Automated solid-state amplification of library constructs for duplex sequencing

Abstract

The present invention relates to methods and compositions for the solid-state linear amplification of macromolecules, such as duplex nucleic acid template constructs that find use in duplex Sequencing by Expansion.

Description

AUTOMATED SOLID-STATE AMPLIFICATION OF LIBRARY CONSTRUCTS FOR DUPLEX SEQUENCINGBACKGROUND OF THE INVENTION

[0001] Commonly used next generation sequencing (NGS) platforms often rely on PCR amplification during library construction to increase the copy number of library fragments in order to meet the needs of the sequencing platforms. However, the most problematic step in sample preparation procedures is amplification. This is due to the fact that 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 methods and compositions for conducting automated solid state amplification of macromolecules, such as paired-end, duplex DNA template constructs. These methods and compositions provide advantages to a number of 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 particularproblem, which in and of itself may also be inventive.BRIEF SUMMARY OF THE INVENTION

[0005] The present disclosure provides improved methods and compositions for automated soldi-state amplification of macromolecules such as 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 solid-state amplification of a duplex nucleic acid template construct, the method including the steps of: (a) providing a duplex nucleic acid template including a double stranded nucleic acid target fragment, in which the double stranded nucleic acid target fragment is joined at a first end to a Y adapter, in which the Y adapter includes a double stranded stem region, a 3’ single stranded arm region and a 5’ single stranded arm region and at a second end to a hairpin adapter, in which the hairpin adapter covalently joins the strands of the double stranded nucleic target fragment; (b) joining the duplex nucleic acid template to a solid support, in which the joining is mediated by the 5’ single stranded arm region of the Y adapter or the 3’ single stranded arm of the Y adapter to produce a support-bound duplex nucleic acid template; (c) contacting the solid support with a nucleic acid extension mixture under nucleic acid hybridization and extension conditions, wherein the nucleic acid extension mixture includes an extension oligonucleotide, a nucleic acid polymerase, dNTPs, and a suitable buffer, in which the extension oligonucleotide specifically hybridizes to the duplex nucleic acid template to provide at free 3’ end, and in which the nucleic acid polymerase synthesizes a complementary copy of the duplex nucleic acid template from the free 3’ end of the extension oligonucleotide; (d) optionally, contacting the solid support with a suitable wash buffer; (e) contacting the solid support with a suitable elution buffer, in which the suitable elution buffer releases the complementary copy from the support-bound duplex nucleic acid template; (f) optionally, contacting the solid support with a suitable wash buffer; and (g) repeating steps (c) through (f) to provide an amplified population of complementary copies of the duplex nucleic acid template.

[0007] In another aspect, the invention provides A method of solid-state printing of a duplex nucleic acid template, the method including the steps of: (a) providing a solid support including an extension oligonucleotide, in which the extension oligonucleotide includes a free 3’ end; (b) providing a duplex nucleic acid template including a double stranded nucleic acid target fragment, in which the double stranded nucleic acid target fragment is joined at a first end to a Y adapter, in which the Y adapter includes a double stranded stem region, a 3’ single stranded arm region and a 5’ single stranded arm region and at a second end to a hairpin adapter, in which thehairpin adapter covalently joins the strands of the duplex nucleic acid template; (c) contacting the duplex nucleic acid template with the solid support including the extension oligonucleotide under nucleic acid hybridization conditions, in which the duplex nucleic acid template specifically hybridizes to the extension oligonucleotide; (d) contacting the solid support with a nucleic acid extension mixture under nucleic acid extension conditions, in which the nucleic acid extension mixture includes a nucleic acid polymerase, dNTPs, and a suitable buffer, and in which the nucleic acid polymerase synthesizes a support-bound complementary copy of the duplex nucleic acid template construct; (e) optionally, contacting the solid support with a suitable wash buffer; (f) contacting the solid support with a suitable elution buffer, in which the suitable elution buffer releases the duplex nucleic acid template from the support-bound complementary copy; (g) optionally, contacting the solid support with a suitable wash buffer; and (h) repeating steps (c) through (g) to provide a population of support-bound complementary copies of the duplex nucleic acid template. In some embodiments, the population of supportbound complementary copies of the duplex nucleic acid template are subjected to one or more polishing steps. In one embodiment, the one or more polishing steps include treatment with an exonuclease enzyme, in which the exonuclease enzyme is capable of digesting free extension oligonucleotide joined to the solid support. In some embodiments, the population of supportbound complementary copies of the duplex nucleic acid template construct are subjected to solid-state amplification according to the method described above. In some embodiments, the first method described above further includes the step of subjecting the support-bound duplex nucleic acid template to a chemical conversion step following step (g), in which the chemical conversion step selectively converts a modified nucleobase of interest, or the native form of a modified nucleobase of interest, to enable detection of the positions of the modified nucleobase of interest in the duplex nucleic acid template. In one embodiment, the modified nucleobase of interest in 5-mC and the chemical conversion step includes treatment of the support-bound duplex nucleic acid template to bisulfite treatment, TET / APOBEC treatment, DNMT1 treatment, or chemoenzymatic treatment. In certain embodiments, the chemical conversion step is followed by a solid-state amplification step. In some embodiments, the nucleic acid polymerase is a strand displacing DNA polymerase. In some embodiments, the duplex nucleic acid template is joined to the solid support by a click chemistry reaction. In one embodiment, the click chemistry reaction is mediated by a terminal azide moiety provided by an end of a single stranded arm region of the Y adapter and a terminal alkyne moiety provided by the solid support. In some embodiments, the nucleic acid synthesis conditions include isothermal conditions. In some embodiments, each round of nucleic acid extension is performed with the same reaction mixtures and solutions. In some embodiments, the double stranded nucleic acid target fragment isprovided by cell-free DNA, genomic DNA, RNA, or a formalin-fixed paraffin-embedded (FFPE) sample. In one embodiment, the FFPE sample is treated with one or more of a FFPE repair step, an end-repair and A-tailing (ERAT) step, an exonuclease treatment step, and a fragmentation step. In yet another embodiment, the FFPE sample is treated with a FFPE repair step including treatment with a thermolabile exonuclease enzyme or a RecJf enzyme.

[0008] In certain embodiments, the solid support includes a flow cell, in which the flow cell is compatible with a system capable of automating the amplification of the duplex nucleic acid template. In some embodiments, the flow cell includes at least one flow channel, the flow channel having a functionalized solid phase surface configured to bind a plurality of duplex nucleic acid templates, and in which each flow channel has an inlet port and an outlet port. In some embodiments, the system includes the flow cell, a sipper in fluidic communication with the flow cell, a heatable mount configured to receive the flow cell, optionally in which a first thermal block is attached to the heatable mount, a holder configured to hold a plurality of reagents, a gantry configured to move in 3 different axis, a pump in fluidic communication with the outlet port of the flow channel, a valve configured to selectively connect the outlet port of the flow channel with a waste reservoir and / or a buffer reservoir, a UV source configured to illuminate the flow channel, and a controller configured to a temperature of the heatable mount, movement of the gantry, selection of a valve, operation of the UV source, and operation of the pump. In some embodiments, the flow cell includes a molded portion and a thin film, in which the molded portion includes the flow channel and the thin film is attached to the molded portion to seal the flow channel and in which the thin film is made from cyclic olefin polymer or cyclic olefin copolymer. In some embodiments, the functionalized solid phase surface provides the terminal alkyne moiety. In certain embodiments, the terminal alkyne moiety is joined to the functionalized surface by a flexible linker.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is a condensed schematic summarizing one embodiment of a method of solid-state linear amplification of a duplex nucleic acid template construct of the present invention in which the 5’ arm of the Y adapter of the construct is bound to a solid support.

[0010] FIG. 2 is a condensed schematic summarizing another embodiment of a method of solid- state linear amplification of a duplex nucleic acid template construct of the present invention in which the 3’ arm of the Y adapter of the construct is bound to a solid support.

[0011] FIG. 3 is a condensed schematic summarizing another embodiment of a method of solid- state linear amplification of a duplex nucleic acid template construct of the present invention that includes a conversion step that enables epigenetic analysis.

[0012] FIG. 4 is a condensed schematic summarizing one embodiment of a method of solid-state linear amplification of a duplex nucleic acid template construct of the present invention that includes an on-support library preparation step.

[0013] FIG. 5 is a condensed schematic summarizing one embodiment of a method of solid-state linear amplification of a duplex nucleic acid template construct of the present invention in which the duplex template construct is printed on the solid support.

[0014] FIG. 6 is a condensed schematic summarizing one embodiment of a method of solid-state linear amplification of a duplex nucleic acid template construct of the present invention in which the duplex template construct is multi-printed on the solid support and the printed daughter strands are subjected to one or more polishing steps prior to linear amplification.

[0015] FIG. 7A and FIG. 7B illustrate an embodiment of a system for automated linear amplification of a macromolecule.

[0016] FIG. 7C illustrates a mixing tube that can be used in the system shown in FIGS. 7A and 7B.

[0017] FIG. 8 A illustrates and embodiment of a flow cell.

[0018] FIG. 8B illustrates a functionalities flow channel surface and how a capture oligonucleotide can be used to immobilize a template construct to the flow channel surface.

[0019] FIG. 8C and FIG. 8D illustrate an embodiment of the flow cell with a thin film bonded to a molded portion of the flow cell.

[0020] FIG. 9 illustrates an embodiment of a mount for receiving the flow cell.

[0021] FIG. 10A illustrates an embodiment of a holder.

[0022] FIG. 10B and FIG. 10C illustrate embodiments of reagent cartridges that can be placed in the holder shown in FIG. 10 A.

[0023] FIG. 11 is a condensed schematic illustrating one embodiment of a method of producing a duplex nucleic acid template construct of the present invention.DETAILED DESCRIPTION OF THE INVENTION

[0024] 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.

[0025] 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).

[0026] 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.

[0027] 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 clearly dictates 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 of the associated items or ideas and one or more other alternative embodiments that include fewer than all of the associated items or ideas.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0032] 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 Automated Solid-State Amplification of Macromolecules

[0033] The methods of the present invention may be used for the amplification (i.e., an increase in copy number) of any suitable macromolecule, including, but not limited to, nucleic acids and proteins. In some embodiments, amplification is performed on a solid support provided by an instrument capable of automating fluidic, thermal and other reaction conditions.

[0034] In certain embodiments, the methods of the present invention may be used for linear amplification of a nucleic acid template, such as a double stranded duplex template construct. Linear amplification, also be referred to herein as isothermal amplification, is well known in the art as a process incorporated into many library preparation protocols. Linear amplification replicates a nucleic acid template (e.g., a target fragment or a library construct) in a non-exponential manner. Accordingly, the original template strand is reused as the template for consecutive replication events, while the newly synthesized complementary copies (i.e., daughter strands) are not replicated as part of the template amplification process. Advantageously, linear amplification prevents errors in DNA replication from being exponentially propagated into the progeny population of complementary copies. In addition, linear amplification is not subject to certain limitations inherent to thermal cycling (i.e., PCR), for example, the dependence on heat- resistant DNA polymerases. This is particularly advantageous when duplexed template constructs are amplified for paired-end sequencing protocols, as heat-resistant DNA polymerases do not possess the robust strand displacement activity required to “un-zip” a duplexed template.

[0035] In certain embodiments, the isothermal conditions may include incubation of an reaction at a temperature from around 25 degrees Celsius to around 90 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 a reaction at a temperature from around 50 degrees Celsius to around 55 degrees Celsius. In one embodiment, the isothermal conditions may include incubation of a reaction at a temperature around 52 degrees Celsius.

[0036] In certain embodiments, the duplex template constructs discussed herein include 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 Template Preparation using Extendable Adapters for Duplexed Sequencing by Expansion”, the contents of which are herein incorporated by references in their entireties.

[0037] In certain embodiments, the methods of the present invention include solid-state linear amplification and / or preparation of duplex template construct libraries. Such methods may be performed on a “chip”, e.g., a flow cell surface, engineered for use with a dedicated samplepreparation instrument, e.g. “system”. Using the capabilities provided by sample preparation instruments described herein, the inventors have found that cyclic manipulations, under isothermal conditions, can be automated, enabling highly efficient preparation of nucleic acid template samples useful for, e.g., synthesis of Xpandomers for duplex sequencing. Because the cyclic reactions are carried out under isothermal conditions, the methods of the present invention avoid the thermal denaturation step(s) that are required by conventional PCR amplification. As mentioned, this offers many advantages, for example, heat-sensitive strand displacing DNA polymerases can be used in the workflow, as they are not subjected to denaturing conditions. In addition, reagents and solutions may be recycled and reused during the amplification process, thus increasing the economy of the library preparation workflow.

[0038] As used herein, the terms “isothermal conditions” and “constant temperature” may be used interchangeably to refer to a set of reaction conditions where the temperature of the reaction is kept essentially constant, or within a smaller ranger relative to conventional PCR, during the course of an amplification reaction. An advantage of isothermal amplification is that the temperature does not need to be cycled between the large range of upper temperatures and lower temperatures characteristic of conventional PCR. The DNA synthesis reaction will work at the same temperature or within a narrow temperature range as the other reactions of the workflow. However, it is not necessary that the temperature be maintained at precisely one temperature.

[0039] One embodiment of the methods of the present invention is depicted in FIG. 1. In this embodiment, duplex template construct 100 is provided, which includes a target nucleic acid fragment (e.g., a library fragment) with parent (+) strand 101a (i.e., a sense strand) and parent (-) strand 101b (i.e., an antisense strand). As used herein, the term “construct” may describe any nucleic acid fragment that is joined to (e.g., covalently bound to or ligated to) a heterologous nucleic acid structure, such as an adapter, so as to introduce features that mediate downstream steps of a workflow, e.g., a nucleic acid sequencing workflow. Here, the library fragment is joined on a first end to hairpin adapter 110, which includes a double stranded stem region joined to 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 joined to a 5’ single stranded arm region and a 3’ single stranded arm region. Likewise, the complementary strands of the double stranded stem portion of the Y adapter are each ligated to one of the strands of the library fragment. In this embodiment, the terminal end of the 5’ single stranded arm region of Y adapter 120 is joined to azide moiety 125 via a linker that, in certain embodiments, may be cleavable, as discussed further herein.

[0040] In step 1, duplex template construct 100 is covalently bound to solid support 130 via support-bound linker 135. In this embodiment, linker 135 provides a terminal alkyne moiety that is capable of reacting with terminal azide moiety 125 to form a triazole group under click reaction conditions, as discussed further herein. In certain embodiments, solid support 130 may be part of a channel disposed in a flow cell (e.g., a chip or a card), which is engineered for automated synthesis of macromolecules, such as Xpandomers.

[0041] Following step 1, a plurality of replication cycles is performed on solid support 130 to amplify duplex template construct 100 in a linear manner. During each cycle, duplex template construct 100 is replicated once to generate a complementary copy of the construct. By repeating the replication cycle under isothermal conditions, many of the reagents required for DNA synthesis can advantageously be reused during subsequent replication cycles. In this embodiment, a single replication cycle includes the following four steps: (1) Step 2a - priming and extension of the duplex template construct; (2) Step 2b - first wash; (3) Step 2c - denaturation and elution of the complementary copy of the duplex template construct; and (4) Step 2d - second wash. As disclosed herein, in certain embodiments, one or more wash steps may be optional, depending on the particular application.

[0042] In step 2a (priming and extension), duplex template construct 100 bound to solid support 130 is contacted with extension oligonucleotide 143 under nucleic acid hybridization conditions and DNA synthesis conditions. Extension oligonucleotide 143 includes a sequence that is complementary to a sequence in the 3’ single stranded arm of Y adapter 120. The DNA synthesis conditions include master mix 140 that provides reagents which, in certain embodiments, may be reused for multiple, or all, cycles of DNA replication. The master mix may include extension oligonucleotide 143, a suitable buffer, a DNA polymerase, and dNTPs. In certain embodiments, the DNA polymerase is a strand displacing polymerase that synthesizes complementary copy 160 of the duplex template construct. An exemplary strand displacing DNA polymerase is BST Large Fragment, commercially available from, e.g., NEB or ThermoFisher. Other suitable strand displacing polymerases may include Phi29, Klenow exo- and the like. An exemplary buffer is the Thermopol buffer (20mM Tris-HCl, lOmM (NH4)2SO4, lOmM KC1, 2mM MgSO4, 0.1% Triton X-100, pH8.8) commercially available from e.g., NEB. In certain embodiments, the master mix may include from around 50mM to around 750mM dNTPs. In one embodiment, the master mix may include around 500mM dNTPs. In certain embodiments, the master mix may also include one or more extension additives. Exemplary extension additives include polymerase enhancing molecules, i.e., PEMs (PEMs are disclosed, e.g., in Applicant’s published PCT applications, WO2019 / 135975 and W02020 / 263703, which are herein incorporated by reference in their entireties), single stranded binding proteins,helicases, PEG (or other crowding agents), alternative nucleotides (e.g., 7-deaza dGTP, N4 Me cytosine and the like), alternative buffers (e.g., HEPES, Tris-Acetate, and the like) and salts (e.g., KC1, NaOAc, (NELfhSC and the like), and alternative cations (e.g., manganese and the like).

[0043] The product of one cycle of replication is complementary copy 160 of duplex template construct 100 that includes a daughter (+) strand and a daughter (-) strand, which are joined by an intervening copy of hairpin adapter 110. Complementary copy 160 also includes a complementary copy of Y adapter 120.

[0044] In step 2b (first wash), the reagents used in step 2a (e.g., master mix 140) are removed from the flow channel and, in certain embodiments, may be retained for subsequent replication cycles. The flow channel is contacted with wash solution 145a, which may include a suitable buffer and salt. In certain embodiments, the wash solution may include around lOmM Tris HC1 and around lOOmM NaCl. In an alternative embodiment, the master mix is not removed from the flow channel prior to washing; in this case, a fresh aliquot of the master mix is used in all subsequent extension cycles.

[0045] In step 2c (denature and elute), the flow channel is contacted with elution mix 150 that enables collection of the newly synthesized complementary copies of duplex template construct 100 (e.g., a single one of 160a, 160b, and 160c for each replication cycle). The elution mix may include a base, such as NaOH (e.g., around lOOmM NaOH) that denatures the hybridized template and complementary copy, thus releasing the complementary copy from the flow channel and into solution. The eluate containing the complementary copy is removed from the flow cell and retained.

[0046] In step 2d (second wash), the flow channel is contacted with wash solution 145b to restore conditions for the next round of priming and extension of the original duplex template construct. In certain embodiments, the wash solution may include a suitable buffer, salt, and chelating agent, e.g., around lOmM Tris HC1, around 5mM NaCl, and around ImM EDTA.

[0047] In certain embodiments, any number of replication cycles may be modified so as to, e.g., adjust dynamic thermal conditions and / or to introduce additional steps or chemistries to achieve a desired replication outcome. For example, single stranded binding proteins and / or blocker oligonucleotides can be included in any suitable step or for any number of replication cycles. In other embodiments, the DNA polymerase component of master mix 140 can be modified for a number of the replication cycles. For example, more than one strand displacing polymerase may be used, e.g., a mixture of strand displacing or strand displacing and non-displacing polymerases may be used.

[0048] Another embodiment of the methods of the present invention is depicted in FIG. 2. In this embodiment, duplex template construct 200 is provided, which includes a target nucleic acidfragment (e.g., a library fragment) with parent (+) strand 201a (i.e., a sense strand) and parent (-) strand 201b (i.e., an antisense strand). The library fragment is joined on a first end to hairpin adapter 210, which includes a double stranded stem region joined to 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 220, which includes a double stranded stem region joined to a 5’ single stranded arm region and a 3’ single stranded arm region. Likewise, the complementary strands of the double stranded stem portion of the Y adapter are each ligated to one of the strands of the library fragment. In this embodiment, the terminal end of the 3’ single stranded arm region of Y adapter 220 is covalently joined to azide moiety 225 via a linker that, in certain embodiments, may be cleavable, as discussed further herein.

[0049] In step 1, duplex template construct 200 is bound to solid support 230 via linker 235. In this embodiment, linker 235 provides a terminal alkyne moiety that is capable of reacting with terminal azide moiety 225 to form a triazole group under click reaction conditions, as discussed further herein. In certain embodiments, solid support 230 is part of a channel disposed in a flow cell (e.g., a chip or a card), which is engineered for automated synthesis of macromolecules, such as Xpandomers.

[0050] As discussed with reference to FIG. 1, following step 1, a plurality of replication cycles is performed on solid support 130 to amplify duplex template construct 200 in a linear manner. During each cycle, duplex template construct 200 is replicated once to generate a complementary copy of the construct. By repeating the replication cycle under isothermal conditions, many of the reagents required for DNA synthesis can be reused during the subsequent replication cycles. In this embodiment, a single replication cycle includes the following four steps: (1) Step 2a - priming and extension of the duplex template construct; (2) Step 2b - first wash; (3) Step 2c - denaturation and elution of the complementary copy of the duplex template construct; and (4) Step 2d - second wash.

[0051] In step 2a (priming and extension), duplex template construct 200 bound to solid support 230 is contacted with extension oligonucleotide 243 under nucleic acid hybridization conditions and DNA synthesis conditions. Extension oligonucleotide 243 includes a sequence that is complementary to a sequence in the 3’ single stranded arm of Y adapter 220. The DNA synthesis conditions include master mix 240 that provides reagents which, in certain embodiments, may be reused for multiple, or all, cycles of DNA replication. As discussed with reference to FIG. 1, the master mix may include extension oligonucleotide 243, a DNA polymerase, or mixture thereof, and dNTPs. In certain embodiments, the DNA polymerase is a strand displacing polymerase that synthesizes complementary copy 260 of the duplex templateconstruct. All complementary copies of duplex template construct 200 include a daughter (+) strand and a daughter (-) strand that are joined by an intervening copy of hairpin adapter 210. The complementary copies also include a complementary copy of Y adapter 220.

[0052] In step 2b (first wash), the reagents used in step 2a (e.g., master mix 240) are removed from the flow channel and retained for subsequent replication cycles. The flow channel is contacted with a suitable wash solution 245a, as disclosed herein.

[0053] In step 2c (denature and elute), the flow channel is contacted with elution mix 250 that enables collection of the newly synthesized complementary copies of duplex template construct 200 (e.g., a single one of 260a, 260b, and 260c for each replication cycle). In certain embodiments, the elution mix may include a base, such as NaOH that denatures the hybridized template and complementary copy, thus releasing the complementary copy from the flow channel and into solution. The eluate containing the complementary copy is removed from the flow cell and retained.

[0054] In step 2d (second wash), the flow channel is contacted with a suitable wash solution 245b, as disclosed herein, to restore conditions for the next round of priming and extension of the original duplex template construct.

[0055] As described herein, in certain embodiments, any number of replication cycles may be modified to, e.g., adjust dynamic thermal conditions and / or to introduce additional steps or chemistries to achieve a desired replication outcome. For example, single stranded binding proteins and / or blocker oligonucleotides can be included in any suitable step or for any number of replication cycles.

[0056] In other embodiments, the methods of the present invention may be used for epigenetic analysis. One such embodiment is depicted in FIG. 3. In this embodiment, duplex template construct 300 is provided, which includes a target nucleic acid fragment (e.g., a library fragment) with parent (+) strand 301a (i.e., a sense strand) and parent (-) strand 301b (i.e., an antisense strand). The target fragment is derived from a biological sample and may include epigenetic modifications of interest, e.g., 5-mC. As disclosed herein, the library fragment is joined on a first end to hairpin adapter 310, which includes a double stranded stem region joined to 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 320, which includes a double stranded stem region joined to a 5’ single stranded arm region and a 3’ single stranded arm region. Likewise, the complementary strands of the double stranded stem portion of the Y adapter are each ligated to one of the strands of the library fragment. In this embodiment, the terminal end of the 3’single stranded arm region of Y adapter 320 is joined to azide moiety 325 via a linker that, in certain embodiments, may be cleavable, as discussed further herein.

[0057] In step 1, duplex template construct 300 is bound to solid support 330 via linker 335. Linker 335 provides a terminal alkyne moiety that reacts with terminal azide moiety 325 of the Y adapter to form a triazole group under click reaction conditions, as discussed herein. In certain embodiments, solid support 330 is part of a channel disposed in a flow cell (e.g., a chip or a card), which is engineered for automated synthesis of macromolecules, such as Xpandomers.

[0058] Following step 1, a plurality of replication cycles is performed on the solid support to amplify the native duplex template construct that includes epigenetic information. This amplification may be referred to herein as “pre-conversion” amplification. During each cycle, duplex template construct 300 is replicated once to generate a complementary copy of the construct. Because the duplex template construct includes an unconverted parental target fragment, each complementary copy will include the nucleotide that normally base pairs with the modified nucleobase of interest, e.g., a “G” at each position of 5-mC in the parental target fragment. By repeating the replication cycle under isothermal conditions, many of the reagents required for DNA synthesis can be reused as duplex template construct 300 is amplified in a linear manner. In this embodiment, a single replication cycle includes the following four steps: (1) Step 2a - priming and extension of the duplex template construct; (2) Step 2b - first wash;(3) Step 2c - denaturation and elution of the complementary copy of the duplex template construct; and (4) Step 2d - second wash.

[0059] In step 2a (priming and extension), duplex template construct 300 bound to solid support 330 is contacted with extension oligonucleotide 343 under nucleic acid hybridization and DNA synthesis conditions. Extension oligonucleotide 343 includes a sequence that is complementary to a sequence in the 3’ single stranded arm of Y adapter 320. The DNA synthesis conditions include master mix 340 that provides reagents which, in certain embodiments, may be retained and reused for multiple, or all, cycles of DNA replication. The master mix may include extension oligonucleotide 343, a DNA polymerase, or mixture thereof, and dNTPs. In certain embodiments, the DNA polymerase is a strand displacing polymerase that synthesizes complementary copy 360 of the duplex template construct. All complementary copies of duplex template construct 300 include a daughter (+) strand and a daughter (-) strand that are joined by an intervening copy of hairpin adapter 310. The complementary copies also include a complementary copy of Y adapter 320.

[0060] In step 2b (first wash), the reagents used in step 2a (e.g., the master mix) are removed from the solid support and retained for subsequent replication cycles. The solid support is contacted with a suitable wash solution 345a, as disclosed herein.

[0061] In step 2c (denature and elute), the solid support is contacted with elution mix 350 that enables collection of the newly synthesized complementary copies (e.g., a single one of 360a, 360b, and 360c for each replication cycle) of duplex template construct 300. As describes herein, in certain embodiments, the elution mix may include a base, such as NaOH, that denatures the hybridized template and complementary copy, thus releasing the complementary copy from the solid support and into solution. The eluate containing the complementary copy is removed from the solid support and retained.

[0062] In step 2d (second wash), the solid support is contacted with a suitable wash solution 345b, as disclosed herein, to restore conditions for the next round of priming and extension of the original duplex template construct.

[0063] In certain embodiments, any number of replication cycles may be modified to, e.g., control the dynamic thermal conditions and / or inclusion of additional steps or chemistries to modify the replication reaction. For example, single stranded binding proteins and / or blocker oligonucleotides may be included in any suitable step or for any number of replication cycles.

[0064] Following “pre-conversion” isothermal amplification, the duplex template construct is subjected to chemical and / or chemo-enzymatic conversion to selectively convert the native form of the modified base of interest (or in other embodiments, to directly convert the modified base of interest) such that its base-pairing properties are altered. For example, if the modified base of interest is 5-mC, the duplex template construct can be subjected to bisulfite treatment to convert native cytosine to uracil, while 5-mC remains unaltered. In this situation, the converted cytosines (uracils) will base pair with thymine, instead of guanosine (see, e.g., Li, Y. and Tollefsbol, Methods Mol Biol. 2011; 719: 11-21).

[0065] Alternative epigenetic detection methods, based on enzymatic conversion chemistries, are also known in the art, such as TET / APOBEC or EM-SEQ (see, e.g., Vaisvila, R. et al., Genome Res. 2021 Jul; 31(7): 1280-1289.). This method detects 5-mC and 5-hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5-mC and 5-hmC into products that cannot be deaminated by APOBEC3 A. In the second reaction, APOBEC3 A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5-mC and 5-hmC. As with the bisulfite method, these conversion chemistries lead to deamination of native cytosine to uracil, while certain modified forms of cytosine remain resistant. Thus, these enzymatic conversion options also reduce the complexity of the genome as native cytosine reads as uracil during a sequencing reaction.

[0066] In other embodiments, the duplex template construct can be subjected to chemo- enzymatic conversion, in which 5-mC is converted to an uracil mimetic with the base pairing properties of natural uracil. In contrast to bisulfite conversion, this strategy selectively converts5-mC residues, while native cytosine residues remain unaltered. Chemo-enzymatic conversion is described in more detail in Applicant’s published PCT application, WO2024 / 083982, entitled, “Detection of Modified Nucleobases”, filed October 19, 2023, the contents of which are herein incorporated by reference in its entirety.

[0067] In certain embodiments, the methods of the present invention may also include a step in which the newly replicated complementary copies, hybridized to the parental template construct are treated with a DNA methyltransferase enzyme, e.g., DNMT1 or DMNT3b prior to the conversion step. Such enzymes are capable of transferring methylation information from a methylated DNA strand to a complementary non-methylated DNA strand, e.g., in regions of CpG islands. In this manner, the methylation information of the parental template construct can be propagated into a population of daughter strands, reducing the likelihood of losing the epigenetic information. Both the parental and daughter template strands can be used as templates for pre-conversions and post-conversion linear amplification.

[0068] One of the advantages offered by the solid-state methods of the present invention is that methylation conversion reactions are not performed in-solution. This increases the stability of enzymes and other macromolecules used in the conversion reaction, which often lose stability when diluted in-solution.

[0069] One embodiment of a conversion cycle is depicted in steps 2e and 2f. In step 2e, the duplex template construct is treated with conversion mix 370. In some embodiments, the conversion cycle may include a plurality of sub-steps. In certain embodiments, during step 2e, the conversion mix converts native cytosine to, e.g., uracil, while 5-mC remains unconverted. Following conversion, in step 2f, the converted duplex template construct is treated with wash solution 345b. Next, a second, post-conversion, isothermal amplification is performed to produce a population of complementary copies of the converted duplex template construct. The nucleotide sequences of populations of pre- and post-conversion complementary copies of the duplex template construct can be compared to identify the positions of the modified nucleobase of interest in the original target fragment.

[0070] Some embodiments of the methods of the present invention include one or more upstream library preparation steps that are also performed on the solid support. One such embodiment is depicted in FIG. 4. In this embodiment, a library fragment is provided, which includes parent (+) strand 400a (i.e., a sense strand) and parent (-) strand 400b (i.e., an antisense strand). In step 1, the library fragment is contacted with hairpin adapter 405 under first nucleic acid ligation conditions. In the embodiment, the loop of the hairpin adapter is joined to a fragment capture moiety (e.g., a biotin moiety) via a cleavable linker. During the first ligation reaction, the complementary strands of the double stranded stem portion of the hairpin adapterare each ligated to one of the strands of the library fragment. Products of the first ligation reaction include asymmetric ligation product 410, in which a single end of the library fragment is joined to a hairpin adapter and symmetric ligation product 409, in which both ends of the library fragment are joined to a hairpin adapter.

[0071] In step 2, solid support 425 is provided that includes support capture moiety 427 (e.g., a streptavidin moiety). In certain embodiments, by-products of the first ligation reactions, e.g., unligated hairpin adapters and library fragments may be removed by art-recognized nucleic acid purification methods. In step 3, the products of the first ligation reaction are contacted with the solid support. In this embodiments, first ligation products 410 and 409 are joined to the solid support via binding of the fragment capture moiety to the support capture moiety. The supportbound first ligation products may be subjected to wash conditions to optimize buffer and or other conditions for downstream steps.

[0072] In step 4, the support-bound first ligations products are contacted with cleavage conditions to release asymmetric ligation product 410a and symmetric ligation product 409a from the solid support by cleavage of the cleavable linker joining the fragment capture moiety to the hairpin adapter.

[0073] In step 5, Y adapter 430 is provided that is joined to solid support 435. In this embodiment, the Y adapter includes a double stranded stem region joined to a 5’ single stranded arm region and a 3’ single stranded arm region. The double stranded end of the Y adapter may include a single 3’ T overhang capable of base pairing with the single 5’ A overhang in the library fragment. The Y adapter may be bound to the solid support using any suitable coupling chemistry, including, but not limited to click chemistry, biotin-strepavidin interactions, and the like.

[0074] As discussed with reference to FIG. 1, in certain embodiments, solid support 437 is part of a channel disposed in a flow cell (e.g., a chip or a card) that is engineered for automated synthesis of macromolecules, such as Xpandomers. The Y adapter is contacted with the first ligation products under second nucleic acid ligation conditions. In this embodiment, only the asymmetric ligation product is capable of being ligated to the Y adapter to product second ligation product 440, e.g., a duplex template construct joined to the solid support.

[0075] n step 6, duplex template construct 440 is contacted with extension oligonucleotide 446 under nucleic acid hybridization and synthesis conditions.

[0076] In step 7, a plurality of replication cycles is carried-out on solid support 430 to amplify the duplex template construct in a linear manner. During each cycle, the duplex template construct is replicated once to generate a complementary copy of the construct. By repeating the replication cycle under isothermal conditions, many of the reagents required for DNA synthesiscan be reused during the subsequent replication cycles. As discussed with references to FIG. 1, a single replication cycle includes the following four steps: (1) Step 2a - priming and extension of the duplex template construct; (2) Step 2b - first wash; (3) Step 2c - denaturation and elution of the complementary copy of the duplex template construct; and (4) Step 2d - second wash. After a suitable number of cycles, amplified population of copies 450 of the duplex template construct may be collected a used as templates for Xpandomer synthesis.

[0077] In some embodiments, the methods of the present invention include a process, referred to herein as “printing,” in which complementary copies of a duplex template construct are synthesized, or “printed”, from an extension oligonucleotide that is covalently bound to a solid support. Such embodiments do not require ligation of the duplex template construct to the solid support. One example of a method that includes a template printing process is depicted in FIG. 5. In this embodiment, duplex template construct 500 is provided, which includes a target nucleic acid fragment (e.g., a library fragment) with parent (+) strand (i.e., a sense strand) and parent (-) strand (i.e., an antisense strand). The library fragment is joined on a first end to a hairpin adapter, which includes a double stranded stem region joined to 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 a Y adapter, which includes a double stranded stem region joined to a 5’ single stranded arm region and a 3’ single stranded arm region. Likewise, the complementary strands of the double stranded stem portion of the Y adapter are each ligated to one of the strands of the library fragment.

[0078] In step 1, solid support 510 is provided, which includes one or more extension oligonucleotide 515 that are bound to the solid support at the 5’ end of the oligonucleotide. In certain embodiments, the extension oligonucleotide may be bound (i.e., covalently linked) to the solid support via maleimide-mediated surface functionalization of the support and click chemistry-mediated linkage of the extension oligonucleotide, as described herein. The extension oligonucleotide includes a sequence that is complementary to a sequence in the single stranded 3’ arm of the Y adapter. The duplex template construct is contacted with the support-bound extension oligonucleotide under nucleic acid hybridization conditions. Duplex template construct 500 is localized to the solid support via hybridization to extension oligonucleotide 515, which also provides a free 3’ for initiation of DNA synthesis. In certain embodiments, solid support 510 is part of a channel disposed in a larger flow cell (e.g., a chip or a card) that is engineered for automated synthesis of macromolecules, such as Xpandomers.

[0079] In step 2, the extension oligonucleotide hybridized duplex template construct is contacted with a strand displacing DNA polymerase under DNA synthesis conditions. The DNApolymerase initiates DNA synthesis from the free 3’ end of the extension oligonucleotide using the duplex template construct as a template to produce newly synthesized complementary copy (i.e., daughter strand) 525 that is covalently joined to solid support 510 via the support-bound extension oligonucleotide. As discussed herein, the newly synthesized daughter strand is described as having been “printed” on the solid support. As such, the printed daughter strand is covalently bound to the solid support. The printed daughter strand may be used as a template for a subsequent linear amplification process.

[0080] In step 3, the hybridized duplex template construct is “melted” off the support by contacting the hybridized strands with denaturing conditions (e.g., a solution of NaOH) to release duplex template construct 500 into solution. In contrast, the newly synthesized daughter copy remains covalently bound to the solid support.

[0081] In step 4, complementary copy 525 of the duplex template construct is contacted with extension oligonucleotide 530 under nucleic acid hybridization and DNA synthesis conditions. Extension oligonucleotide 530 includes a sequence that is complementary to a sequence at the 3’ end of complementary copy 525. A DNA polymerase initiates DNA synthesis from the 3’ end of the extension oligonucleotide to produce newly synthesized duplex template construct copy using the daughter strand as a template. As discussed herein, repeated replication cycles can be carried out under isothermal conditions to generate a plurality of duplex template construct copies 550 that can be recovered in solution.

[0082] In certain embodiments, the methods may include multiple cycles of template printing to increase the copy number of support-bound templates for linear amplification. For example, a duplex template construct may be printed on a support multiple times to yield a plurality of complementary copies on the support that provide templates for subsequent linear amplification. One example of multi -template printing, followed by linear amplification, is depicted in FIG. 6. As shown in FIG. 6, solid support 610 is provided that includes a plurality of extension oligonucleotides 615 covalently bound to the support via linkers 617. The sequence of the extension oligonucleotide is designed to be complementary to a sequence in a single stranded arm region of a Y adapter. Prior to template printing, the solid support is subjected to a pre-wash step with a suitable wash solution. As discussed with reference to FIG. 5, in step 1, duplex template construct 600 is provided in-solution and contacted with the support-bound extension oligonucleotides under nucleic acid hybridization conditions. As shown, the terminal Y adapter of the duplex template construct specifically hybridizes to the support-bound extension oligonucleotide. In step 2, the hybridized duplex template construct is contacted with a strand displacing DNA polymerase under DNA synthesis conditions. The DNA polymerase initiates DNA synthesis from the free 3’ end of the extension oligonucleotide using the duplex templateconstruct as a template to produce newly synthesized complementary copy (i.e., daughter strand) 620 that is covalently joined to solid support 610 via the support-bound extension oligonucleotide. In step 3, following the extension reaction, hybridized duplex template construct 600 and complementary copy 620 strands bound to the solid support are washed with a suitable wash solution. In step 4, the hybridized duplex template construct is “melted” off the support by contacting the hybridized strands with denaturing conditions (e.g., a solution of NaOH) to release duplex template construct 600 into solution. In contrast, newly synthesized daughter copy 620 remains covalently bound to the solid support. The released duplex template construct is retained and reused for subsequent rounds of template printing. In certain embodiments, the template printing process may include from around 5 to around 10 cycles, each cycle using the same duplex template construct as the template.

[0083] In one embodiment, a multi-print cycling process may include the following steps: a) a chip is functionalized with a maleimide reaction mixture at around 23° C for around 20 minutes with UV light; b) the chip is washed with a maleimide wash buffer; c) an extension oligonucleotide is bound to the functionalized chip using a click chemistry reaction at around 37° C for around 10 minutes (an exemplary extension oligonucleotide may have the following sequence: R-TTTTTDDDDDDDDDD-TTCAGACGTGTGCTCTTCCGATCT, where “R” represents an azide group and “D” represents PEG3; and the nucleotide sequence is designed to be complementary to a sequence in the Y adapter of the duplex template construct); d) the chip is washed with a click wash buffer; e) for printing, the temperature of the support is set to around 37° C; f) the support is washed with a pre-wash buffer; g) a hybridization mix (including duplex template construct input) is flowed over the support at around 37° C; h) the temperature is ramped up to around 90° C and held at that temperature for around 2 minutes; i) the temperature is ramped down to around 37° C and held at that temperature for around 2 minutes; j) an extension “master-mix” of regents (including, e.g., Bst DNA polymerase) is flowed over the support and the temperature is ramped up to around 55° C and held at that temperature for around 10 minutes; k) the support is washed with a post-wash buffer; 1) the print cycle (steps e) through k) is repeated from around 6 to around 10 times; and m) the support is washed with around lOOmM NaOH to denature and remove the duplex template construct input.

[0084] In step 5, the support-bound, newly printed complementary copies of the duplex template construct may be subjected to one or more “polishing” and / or “capping” steps prior to linear amplification. For example, in one step, any remaining unextended extension oligonucleotide may be removed from the solid support by exonuclease treatment. Suitable exonucleases for this application include, e.g., Exonuclease I, which has 3’ -> 5’ exonuclease activity and specifically digests single stranded DNA. In certain embodiments, prior toexonuclease treatment, the free 3’ end of the daughter strands may be protected, or blocked, from exonuclease-mediated digestion. In one embodiment, terminal deoxynucleotidyl transferase (TdT) can be used to join a terminal ddNTP to the free 3’ end of the complementary copy strands to protect them. In another embodiment, terminal ddNTPs may be added by polymerase- mediated extension of a short oligonucleotide that is hybridized near the 3’ end of the printed daughter strands. In other embodiments, the 3’ ends of the daughter strands may be joined to a capping oligonucleotide to protect the newly printed strands from exonuclease digestion. The capping oligonucleotide may have a sequence that hybridizes to a short oligonucleotide that itself hybridizes to the 3’ end of the daughter strand with a 5’ overhang that provides a hybridization site for the capping oligonucleotide, which can then be ligated to the 3’ end of the daughter strand.

[0085] In one embodiment, an exemplary blocking and polishing method may include the following steps: a) a printed chip is washed with a pre-wash buffer; b) a TdT reaction mixture including ddNTPs is flowed on the chip and incubated at around 37° C for around 1.5 hours; c) the chip is heated to around 85° C for around 10 minutes to inactive the TdT enzyme; d) the chip is washed with a post-wash buffer at around 85° C for around 2 minutes; e) the temperature of the chip is ramped down to around 55° C; f) the chip is washed with around lOOmM NaOH; g) the chip is washed with pre-wash buffer; g) a hybridization mix is flowed on the chip at around 37° C for around 1 minute to block exonuclease-mediated digestion of the daughter strands; h) a solution including an exonuclease is flowed on the chip and incubated at around 37° C for around 10 minutes; i) the temperature of the chip is ramped up to around 80° C to inactivate the exonuclease; j) a post-wash solution is flowed on the chip; k) the temperature of the chip is ramped down to around 55° C and the chip is washed with around lOOmM NaOH; and 1) the chip is washed with a pre-wash buffer.

[0086] Following the template printing and polishing steps, linear amplification of the printed templates can be conducted as described herein. For example, from around 20 to around 100 cycles of template extension reactions can be performed to provide an amplified pool of newly synthesized duplex template constructs for subsequent use in, e.g., synthesis of Xpandomers for nanopore sequencing. Advantageously, the combination of a multi-printing protocol and a linear amplification protocol improves template yield in the final amplicon pool. This is particularly advantageous when the copy number in the library construct input is low, for example, when cell-free DNA is the source of the library fragments.System for Automated Solid-State Amplification of Macromolecules

[0087] The methods described herein are adapted for automation using a consumable cartridge substrate (e.g., a chip or flow cell) under the control of dedicated sample preparation instrumentation. Suitable cartridges and instruments for the practice of the present invention are disclosed in Applicant’s PCT Application No. PCT / IB2024 / 059484, filed February 15, 2024, entitled, “System and Methods for Automated Expandomer Synthesis”, filed September 27, 2024, the contents of which are herein incorporated by reference in its entirety.

[0088] One embodiment of a suitable sample preparation instrument is depicted FIG. 7A and FIG. 7B. Here, a system 100 is used for macromolecule synthesis, e.g., linear amplification of a library of duplexed nucleic acid template constructs. The system 100 includes a removable and disposable flow cell (i.e., cartridge or chip) 200, a mount 300 (including a thermal block described below) for receiving the flow cell 200, a holder / vial rack 400, an XYZ gantry 500, a pump (e.g., syringe pump) 600, a flow and pressure gauge 602, a waste container 604, a buffer container 606, a selectable valve (e.g., rotary valve) 608, a UV source 700, and a controller (not shown). The holder 400 can also be used to hold sample and the end product or anything else that can be contained in a tube, container, vial, or reservoir that can be placed in the holder.

[0089] In some embodiments as shown in FIG. 7A and FIG. 7B, a pump 600 can be used in a pull mode to draw fluid into the inlet port of the flow cell 200 via the sipper(s) 22, and the fluid can be drawn out of the flow cell 200 through the outlet ports of the flow cell 200 and into a waste reservoir 604. The pump 600 can also be used in a push mode to pump fluid, such as a wash solution or elution buffer, from a buffer reservoir 606 and into the flow cell 200 through the outlet port, and then the fluid can be pushed out of the flow cell 200 through the inlet port and through the sipper(s) into a product collection tube. A rotary valve 602 or other type of valve can be used with the pump 600 to switch between the waste reservoir 604 and the buffer reservoir 606 in order to allow the pump to operate in both a push and pull configuration. The pump 600 can be a syringe pump or other type of pump that is capable of precisely metering out very small amounts of fluid (i.e., in the microliter to milliliter range).

[0090] In some embodiments, the controller can control the XYZ gantry 500 and pump 600 to perform liquid mixing operations within the flow cell 200 or directly on / in the holder 400. Consumable device / cartridge

[0091] FIG. 8A illustrates a perspective view of one embodiment of a flow cell 200 that the user can load into the system. The flow cell 200 can be a consumable device or cartridge that is disposed of after use. The flow cell 200 encloses at least one flow channel 202 that provides a functionalized solid phase surface to which a plurality of capture molecules, e.g., oligonucleotides or Y adapters, can be attached. In some embodiments, the flow cell 200 has aplurality of channels, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 flow channels. In some embodiments, the flow channel 202 can be made of a substrate that is directly functionalized, instead of being coated with a material that is then functionalized. The flow channels can be fabricated using microfluidic techniques and can be used to perform reactions involving fluid volumes in the microliter range. In some embodiments, the flow cannel 202 can be serpentine to increase the flow length and surface area available to carry out the reactions and / or to aid in mixing.

[0092] In some embodiments, the flow cell 200 can be fabricated from a single injection molded portion 201 that is bonded to a thin film 203, as shown in FIG. 8C and FIG. 8D. For example, the injection molded portion can include the base substrate, the inlet ports, the outlet ports, and a portion of the flow channels 202. The thin film 203 can be disposed over the base substrate and flow channels 202 to enclose and complete the formation of the flow channels 202. The thin film that forms the base of the flow cell 200 can be placed against a thermal block, as further described below. The thin film can have a low thermal resistance which allows efficient heat transfer from a thermal block to the flow cell 200, which allows the rapid heating and cooling of the flow cell 200 to facilitate the different temperature reactions of the workflow. In some embodiments, the thin film can be made of a cyclic olefin polymer (COP) or a cyclic olefin copolymer (COC). Other types of polymers may also be used to form the thin film, such UV transparent polymers that can be functionalized (i.e., proton abstractable polymers) and bonded to the injection molded portion of the flow cell. Other polymers that can be used include but are not limited to polypropylene and polyethylene. The thin film can be bonded to the molded portion using a variety of techniques, such as thermal bonding, laser welding, or chemical bonding.

[0093] Each flow channel 202 of the flow cell 200 has an inlet port 220 and an outlet port 222 that provide access to the flow channel 202. Affixed to the inlet port 220 is a sipper 224 that can be used to draw reagents, buffers, wash solution, sample, etc. into the flow cell 200 when the pump is operated in a pull mode. The sipper 224 can also be used to deposit the finished product from the flow cell 200 into a collect tube when the pump is operated in a push mode. In some embodiments, the sipper 224 is pre-attached to the inlet port 220 so that the end user does not need to attach the sipper 224 to the flow cell 200 before use. Instead, the end user can simply insert the preassembled flow cell 200 into the mount 300 as shown in FIGS. 7A and 9. Reducing or minimizing the length and volume of the sipper 224 is advantageous in reducing the loss and / or carryover of precious reagents, sample, and the synthesized molecule (e.g., the amplified library construct) during the synthesis process. The sipper 224 configuration also provides a direct path to the flow cell that does not need to pass through a valve, which can reduce contamination and leak issues, especially when using corrosive reagents.

[0094] FIG. 8B illustrates an embodiment of the flow channel surface 204 that has been functionalized to bind capture oligonucleotides or 206 that can hybridize to a portion of the sample. For example, the capture oligonucleotide 206 can include a polynucleotide sequence that is complementary to and can hybridize with a sequence in a binding region of the sample polynucleotide 208.

[0095] FIG. 9 illustrates an embodiment of the mount 300 for receiving the flow cell 200. As shown, the mount 300 can receive two flow cells 200. In other embodiments, the mount can receive 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 flow cells. A thermal block 302 is thermally coupled to the mount 300 and is used to control the temperature of the flow cell. The thermal block 302 can be quickly cooled or heated as desired so that the reactions in the flow cell can be performed at temperatures between about 20 Celsius and 60 Celsius. In other embodiments, the temperature range can between about 5 Celsius and 90 Celsius.

[0096] As shown in FIG. 9, the thermal block 302 can include fins to radiate heat and a fan to aid in heat transfer from the thermal block. The thermal block 302 can be thermally attached to the mount 300 or can be integrally formed with the mount 300. For example, one face of the thermal block 302 can be formed to receive the flow cells 200. In some embodiments, the controller can provide real-time thermal control of the thermal block.

[0097] In some embodiments, a thermal pad can be placed between the mount 300 and flow cell 200 to improve heat transfer from the thermal block 302 to the flow cell 200. In some embodiments, the thermal pad can be placed on or pre-attached to the mount. In other embodiments, the thermal pad can be place on or pre-attached to the base of the flow cell 200. In some embodiments, a thermal paste or gel can be used instead of a thermal pad.

[0098] In some embodiments, a portion of the sipper 224 can be disposed against the thermal block 302 or mount 300 in order preheat the fluids as they are drawn into the flow cell 200. In some embodiments, the portion of the sipper 224 that is disposed against the thermal block 302 or mount 300 can be serpentine.

[0099] In some embodiments, when the flow cell 200 has more than one flow channel and more than one sipper, the spacing between the sippers 224 after being secured to the mount 300 is compatible for use with 96 well plates, which allows the reagents and / or sample to be stored in a 96 well plate, which can be loaded onto the holder 400.

[0100] FIG. 10A illustrates an embodiment of a holder 400. The holder 400 can hold reagents, samples, buffers, and other liquids used in the synthesis method. As shown, a 96 well plate 402 (or plate with another well count) and vial or tube holder 404 can be included in the holder 400. Reagent trays, bottles, and other containers for holding liquids can also be secured to the holder 400. The openings of the containers, such as the openings of the tubes in the tubeholder 404 and the wells of the 96 well plate 402 can all be located on the same plane or height. This makes it easier for a covering to be applied over the openings and pierced when needed with a piercing tool 406. Height adapters can be used if needed to adjust the heights of openings so that they are level. Alternatively, custom vial or tube racks and well plates with preadjusted heights can be used with the holder 400 to simplify user operation and reduce user error. The liquid holders can be removably attached to the holder 400 to allow for user customization.

[0101] The holder 400 can be moved in all three axes with the XYZ gantry 500, as shown in FIG. 7A. This configuration allows the flow cell 200 and sipper 224 and the piercing tool 406 to remain in a fixed position while the holder 400 is moved by the gantry 500 to the piercing tool 406 when the covering over the openings needs to be pierced and then to the sippers 224 once the coverings have been pierced. To access different reagents and samples, the gantry 500 can move the holder away from the sippers 224 and then align the sippers 224 with the new reagents and / or samples. One benefit from holding the flow cell 200 in a fixed position is that it reduces the movement of wiring and tubing attached to the flow cell, thermal block, UV source, and other associated components. Repeated movement of these components may cause disconnects or damage to these sensitive components, and therefore, it may be beneficial to adopt a configuration where these components remain stationary.

[0102] In other embodiments, the gantry 500 can be used to move the flow cell 200 and piercing tool 406 while the regent holder and reagents are stationary. This can be accomplished by attaching the mount 300 and flow cell 200 to the gantry 500, while the holder can remain fixed in place on the deck of the instrument. The UV source can still optionally remain fixed in one location since the flow cell 200 can be moved in position in front of the UV source when needed.

[0103] FIG. 10B and FIG. 10C illustrate a reagent cartridge 410 that can be loaded onto the holder 400. The reagent cartridge can include a plurality of different types of liquid reservoirs 412 of different sizes and / or shape as well as a cover 414 that can be used to seal the reagent cartridge. In some embodiments, the gantry 500 can move the reagent cartridge 410 and press the reagent cartridge against the cover 414, which can be held in a fixed horizontal position in order to seal reagent cartridge. In some embodiments, the reagent cartridge 410 can also include drop-in locations to receive tubes 416 or vials (e.g., Eppendorf tubes). Although described as tubes 416, other containers or reservoirs can be used instead to hold the liquids and reagents. In some embodiments, these tubes can be provided by the manufacturer to contain premade Library or master mix solutions. In addition, a tube can be used to collect the synthesized molecule.

[0104] The gantry 500 can also be optionally used with the sippers 224 or a separate mixing tube 700 to perform liquid mixing and / or bubble mixing operations on the reagent cartridge if desired, as shown in FIG. 7C. The mixing tube 700 can be attached to a dedicated pump 600 and can optionally have an internal volume that is sufficient to aspirate all the fluid in the tube 416 that needs to be mixed. In some embodiments, to achieve the internal volume needed, the mixing tube 700 can optionally have a coiled portion 702 to accommodate a relatively long length of tubing in a small space within the chassis of the device. The inlet of the mixing tube 700 can be fixed to a specific location within the chassis of the device so that the gantry can move tubes 416 to the inlet of the mixing tube 700 when needed.Duplex Template Construct Libraries

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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 invention allow for linear amplification of duplex library constructs prior to synthesis of duplexed Xpandomers for nanopore sequence determination.

[0109] 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.

[0110] 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 to enable high accuracy duplex and methylome Sequencing by Expansion are discussed herein.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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, hybridizationbased enrichment, or chemical labeling-based enrichment.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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).

[0122] 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.

[0123] 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.

[0124] 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 availablefrom, 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 Red 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.

[0125] 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.

[0126] 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 RecJf, 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 RecJf into the FFPE step may improve sequence coverage and lower the percentage of clipped bases seen in sequence analysis.

[0127] 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., stickyends). 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 for bioinformatic analysis (e.g., clustering of related sequences into families based on shared unique molecular identifier barcodes, UMIs).

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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 the two 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.

[0132] 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 forms a stable duplex under standard reaction conditions for the enzyme-catalyzed nucleic acid ligation reaction.

[0133] 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.

[0134] 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.

[0135] “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.

[0136] 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.

[0137] 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.

[0138] The ligation of adapters to both free ends of the double stranded DNA target fragments give rise to a pool of adapter-ligated double stranded DNA target fragments with adapters at the 5’ and 3’ ends of the target.

[0139] 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.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.

[0140] One method of generating a duplex (parent-parent) template construct according to the present invention 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, target fragment 1100 is contacted with hairpin adapter 1110, 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 1125 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 the library fragment may be optimized to preferentially generate asymmetric duplex template construct 1125. In step 2, Y adapter 1130 is immobilized on solid support 1133 via linker 1135 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 1135 includes a poly dU sequence. The ligation products of step 1, including asymmetric duplexed template construct 1125 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 1133. In contrast, asymmetric duplexed template construct 1125 may be ligated to Y adapter 1130 to form Y adapter-ligated duplexed template construct 1140, bound to solid support 1133 via linker 1135. In step 3, Y adapter-ligated duplexed template construct 1140 is treated with, e.g., a USER enzyme to cleave the polyU sequence in linker 1135 and release the Y adapter-ligated duplexed template construct from the solid support. In other embodiments, linker 1135 may include other suitable selectively-cleavable moi eties known in the art, such as a photocleavable moiety.

[0141] 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, reaction products may be denatured, such that only library fragments with strands that are covalently paired by ligation toan 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.Solid-Phase Synthesis

[0142] As discussed herein, 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", 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 some embodiments 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. In some embodiments, a chip may be referred to as a flow cell. 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.

[0143] 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’ or 3’ 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 biotinylatedmolecule to the avidin or streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions.

[0144] 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 end of single stranded DNA fragment (or oligonucleotide) is bound to the linker moiety. Attachment is mediated by a streptavidin moiety provided by the 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.

[0145] Alternatively, immobilization of a capture moiety or oligonucleotide (e.g., an extension oligonucleotide) to a solid support may be accomplished by covalent linkage of the capture 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. A terminal alkyne moiety provided by the end of the linker distal to the substrate is capable of reacting with a terminal 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.

[0146] In certain instances, the linkage between the capture moiety and the solid support is cleavable, enabling primer extension products or library constructs 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.

Claims

CLAIMSWhat is claimed is:

1. A method of solid-state amplification of a duplex nucleic acid template, the method comprising the steps of:(a) providing a duplex nucleic acid template comprising a double stranded nucleic acid target fragment, wherein the double stranded nucleic acid target fragment is joined at a first end to a Y adapter, wherein the Y adapter comprises a double stranded stem region, a 3’ single stranded arm region and a 5’ single stranded arm region and at a second end to a hairpin adapter, wherein the hairpin adapter covalently joins the strands of the double stranded nucleic target fragment;(b) joining the duplex nucleic acid template to a solid support, wherein the joining is mediated by the 5’ single stranded arm region of the Y adapter or the 3’ single stranded arm of the Y adapter to produce a support-bound duplex nucleic acid template;(c) contacting the solid support with a nucleic acid extension mixture under nucleic acid hybridization and extension conditions, wherein the nucleic acid extension mixture comprises an extension oligonucleotide, a nucleic acid polymerase, dNTPs, and a suitable buffer, wherein the extension oligonucleotide specifically hybridizes to the duplex nucleic acid template to provide at free 3’ end, and wherein the nucleic acid polymerase synthesizes a complementary copy of the duplex nucleic acid template from the free 3’ end of the extension oligonucleotide;(d) optionally, contacting the solid support with a suitable wash buffer;(e) contacting the solid support with a suitable elution buffer, wherein the suitable elution buffer releases the complementary copy from the support-bound duplex nucleic acid template;(f) optionally, contacting the solid support with a suitable wash buffer; and(g) repeating steps (c) through (f) to provide an amplified population of complementary copies of the duplex nucleic acid template.

2. A method of solid-state printing of a duplex nucleic acid template, the method comprising the steps of:- 38 -(a) providing a solid support comprising an extension oligonucleotide, wherein the extension oligonucleotide comprises a free 3’ end;(b) providing a duplex nucleic acid template comprising a double stranded nucleic acid target fragment, wherein the double stranded nucleic acid target fragment is joined at a first end to a Y adapter, wherein the Y adapter comprises a double stranded stem region, a 3’ single stranded arm region and a 5’ single stranded arm region and at a second end to a hairpin adapter, wherein the hairpin adapter covalently joins the strands of the duplex nucleic acid template;(c) contacting the duplex nucleic acid template with the solid support comprising the extension oligonucleotide under nucleic acid hybridization conditions, wherein the duplex nucleic acid template specifically hybridizes to the extension oligonucleotide;(d) contacting the solid support with a nucleic acid extension mixture under nucleic acid extension conditions, wherein the nucleic acid extension mixture comprises a nucleic acid polymerase, dNTPs, and a suitable buffer, and wherein the nucleic acid polymerase synthesizes a support-bound complementary copy of the duplex nucleic acid template construct;(e) optionally, contacting the solid support with a suitable wash buffer;(f) contacting the solid support with a suitable elution buffer, wherein the suitable elution buffer releases the duplex nucleic acid template from the support-bound complementary copy;(g) optionally, contacting the solid support with a suitable wash buffer; and(h) repeating steps (c) through (g) to provide a population of support-bound complementary copies of the duplex nucleic acid template.

3. The method of claim 2, wherein the population of support-bound complementary copies of the duplex nucleic acid template are subjected to one or more polishing steps.

4. The method of claim 3, wherein the one or more polishing steps comprise treatment with an exonuclease enzyme, wherein the exonuclease enzyme is capable of digesting free extension oligonucleotide joined to the solid support.

5. The method of claim 2 or 3, wherein the population of support-bound complementary copies of the duplex nucleic acid template construct are subjected to solid-state amplification according to the method of claim 1.

6. The method of claim 1, further comprising the step of subjecting the support-bound duplex nucleic acid template to a chemical conversion step following step (g), wherein the chemical conversion step selectively converts a modified nucleobase of interest, or the native form of a modified nucleobase of interest, to enable detection of the positions of the modified nucleobase of interest in the duplex nucleic acid template.

7. The method of claim 6, wherein the modified nucleobase of interest in 5-mC.

8. The method of claim 6 or 7, wherein the chemical conversion step comprises treatment of the support-bound duplex nucleic acid template to bisulfite treatment, TET / APOBEC treatment, DNMT1 treatment, or chemoenzymatic treatment.

9. The method of any one of claims 6 to 8, wherein the chemical conversion step is followed by a solid-state amplification step.

10. The method of any one of claims 1 to 9, wherein the nucleic acid polymerase is a strand displacing DNA polymerase.

11. The method of any one of claim 1 to 3, wherein the duplex nucleic acid template is joined to the solid support by a click chemistry reaction.

12. The method of claim 11, wherein the click chemistry reaction is mediated by a terminal azide moiety provided by an end of a single stranded arm region of the Y adapter and a terminal alkyne moiety provided by the solid support.

13. The method of any one of claims 1 to 12, wherein the nucleic acid synthesis conditions comprise isothermal conditions.

14. The method of claim 13, wherein each round of nucleic acid extension is performed with the same reaction mixtures and solutions.

15. The method of any one of claims 1 to 14, wherein the double stranded nucleic acid target fragment is provided by cell-free DNA, genomic DNA, RNA, or a formalin-fixed paraffin-embedded (FFPE) sample.

16. The method of claim 15, wherein the FFPE sample is treated with one or more of a FFPE repair step, an end-repair and A-tailing (ERAT) step, an exonuclease treatment step, and a fragmentation step.

17. The method of claim 16, wherein the FFPE sample is treated with a FFPE repair step comprising treatment with a thermolabile exonuclease enzyme or a RecJf enzyme.

18. The method of any one of claims 1 to 17, wherein the solid support comprises a flow cell, wherein the flow cell is compatible with a system capable of automating the amplification of the duplex nucleic acid template.

19. The method of claim 18, wherein the flow cell comprises at least one flow channel, the flow channel having a functionalized solid phase surface configured to bind a plurality of duplex nucleic acid templates, and wherein each flow channel has an inlet port and an outlet port.

20. The method of claim 18 or 19, wherein the system comprises the flow cell, a sipper in fluidic communication with the flow cell, a heatable mount configured to receive the flow cell, optionally wherein a first thermal block is attached to the heatable mount, a holder configured to hold a plurality of reagents, a gantry configured to move in 3 different axis, a pump in fluidic communication with the outlet port of the flow channel, a valve configured to selectively connect the outlet port of the flow channel with a waste reservoir and / or a buffer reservoir, a UV source configured to illuminate the flow channel, and a controller configured to a temperature of the heatable mount, movement of the gantry, selection of a valve, operation of the UV source, and operation of the pump.

21. The method of any one of claims 18 to 20, wherein the flow cell comprises a molded portion and a thin film, wherein the molded portion comprises the flow channel and the thin film is attached to the molded portion to seal the flow channel and wherein the thin film is made from cyclic olefin polymer or cyclic olefin copolymer.

22. The method of claim 19, wherein the functionalized solid phase surface provides the terminal alkyne moiety.

23. The method of claim 22, wherein the terminal alkyne moiety is joined to the functionalized surface by a flexible linker.

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