissue.

[0131] In certain embodiments, the methods of the present invention include strategies to overcome or mitigate the degradation of nucleic acids derived from FFPE samples prior to duplex library preparation. Accordingly, the methods may include one or more of the following nucleic acid sample treatment steps: A) FFPE repair; B) end-repair and A-tailing (ERAT), C) other forms of damage treatment, and D) fragmentation. The methods of the present invention may include any one of these treatment steps in any combination and in any order.

[0132] Commercial kits and proteins or other protocols available to enable individual treatment steps A), B), C), and D) are known in the art. For example, an FFPE repair kit is commercially available from, e.g., New England Biolabs, an ERAT kit is commercially available from, e.g., Roche Sequencing Solutions, Inc., thermolabile exonucleases are commercially available from, e.g., New England Biolabs, and FragTail fragmentation kit is commercially available from, e.g., Roche Sequencing Solutions, Inc. However, the inventors have found that one or more of these commercial products can be optimized or improved for the practice of the methods of the present invention. For example, in certain non-limiting embodiments, a commercially available FFPE repair kit may be improved by including a thermolabile exonuclease enzyme (e.g., “TL exol”) and / or a RecJf enzyme. RecJf is known in the art as a single-stranded DNA specific exonuclease that degrades DNA from the 5' end. In certain embodiments, it is a fusion of the RecJ protein with maltose binding protein (MBP), enhancing its solubility. The inventors have unexpectedly discovered that adding either TL exol or RecJf to a FFPE kit significantly reduces the occurrence of polymerase errors (e.g., “U-turns”) during replication of a duplex template construct. In other embodiments, any suitable exonuclease enzyme or other nucleic acid “clean-up” enzyme (e.g., enzymes involved in base excision repair,double stranded DNA repair or removal of single stranded DNA) may be included in an FFPE repair kit.

[0133] The inventors have also unexpectedly discovered that the order of addition of any one of steps A) - D) can also improve the quality of the nucleic acids extracted from FFPE samples prior to library preparation. One of ordinary skill in the art will recognize that the optimal number of steps and order of addition will depend on the specific library preparation and sequencing protocols required for a particular application.

[0134] Non-limiting examples of exemplary FFPE treatment protocols include the following steps. Protocol E: step 1) FFPE repair, step2) SPRI clean-up, step 3) Fragtail, step 4) SPRI clean-up; Protocol 2: step 1) Fragtail, step 2) SPRI clean-up, step 3) FFPE repair plus exo, step 4) TL protK, step 5) ERAT; Protocol 3: step 1) Fragtail, step 2) SPRI clean-up, step 3) FFPE repair plus Reclf, step 4) TL protK , step 5) ERAT. As disclosed herein, the methods may be optimized by addition, removal, or changing the order of any one or more of the disclosed treatment steps. In certain applications, the addition of the ERAT step may improve genomic recovery from FFPE samples. In addition, introduction of TL exol or Reclf into the FFPE step may improve sequence coverage and lower the percentage of clipped bases seen in sequence analysis.

[0135] For epigenetic analysis, in certain embodiments, the DNA target fragments may be any DNA fragment, derived from a biological sample, having a sequence of interest that may or may not include epigenetic modifications or DNA damage to one or more nucleobases. In some aspects, the DNA target fragments may include cytosine modifications (i.e., 5-mC, 5-hmC, 5-fC, and / or 5-caC). The DNA target fragments can be a single DNA molecule in the sample or may be the entire population of DNA molecules in a sample (or a subset thereof) having, e.g., a cytosine modification. The DNA target fragments can comprise a plurality of DNA sequences such that the methods described herein may be used to generate a library of DNA target fragments that can be analyzed individually (e.g., by determining the sequence of individual targets) or in a group (e.g., by multiplexed DNA sequencing methodologies).

[0136] In some embodiments, the methods described herein include the step of adding adapter DNA molecules to double stranded DNA target fragments. An adapter DNA, or DNA linker, is a short, chemically synthesized, single- or double-stranded oligonucleotide that can be ligated to one or both ends of other DNA molecules. Double-stranded adapters can be synthesized so that each end of the adapter has a blunt end or a 5' or 3' overhang (i.e., sticky ends). DNA adapters are ligated to the DNA target fragments to provide sequences for, e.g., primer extension reactions and sequencing reactions with complimentary primers and / or forbioinformatic analysis (e.g., clustering of related sequences into families based on shared unique molecular identifier barcodes, UMIs).

[0137] Prior to ligation of adapters, the ends of the DNA fragments can be prepared for ligation. For example, by end repair and creating blunt ends with 5’ phosphate groups. Fragmented DNA may be rendered blunt-ended by a number of methods known to those skilled in the art. In a particular method, the ends of the fragmented DNA are “polished” with T4 DNA polymerase and Klenow polymerase, a procedure well known to skilled practitioners, and then phosphorylated with a polynucleotide kinase enzyme. A single ‘A’ deoxynucleotide is then added to both 3' ends of the DNA molecules using Taq polymerase or Klenow exo minus polymerase enzyme, producing a one-base 3' overhang that is complementary to the one-base 3' ‘T’ overhang on the double-stranded end of an adaptor.

[0138] In some instances, the adapters may include two oligonucleotides that are partially complementary such that they hybridize to form a region of double stranded sequence, but also retain a region of single stranded, non-hybridized sequence. The region of single stranded sequence may include “universal” oligonucleotide binding sequences, enabling all target fragments in a library to bind to the same oligonucleotide, which may be a capture oligonucleotide, to localize target fragments to a solid support, an oligonucleotide primer for a primer extension reaction, a PCR primer, sequencing primer, or combinations thereof. In certain instances, the adapters may include two regions of single-stranded, non-hybridized sequence (i.e., a first, 5’ single stranded region and a second, 3’ single stranded region). This configuration is known in the art as a “Y” adapter. The first and second single stranded regions of a Y adapter are not complementary and may include different primer hybridization sequences and other features.

[0139] The portions of the two single stranded regions of the adapters typically include at least 10, or at least 15, or at least 20 consecutive nucleotides on each strand. The lower limit on the length of the single stranded regions will typically be determined by function, for example, the need to provide a suitable sequence for binding of a primer for primer extension, PCR and / or sequencing. Theoretically there is no upper limit on the length of the single stranded regions, except that in general it is advantageous to minimize the overall length of the adapter, for example, in order to facilitate separation of unbound adapters from adapter-ligated double stranded DNA target fragments following the ligation step. Therefore, it is preferred that the single stranded regions should be fewer than 50, or fewer than 40, or fewer than 30, or fewer than 25 consecutive nucleotides in length on each strand.As used throughout, the term “hairpin adapter” refers to a nucleic acid sequence that has two complementary regions that hybridize to one another to form a double-stranded region with thetwo complementary regions being connected by a single-stranded loop. The hairpin adapters described herein can be of any length suitable for use in the provided methods. For example, the hairpin adapters can be at least 10, at least 20, at least 30, at least 40, or at least 50, nucleotides in length or longer. Optionally, the hairpin adapters are 15 to 40 base pairs in length.

[0140] The double stranded region of the adapter is a short double stranded region, typically comprising 5 or more consecutive base pairs, formed by annealing of the two partially complementary polynucleotide strands. Generally, it is advantageous for the double stranded region to be as short as possible without a loss of function. By “function” in this context is meant that the double stranded regions form a stable duplex under standard reaction conditions for the enzyme-catalyzed nucleic acid ligation reaction.

[0141] The precise nucleotide sequence of the adapters is generally not material to the invention and may be selected by the user such that the desired sequence elements are ultimately included in the common sequences of the library of adapter-ligated double stranded DNA target fragments. Additional sequence elements may be included, for example, to provide binding sites for primers which will ultimately be used in sequencing of complementary copy strands of the DNA target fragments. The adapters may further include “tag” sequences, unique molecular identifiers (UMI), and / or sample identifier sequences, which can be used to tag, track, and differentiate target fragments and complementary copies thereof derived from a particular source. The general features and use of such sequences is well known in the art.

[0142] The ends of the single stranded regions of the adapters may be biotinylated or bear another functionalities that enables it to be captured, or immobilized, on a surface, such as a solid support. Alternative functionalities other than biotin are known in the art, e.g., as described in Applicant’s published Patent Application no. WO2020 / 172479 entitled, “Methods and Devices for Solid-Phase Synthesis of Xpandomers for use in Single Molecule Sequencing”, which is herein incorporated by reference in its entirety.

[0143] “Ligation” of adapters to the 5' and 3' ends of each fragmented double stranded nucleic acid target fragment involves joining of the two polynucleotide strands of the adapter to the double-stranded target polynucleotide such that covalent linkages are formed between both strands of the two double-stranded molecules. Preferably such covalent linking takes place by formation of a phosphodiester linkage between the two polynucleotide strands but other means of covalent linkage (e.g., non-phosphodiester backbone linkages) may be used. However, it is an essential requirement that the covalent linkages formed in the ligation reactions allow for read- through of a polymerase, such that the resultant construct can be copied in a primer extension reaction using primers which bind to sequences in the regions of the adapter-target construct that are derived from the adapter molecules.

[0144] In some instances, the adapters and DNA target fragments may be incubated with a ligase to covalently link the adapters and DNA target fragments. Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3 ' hydroxyl termini in duplex DNA or RNA. The enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA. An exemplary ligase is T4 ligase, which is the most frequently used enzyme for cloning. Another ligase that may be used is E. coli DNA ligase, which preferentially connects cohesive double-stranded DNA end but is also active on blunt ends DNA in the presence of Ficoll or polyethylene glycol. Another ligase that may be used is DNA ligase Ilia, which is known to function in mitochondria.

[0145] The products of the ligation reaction may be subjected to purification steps in order to remove unbound adapter molecules before the adapter-target constructs are processed further.

[0146] The ligation of adapters to both free ends of the double stranded DNA target fragments gives rise to a pool of adapter-ligated double stranded DNA target fragments with adapters at the 5’ and 3’ ends of the target.

[0147] There are several standard methods for separating the strands of an adapter-ligated double stranded DNA target fragment by denaturation, including thermal denaturation, or chemical denaturation in either 100 mM sodium hydroxide solution or formamide solution. The pH of a solution of single-stranded DNA fragments can be neutralized by adjusting with an appropriate solution of acid, or preferably by buffer-exchange through a size-exclusion chromatography column pre-equilibrated in a buffered solution, including SPRI and others for buffer exchange or size selection.

[0148] As used herein, the term “complementary” refers to nucleic acid sequences that are capable of forming Watson-Crick base-pairs. For example, a complementary sequence of a first sequence is a sequence which is capable of forming Watson-Crick base-pairs with the first sequence. The term “complementary” does not necessarily mean that a sequence is complementary to the full-length of its complementary strand, but the term can mean that the sequence is complementary to a portion thereof. Thus, in some embodiments, complementarity encompasses sequences that are complementary along the entire length of the sequence or a portion thereof. For example, two sequences can be complementary to each other along at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the sequence. Here, the term “sequence” encompasses, but is not limited to, nucleic acid sequences, polynucleotides, oligonucleotides, probes, primers, primer-specific regions, and target-specific regions. Despite any mismatches, the two sequences should have the ability to selectively hybridize to one another under appropriate conditions.

[0149] One embodiment of a method of the present invention is set forth in FIG. 7. As shown in FIG. 7A, a double stranded target fragment 700 is derived from genomic DNA or some other nucleic acid source. The double stranded target fragment includes parent (+) strand 705A (i.e., a sense strand) and parent (-) strand 705B (i.e., an antisense strand). In some embodiments, the double stranded target fragment can be contacted with reagents that repair common forms of DNA damage. For example, a commercially available repair kit, such as the PreCR® repair mix, (available from NEB) may be included in the DNA synthesis conditions. In Step 1, adapters 707 and 709 each are joined to an opposite end of the double stranded target fragment. As shown in this embodiment, the adapters are Y adapters, and each includes a region of double stranded DNA and two regions of single stranded DNA. The adapters are joined to the double stranded target fragment such that one strand of the double stranded region of the adapter is ligated to one strand of the double stranded target fragment, while the other strand of the double stranded region of the adapter is not ligated to the target fragment. In this embodiment, the joining of the adapters to the double stranded target fragment is referred to as “partial ligation”. Partial ligation may be achieved, for example, by blocking the 5’ end of each strand of the target fragment to prevent ligation to the 3’ ends of the double stranded regions of the Y adapters. In this embodiment, blocking is achieved by dephosphorylation of the 5’ ends of target fragment, as represented by solid circles 706a and 706b. The adapter-ligated double stranded target fragment product 710 includes a single free 3’ end in each strand of the double stranded portion of the ligation product, represented by arrows 711a and 71 lb at the unligated junctions between the adapter strands and the target fragment strands.

[0150] While step 1 in FIG. 7A relies on dephosphorylation of the double-stranded target fragment to produce free 3’ ends 711a and 71 lb in the adapter ligation product, it is noted here that any method for producing a free 3’ end, single stranded nick, or other site for initiation of nucleic acid synthesis, may be used. For example, a ligation product with no free 3’ ends at the ligation site (i.e., where ligation of compatible ends is complete) can be treated with one or more enzymes that create one or more single strand nick. As another example, the adapters and the double-stranded target fragments can have ends that are not fully overlapping, thereby leaving a spacer region upon ligation, which provides a free 3’ end and serve as a site for polymerase binding and nucleic acid synthesis, as described below. It is further noted that the nick / spacer region need not be precisely at the ligation site for the subsequent steps of the method. No limitation with respect to free 3’ initiation sites or their location in the adapter-ligated target fragment. As such, any method for obtaining or generating adapter-ligated target fragments of the general structure of ligation product 710 or a similar structure with at least one free 3’ end may be used, such as those discussed with reference to FIGS. 8-11.

[0151] In certain embodiments, the adapters may include sequences, or other features, that mediate downstream steps of the workflow. For example, the adapters may include nucleic acid sequences or other chemical moieties for immobilization of the templates on a solid support, sequences for hybridization of oligonucleotide primer(s), sequences enabling bioinformatic analysis of DNA sequence information (e.g., unique molecular identifier bar codes [UMI], sample identifiers [SID]), and the like. In certain embodiments, the structures of adapters 707 and 719 may be identical or different, depending on the particular application. Any of the features discussed herein may be include in either one or both adapters, depending on the particular application(s). In certain embodiments, UMI sequences are not required for downstream steps of the duplex sequencing workflow (e.g., bioinformatic error correction or pairing of sequences derived from both strands of a double stranded DNA target fragment). Advantageously, the complexity of adapter design may thus be reduced.

[0152] As shown in FIG. 7B, in step 2, the free 3’ ends, 711a and 711b provide initiation sites for a nucleic acid synthesis reaction. The adapter-ligated target fragments 700 are contacted with a strand displacing DNA polymerase, which binds to free 3’ ends, 711a and 711b. Any suitable strand displacing polymerase may be employed, such as Klenow fragment. When placed under nucleic acid synthesis (i.e., extension) conditions, the DNA polymerase begins nucleic acid synthesis from the free 3' OH group at sites 711a and 711b using the opposite strand of the target fragment as a template, while simultaneously displacing the complementary strand of the target fragment in a 5' to 3' direction.

[0153] In some embodiments, the DNA synthesis conditions can include a Y family DNA polymerase, such as DPO4, that has the ability to preferentially incorporate the correct cytosine nucleotide opposite 8-oxo-G, which is known in the art as an oxidized form of natural guanine, instead of mispairing 8-oxo-G with A.

[0154] As the polymerases proceed along the target fragment strands in opposite directions (and on different strands of the target fragment), the two strands of the target fragment are separated, finally resulting in two nucleic acid extension products 715 and 717. Each of the nucleic acid extension products includes a strand of the original target fragment (e.g., parent strands, 705a and 705b) and a newly synthesized complementary copy stand (e.g., daughter strands 719b and 719a). Each of the nucleic acid extension products includes a fully ligated adapter at one end (i.e., there are no nicks or gaps between the strands of the target fragment and strands of the adapter) and either a blunt or overhang terminus at the opposite end. In nucleic acid extension products, the end opposite the adapter ligated end (i.e., lacking an adapter) is expected to be blunt if the polymerase traversed the entire target fragment strand, while it will have a single-stranded 5' overhang in the parent strand if nucleic acid synthesis terminated beforethe polymerase reached the end of the target fragment parent strand. Further, if the polymerase employed has terminal transferase activity, there may be a 3' overhang instead of a blunt end if the polymerase traverses the entire target fragment. No limitation in this regard is intended. In the embodiment depicted in FIG. 7B, the ends of the nucleic acid extension products 715 and 717 include single 3’ A overhangs.

[0155] In certain embodiments, the extension reaction may include modified nucleotides that weaken the duplex strength of the extension products, i.e., the strength of the hydrogen bonds between the parent template strand and the newly synthesized daughter strand. Exemplary, nonlimiting, modified nucleotides include N4-Me dCTP and 7-deaza dGTP. In embodiment illustrated in FIG. 7B, daughter strands 719a and 719b incorporate such modified dGTP and dCTP analogs, as denoted by the circles around “G” and “C” in these strands.

[0156] As shown in FIG. 7C, in step 3, in one embodiment, 5’ ends 706a and 706b of the parent strands of the extension products are activated by PNK-dependent phosphorylation. Other means of “masking” (i.e., preventing ligation) and “unmasking” (i.e., enabling ligation) the ends of a nucleic acid strand to either activate or block ligation of two fragments are contemplated by the present invention. For example, chemical masking / unmasking using base protection strategies known in the art may be used in certain embodiments. Hairpin adapters 719 are then contacted with the extension products and ligated to the double stranded ends to produce first duplexed template construct 721 and second duplexed template construct 723. Ligation of hairpin adapters is facilitated by a single 3’ T overhand in the hairpin adapters that is capable of base-pairing with the single A overhang in the daughter stand of the extension products. In each duplexed template construct, the daughter strand is covalently coupled (i.e., covalently bound) to the parent strand by ligation to the intervening hairpin adapter.

[0157] As shown in FIG. 7D, in step 4, the duplexed template constructs 721 and 723 are prepared for the direct synthesis of Xpandomers. Advantageously, the duplexed template constructs can be directly copied into Xpandomer products without prior PCR amplification. (Although, in certain applications, an amplification step may be included in the methods disclosed herein). This reduces the likelihood of sequencing errors due to nucleotide misincorporations during amplification, particularly in homopolymer sequences in the target fragment. Here, primers 725 and 727 are hybridized to sequences in the 3’ ends of the single stranded regions of the Y adapters and provide initiation sites for Xpandomer synthesis. In certain embodiments blocker oligonucleotides 727 and 729 may be, optionally, hybridized to sequences in the 5’ ends of the single stranded regions of the Y adapters and function to terminate Xpandomer synthesis.

[0158] Xpandomer synthesis reactions are carried out in which Xpandomers are synthesized from primers 725 and 727 in the 5’ to 3’ direction, as the parent and daughter strands of the template are “unzipped”. In this embodiment, unzipping is facilitated by weak hydrogen bonding the strands due to the modified G and C nucleotides incorporated into the daughter strands. In other embodiments, any suitable nucleotide analogs may be used to either weaken a base pair, or, alternatively, to strengthen a base pair. For example, in some embodiments, DAP or an analog of thymidine may be used to modify the strength of an A:T base pair. Further details of the Xpandomer synthesis reactions are discussed herein.

[0159] In other embodiments, the methods of the present invention may include alternative adapter designs to generate the duplexed template constructs. For example, in one embodiment, an alternative adapter design may include a pre-nicked hairpin adapter, as shown in FIG. 8A. The nick may be introduced into the hairpin adapter according to any suitable method, such as those discussed with reference to FIG. 7A, as well as by using chemical methods known in the art. Here, pre-nicked hairpin adapter 800 includes double stranded region 805 and single stranded loop region 807. The double stranded region 805 includes a single stranded nick 810 in one of the strands. In certain embodiments, the sequence of the double stranded region of the adapter may be designed to include a single stranded nickase site. Following synthesis of the adapter, the hairpin may be contacted with a nickase enzyme, followed by a phosphatase enzyme, to generate a single stranded nick in one strand that is refractory to ligation due to the absence of a 5’ phosphate. The single stranded nick therefore provides a free 3’ end capable of being extended by a DNA polymerase. However, the free 3’ end is not capable of being religated to the free 5’ end by a DNA ligase.

[0160] In step 1, a double stranded target fragment 820 is derived from genomic DNA or some other nucleic acid source. The double stranded target fragment 820 includes parent (+) strand 820a (i.e., a sense strand) and parent (-) strand 820b (i.e., an antisense strand). In Step 1, pre-nicked hairpin adapters 800a and 800b are joined to opposite ends of the double stranded target fragment. The adapters are joined to the double stranded target fragment such that each strand of the double stranded region of the adapter is ligated to a strand of the double stranded target fragment. In this embodiment, the joining of the adapters to the double stranded target fragment is referred to as “complete ligation”, in contrast to the partial ligation discussed with reference to FIG. 7A. The adapter-ligated double stranded target fragment product 830 includes a single stranded nick in each strand that provides a free 3’ end, represented by arrows 810a and 810b.

[0161] In certain embodiments, the adapters may include sequences, or other features, that mediate downstream steps of the workflow. For example, the adapters may include nucleic acidsequences or other chemical moieties for immobilization of the templates on a solid support, sequences for hybridization of oligonucleotide primer(s), sequences enabling bioinformatic analysis of DNA sequence information (e.g., unique molecular identifier bar codes [UMI], sample identifiers [SID]), and the like. In certain embodiments, the structures of the adapters may be identical or different, depending on the particular application. Advantageously, in certain embodiments, UMI sequences are not required for downstream steps of the workflow (e.g., bioinformatic error correction or pairing of sequences derived from both strands of a double stranded DNA target fragment) and thus reduce the complexity of adapter design.

[0162] As shown in step 2, adapter-ligated double stranded target fragment is treated with conditions that denature the double stranded target fragment (e.g., heat or alkaline treatment) to yield two adapter-ligated single stranded target fragments. In the depiction shown in FIG. 8A, only a single adapter-ligated single stranded target fragment 833 is shown, for the sake of simplicity. Adapter-ligated single stranded target fragment 833 includes hairpin adapter 800a, single stranded target fragment 820b (i.e., the antisense strand) and extendable 3’ end 810a.

[0163] As shown in FIG. 8B, in step 3, the adapter-ligated single stranded target fragments are contacted with a DNA polymerase under nucleic acid extension conditions, which uses free 3’ end 810a to initiate synthesis of complementary daughter strand 840a, using target fragment strand 820b as a template. Synthesis of a full-length complementary daughter strand copy of the target fragment yields adapter-ligated double stranded fragment 850, which includes a strand of the original parental target fragment covalently coupled to a newly synthesized daughter strand by the hairpin adapter. In certain embodiments, the full-length complementary daughter strand copy of the target fragment will include a single 3’ A overhand.

[0164] In an alternative embodiment, step 2 may be omitted from the method and adapter- ligated double stranded target fragment product 830 may be directly contacted with a stranddisplacing DNA polymerase to initiate complementary copy strand synthesis off free 3’ ends provided by singe stranded nicks 810a and 810b. In this instance, DNA synthesis proceeds as discussed with reference to FIG. 7B, step 2, until two separate adapter-ligated double stranded fragments are produced, each including one parental strand derived from the original double stranded target fragment and one daughter complementary copy strand covalently joined to the parental strand via the hairpin adapter.

[0165] In certain embodiments, as discussed herein, the extension reaction may include modified nucleotides that weaken the duplex strength of the extension products, i.e., the strength of the hydrogen bonds between the parent template strand and the newly synthesized daughter strand. Exemplary, non-limiting modified nucleotides are N4-Me dCTP and 7-deaza dGTP.

[0166] As shown in step 4, Y adapters 855 are then contacted with the adapter-ligated double stranded fragment 850 and ligated to the double stranded end to produce duplexed template constructs, as discussed with reference to FIG. 7D. Ligation of the Y adapters is facilitated by a free 3’ T overhang in the Y adapters that is capable of base-pairing with the single A overhang in the daughter stand of the extension products. In each duplexed template, the daughter strand is covalently coupled (i.e., covalently bound) to the parent strand by ligation to the intervening hairpin adapter. The duplexed template constructs may then be utilized for Sequencing by Expansion, as discussed with reference to FIG. 7.

[0167] Another embodiment of a method of the present invention is set forth in FIG. 9. This embodiment provides the advantages of solid-state synthesis of duplex template constructs. As shown in FIG. 9A, a double stranded target fragment 900 is derived from genomic DNA or some other nucleic acid source. The double stranded target fragment 900 includes parent (+) strand 905A (i.e., a sense strand) and parent (-) strand 905B (i.e., an antisense strand). Prior to step 1, adapters 907 and 909 are joined to the ends of the double stranded target fragment. In this embodiment, adapters 907 and 909 include features that enable solid state synthesis. For example, the adapters may include biotin moi eties 907a and 909a joined to the 3’ ends of the single stranded portion of the Y adapters and cleavable linkers 907b and 909b juxtaposed at the 5’ end of the biotin moi eties.

[0168] In certain embodiments, joining of the adapters to the double stranded target fragment may be facilitated by including a 3’ single base overhang in one strand of the Y adapters (e.g., a single T overhang) and a complementary 3’ single base overhang in the opposite strand of the double stranded target fragment (e.g., a single A overhang). Base pairing of the T and A overhangs thus brings the adapter and target fragment into alignment for ligation. As shown in this embodiment, the adapters are Y adapters, and each includes a region of double stranded DNA and two regions of single stranded DNA. The adapters are joined to the double stranded target fragment such that one strand of the adapter is ligated to one strand of the target fragment, while the other strand of the adapter is not ligated to the target fragment. In this embodiment, the joining of the adapters to the double stranded target fragment is referred to as “partial ligation”. Partial ligation may be achieved, for example, by blocking the 5’ end of each strand of the target fragment to prevent ligation to the 3’ ends of the double stranded regions of the Y adapters. Blocking may be achieved in certain embodiments by phosphorylation of the 3’ ends of target fragment, as represented by solid circles 907c and 909c at the 5’ ends of each strand of the target fragment. The adapter-ligated double stranded target fragment product will include a single free 3’ end in each strand of the double stranded portion of the ligation product.

[0169] In step 1, following ligation of the adapters to the double stranded target fragments, the single strands of Y adapters 907 and 909 that include terminal blocked ends 907c and 909c are released from the adapter-ligated target fragments. In some embodiments, this may be accomplished by applying denaturing the double stranded portion of the Y adapters. The opposite strands of the Y adapters that include terminal biotin moieties 907a and 909a, however, remain covalently associated with the double stranded target fragment. The adapter-ligated double stranded target fragment 910 may then be captured on a streptavidin-coated solid support 915. In other embodiments, the adapter-ligated double stranded target fragments are first captured on the solid support; subsequently, the single strands of the Y adapters are released via denaturation.

[0170] As shown in FIG. 9B, prior to step 2, the adapter-ligated double stranded target fragment may be treated to denature the two strands of the target fragment and produce single stranded adapter-ligated parent (+) strand 910a and single stranded adapter-ligated parent (-) strand 910b. The blocked adapter strands that were removed from the double stranded target fragments in step 1 may then be replaced with extendable primers 911a and 911b. In this embodiment, the extendable primer includes 1) a 3’ end (911c and 91 Id) that is designed to hybridize to the adapter strand portion that remains ligated to the target fragment strand and 2) a 5’ end that is single stranded.

[0171] In step 2, extendable primers, 911a and 911b, hybridized to single stranded adapter- ligated parent (+) strand 910a and single stranded adapter-ligated parent (-) strand 910b, are contacted with a DNA polymerase under nucleic acid extension conditions. Nucleic acid synthesis is initiated from the free 3' OH group of the primers, using the single stranded parent fragments as templates. In certain embodiments, the extension reaction may include modified nucleotides that weaken (or strengthen, depending on the application) the strength of the hydrogen bonds between the parent template strand and the newly synthesized daughter strand. Exemplary, non-limiting modified nucleotides are N4-Me dCTP and 7-deaza dGTP. In embodiment illustrated in FIG. 3B, daughter strands 315a and 315b incorporate modified dGTP and dCTP analogs, as denoted by the circles around “G” and “C” in these strands.

[0172] The DNA polymerases proceed along the target fragment strands in opposite directions, finally in two adapter-ligated double stranded copies of the target fragment, 920 and 925, each including one strand of the original double stranded target fragment (i.e., the parent strand) and one newly synthesized complementary copy strand (i.e., the daughter strand). Each of the double stranded copies includes a fully ligated adapter at one end (i.e., there are no nicks or gaps between the fragment and the adapter) and either a double stranded blunt end or single stranded overhang terminus at the opposite end. For example, the end opposite the adapterligated end (i.e., lacking an adapter) is expected to be blunt if the polymerase traversed the entire target fragment strand, while it will have a single-stranded 5' overhang in the parent strands if nucleic acid synthesis terminated before the polymerase reached the end of the target fragment parent strand. Further, if the polymerase employed has terminal transferase activity, there may be a 3' overhang instead of a blunt end if the polymerase traverses the entire target fragment. No limitation in this regard is intended. In the embodiment depicted in FIG. 9, each adapter-ligated double stranded product is bound to the solid support via the biotin moiety joined to the single strand 3 ’ end of the Y adapter.

[0173] As shown in FIG. 9C, in step 3, hairpin adapters 927 are then contacted with the extension products and ligated to the double stranded ends to produce first duplexed template construct 930 and second duplexed template construct 935. Ligation of the hairpin adapters may be facilitated as discussed with reference to FIG. 7C. In each duplexed template construct, the daughter strand is covalently coupled (i.e., covalently bound) to the parent strand via ligation of each strand to opposite ends of the intervening hairpin adapter. In certain embodiments, the duplexed template constructs may be released from the solid support by breaking the cleavable bond positioned proximal to the terminal biotin moieties. For example, UV light may be applied to cleave a photosensitive bond, as discussed further herein and depicted by the lightning bolts illustrated in FIG. 9C.

[0174] The duplexed template constructs for Sequencing by Expansion, discussed with reference to FIG.7 - FIG.9, each includes a parental strand derived from the original double stranded DNA target fragment and a newly synthesized daughter strand copy in which these two strands are covalently coupled by the intervening hairpin adapter. In certain alternative embodiments, the methods of the present invention include synthesis of a duplexed template construct in which both strands of the original double stranded DNA target fragment are covalently couple by an intervening hairpin adapter. Such duplexed templates are referred to herein as “parent-parent” templates.

[0175] One method of generating a parent-parent template construct according to the present invention is depicted in FIG. 10. In this embodiment, library fragment (i.e., the double stranded DNA target fragment) 1000 includes parental sense strand 1000a and parental antisense strand 1000b. The library fragment is end repaired and A-tailed to generate single 3’ A overhangs in each strand, as discussed herein. In step 1, target fragment 1000 is contacted with hairpin adapter 1010, which includes a single T overhang to facilitate alignment with the target fragment, and a DNA ligase enzyme. The desired product of the ligation reaction is asymmetric duplexed template construct 1025 that includes a single hairpin adapter ligated to one end of the double stranded target fragment. In certain embodiments, the ratio of the hairpin adapter to thelibrary fragment may be optimized to preferentially generate asymmetric duplex template construct 1025. In step 2, Y adapter 1030 is immobilized on solid support 1033 via linker 1035 that is covalently bound at one end to the single stranded 5’ end of the Y adapter and at the other end to the solid support. In this embodiment, linker 1035 includes a poly dU sequence. The ligation products of step 1, including asymmetric duplexed template construct 1025 are then contacted with the immobilized Y adapter and a DNA ligase enzyme. In some embodiments, the products of the ligation of step 1 are first denatured, followed by size selection purification, to remove smaller side products. Other side products of the ligation reaction of step 1, including any symmetric duplex templates (i.e., constructs with hairpin adapters ligated to both double stranded ends of the target fragment) are not capable of ligating to the Y adapter, and thus will not be associated with solid support 1033. In contrast, asymmetric duplexed template construct 1025 may be ligated to Y adapter 1030 to form Y adapter-ligated duplexed template construct 1040, bound to solid support 1033 via linker 1035. In step 3, Y adapter-ligated duplexed template construct 1040 is treated with, e.g., a USER enzyme to cleave the polyU sequence in linker 1035 and release the Y adapter-ligated duplexed template construct from the solid support. In other embodiments, linker 1035 may include other suitable selectively-cleavable moi eties known in the art, such as a photocleavable moiety.

[0176] In certain embodiments, synthesis of the parent-parent duplex template construct may be carried out entirely in-solution. For example, the synthesis of the duplex template construct may include a first ligation reaction in which a hairpin adapter is ligated to a first end of a library fragment in-solution and a second ligation in which a Y adapter is ligated to a second end of the library fragment in-solution. After the first ligation reaction, products may be denatured, such that only library fragments with strands that are covalently paired by ligation to an intervening hairpin adapter will remain physically associated for the subsequent second ligation. Likewise, for the second ligation reaction, the only constructs capable of ligation to the Y adapter are those in which the library fragment has a single free double stranded end. In certain embodiments, a step of exonuclease-mediated clean-up to remove single stranded side products may be included after the second ligation step. Additional purification steps may be included in the workflow, e.g., one or more SPRI bead purification steps.

[0177] An alternative method of generating a parent-parent template construct is depicted in FIG. 11. In this embodiment, library fragment (i.e., the double stranded DNA target fragment) 1100 includes parental sense strand 1100a and parental antisense strand 1100b. The library fragment is end repaired and A-tailed to generate single 3’ A overhangs in each strand, as discussed herein. In step 1, the target fragment 1100 is contacted with hairpin adapters 1110aand 1110b, which include a single T overhang to facilitate alignment with the target fragment, and a DNA ligase enzyme. In this embodiment one of the two hairpin adapters, e.g., adapter 1110a, is designed to include a single stranded cleavage site in the sequence of each strand of the double stranded portion of the hairpin adapter. The sites are staggered in position such that cleavage of both sites leaves a single stranded overhang in one strand of the double stranded portion of the hairpin adapter. Ligation product 1120 is a dual hairpin adapter-ligated double stranded target fragment with cleavable hairpin adapter 1110a joined to one end and non- cleavable hairpin adapter 1110b joined to the opposite end of the target fragment.

[0178] In step 2, the dual hairpin adapter-ligated double stranded target fragment 1120 is treated with conditions that generate single stranded breaks at the opposing cleavage sites in each strand of the double stranded region of the cleavable hairpin adapter 1110a then denatured, e.g., by treatment with a strong base, heat or a combination thereof. The resulting product, asymmetric adapter-ligated double stranded target fragment 1130 includes a single stranded overhang 1135 that is derived from the sequence of cleavable hairpin adapter 1110a.Asymmetric adapter-ligated double stranded target fragment 1130 is contacted with Y adapter 1140 that includes a single stranded overhang 1145 in the double stranded portion of the adapter that is designed to be complementary to single stranded overhang 1135 in the asymmetric adapter-ligated double stranded target fragment 1130 and a DNA ligase. Ligation of Y adapter 1140 to asymmetric adapter-ligated double stranded target fragment 1130 generates duplexed template construct 1150 with Y adapter 1140 ligated to one end of the double stranded target fragment and hairpin adapter 1110b ligated to the other end of the double stranded target fragment. Thus, duplex template construct 1150 includes both strands of the original parental template covalently joined by intermediary non-cleavable hairpin adapter 1110b.

[0179] Other methods and compositions for producing libraries of duplex template constructs for use in Sequencing by Expansion are disclosed in Applicants published PCT application no.s WO / 2025132779 and WO / 2025132788, the entire contents of which are herein incorporated by reference in their entireties.Solid-Phase Synthesis

[0180] In certain embodiments, one or more steps of generating and / or amplifying the duplexed template constructs and / or Xpandomer synthesis may be conducted on a solid support. As used herein, the terms "solid support", “solid-state”, "solid-phase", “on-support” and "substrate" may be used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, e.g., a surface of a polymeric microfluidic card or chip. In someembodiments it may be desirable to physically separate regions of a card or chip for different reactions with, for example, etched channels, trenches, wells, raised regions, pins, or the like. According to other embodiments, the solid support(s) will take the form of insoluble beads, resins, gels, membranes, microspheres, or other geometric configurations composed of, e.g., controlled pore glass (CPG) and / or polystyrene.

[0181] The invention encompasses solid-phase synthesis methods in which a capture moiety is immobilized on a solid support. In certain instances, the capture moiety includes a first end covalently bound to the solid support and a second end that provides a functional group capable of binding to the 5’ end of a single stranded target sequence. As used herein, the term "immobilized", refers to the association, attachment, or binding between a molecule (e.g., linker, adapter, or oligonucleotide) and a support in a manner that provides a stable association under the conditions of elongation, amplification, ligation, and other processes as described herein. Such binding can be covalent or non-covalent. Non-covalent binding includes electrostatic, hydrophilic and hydrophobic interactions. Covalent binding is the formation of covalent bonds that are characterized by sharing of pairs of electrons between atoms. Such covalent binding can be directly between the molecule and the support or can be formed by a cross linker or by inclusion of a specific reactive group on either the support or the molecule or both. Covalent attachment of a molecule can be achieved using a binding partner, such as avidin or streptavidin, immobilized to the support and the non-covalent binding of the biotinylated molecule to the avidin or streptavidin. Immobilization may also involve a combination of covalent and non- covalent interactions.

[0182] Any suitable covalent attachment means known in the art may be used for these purposes. The chosen attachment chemistry will depend on the nature of the solid support and any derivatization or functionalities applied thereto. The extension oligonucleotide may include a moiety, which may be a non-nucleotide chemical modification, to facilitate attachment. Certain exemplary embodiments of suitable surface chemistries include conventional streptavidin / biotin interaction chemistry and involve functionalization of a solid support, e.g., with a linker moiety that includes terminal a biotin moiety. In this embodiment, the 5’ end of single stranded DNA fragment (or oligonucleotide) is bound to the linker moiety. Attachment is mediated by a streptavidin moiety provided by the 5’ end of the single stranded DNA fragment. The linker moieties disclosed herein may be of sufficient length to connect the single stranded DNA fragment to the support such that the support does not significantly interfere with primer extension reaction.

[0183] Alternatively, immobilization of a capture moiety or oligonucleotide (e.g., an extension oligonucleotide) to a solid support may be accomplished by covalent linkage of thecapture oligonucleotide to the solid support via a click reaction. In this embodiment, the covalent linkage may be mediated by a maleimide-PEG-alkyne linker that is crosslinked to the solid support. An alkyne moiety provided by the end of the linker distal to the substrate is capable of reacting with an azide group provided by the 5’ end of the capture oligonucleotide. Methods of functionalizing a solid support with maleimide-linker polymers is provided in Applicant’s published Patent Application No. WO2020 / 172479, which is herein incorporated by reference in its entirety.

[0184] In certain instances, the linkage between the capture moiety and the solid support is cleavable, enabling primer extension products to be released from the support following synthesis. Cleavable linkers and methods of cleaving such linkers are known and can be employed in the provided methods using the knowledge of those of skill in the art. For example, the cleavable linker can be cleaved by an enzyme, a catalyst, a chemical compound, temperature, electromagnetic radiation or light. Optionally, the cleavable linker includes a moiety hydrolysable by beta-elimination, a moiety cleavable by acid hydrolysis, an enzymatically cleavable moiety, or a photo-cleavable moiety. In some embodiments, a suitable cleavable moiety is a photocleavable (PC) spacer or linker phosphoramidite available from Glen Research. Kits

[0100] The present disclosure also provides for kits including any of the linear amplification reagents of the present disclosure, and one or more additional components. In some embodiments, the kits include one or more of (i) an oligonucleotide primer, (ii) an isothermal amplification buffer (typically including Tris-HCl, (NH^SCU, KC1, MgSCU, and Tween 20, providing the appropriate ionic strength and pH for optimal polymerase activity during isothermal amplification), (iii) a dNTP mix, (iv) a pyrophosphatase, (v) a single stranded binding protein, a strand displacing DNA polymerase, and (vi) a nicking endonuclease. In some embodiments, a kit may include a proteinase K enzyme. Non-limiting examples each of these components are disclosed herein.

[0101] In some embodiments, the kit may provide a concentrated linear amplification master mix including one or more of (i) around 5pM primer (to provide around 4pmol of primer in the final reaction), (ii) around 10X isothermal amplification buffer, (iii) around 4mM of each dNTP, including 100% 7-deaza dGTP, (iv) around 0.6U / pL pyrophosphatase, (v) around lOpg / pL single stranded binding protein (e.g., gp32), around 6U / pL Bst DNA polymerase, and around 10U / .L nickase endonuclease (e.g., Nt.BspQI).

[0102] Typically, the reagent mixture(s) of the kit are concentrated, so that an aliquot is added to the final reaction volume, along with a duplex template construct (i.e., a doublestranded nucleic acid template). In a non-limiting, exemplary linear amplification reaction, around 0.5pmol nucleic acid template is added to a IX linear amplification mix.

[0103] In some embodiments, the kit further includes one or more buffer solutions and / or wash solutions. In some embodiments, the kit further includes beads having a functionalized surface. In some embodiments, the kit further includes SPRI beads.Sequencing by Expansion

[0185] One nucleic acid sequencing methodology that may be implemented with the methods of the present invention is “Sequencing by Expansion” (SBX®), developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion", which is herein incorporated by reference in its entirety). As previously discussed, SBX® uses biochemical polymerization to transcribe the sequence of a DNA template, e.g., a duplex template construct, onto a measurable polymer called an “Xpandomer”. SBX® is based on the polymerization of highly modified, non-natural nucleotide analogs, referred to as “XNTPs”. XNTPs are expandable, 5' triphosphate modified non-natural nucleotide analogs compatible with template dependent enzymatic polymerization. The XNTP has two distinct functional regions; namely, a selectively cleavable phosphoramidate bond, linking the 5’ a-phosphate to the nucleobase, and a symmetrically synthesized reporter tether (SSRT) that is attached within the nucleoside triphosphoramidate at positions that allow for controlled expansion by cleavage of the phosphoramidate bond. XNTPs are described in further details in Applicant’s U.S. patent no.s 10,301,345 and 10,774,105, which are herein incorporated by reference in their entireties. The SSRT includes linkers separated by the selectively cleavable phosphoramidate bond. Each linker attaches to one end of a reporter code. XNTP substrates incorporated into daughter strand products of template-dependent polymerization are in the “constrained” configuration. The constrained configuration of polymerized XNTPs is the precursor to the expanded configuration, as found in Xpandomer products.

[0186] The transition from the constrained configuration to an expanded configuration results from cleavage of the selectively cleavable phosphoramidate bonds within the primary backbone of the Xpandomer. In this embodiment, the SSRTs include one or more reporters or reporter codes, specific for the nucleobase to which they are linked, thereby encoding the sequence information of the template. In this manner, the SSRT provides a means to expand the length of the Xpandomer and lower the linear density of the sequence information of the parent strand.

[0187] The SSRT (i.e., “tether”) of the XNTP includes several distinct functional elements, or features, such as polymerase enhancement regions, reporter codes, and translationcontrol element (TCEs). These features are discussed in further details in Applicant’s published PCT application WO2020 / 236526, which is herein incorporated by reference in its entirety. Each of these features performs a unique function during translocation of the Xpandomer through a nanopore to produce a series of unique and reproducible electronic signal. The SSRT is designed for controlling the rate of Xpandomer translocation by the TCE through a combination of sterics and / or electrorepulsion, Different reporter codes are sized to block ion flow through a nanopore at different measurable levels. In certain embodiments, reference is made to the “reporter construct” of the XNTP, which includes, from a proximal end to a distal end, the TCE, a symmetrical Y brancher, and two symmetric reporter code, each joined to an end of the Y brancher distal to the TCE. The reporter construct is a feature of the larger SSR structure.

[0188] Specific SSRT polymeric sequences can be efficiently synthesized using phosphoramidite chemistry typically used for oligonucleotide synthesis. Reporter codes and other features can be designed by selecting a sequence of specific phosphoramidites from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycol with lengths of 1 to 12 or more ethylene glycol units and aliphatic polymers with lengths of 1 to 12 or more carbon units. In certain embodiments, the SSRTs include features referred to as “polymerase enhancement regions” at the ends of the SSRTs proximal to the nucleotide triphosphoramidate diester. Polymerase enhancement regions may include positively charged polyamine spacers (e.g., primary, secondary, tertiary, or quaternary amines) or triamine spacers (three secondary amines each separated by three carbons) that facilitate incorporation of XNTP structures by a nucleic acid polymerase. In certain embodiments, the polymerase enhancement region includes two repeat units spermine

[0189] In certain embodiments, an XNTP may be a compound having the generalized structure depicted in FIG. 14.

[0190] In one embodiment, R may be H, for example, when the compounds are used to sequence a DNA template.

[0191] In certain embodiments, nucleobase is adenine, cytosine, guanine, thymine, uracil or a nucleobase analog. As one of skill in the art will appreciate, adenine, cytosine, guanine, thymine, and uracil are naturally occurring nucleobases. As used herein, the term “nucleobase analog” refers to non-naturally occurring nucleobases that are capable of forming Watson and Crick base pair with a complementary nucleobase on an adjacent single-stranded nucleic acid template.

[0192] To obtain sequence information, an Xpandomer is translocated through a nanopore, from the cis reservoir to the trans reservoir. As the Xpandomer translocates, a reporterenters the stem until its translocation control element stops at the stem entrance. The reporter is held in the stem until the TCE is enabled to pass into and through the stem, whereupon translocation proceeds to the next reporter. Upon passage through the nanopore, each of the reporter codes of the linearized Xpandomer generates a distinct and reproducible electronic signal, specific for the nucleobase to which it is linked.

[0193] In certain embodiments, Xpandomers produced by the SBX chemistry may be analyzed using a nanopore-based sequencing chip. A nanopore-based sequencing chip can incorporate a large number of sensor cells configured as an array. For example, the chip may include an array of one million cells configured in 1000 rows by 1000 columns of cells. Each cell in the array may include a control circuit integrated on a silicon substrate. Such nanoporebased sequencing chips, devices, and systems are described, e.g., in Applicant’s published patent application no. WO2021 / 219795, which is herein incorporated by reference in its entirety.

[0194] Proprietary in-house bioinformatics pipelines are typically used to process sequencing reads. In certain embodiments, the methods disclosed herein may leverage UMIs to enable pairing of related sequence reads. Read pairs may be quality filtered and trimmed of adapter and primer sequences. UMI sequences may be clustered together, defining UMI-families (all reads originating from a single DNA template). In other embodiments, sequencing reads may be processed without UMI-based pairing, for example, when a single read includes information derived from both strands of a double stranded DNA target fragment, e.g., from a duplex template construct, as disclosed herein.Xpandomer Synthesis Reaction

[0195] The Xpandomer synthesis reaction represent a critical step in SBX®, as it is responsible for accurately transcribing the sequence of the DNA template of interest into the sequence of the Xpandomer, which is the polymer directly read by the nanopore sensor.Through trial and error, the inventors have developed a complex reaction mixture for Xpandomer synthesis, which includes a DNA polymerase and many additives that enable incorporation of the bulky XNTP substrates by the polymerase into the very large Xpandomer structure.

[0196] In certain embodiments, a non-limiting Xpandomer synthesis reaction mixture may include the following reagents: a buffer / salt system, polymerase cofactors, polymerase enhancing moieties (PEMs), a DNA polymerase, XNTP substrates, a phosphate shield molecule, a solvent, a crowding agent, and optionally, additional additives. In some embodiments, the buffer / salt system may include TrisCi and NaCl; the polymerase cofactors may include MnCE formulated in MES; the PEMs may include molecules disclosed in Applicant’s published PCT applications, WO2019 / 135975 and W02020 / 263703 and PCT application no.PCT / US24 / 061051, which are herein incorporated by reference in their entireties; the DNA polymerase may include a variant of DP04 polymerase as disclosed in Applicant’s U.S. patent no.s 11,299,725, 11,708,566, 11,530,392, published PCT application no. WO / 2025 / 082960 and U.S. provisional patent application no. 63 / 788,330, filed April 14, 2025, each of which is herein incorporated by reference in their entireties; the phosphate shield molecule may include hexametaphosphate (HMP); the solvent may include NMP and DMSO; the crowding agent may include PEG8k; and the additional additives may include imidazole and betaine.

[0197] In certain embodiments, the Xpandomer synthesis reaction comprises a variant of wildtype DPO4 polymerase, designated C7326, with the following amino acid substitutions: F37T D39L K56 Y_A57S_I59M_E63R_M76W_K78E_E79P_Q82W_Q83 G_S 86E K 152 A_11 53V_A155G_D156S_M157K_D179N_P184Q_G187P_N188Y_I189F_E192Q_I248T_S272C_ V289W_T290R_E291S_D292R_L293W_D294N_I295S_V296Q_S297Y_G299W_R300S_T30 lW_K321Q_E324K_E325K_E327KA341-352 (SEQ ID NO:2). The amino acid sequence of wildtype DPO4 polymerase is set forth in SEQ ID NO: 1, while the amino acid sequence of variant C7326 is set forth in SEQ ID NO:2. In some embodiments, a variant of DPO4 polymerase suitable for the practice of the present invention may be a variant that is at least 85% identical to SEQ ID NO:2.

[0198] One of the challenges encountered by the DNA polymerase when replicating the duplex template constructs of the present invention are double stranded regions formed when the two complementary strands of the target fragment are hybridized. DPO4 polymerase does not possess robust strand displacement activity and, as such, it does not efficiently extend through double stranded regions in a template. Indeed, the inventors have observed that the DPO4 variants commonly used in Xpandomer synthesis will prematurely “jump” from one template strand to the other as the duplex template construct is copied, thereby synthesizing incomplete Xpandomer copies of the two template strands. This phenomenon is referred herein to as a polymerase “U-turn”.

[0199] In optimizing Xpandomer synthesis conditions using the duplex template constructs of the present invention, the inventors have tested numerous biological additives and other physical forces or manipulations to reduce the occurrence of polymerase U-turns and increase the percentage of full-length duplexed Xpandomers synthesized. The following classes of additives were observed to reduce the rate of polymerase U turns during replication of duplexed template constructs: 1) single stranded binding proteins (SSBs), which are proteins that bind to and help stabilize single stranded regions of DNA and prevent formation of more stable secondary structures; 2) proteins, or enzymes, known to participate in DNA recombinationprocesses, or to otherwise manipulate regions of single stranded DNA; 3) non-protein additives known to beneficially impact Xpanodmer synthesis (e.g., PEMs or polyphosphate analogs); 4) the biochemistry conditions of the Xpandomer synthesis reaction, including order of addition (e.g., pre-treatment of the template with an additive such as SSB prior to the Xpandomer synthesis reaction) ; 4) stretching forces proposed to enhance solid-state replication of duplexed template constructs. For example, in certain embodiments a duplex template construct may be associated with a solid support through hybridization of a sequence in its 3’ end with an extension oligonucleotide that is covalently bound to the support. The 3’ end of the extension oligonucleotide provides an initiation site for Xpandomer synthesis by a DNA polymerase, as disclosed herein. A blocker oligonucleotide can be designed to hybridize to a sequence in the opposite 5’ end of the duplex template construct. In certain embodiments, the 3’ end of the blocker oligonucleotide can be joined to a moiety that is susceptible to, e.g., an applied external force that “stretches” apart double stranded regions by overcoming the strength of the hydrogen bonds between the two complementary strands of the duplex template construct.

[0200] A non-limiting list of SBX® synthesis enhancers according to the present invention is set forth in Table 2. It is to be emphasized that the following list of exemplary additives is intended to merely illustrate one of many suitable possibilities of the larger genus (i.e., class) of additives, and other forces recited in Table 2, contemplated by the present invention.Table 2SBX® synthesis enhancersEXAMPLESExample 1 Linear Amplification of a Duplex Template Construct

[0201] This Example provides a protocol / workflow for the linear amplification of a HD template construct (e.g., a duplex template construct).

[0202] First, an initial extension reaction is conducted in which the HD template construct is replicated to generate a complementary copy hybridized to the parental HD template. A hybridization mix is provided that includes the following reagents: 20 M primer and 0.4 mM each dNTP (with 100% 7-deaza dGTP and 100% IS Me dCTP) and 0.5ng / L HD template construct in IX amplification buffer. The primer is allowed to anneal to the HD template construct by incubating the hybridization mix at 90°C / 105°C for 30 seconds then room temperature for 3 minutes.

[0203] Then, an enzyme mix is provided that includes the following reagents: 5pg single strand binding protein and 4U Bst polymerase. For the extension reaction, 2pLof the enzyme mix is added to 16 L of the hybridization mix, and the extension reaction is incubated at 37°C for 1 hr.

[0204] Next the linear amplification reaction is performed as follows: a diluted nickase sample is provided that includes 5.00U / g / pL of nickase enzyme in water; 2 L of the diluted nickase sample is added to the extension reaction and the linear amplification reaction is run for 24 hr at 52°C.

[0205] Following the linear amplification reaction, the sample is denatured by adding a denaturation mix including 0.10M NaOH and 0.10% SDS; the denaturation reaction is run for 20 minutes at 65°C. The sample is allowed to cool and is then the nucleic acids are purified using the KAPA purification kit available from Roche Sequencing Solutions, following the manufacturer’s protocol.

[0206] The products of the linear amplification reaction can be assessed by running an aliquot of the sample on a “hyb gel”. Briefly, a hyb buffer is provided that includes thefollowing reagents: 50mM TrisCi, 200mM NaCl, 0.20pM SIMA labeled oligonucleotide (that is capable of hybridizing to a sequence in the Y adapter portion of the HD duplex construct), and 2% PEG 8K. 9pL of the hyb buffer is added to IpL of the extension reaction and 15pL of the hybridization reaction is run on a 4-12% non-denaturing gel; products are visualized by illumination of the SIMA labeled oligonucleotide.Example 2 Library Preparation for Duplex Sequencing by Expansion

[0207] This Example provides an exemplary workflow for library preparation for duplex Sequencing by Expansion; this workflow includes the following general steps: 1) DNA fragmentation, adapter ligation, and SPRI size selection; 2) amplification and purification of template constructs; 3) quantification and QC of template constructs; and 4) template pooling and preparation for Xpandomer synthesis.

[0208] A kit for performing this workflow may include the following reagents: 1) Fragmentation Ready Mix; 2) Ligation Mix 1 (including 36 unique hairpin SID adapters); 3) Ligation Mix 2 (including Y adapters, ligation digest, SPRI beads, and elution buffer); 4) Amplification Mix (including amplification initiator, and amplification digest); 5) Elution Buffer 2; and 6) Quantification Assay.

[0209] Step 1) DNA fragmentation, adapter ligation, and SPRI size selection

[0210] DNA fragmentation is performed as follows: a) 50ng of an unsheared DNA sample is diluted to 17.5pL with lOmM Tris Cl, pH 8 and stored on ice; b) 12.5pL of Fragmentation Ready Mix is added to the DNA sample to a final volume of 30pL; the sample is mixed with a pipet 15-20 times; c) the sample is incubated in a thermocycler for 25 minutes at 37 degrees C, 30 minutes at 55 degrees C and then held at 4 degrees C.

[0211] Ligation 1 (hairpin SID) is performed as follows: a) a HP. SID sample is prepared by transferring 12pL of HP. SID to a 0.2mL PCR tube and hybridizing the adapters in a thermocycler for 1 minute at 95 degrees C, 25 minutes at 37 degrees C, 30 minutes at 55 degrees C, and holding at 4 degrees C.; b) lOpL of hybridized HP. SID is added to 30pL of fragmented library in a 0.2mL PCR tubes and mixed; c) 20pL of Ligation Mix 1 is added to the tube; and d) the sample is incubated in a thermocycler for 30 minutes at 23 degrees C, 4 minutes at 95 degrees, and held at 23 degrees.

[0212] Ligation 2 and clean-up is performed as follows: a) 5pL Y adapter and 45pL Ligation Mix 2 are added to Ligation 1, mixed and incubated at 23 degrees C for 30 minutes; b)2pL of Ligation Digest is added to the sample; c) the sample is mixed and incubated in a thermocycler for 10 minutes at 55 degrees C, 3 minutes at 95 degrees C, and held at 23 degrees; d) the Ligation 2 sample is transferred to a tube containing Ligation SPRI beads, mixed and incubated at room temperature for 10 minutes; e) the beads are captured against a magnet and the supernatant is collected and discarded; f) the beads are washing with 800 L 80% ethanol; g) the ethanol wash step is repeated; h) the ethanol is removed and the beads are allowed to air dry for 2 minutes; i) the beads are resuspended in 40 L EB1 and incubated at room temperature for 5 minutes; and j) the beads are captured against a magnet and 20 L ligation supernatant is collected into a fresh 0.2mL tube.

[0213] Step 2) Amplification and Clean-Up

[0214] Amplification is performed as follows: a) 40 L of Amplification Mix is added to 40 L Ligation sample on ice and mixed; b) the sample is incubated in a thermocycler for 15 minutes at 4 degrees C and I hour at 37 degrees C; c) 2 L Amplification Initiator is added to the sample and mixed; and d) 42 L of Amplification reaction is incubated at 52 degrees C for 16-24 hours.

[0215] Amplification clean-up is performed as follows: a) 2 L of Amplification Digest is added to the Amplification Reaction and incubated at 55 degrees C for 10 minutes; b) 90 L SPRI beads is added to the sample and incubated at room temperature for 10 minutes; c) the beads are captured against a magnet and the supernatant is discarded; d) the beads are washed with 150pL 80% ethanol for 30 seconds at room temperature; e) the wash step is repeated; f) the beads are air dried for two minutes at room temperature; g) the beads are resuspended in 50 L EB2 and incubated three minutes at room temperature; and h) the beads are captured on a magnet and 50 L supernatant is collected to a new tube.

[0216] Step 3) Quantification and Quality Control

[0217] Initial Set-Up is performed as follows: a) the Qubit Assay USB drive is inserted into the USB port on the back of a Qubit Fluorometer; b) in the Settings menu, press “Import New Assay”; c) press “Library Quant”; d) press “New Folder”; e) enter “Custom” for folder name; f) press “Save” and “Done”.

[0218] Calibration is performed as follows: a) molecular beacon standards (STI and ST2) are brought to room temperature; b) the standards are incubated in a heat block at 55 degrees C for 5 minutes; c) the standards are cooled at room temperature for 5 minutes; d) the standard is inserted into the Qubit Fluorometer; e) press “Custom” on the home screen menu; f) on the Choose Assay Screen, press “Library Quant”; g) press “Run Standards”; and h) follow the prompts on the screen.

[0219] Quantification is run as follows: a) I pL of amplified product is added to_the Molecular Beacon Quantification Assay and incubated in a heat block for 5 minutes at 55 degrees C; b) the sample is cooled for five minutes at room temperature; c) the sample is inserted into the Qubit Fluorometer; d) from the home screen menu, press “Library Quant”; e) press “Run Samples” f) select I L; and g) press “Read Tube”.

[0220] Step 4) Template Pooling and Preparation for Xpandomer Synthesis

[0221] Template pooling and preparation is performed as follows: a) library samples are combined based on volumes calculated from the table; b) the total volume of pooled libraries is brought to 37.5 L with EB2; and c) the samples are stored at -20 degrees C until use.

Claims

CLAIMSWhat is claimed is:

1. A method of amplifying a duplex nucleic acid template, the method comprising the steps of:(a) providing a first double stranded nucleic acid product, wherein a first strand of the first double stranded nucleic acid product comprises the duplex nucleic acid template and a second strand of the first double stranded nucleic acid product comprises a copy of the duplex nucleic acid template, and wherein the copy of the duplex nucleic acid product comprises a nickase endonuclease cleavage site;(b) contacting the double stranded nucleic acid product with a nickase endonuclease under endonuclease conditions, wherein the nickase endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the copy of the duplex nucleic acid template;(c) contacting the free 3’ end in the copy of the duplex nucleic acid product with a strand displacing nucleic acid polymerase under nucleic acid synthesis conditions, wherein the strand displacing nucleic acid polymerase synthesizes a new copy of the duplex nucleic acid template, wherein the new copy displaces the copy of step (a) from the first double stranded nucleic acid product; and(d) repeating steps (a) through (c) to provide an amplified population of copies of the duplex nucleic acid template.

2. The method of claim 1 further comprising the steps of:(e) contacting the amplified population of copies of the duplex nucleic acid template with an oligonucleotide probe under nucleic acid hybridization conditions, wherein the oligonucleotide probe comprises the nucleic acid target sequence, and wherein the oligonucleotide probe is bound to a solid support;(f) washing the solid support to selectively retain an enriched copy of the duplex nucleic acid template, wherein the enriched copy comprises the nucleic acid target sequence, and wherein the enriched copy is specifically hybridized to the oligonucleotide probe;(g) releasing the enriched copy of the duplex nucleic acid template from the solid support;(h) providing a second double stranded nucleic acid product wherein a first strand of the second double stranded nucleic acid product comprises the enriched copy of the duplex nucleic acid template and a second strand of the second double stranded- 64 -nucleic acid product comprises a copy of the enriched copy, wherein the copy of the enriched copy of the duplex nucleic acid template comprises a nickase endonuclease cleavage site;(i) contacting the second double stranded nucleic acid product with a nickase endonuclease under endonuclease conditions, wherein the nickase endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the copy of the enriched copy;(j) contacting the free 3’ end in the copy of the enriched copy of the duplex nucleic acid template with a strand displacing nucleic acid polymerase under nucleic acid synthesis conditions, wherein the strand displacing DNA polymerase synthesizes a new copy of the enriched copy of the duplex nucleic acid template, and wherein the new copy displaces the copy of step (h); and(k) repeating steps (h) through (j) to provide an amplified population of copies of the enriched copy of the duplex template construct comprising the nucleic acid target sequence.

3. The method of claim 1 further comprising the steps of:(e) contacting the amplified population of copies of the duplex nucleic acid template with an oligonucleotide primer under nucleic acid hybridization conditions, wherein the nucleic acid sequence of the oligonucleotide primer is complementary to a nucleic acid sequence in the amplified copies of the duplex nucleic acid template, and wherein the oligonucleotide primer is bound to a solid support, and wherein the amplified copies of the duplex nucleic acid template hybridize to the oligonucleotide primer bound to the solid support;(f) contacting the oligonucleotide primer with a nucleic acid polymerase under nucleic acid synthesis conditions, wherein the nucleic acid polymerase extends the oligonucleotide primer to form a second double stranded nucleic acid product, wherein the second double stranded nucleic acid product comprises a copy of the amplified copy of the duplex nucleic acid template bound to the solid support;(g) contacting the second double stranded nucleic acid product with denaturing conditions, wherein the denaturing conditions produce a single stranded nucleic acid product bound to the solid support, wherein the single stranded nucleic acid product comprises the copy of the amplified copy of the duplex nucleic acid template;(h) contacting the single stranded nucleic acid product with an oligonucleotide primer under nucleic acid hybridization conditions, wherein the oligonucleotide primer comprises a nucleic acid sequence complementary to a nucleic acid sequence in thesingle stranded nucleic acid product and a nucleic acid polymerase under nucleic acid synthesis conditions, to produce a third double stranded nucleic acid product, wherein the third double stranded nucleic acid product comprises a copy of the single stranded nucleic acid product; and(i) contacting the third double stranded nucleic acid product with denaturing conditions to produce an amplified population of copies of the duplex nucleic acid template released from the solid support.

4. The method of any one of claims 1 to 3, wherein the nickase endonuclease is selected from the group consisting of Nb. BbvCl, Nb. Bsml, Nt. BstNBI, Nt. BspQI, Nt. BspD61, Nt. Bst9I, Nt. BstSEI, Nt. BsmAI, Nt. AIwI, Nb. BsrDl, and Nt. CviPII, or variants thereof.

5. The method of any one of claims 1 to 3, wherein the strand displacing nucleic acid polymerase is a strand displacing DNA polymerase selected from the group consisting of Bst wildtype, Bst 2.0, Bst 3.0, Bsu, and Klenow fragment, or variants thereof.

6. The method of any one of claims 1 to 5, wherein the nickase endonuclease conditions and the DNA synthesis conditions are provided in the same reaction mixture.

7. The method of claim 6, wherein the reaction mixture comprises a nucleotide analog, wherein the nucleotide analog comprises N4-Me dCTP or 7-deaza dGTP.

8. The method of claim 6 or 7, wherein the reaction mixture comprises one or more of a single stranded binding protein, a pyrophosphatase, a DPO4 polymerase or a variant thereof, and a translesion repair enzyme.

9. The method of any one of claims 6 to 8, wherein the nickase endonuclease conditions and the DNA synthesis conditions comprise isothermal conditions, wherein the isothermal conditions comprise incubation at around 50 degrees Celsius to around 55 degrees Celsius for around 5 hours to around 24 hours.

10. The method of claim 3, wherein the nucleic acid synthesis conditions of step (f) comprise the use of native nucleotides.

11. The method of any one of claims 1 to 10, wherein the duplex nucleic acid template comprises a double stranded nucleic acid fragment joined on a first end to a Y adapter and joined on a second end to a hairpin adapter, wherein the hairpin adapter covalently joins the two strands of the double stranded nucleic acid fragment.

12. The method of claim 11, wherein the Y adapter comprises a nickase endonuclease recognition site.

13. The method of claim 11 or 12, wherein the double stranded nucleic acid fragment is provided by a formalin-fixed, paraffin embedded (FFPE) sample.

14. The method of claim 13, wherein the double stranded nucleic acid fragment is treated with one or more of an FFPE repair kit, an end-repair A-tail (ERAT) kit, an exonuclease enzyme, or an enzymatic fragmentation (FragTail) kit.

15. The method of claim 14, wherein the FFPE repair kit comprises a thermolabile exonuclease enzyme or a RecJf enzyme.

16. The method of claim 14, wherein the double stranded nucleic acid fragment is treated with a fragmentation kit, a SPRI purification kit, a FFPE repair kit, a RecJf enzyme, a thermolabile proteinase K, and an ERAT kit.

17. The method of any one of claims 1 to 16, further comprises the step of purifying the amplified population of copies of the duplex template construct with SPRI beads.

18. The method of claim 17, wherein the purified copies of the duplex template construct are eluted from the SPRI beads with an elution buffer comprising one or more of a Tris buffer, a sodium salt, EDTA, and PEG8k.

19. The method of any one of claims 1 to 18, further comprising the step of real-time quantification of the amplified population of copies of the duplex nucleic acid template using a molecular beacon probe.

20. The method of any one of claims 11 to 19, wherein the step of providing a first double stranded nucleic acid product comprises contacting the duplex nucleic acid template with an oligonucleotide primer under nucleic acid hybridization conditions, wherein the oligonucleotide primer comprises a nucleic acid sequence complementary to a sequence a single stranded arm region of the Y adapter and a nucleic acid polymerase under nucleic acid synthesis conditions.

21. The method of any one of claims 1 to 18, further comprising contacting the amplified population of duplex nucleic acid templates with an extension oligonucleotide under nucleic acid hybridization conditions, wherein the extension oligonucleotide comprises a nucleic acid sequence complementary to the duplex nucleic acid template, and with a variant of DPO4 polymerase under Xpandomer synthesis conditions.

22. The method of claim 21, wherein the Xpandomer synthesis conditions comprise a buffer / salt system, polymerase cofactors, polymerase enhancing moi eties (PEMs), XNTP substrates, a phosphate shield molecule, a solvent, a crowding agent, and a single stranded binding protein.

23. The method of claim 20 or 21, wherein the extension oligonucleotide is bound to a solid support.

24. A kit for linear amplification of a duplex template construct comprising one or more of an oligonucleotide primer, an isothermal amplification buffer, dNTPs, a pyrophosphatase,a single stranded binding protein, a strand displacing DNA polymerase, and a nicking endonuclease.

25. The kit of claim 24, wherein the isothermal amplification buffer comprises one or more of Tris-HCl, (NH^SCU, KC1, MgSCU, and Tween 20, the dNTPs comprise 7-deaza dGTP, the single stranded binding protein is selected from the group consisting of gp32,TTH, KOD, RPA, NCp7, RecA, and UvrD, the strand displacing DNA polymerase is a Bst polymerase or a variant thereof, and the nicking endonuclease is selected from the group consisting of Nb. BbvCl, Nb. Bsml, Nt. BstNBI, Nt. BspQI, Nt. BspD61, Nt. Bst9I, Nt. BstSEI, Nt. BsmAI, Nt. AIwI, Nb. BsrDl, and Nt. CviPII, or variants thereof.- 68 -

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