Methods and compositions for strand enrichment and template preparation for sequencing by expansion

Abstract

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

Description

Attorney Docket P39649-WO METHODS AND COMPOSITIONS FOR STRAND ENRICHMENT AND TEMPLATE PREPARATION FOR SEQUENCING BY EXPANSION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims the benefit of the filing date of U.S. Provisional Application No.63 / 694233 filed on September 13, 2024; the disclosure of which is hereby incorporated by reference herein in its entirety. BACKGROUND

[0002] Measurement of biomolecules is a foundation of modern medicine and is broadly used in medical research, and more specifically in diagnostics and therapy, as well in drug development. Nucleic acids encode the necessary information for living things to function and reproduce, and are essentially a blueprint for life. Determining such blueprints is useful in pure research as well as in applied sciences. In medicine, sequencing can be used for diagnosis and to develop treatments for a variety of pathologies, including cancer, heart disease, autoimmune disorders, multiple sclerosis, and obesity. In industry, sequencing can be used to design improved enzymatic processes or synthetic organisms. In biology, this tool can be used to study the health of ecosystems, for example, and thus have a broad range of utility. Similarly, measurement of proteins and other biomolecules has provided markers and understanding of disease and pathogenic propagation.

[0003] An individual's unique DNA sequence provides valuable information concerning their susceptibility to certain diseases. It also provides patients with the opportunity to screen for early detection and / or to receive preventative treatment. Furthermore, given a patient's individual blueprint, clinicians will be able to administer personalized therapy to maximize drug efficacy and / or to minimize the risk of an adverse drug response. Similarly, determining the blueprint of pathogenic organisms can lead to new treatments for infectious diseases and more robust pathogen surveillance. Low cost, whole genome DNA sequencing will provide the foundation for modern medicine. To achieve this goal, sequencing technologies must continue to advance with respect to throughput, accuracy, and read length.

[0004] Over the last decade, a multitude of next generation DNA sequencingAttorney Docket P39649-WO technologies have become commercially available and have dramatically reduced the cost of sequencing whole genomes. These include sequencing by synthesis ("SBS") platforms (Illumina, Inc., 454 Life Sciences, Ion Torrent, Pacific Biosciences) and analogous ligation based platforms (Complete Genomics, Life Technologies Corporation).

[0005] Nanopore based nucleic acid sequencing is a compelling approach that has been widely studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93: 13770-13773, 1996) characterized single-stranded polynucleotides as they were electrically translocated through an alpha hemolysin nanopore embedded in a lipid bilayer. It was demonstrated that during polynucleotide translocation partial blockage of the nanopore aperture could be measured as a decrease in ionic current. Polynucleotide sequencing in nanopores, however, is burdened by having to resolve tightly spaced bases (0.34 nm) with small signal differences immersed in significant background noise. The measurement challenge of single base resolution in a nanopore is made more demanding due to the rapid translocation rates observed for polynucleotides, which are typically on the order of 1 base per microsecond. Translocation speed can be reduced by adjusting run parameters such as voltage, salt composition, pH, temperature, and viscosity, to name a few. However, such adjustments have been unable to reduce translocation speed to a level that allows for single base resolution.

[0006] Stratos Genomics has developed a method called Sequencing by Expansion ("SBX") that uses a biochemical process to transcribe the sequence of DNA onto a measurable polymer called an "Xpandomer" (Kokoris et al., U.S. Pat. No.7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion"). The transcribed sequence is encoded along the Xpandomer backbone in high signal-to-noise reporters that are separated by ~10 nm and are designed for high-signal-to-noise, well-differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA. Xpandomers can enable several next generation DNA sequencing detection technologies and are well suited to nanopore sequencing.

[0007] The Sequencing by Expansion nucleic acid sequencing workflow uncouples the biochemical steps of synthesizing the Xpandomer from the downstream measurement steps, in which the entire Xpandomer molecule is passed through a nanopore sensor for base calling. These differences relative to traditional sequencing by synthesis workflowsintroduce SBX -specific requirements for certain pre-measurement steps, e.g., thepreparation of single stranded template for Xpandomer synthesis. While significantadvances have been made in this field, commercially viable implementation of SBX wouldAttorney Docket P39649-WO benefit from improvements that address the need, under certain circumstances, to provide single stranded DNA templates from a library of double stranded constructs. The present invention fulfills these needs and provides further related advantages as discussed below.

[0008] All of the subject matter discussed in the Background section is not necessarily prior art and should not be assumed to be prior art merely as a result of its discussion in the Background section. Along these lines, any recognition of problems in the prior art discussed in the Background section or associated with such subject matter should not be treated as prior art unless expressly stated to be prior art. Instead, the discussion of any subject matter in the Background section should be treated as part of the inventor’s approach to the particular problem, which in and of itself may also be inventive. BRIEF SUMMARY OF THE INVENTION

[0009] In one aspect, the invention provides a method for preparing a sample enriched for single stranded nucleic acid templates, including the steps of: a) providing a nucleic acid library construct, in which the nucleic acid library construct includes a double stranded nucleic acid library fragment joined on a first end and on a second end to a Y adapter; b) providing an asymmetric PCR reaction mixture, in which the asymmetric PCR reaction mixture includes a forward primer and a reverse primer, in which the forward and reverse primers are capable of hybridizing to the Y adapter, and in which one of the forward primer or the reverse primer is present in molar excess relative to the other primer; c) performing a PCR reaction using the nucleic acid library construct as a template; and d) collecting a sample enriched for single stranded nucleic acid templates, in which the single stranded acid templates include the sequence of the primer present in molar excess in the asymmetric PCR reaction. In one embodiment, the molar ratio of one primer to the other primer is from 1:20 to 1:40. In another embodiment, the asymmetric PCR reaction includes from 15 to 40 PCR cycles. In another embodiment, the asymmetric PCR reaction includes 7-deaza-dGTP.

[0010] In another aspect, the invention provides a method for preparing a sample enriched for single stranded nucleic acid templates, including the steps of: a) providing a nucleic acid library construct, in which the nucleic acid library construct includes a double stranded nucleic acid library fragment joined on a first end and on a second end to a Y adapter; b) providing a PCR reaction mixture, in which the PCR reaction mixture includes a forward primer and a reverse primer, in which the forward primer and the reverse primers are capable of hybridizing to the Y adapter, and in which one of the forward primer or the reverse primer includes a 5’ biotin moiety; c) performing a PCR reaction using the nucleic acidAttorney Docket P39649-WO library construct as a template to provide a sample of double stranded nucleic acid amplicons, in which one of the strands of the double stranded nucleic acid amplicons includes the 5’ biotin moiety; d) contacting the double stranded nucleic acid amplicons with streptavidin- coated beads to provide a sample of double stranded nucleic acid amplicons bound to streptavidin-coated beads; e) contacting the double stranded nucleic acid amplicons with denaturing conditions, in which the denaturing conditions separate the two strands of the double stranded nucleic acid amplicons; and f) collecting a sample enriched for single stranded nucleic acid templates, in which the single stranded nucleic acid templates lack the 5’ biotin moiety. In one embodiment, the primer including the 5’ biotin moiety includes two biotin moieties and one or more PEG6 spacer moieties. In another embodiment, the denaturing conditions include around 200mM NaOH.

[0011] In another aspect, the invention provides method for preparing a sample enriched for single stranded nucleic acid templates, including the steps of: a) providing a nucleic acid library construct, in which the nucleic acid library construct includes a double stranded nucleic acid library fragment joined on a first end and on a second end to a Y adapter; b) providing a PCR reaction mixture, in which the PCR reaction mixture includes a forward primer and a reverse primer, in which the forward primer and the reverse primer are capable of hybridizing to the Y adapter, and in which one or both of the forward primer and the reverse primer includes a phosphorthioate bond; c) performing a PCR reaction using the nucleic acid library construct as a template to provide a samples of double stranded nucleic acid amplicons, in which one or both of the strands of the double stranded nucleic acid amplicons includes a phosphorthioate bond; and d) contacting the sample of double stranded nucleic acid amplicons with a 5’ to 3’ exonuclease enzyme, in which the 5’ to 3’ exonuclease enzyme digests the 5’ end of a stand of the nucleic acid amplicons up to the position of the phosphothioate bond to produce a region of single stranded nucleic acid at the 3’ end of the opposite strand of the nucleic acid amplicons. In one embodiment, both the forward and the reverse primer include a phosphothioate bond. In another embodiment, the region of single stranded nucleic acid at the 3’ end of the opposite strand includes a binding site for an extension oligonucleotide. In another embodiment, the 5’ to 3’ exonuclease is a lambda exonuclease. In other embodiments, one of the forward and reverse primers includes the phosphorthioate bond, and in which the strand lacking the phosphorthioate bond is completely digested by the 5’ to 3’ exonuclease enzyme to produce a sample enriched for the single stranded nucleic acid templates.Attorney Docket P39649-WO

[0012] In another aspect, the invention provides a method for preparing a sample enriched for single stranded nucleic acid templates, including the steps of: a) providing a nucleic acid library construct, in which the nucleic acid library construct includes a double stranded nucleic acid library fragment joined on a first end and on a second end to a Y adapter and in which the heterologous nucleic acid adapter includes a nickase endonuclease cleavage site; b) providing an extension reaction mixture, in which the extension reaction mixture includes an extension oligonucleotide and a strand displacing nucleic acid polymerase, in which the extension oligonucleotide is capable of hybridizing to the Y adapter; c) performing an extension reaction using the nucleic acid library construct as a template, in which the extension reaction produces a double stranded nucleic acid product, in which the double stranded nucleic acid product includes a complementary copy of the nucleic acid library construct; d) contacting the double stranded nucleic acid product with a nicking endonuclease under endonuclease conditions, in which the nicking endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the complementary copy of the nucleic acid library construct; e) contacting the free 3’ end with the strand displacing nucleic acid polymerase under nucleic acid synthesis conditions, in which the strand displacing nucleic acid polymerase synthesizes a new complementary copy of the nucleic acid library construct, in which the new complementary copy of the nucleic acid library construct displaces the complementary copy of step c) from the double stranded nucleic acid product; and f) repeating steps d) and e) to provide an amplified population of single stranded nucleic acid templates, in which the single stranded nucleic acid templates includes the sequence of the complementary copy of the nucleic acid library construct. In one embodiment, the nickase endonuclease is selected from the group consisting of Nb. BbvCl, Nb. Bsml, Nt. BstNBI, Nt. BspQI, Nt. BspD61, Nt. Bst9I, Nt. BstSEI, Nt. BsmAI, Nt. AIwI, Nb. BsrD1, and Nt. CviPII, or variants thereof. In another embodiment, the strand displacing nucleic acid polymerase is a strand displacing DNA polymerase selected from the group consisting of Bst wildtype, Bst 2.0, Bst 3.0, Bsu, and Klenow fragment, or variants thereof. In another embodiment, the nickase endonuclease conditions and the DNA synthesis conditions are provided in the same reaction mixture. In some embodiments, the reaction mixture includes a nucleotide analog, in which the nucleotide analog comprises N4-Me dCTP or 7-deaza dGTP. In some embodiments, the reaction mixture includes one or more of a single stranded binding protein, a pyrophosphatase, a DPO4 polymerase or a variant thereof, and a translesion repair enzyme. In some embodiments, the extension reaction further includes a blocker primer, inAttorney Docket P39649-WO which the blocker primer hybridizes to the single stranded copies of the nucleic acid library construct, and in which the blocker primer provides an initiation site for nucleic acid synthesis. In one embodiment, which the blocker primer hybridizes to a sequence derived from the heterologous adapter. In further embodiments, the heterologous nucleic acid adapter of any of the methods disclosed herein is a Y adapter, and in which the Y adapter includes one or more of an SID and a UMI sequence. In certain embodiments, the double stranded nucleic acid library fragment of any of the methods disclosed herein includes a cDNA copy of single cell RNA (scRNA).

[0013] In another aspect, the invention provides a method of synthesizing an Xpandomer, including the steps of: a) providing the sample enriched for single stranded nucleic acid templates according to any of the above methods; b) providing an Xpandomer synthesis reaction mixture; and c) providing Xpandomer synthesis conditions. In one embodiment, the Xpandomer synthesis conditions include providing an extension oligonucleotide bound to a solid support, in which the extension oligonucleotide includes a sequence complementary to a sequence in the heterologous adapter of the double stranded nucleic acid constructs, and in which the single stranded template is capable of hybridizing to the extension oligonucleotide bound to the solid support. In some embodiments, the Xpandomer synthesis reaction mixture includes one or more of XNTP substrates, a variant of DPO4 polymerase, a manganese salt, a PEM, and a single stranded binding protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG.1 is a condensed schematic illustrating one embodiment of an Xpandomer synthesis process carried out on a solid support using a single stranded nucleic acid as a template.

[0015] FIG.2 is a condensed flowchart illustrating one embodiment of a method that incorporates a strand enrichment step with a library preparation process to enable Xpandomer synthesis.

[0016] FIG.3 is a simplified depiction of two exemplary embodiments of Y adapters.

[0017] FIG.4 is a condensed schematic summarizing one embodiment of a library and template preparation workflow for Xpandomer synthesis that includes a linear amplification step and means to prevent cross-hybridization of single stranded amplicons.Attorney Docket P39649-WO DETAILED DESCRIPTION OF THE INVENTION

[0018] 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.

[0019] 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).

[0020] 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.

[0021] 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.

[0022] Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” andAttorney Docket P39649-WO “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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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 toAttorney Docket P39649-WO 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.Sequencing by Expansion (SBX ) – Overview

[0027] The nucleic acid sequencing methodology compatible with the present invention is “Sequencing by Expansion” (SBX®), developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No.7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion", which is herein incorporated by reference in its entirety). Briefly, SBX® uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an “Xpandomer”. SBX® is based on the polymerization of highly modified, non- natural nucleotide analogs, referred to as “XNTPs”. XNTPs are expandable, 5' triphosphate modified nucleotide analogs compatible with template dependent enzymatic polymerization. The XNTP has two distinct functional regions; namely, a selectively cleavable phosphoramidate bond, linking the 5’ -phosphate to the nucleobase, and a symmetrically synthesized reporter tether (SSRT) that is attached within the nucleoside triphosphoramidate at positions that allow for controlled expansion by cleavage of the phosphoramidate bond. XNTPs are described in further details in Applicant’s U.S. Patent No.s 10,301,345 and 10,774,105, which are herein incorporated by reference in their entireties. The SSRT includes linkers separated by the selectively cleavable phosphoramidate bond. Each linker attaches to one end of a reporter code. XNTP substrates incorporated into daughter strand products of template-dependent polymerization are in the “constrained” configuration. The constrained configuration of polymerized XNTPs is the precursor to the expanded configuration, as found in Xpandomer products.

[0028] The transition from the constrained configuration to an expanded configuration results from cleavage of the selectively cleavable phosphoramidate bonds within the primary backbone of the daughter strand. In this embodiment, the SSRTs include one or more reporters or reporter codes, specific for the nucleobase to which they are linked, thereby encoding the sequence information of the template. In this manner, the SSRTs provide a means to expand the length of the Xpandomer and lower the linear density of the sequence information of the parent strand.Attorney Docket P39649-WO

[0029] The SSRT (i.e., “tether”) of the XNTP includes several distinct functional elements, or features, such as polymerase enhancement regions, reporter codes, and translation control element (TCEs). These features are discussed in further details in Applicant’s published PCT application WO2020 / 236526, which is herein incorporated by reference in its entirety. Each of these features performs a unique function during translocation of the Xpandomer through a nanopore to produce a series of unique and reproducible electronic signal. The SSRT is designed for controlling the rate of Xpandomer translocation by the TCE through a combination of sterics and / or electrorepulsion, Different reporter codes are sized to block ion flow through a nanopore at different measurable levels. In certain embodiments, reference is made to the “reporter construct” of the XNTP, which includes, from a proximal end to a distal end, the TCE, a symmetrical Y brancher, and two symmetric reporter code, each joined to an end of the Y brancher distal to the TCE. The reporter construct is a feature of the larger SSR structure.

[0030] To obtain sequence information, an Xpandomer is translocated through a nanopore, from the cis reservoir to the trans reservoir. As the Xpandomer translocates, a reporter enters the stem until its translocation control element stops at the stem entrance. The reporter is held in the stem until the TCE is enabled to pass into and through the stem, whereupon translocation proceeds to the next reporter. Upon passage through the nanopore, each of the reporter codes of the linearized Xpandomer generates a distinct and reproducible electronic signal, specific for the nucleobase to which it is linked.

[0031] In certain embodiments, Xpandomers produced by the SBX chemistry may be analyzed using a nanopore-based sequencing chip. A nanopore-based sequencing chip can incorporate a large number of sensor cells configured as an array. For example, the chip may include an array of one million cells configured in 1000 rows by 1000 columns of cells. Each cell in the array may include a control circuit integrated on a silicon substrate. Such nanopore-based sequencing chips, devices, and systems are described, e.g., in Applicant’s published patent application no. WO2021 / 219795, which is herein incorporated by reference in its entirety. The Xpandomer Synthesis Process

[0032] The Xpandomer molecule is a central features of Sequencing by Expansion.SBX is distinguished from many next generation sequencing methodologies in that thebiochemical steps of Xpandomer synthesis are uncoupled from the nanopore-basedAttorney Docket P39649-WO measurement steps. FIG.1 illustrates a simplified depiction of an Xpandomer synthesis process. In this embodiment, an Xpandomer synthesis reaction is carried out on a solid support, e.g., a flow channel surface. In other embodiments, the Xpandomer synthesis process may be carried out in solution. Here, flow channel surface 104 has been functionalized to enable covalent linkage (i.e., joining or association) of capture probes. In this embodiment, a capture probe may be an extension oligonucleotide to initiate Xpandomer synthesis. Significantly, extension oligonucleotides are capable of hybridizing to single stranded nucleic acid templates, or single stranded portions of double stranded templates. As shown in FIG.1, in step i) the flow channel surface may be functionalized with alkyne- maleimide linker 104a. The linker provides a terminal maleimide moiety that can be converted into a reactive group and subsequently crosslinked to the flow channel surface (e.g., a polyolefin substrate), via a catalyst-free photochemical proton abstraction reaction. Advantageously, an alkyne moiety provided by the end of the linker distal to the substrate is capable of chemically reacting with a polymer, e.g., an extension oligonucleotide, that includes a free azide group.

[0033] As further shown in FIG.1, step i) extension oligonucleotide 106 may be covalently joined to the flow channel surface via formation of triazole group 107 between the terminal alkyne moiety of linker 104a and a terminal azide moiety provided by extension oligonucleotide 106, via a conventional copper-catalyzed click reaction. Thus, in certain embodiments, an extension oligonucleotide may be conveniently “clicked” to an alkyne- functionalized flow channel surface.

[0034] In certain embodiments, the substrate-linked extension oligonucleotide may include the following features, from the 5’ to the 3’ direction: a polymeric spacer, a photocleavable moiety (e.g., derived from a photocleavable spacer, or modifier, phosphoramidite commercially available from Glen Research), a leader sequence (here depicted as 106a), a concentrator sequence (here depicted as 106b), and an oligonucleotide primer (here depicted as 106c).

[0035] As shown in FIG.1, in step ii), single stranded polynucleotide 108 is hybridized to extension oligonucleotide 106. The single stranded polynucleotide includes a 5’ sequence that is complementary to the sequence of oligonucleotide primer 106c. In this embodiment, single stranded polynucleotide 108 serves as a template for Xpandomer synthesis, which is initiated from primer 106c. In certain embodiments, the polynucleotide template includes a 5’ heterologous sequence, derived from, e.g., a conventional Y adapter, that provides theAttorney Docket P39649-WO sequence complementary to the oligonucleotide primer. The polynucleotide template may be provided in a hybridization solution compatible with nucleic acid hybridization, e.g., a solution including a buffer and a suitable concentration of salt. The polynucleotide template may be produced from a library of double stranded nucleic acid template constructs, as discussed further herein.

[0036] The Xpandomer synthesis process is illustrated in FIG.1, step iii). Xpandomer synthesis is initiated from the 3’ end of the oligonucleotide primer using polynucleotide strand 108 as a template, as discussed herein. An Xpandomer synthesis reaction includes a nucleic acid polymerase, e.g., a modified DNA polymerase, and XNTP substrates to synthesize an Xpandomer copy of polynucleotide template 208. The Xpandomer synthesis reaction is described in further detail herein. Following synthesis, the Xpandomer molecules are subjected to certain processing steps, including treatment with an acid solution to cleave the selectively cleavable phosphoramidate bonds in the XNTPs so as to transition the Xpandomer from the constrained to the elongated configuration.

[0037] As shown in step iv), after the Xpandomer extension reaction is complete, UV light can be used to cleave the photocleavable moiety in extension oligonucleotide 106, which releases Xpandomer molecule 110 from flow channel surface 104. A sample of released Xpandomers may be eluted from the flow cell for, e.g., downstream nanopore sequence determination. Library Preparation and Processing for Xpandomer Synthesis

[0038] In certain embodiments, the template used for Xpandomer synthesis is a single stranded DNA molecule, as depicted in FIG.1. As such, conventional library preparation workflows may require one or more additional steps to generate a sample enriched for single stranded DNA template molecules. FIG.2 illustrates an exemplary workflow that includes a step to enrich for single stranded DNA templates, following the initial steps to prepare the DNA library.

[0039] Here, in step 201, the general method typically begins with DNA from a biological sample. The DNA obtained or provided from the biological sample may be genomic DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), cDNA or a combination thereof.

[0040] 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,Attorney Docket P39649-WO 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.

[0041] 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.

[0042] In some embodiments, the DNA sample is circulating cell-free DNA (cfDNA), which is DNA found in the blood and is not present within a cell. cfDNA can be isolated from blood or plasma using methods known in the art. Commercial kits are available for isolation of cfDNA including, for example, the Circulating DNA Kit (Qiagen). The DNA sample may result from an enrichment step, including, but is not limited to antibody immunoprecipitation, chromatin immunoprecipitation, restriction enzyme digestion-based enrichment, hybridization-based enrichment, or chemical labeling-based enrichment.

[0043] In step 203, 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. 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 beAttorney Docket P39649-WO 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.

[0044] 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 non- specific 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.

[0045] 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.

[0046] 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 moreenzyme 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 DNApolymerase).Attorney Docket P39649-WO

[0047] 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’ deoxynucleotideis then added to both 3 ends of the DNA molecules using Taq polymerase or Klenow exominus polymerase enzyme, producing a one-base 3 overhang that is complementary to theone-base 3 ‘T’ overhang on the double-stranded end of an adaptor. These steps arecommonly referred to in the art as ERAT.

[0048] In certain embodiments, a DNA sample may be generated from an RNA sample extracted from a single cell, e.g., for single cell RNA (scRNA) sequencing. Methods for single cell RNA sequencing are known in the art and typically include the steps of preparation of a suspension of single cells, isolation of single cells, cell lysis, reverse transcription of RNA into cDNA, cDNA amplification, and library preparation.

[0049] In certain embodiments, a tissue sample is processed into a single cell suspension using a system that can utilize mechanical and enzymatic or chemical processes on a solid or liquid tissue sample and thus process the sample into single cells, nuclei, organelles, and biomolecules. In some embodiments, the tissue processing system performs affinity or other purifications to enrich or deplete cell types, organelles such as nuclei, mitochondria, ribosomes, or other organelles, or extracellular fluids. As used herein, the term “tissue” refers to any biological specimen obtained from any source such as a human, animal, or plant tissue. Examples of tissues include, without limitation, a biopsy sample, a cellular conglomerate, an organ fragment, whole blood, bone marrow, a fine needle aspirate, or any other solid, semi-solid, gelatinous, frozen or fixed three dimensional or two dimensional cellular matrix of biological origin.

[0050] In certain embodiments, a single cell suspension may be obtained enzymatically using trypsin or papain to digest proteins connecting cells in tissue samples or releasing adherent cells in culture, or mechanically separating cells in a sample. Single cells can be placed in any suitable reaction vessel in which single cells can be treated individually. For example, a 96-well plate, 384-well plate, or a plate with any number of wells such as 1000, 2000, 4000, 6000, 10000 or more. The multi-well plate can be part of a chip and / or device. For example, a square chip may include 125 by 125 nano-wells, with a diameter of 0.1 mm.

[0051] According to some embodiments, single cells can be isolated using a microfluidic device that includes a droplet generator. For example, a population of single cells may be flowed through a channel of a microfluidic device, the microfluidic device including a dropletAttorney Docket P39649-WO generator in fluid communication with the channel, under conditions sufficient to effect inertial ordering of the cells in the channel, thereby providing periodic injection of the cells into the droplet generator to encapsulate single cells in individual droplets. In some embodiments, the method of encapsulating single cells in droplets includes the addition of an immiscible phase fluid, e.g., oil, to generate an emulsion of droplets each containing a single cell. Additional description of cell encapsulation using microfluidic droplet generators is found, e.g., in U.S. Patent Application Publication No.20150232942.

[0052] According to some embodiments, the cells are isolated using Fluorescence activated cell sorting (FACS) or Flow cytometry. According to some embodiments, the cells are isolated using micropipetting or micromanipulation. According to additional embodiments, the cells are isolated using microscope-guided capillary pipettes, or by other standard means.

[0053] Following isolation of single cells, mRNA can be released from the cells by lysing the cells. Lysis can be achieved by, for example, heating or freeze-thaw of the cells, or by the use of detergents or other chemical methods, or by a combination of methods. However, any suitable lysis method can be used. A mild lysis procedure can advantageously be used to prevent the release of nuclear chromatin, thereby avoiding genomic contamination of the cDNA library, and to minimize degradation of mRNA. For example, heating the cells at 72° C. for 3 minutes in the presence of triton x100 is sufficient to lyse the cells while resulting in no detectable genomic contamination from nuclear chromatin. Alternatively, cells can be heated to 65° C. for 10 minutes in water or 70° C. for 90 seconds in PCR buffer II (Applied Biosystems) supplemented with 0.5% NP-40; or lysis can be achieved with a protease such as Proteinase K or by the use of chaotropic salts such as guanidine isothiocyanate.

[0054] mRNA purification can be achieved by any method known in the art, for example, by binding the mRNA to a solid phase. Commonly used purification methods include magnetic or paramagnetic beads (e.g., of Dynabeads® BcMag®, and MagaCell®). Alternatively, specific contaminants, such as ribosomal RNA can be selectively removed using affinity purification.

[0055] A reverse transcription (RT) reaction may include several components to synthesize complementary DNA (cDNA) from an RNA template. These include the RNA template, a reverse transcriptase enzyme, primers, deoxynucleotide triphosphates (dNTPs), buffer, and potentially other factors like RNase inhibitors. Commonly used reverse transcriptase enzymes include AMV or M-MLV reverse transcriptase.Attorney Docket P39649-WO

[0056] In some embodiments, the step of reverse transcription uses RT primers including, in certain embodiments one or more of poly dTs, cell barcode, UMI, and primer binding sequences. The poly dT stretch is designed to prime the reverse transcriptase at the poly A tail of the mRNA molecules. The cells' barcodes are a domain that uniquely identifies the sample source of the nucleic acid being sequenced to enable sample multiplexing by marking every molecule from a given sample (e.g. a single cell within a well) with a specific barcodeor “tag”. The primer binding sequence, located at the 5 end of the reverse transcriptionprimer, are primers used for amplification following reverse transcription. Other application dependent features may be included in the RT primer, depending on particular applications.

[0057] The cDNA products of reverse transcription may then be amplified by a PCR reaction to provide a sample of double stranded DNA for subsequence library prep.

[0058] In step 205, in some embodiments, the methods described herein include the step of providing adapter DNA molecules for ligation 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 endor a 5 or 3 overhang (i.e., sticky ends). DNA adapters are ligated to (i.e., joined to) the DNAtarget 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).

[0059] 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 that enable, e.g., PCR or linear amplification of a DNA construct or library of constructs.Attorney Docket P39649-WO

[0060] 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.

[0061] 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.

[0062] 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.

[0063] The adapter may be formed by annealing, or hybridizing, two single-stranded oligonucleotides prepared by conventional automated oligonucleotide synthesis. Theoligonucleotides are partially complementary such that the 3 end of a first oligonucleotide iscomplementary to the 5 end of a second oligonucleotide. The 5 end of the firstoligonucleotide and the 3 end of second oligonucleotide are not complementary to eachother. When the two strands are annealed, the resulting structure is double stranded at one end (the double-stranded region) and single stranded at the other end (the unmatched region)Attorney Docket P39649-WO and is referred to herein as a “Y-shaped adapter”. The double-stranded region of the Y- shaped adapter may be blunt-ended or it may have an overhang. In the latter case, theoverhang may be a 3 overhang or a 5 overhang, and may comprise a single nucleotide ormore than one nucleotide. The Y-shaped adapter is phosphorylated at its 5 end and thedouble-stranded portion of the duplex contains a single base 3 overhang comprising a ‘T’deoxynucleotide. The adapters are then ligated using T4 Ligase, rATP, to the ends of doublestranded template molecules containing a single base 5 overhand of an ‘A’ nucleotide.

[0064] 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.

[0065] In certain embodiments, a Y adapter may be a “YSU” adapter that includes one or more primer hybridization sequences, one or more nickase recognition and cleavage sequences, a UMI sequence, and a SID sequence. In other embodiments, a Y adapter may be a “YS” adapter that includes one or more primer hybridization sequences, one or more nickase recognition sequences, and a SID sequence. YSU adapters are disclosed in Applicant’s published PCT application no. WO / 2025 / 132779, which is hereby incorporated by reference in its entirety.

[0066] Non-limiting examples of these adapter configurations are illustrated in FIG.3. In certain embodiments, YS adapter 310 may include one or more of the following features: extension oligonucleotide hybridization sequence 311, here positioned in the 5’ single stranded arm of the Y adapter; amplification primer hybridization sequence 312, here positioned in the 3’ single stranded arm of the Y adapter; SID sequence 313, here positioned in the double stranded stem region of the Y adapter; first nickase site 314, here positioned in the 3’ single stranded arm of the Y adapter; Xpandomer synthesis runway sequence 315, here positioned in the 5’ single stranded arm of the Y adapter. The Xpandomer runway sequence is the first sequence that is copied into the Xpandomer and is designed to facilitate initiation of Xpandomer synthesis by the polymerase. In certain embodiments, the sequence includes bases that are easily recognized and copied by the polymerase. In one embodiment, the Xpandomer runway sequence may be CAACAA or a variant thereof. The YS adapter mayAttorney Docket P39649-WO further include blocker / cap hybridization sequence 316, here positioned in the 3’ single stranded arm of the Y adapter.

[0067] In certain embodiments, YSU adapter 320 may include one or more of the following features: extension oligonucleotide hybridization sequence 321, here positioned in the 5’ single stranded arm of the Y adapter; amplification primer hybridization sequence 322, here positioned in the 3’ single stranded arm of the Y adapter; SID sequence 323, here positioned in the double stranded stem region of the Y adapter; first nickase site 324, here positioned in the 3’ single stranded arm of the Y adapter; second nickase site 325, here positioned in the 5’ single stranded arm of the Y adapter; UMI sequence 326, here positioned in the double stranded stem region of the Y adapter; Xpandomer synthesis runway sequence 327, here positioned in the 5’ single stranded arm of the Y adapter; blocker / cap hybridization sequence 328, here positioned in the 3’ single stranded arm of the Y adapter; and capping runway sequence 329, here positioned in the 3’ single stranded arm of the Y adapter. The capping runway sequence is the last sequence that is copied by the polymerase into the Xpandomer and is designed to facilitate joining of the blocker oligonucleotide to the Xpandomer by the polymerase. In certain embodiments, the capping runway sequence may be AAA or the like.

[0068] In step 207, “ligation” of adapters to the 5 and 3 ends of each fragmented doublestranded 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.

[0069] In some instances, the adapters and DNA target fragments may be incubated with a ligase to covalently link (i.e., join) 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. coliAttorney Docket P39649-WO 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.

[0070] 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.

[0071] The ligation of adapters to both free ends of the double stranded DNA target (e.g., library) fragments gives rise to a pool of adapter-ligated DNA target (e.g., library) fragments with adapters at the 5’ and 3’ ends (i.e., a first end and a second end) of the target insert. The adapter-ligated DNA target fragments may be interchangeably referred to herein as nucleic acid library constructs, double stranded library constructs, or double stranded target fragments. The skilled artisan will recognize that, in certain embodiments, the insert is a mostly double stranded nucleic acid molecule, while the terminal heterologous adapters include a double stranded stem region ligated to the insert and two single stranded arm regions.

[0072] In step 209, in some embodiments, the adapter ligated library fragments may be optionally amplified. The expression “amplification” or “amplifying” refers to a process by which extra or multiple copies of a particular polynucleotide are formed. Amplification includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are known and widely practiced in the art. See, e.g., U.S. Pat. Nos.4,683,195 and 4,683,202 and Innis et al., “PCR protocols: a guide to method and applications” Academic Press, Incorporated (1990) (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific sequences within a DNA sample (or library), e.g., sequences present in a single stranded arm region of a Y adapter, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0073] In some embodiments, primers have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skillAttorney Docket P39649-WO in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2ndEdition (Cold Spring Harbor Press, New York, 2003).

[0074] Inclusion of PCR amplification to form complementary copies of the adapter- target constructs is advantageous, for several reasons. Firstly, inclusion of the primer extension step, and subsequent PCR amplification, acts as an enrichment step to select for adapter-target constructs with adapters ligated at both ends, especially in the case of methods of the disclosure, as non-desired transcripts are not amplified in the PCR reaction. Only target constructs with adapters ligated at both ends provide effective templates for PCR using common or universal primers specific for primer-binding sequences in the adapters, hence it is advantageous to produce a template library comprising only double-ligated targets prior to PCR amplification.

[0075] In step 211, in certain embodiments, the amplified library may be optionally subjected to one or more target enrichment steps. In general terms, target enrichment enables, e.g., targeted sequencing of just the coding regions or specific genes or segments of chromosomes that are relevant to a particular disease. With this approach, the rest of the whole genome can be disregarded, simplifying downstream bioinformatics analysis and making it more efficient and affordable.

[0076] Various target enrichment methods are known in the art, including hybridization capture based target enrichment and primer extension based target enrichment (see, e.g., the KAPA HyperCap work-flow and the KAPA HyperPETE work-flow, both commercially available from Roche Sequencing Solutions). Dual primer extension based target enrichment (PETE) is described in greater detail in Applicant’s U.S. Patent No.11,773,388, the contents of which are herein incorporated by reference in their entirety.

[0077] In step 213, in certain embodiments, the enriched library constructs may optionally be subjected to a second amplification step. In some embodiments, the second amplification step may be a second PCR amplification step, as discussed with reference to step 209. The second PCR amplification step increases the amount of enriched library construct DNA and, in some cases, is essential to obtain enough coverage for reliable sequencing for samples with small amounts of starting material.

[0078] In step 215, a sample enriched for single stranded DNA is produced from the library of double stranded constructs. There are several standard methods for separating the strands of a double stranded DNA fragment, e.g., by denaturation, including thermalAttorney Docket P39649-WO denaturation, or chemical denaturation, e.g., in either around 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 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.

[0079] Under certain conditions, the inventors have found that strand enrichment methods based on simple denaturation of double stranded constructs have certain disadvantages in providing single stranded nucleic acid (e.g., DNA) templates for Xpandomr synthesis. For example, when double stranded DNA is denatured, even though the single strands may be capable of hybridizing to the extension oligonucleotide, as desired, the remainder of the single strand may also hybridize to its complement strand present in the sample. This has been observed to arrest extension of the Xpandomer molecule, possibly through formation of regions of secondary or other structures that impede progression of the DNA polymerase. In short, it was found that samples of denatured double stranded constructs are too complex to efficiently enable synthesis of the Xpandomer product (i.e., step 217 of the method of FIG.2). Based on the foregoing, the inventors have devised several alternative methods for template strand enrichment for Xpandomer synthesis, which are described in further detail below. Alternative Methods of Strand Enrichment and Xpandomer Template Preparation

[0080] I. Asymmetric PCR (aPCR).

[0081] Asymmetric PCR is known in the art as a variation of the polymerase chain reaction (PCR) that amplifies one strand of DNA more than another. As used herein, aPCR is used to create single-stranded DNA from double stranded DNA, which is then used as a template in the Xpandomer synthesis reaction, as discussed with reference to FIG.1. Asymmetric PCR is a convenient way to generate single stranded DNA template molecules because it doesn't involve physical, chemical, or enzymatic denaturation.

[0082] In certain embodiments, asymmetric PCR uses different concentrations of primers for amplification, usually in molar ratios of 1:5 to 1:100. In the early stages of amplification, both primers are available, and amplification occurs exponentially, producing double- stranded amplicons (ds-amplicons). As the PCR reaction progresses, the lower concentration limiting primer is used up, and the excess primer creates linear synthesis of the targeted single-stranded DNA.Attorney Docket P39649-WO

[0083] In certain embodiments, strand (i.e., template) enrichment by aPCR may include the following steps:

[0084] (a) treating a nucleic acid with deoxyribonucleaside-5’-triphosphates, an agent for polymerization, and a pair of oligonucleotide primers under hybridization conditions such that an extension product of a first primer of the primer pair is synthesized that is complementary to a nucleotide sequence in the nucleic acid, in which the extension product of the first primer can serve as a template for synthesis of an extension product of a second primer of the pair; (b) denaturing the extension products of the primers formed in step (a) from the templates on which they were synthesized; and (c) treating the products of step (b) with the primers and under conditions of step (a), in which one of the first and second primers is present in limiting concentrations. The single stranded DNA generating in the reaction is the extension product of the primer present in the highest concentration.

[0085] As mentioned, depletion of the limiting primer during the exponential amplification results in the linear synthesis of the strand extended from the excess primer. As such, aPCR requires optimization to identify the proper primer ratios, the amounts of starting material, and the number of amplification cycles that can generate a reasonable amount of template for Xpandomer synthesis.

[0086] In certain embodiments, the molar ratio for aPCR primers may be from 1:2 to 1:100, from 1:5 to 1:75, from 1:10 to 1:50, around 1:40, around 1:25, around 1:20, or around1:10. For example, in one embodiment, an aPCR reaction may include 0.5 M of one primerand 10 M of the other primer.

[0087] In certain embodiments, the template input may be double stranded DNA amplicons produced in a first amplification reaction (e.g., step 209 of FIG.2). In some embodiments, the template input may be from 5000pg to 0.5pg, from 2500pg to 10pg, from 100pg to 20pg of amplicon (e.g., amplified ligated library constructs). In some embodiments, the template input may be 0.5pg or less of ligated library constructs. In other embodiments, the product of the ligation reaction (e.g., step 207 of FIG.2) may be directly used as the template for aPCR, without having first subjection the ligates to a first PCR reaction (e.g., step 209 of FIG.2). In this embodiment, no target enrichment step (e.g., step 211 of FIG.2) is performed prior to aPCR.

[0088] In certain embodiment, the number of cycles run for an aPCR amplification may be from 10 to 50, from 15 to 40, or around 20 or fewer cycles. In one embodiment the number of cycles may be around 40 cycles.Attorney Docket P39649-WO

[0089] In some embodiments, the aPCR reaction may include alternative nucleotide substrates. In certain embodiments, the aPCR reaction may include 7-deaza-2’-deoxy- dGTP (to replace all or some of natural dGTP) or diaminopurine (to replace all or some of natural dATP). In some embodiments, the aPCR reaction may include equimolar concentrations of 7-deaza-2’-deoxy-dGTP and natural dGTP. In other embodiments, the aPCR reaction may include an excess concentration of 7-deaza-2’-deoxy-dGTP relative to dGTP. In other embodiments, the aPCR reaction may include an excess concentration of dGTP relative to 7- deaza-2’-deoxy-dGTP.

[0090] In one exemplary embodiments, a 50 L aPCR reaction may include the followingreagents: 0.5pg amplicon input; 10 M forward primer; 0.5 M reverse primer; 0.02U / L ofpolymerase (e.g., KAPA HiFi HotStart, commercially available from Roche Sequencing); and 1X KAPA HiFi buffer (100mM Tris-SO4, 80mM TMAC, 2.5mM MgCl2, 0.04% gelatin, 0.3mM dNTPs with 50% 7-deaza dGTP, 6% glycerol, 1mM Tris base, 1mM KCl, 0.004%Detergent N, and 2 M EDTA). The aPCR reaction may include the following steps: a) initialdenaturation at around 98 degrees C. for around 45 seconds; b) 30 cycles of denaturation at around 98 degrees C for around 25 seconds, annealing at around 64 degrees C.for around 30 seconds, and extension at around 72 degrees C. for around 60 seconds; and c) a final extension at around 72 degrees C. for around 60 seconds.

[0091] In certain embodiments, the products of an aPCR reaction may be verified by conventional gel electrophoretic techniques.

[0092] In certain embodiments, the crude aPCR reaction may be added directly into the Xpandomer synthesis reaction workflow, as described with reference to FIG.1. In other embodiments, the crude aPCR reaction may first be subjected to one or more nucleic acid purification steps, using, e.g., the KAPA Hyper beads, as described herein, prior to the SBX synthesis reaction.

[0093] In some embodiments, the products of the aPCR reaction may be mixed with a blocker oligonucleotide prior to hybridization with the extension oligonucleotide. In certain embodiments, a molar excess of blocker may be added, e.g., an excess of 2pmol to 20pmol blocker oligonucleotide.

[0094] In certain embodiments, the products of the aPCR reaction and blocker oligonucleotide may be added to a hybridization buffer. An exemplary hybridization buffer may include a buffer, e.g., 50mM Tris HCl; a salt, e.g., 500mM NaCl or NH4OAc; 2% PEG, and IM urea. In some embodiments, the aPCR reaction may be quenched with a bufferAttorney Docket P39649-WO including EDTA, proteinase K, and SDS and heated at up to around 90 degrees C. for around 10 minutes prior to adding the reaction to a hybridization buffer.

[0095] II. Amplicon Immobilization Melt (AIM)

[0096] According to this method, a sample enriched for single stranded DNA is prepared by melting double stranded PCR amplicons. As such, a sample of PCR amplicons is immobilized on a solid support via attachment of one of the strands to the support, while the other strand remains unattached. When the immobilized amplicons are treated with melting conditions, the strand that is unattached from the support is released into solution and may be collected as a sample enriched for single stranded templates. As used herein, the term “melt” refers to the process of separating a double stranded DNA fragment into single strands, which can be achieved by, e.g., base-mediated denaturation or heating a sample of double stranded DNA.

[0097] In certain embodiments, strand (i.e., template) enrichment by AIM may include the following steps: (a) performing a PCR amplification reaction on a sample of double stranded DNA constructs with a pair of primers, in which one of the primers includes a 5’ biotin moiety; (b) performing a purification step to isolate the amplicons produced in step (a) from, e.g., free primers; (c) binding the isolated amplicons to streptavidin-coated beads; (d) contacting the bead-bound amplicons with denaturing (i.e., “melt”) conditions to separate the two strands of each amplicon and produce a solution enriched for single stranded DNA molecules, in which the single strands lack a 5’ biotin moiety; (e) purifying single stranded DNA molecules from the solution.

[0098] In certain embodiments, the pair of primers includes a forward primer that lacks a 5’ biotin moiety and a reverse primer that includes a biotin moiety. In some embodiments, the reverse primer includes more than one biotin moiety at the 5’ end. In another embodiment, the reverse primer includes two or more biotin moieties at the 5’ end. In other embodiments, the reverse primer includes one or more PEG 6 spacer moieties interposed between the 5’ biotin moiety and the primer sequence. In some embodiments, the reverse primer includes three PEG 6 spacer moieties interposed between two biotin moieties at the 5’ and the primer sequence. In other embodiments, the bond between the ultimate and pentultimate nucleotides of the forward primer is a phosphorothioate bond. In certain embodiments, the pair of primers includes a forward primer that includes a 5’ biotin moiety and a reverse primer that lacks a biotin moiety.Attorney Docket P39649-WO

[0099] In certain embodiments, an exemplary PCR reaction may include the followingreagents: around 3pg of DNA template; around 1 M of each primer (F and R); a PCR mastermix, e.g., HiFi HotStart Master Mix, including a DNA polymerase, commercially available from Roche Sequencing; and around 5mM EDTA.

[0100] In certain embodiments, an exemplary PCR amplification step to produce amplicons of a template with a length of around 437 base pairs may include the following series of sub-steps: (a) an initial denaturation step at around 95 degrees C. for around two minutes; (b) around 33 cycles of: denaturation at around 95 degrees C. for around 10 seconds; annealing at around 67 degrees C. for around 20 seconds; and extension at around 72 degrees C. for around 30 seconds; and (c) a final extension step at around 72 degrees C. for around 3 minutes. It is to be understood that these conditions are exemplary in nature and are intended to illustrate one embodiment of producing amplicons and are not intended to be limiting in any fashion. One of skill in the art will recognize that PCR conditions need to be optimized based on many factors, including the length and nucleotide composition of the DNA template.

[0101] In certain embodiments, an exemplary purification step to isolate PCR amplicons may include the following series of sub-steps: (a) adding around an equal volume of KAPA HyperPure beads, commercially available from Roche Sequencing, to the crude PCR reaction and incubating at room temperature for around 5 minutes; (b) placing the sample tube on a magnet and removing the supernatant; (c) washing the beads twice with around 80% ethanol;(d) allowing the beads to nearly dry; (e) resuspending the beads around 15 L of TE andincubating for around 2 minutes at room temperature; and (f) pelleting the beads and removing the supernatant to a fresh tube.

[0102] In certain embodiments, the step of binding the isolated amplicons to streptavidin- coated beads may include the following series of sub-steps: (a) providing a sample of streptavidin-coated beads, e.g., MyOne T1 streptavidin beads commercially available fromThermo Fisher Scientific; (b) adding around 25 L of MyOne T1 beads for every microgramof pure PCR product to the sample of purified amplicons and gently agitating the sample for around 30 minutes at room temperature; (c) collecting the beads on a magnet and discarding the supernatant; (d) washing the beads in a wash buffer; and (e) pelleting the beads and discarding the supernatant.

[0103] In certain embodiments, the step of contacting the bead-bound amplicons with denaturing (e.g., “melt”) conditions to separate the two strands of each amplicon and produceAttorney Docket P39649-WO a solution enriched for single stranded DNA molecules in which the single strands lack a 5’ biotin moiety may include the following series of sub-steps: (a) adding an equal volume of 200mM NaOH to the amplicon bound streptavidin-coated beads; (b) vortexing the sample for 4 seconds every 2 minutes for a total of 10 minutes; (c) pelleting the beads and removing the supernatant containing the melted-off sample of single DNA strands to a fresh tube; and (d) adding an equal volume of around 200mM Tris, pH 8.0 to neutralize the solution.

[0104] In certain embodiments, the step of purifying the single stranded DNA molecules from the solution may include the following series of sub-steps: (a) adding around 3x volume of KAPA pure beads to the neutralized solution and incubating at room temperature for around five minutes; (b) collecting the beads on a magnet and discarding the supernatant; (c) washing the beads twice with around 80% ethanol; (d) letting the beads dry at room temperature; (e) resuspending the beads in 1X TE and incubating at room temperature for around 2 minutes; and (f) pelleting the beads and removing the supernatant containing the sample of single stranded DNA templates to a fresh tubes.

[0105] III. Exonuclease-Mediated Strand Digestion

[0106] The embodiments described herein are based on the use of partial or complete exonuclease-mediated digestion of at least one strand of a double stranded library fragment to produce a single stranded region for hybridization of the SBX extension oligonucleotide. Such protocols offer many benefits, including a reduction in template loss and removal of time-consuming purification steps. Exonuclease digestion of one strand of a double stranded library fragment and can, in certain embodiments, be partial removal of the 5’ end of one or both strands of an double stranded DNA construct or complete removal of one strand in the 5’ to 3’ direction. The degree of partial digestion (e.g., the number of nucleotides removed) and the strand targeted for digestion can be controlled as described below.

[0107] Examples of 5' to 3' exonucleases include: T5 exonuclease, Lambda exonuclease, E. coli DNA polymerase I (partially), Taq DNA polymerase (which has a 5' to 3' exonuclease domain), and the exonuclease activity associated with the FEN-1 protein; these enzymes all cleave nucleotides from the 5' end of a DNA strand, removing them one at a time.

[0108] In one embodiment of the present invention, the double stranded DNA construct is subject to partial 5’ to 3’exonuclease mediated digestion. This process is referred to herein as “chew-back”. According to one aspect of this embodiment, internal phosphorothiate (Ps) bonds are introduced into the double-stranded DNA construct during PCR amplification with one or more modified PCR primers. This places the phosphorothiate bonds near the 5’ end ofAttorney Docket P39649-WO one or both strands of the construct amplicon. The phosphorothioate (PS) bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligo. This modification renders the internucleotide linkage resistant to nuclease degradation.^

[0109] The length of the chew-back region will be determined by the positioning of the modified phosphorothiate nucleotides within the PCR primers. Exonuclease digestion of the Ps modified amplicons (with, e.g., lambda exonuclease) commences at the 5’ end of the amplicon strands and, importantly, terminates when the exonuclease reaches the Ps bonds. The digested amplicon product thus provides free single-stranded regions at the 3’ end of each strand for hybridization of an extension oligonucleotide.

[0110] In some embodiments, the forward and / or reverse PCR primer may be designed with the following sequence: 5’ Xn1Yn2 Zn33’, where X is an oligonucleotide sequence in which consecutive nucleotides are joined by phosphodiester bonds, Y is an oligonucleotide sequence in which consecutive nucleotides are joined by phosphorothiate bonds and Z is an oligonucleotide sequence in which consecutive nucleotides are joined by phosphodiester bonds. In some embodiments n1 may be from around 10 to around 50, from around 15 to around 45, from around 20 to around 40, or from around 25 to around 35 nucleotides. In some embodimentsn2may be from around 2 to around 5 nucleotides, from around 3 to around 4 nucleotides, or around 3 nucleotides. In some embodiments,n3may be from around 5 to around 30, from around 7 to around 25, from around 9 to around 20, or from around 10 to around 15 nucleotides.

[0111] In some embodiments, the 5’ ends of both strands of the construct amplicons are phosphorylated. This is advantageous when both strands are intended to be digested by the 5’ to 3’ exonuclease.

[0112] In certain embodiments, one PCR primer may include a modification at the 5’ end that renders one strand resistant to exonuclease digestion. For example, one primer may include a 5’ fluorescent moiety, such as a SIMA dye.

[0113] In other embodiments, one complete strand of a double stranded library construct amplicon may be digested by an exonuclease, while the other strand remains intact to serve as a template for synthesis of the Xpandomer. This is referred to herein as “strand selective exonuclease” treatment. Any suitable 5’ – 3’ single stranded exonuclease, e.g., lambda exonuclease may be used for the present method. In one embodiment, the 5’ end of one strand of an amplicon is phosphorylated, while the other strand lacks a 5’ phosphate. The strand lacking the 5’ phosphate will be resistant to 5’ – 3’ exonuclease activity and may beAttorney Docket P39649-WO retained as a single stranded template. Strand selective 5’ phosphorylation may be achieved by art-recognized techniques, e.g., 5’ phosphorylation of one of either the forward or reverse PCR primer.

[0114] 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. IV) Linear / Isothermal Amplification

[0115] Linear amplification, also be referred to herein as isothermal amplification, is well known in the art (see, e.g., Joneja and Huang.2011. Anal. Biochem. July 1; 414(1):58-69, the entire contents of which is herein incorporated by reference in its entirety). Linear amplification replicates a nucleic acid template in a non-exponential manner. Accordingly, the original template strand (i.e. the parental strand) serves as a template for all subsequent replication events, while the newly synthesized complementary copy strands (i.e. the daughter strands) are not replicated as part of the template amplification process. Advantageously, linear amplification prevents errors in replication from being exponentially propagated into the progeny population of complementary copy strands. Linear amplification is also advantageous in that it is not limited by constraints of thermal cycling well known in the art, e.g., dependence on heat-resistant DNA polymerases. In particular, many of the heat- resistant DNA polymerase used for conventional PCR do not possess robust strand displacement activity.

[0116] In certain embodiments, the methods of the present invention may be used for the linear amplification of any of the DNA libraries or constructs disclosed herein to provideAttorney Docket P39649-WO templates for Xpandomer synthesis. Certain embodiments of linear amplification methods used to prepare templates for Xpandomer synthesis are disclosed in Applicant’s U.S. provisional patent application no.63 / 680112, filed August 7, 2024, which is herein incorporated by reference in its entirety.

[0117] In certain embodiments, the methods of the present invention for linear amplification employ nicking endonuclease-enabled strand displacement by a DNA polymerase. In certain embodiments, the methods include the following steps: (1) a nicking enzyme recognizes and cleaves a specific site in a single strand of a double stranded region of DNA, creating a free 3’ end; (2) a strand displacing DNA polymerase extends a new strand from the newly created free 3’ end, the new strand including the recognition site for the nicking enzyme; (3) the non-template strand is displaced from the template strand by the nascent strand, e.g., the non-template strand is displaced as a free single strand in solution; (4) the newly synthesized strand includes the renewed nickase site for subsequent rounds of amplification using the original template strand as the template for the repeated cycles of nicking and extension.

[0118] As used herein, “nicking” refers to the cleavage of only one strand of a double- stranded portion of a fully or partially double-stranded nucleic acid. The position where the nucleic acid is nicked is referred to as the nicking site or nickase cleavage site. The recognition sequence that the nicking enzyme recognizes is referred to as the nicking enzyme binding or recognition site. “Capable of nicking” refers to an enzymatic capability of a nicking enzyme.

[0119] As used interchangeably herein, a nicking enzyme, a nickase, or a nicking endonuclease is a protein that binds to double-stranded DNA and cleaves one strand of a double-stranded duplex. The nicking enzyme may cleave either upstream or downstream of the binding site, or nicking enzyme recognition site. When used in connection with a nicking endonuclease, the letters “Nt” or “Nb” indicate whether it is the top (Nt) or bottom (Nb) strand that is cut by the enzyme. In certain embodiments, a suitable nickase will be one that has a relatively long recognition site in order to minimize the occurrence of cleavage at undesired sites in the library fragment. In other embodiments, single stranded cleavage may be achieved by use of a CRISPR- cas enzymatic system.

[0120] In certain embodiments, the nicking enzyme may be selected from the group consisting of one or more of the nicking enzymes listed in Table 1. Those of ordinary skill in the art will recognize that various nicking enzymes other than those mentioned specificallyAttorney Docket P39649-WO herein may be used in the present methods. Nicking enzymes are available from many commercial sources, for example, New England Biolabs (NEB). In certain embodiments, a suitable nicking enzyme is Nt.BspQ1, commercially available from NEB.

[0121] In other embodiments, a suitable nicking enzyme may be a variant of any of the enzymes set forth in Table 1. In certain embodiments, the suitable variant may have from around 95% to around 99% identity, from around 90% to around 95%, from around 85% to around 90%, from around 80% to around 85% or less than around 80% identity to an enzyme set forth in Table 1. Table 1 Exemplary Nicking Enzymes Enzyme Recognition sequence Isolation source 3’- GGAGTXCG-5’ Bacillus brevis strain C Nb. BbvCI 3’-CTTACXGN-5’ Bacillus stearothermophilus NUB 36 Nb. BsmI 5’-GAGTCNNNNVN-3’ Bacillus stearothermophilus Nt. BstNBI 5’-GCTCTTCNVN-3’ Bacillus sp. strain Q Nt. BspQI 5’-GAGTCNNNNVN-3’ Bacillus sp. strain D6 Nt. BspD6I 5’-GAGTCNNNNVN-3’ Bacillus stearothermophilus strain 9 Nt. Bst9I 5’-GAGTCNNNNVN-3’ Bacillus stearothermophilus Nt. BstSEI 5’-GTCTCNVN-3’ Bacillus stearothermophilus A664 Nt. BsmAI 5’-GGATCNNNNVN-3’ Engineered endonuclease Nt. AlwI 5’-GCAATGXNN-3’ Bacillus stearothermophilus D70 Nb. BsrDI 5’-CXCD-3’ Chlorella virus NYs-1 Nt. CviPII 5’-RXAG-3’ Chlorella virus NY-2A Nt. CviQIIAttorney Docket P39649-WO (note: D=A or G or T; N=A or G or C or T; “X” indicates that the complementary strand of the sequence is recognized and cut; “V” indicates that the sequence itself is recognized and cut).

[0122] According to the methods of the present invention, DNA polymerases with strand displacement activity are particularly advantageous. In certain embodiments, a suitable stand displacing DNA polymerase is a Bst DNA polymerase. In other embodiments suitable strand displacing DNA polymerases may include Bsu, Klenow Fragment, Phi29, Phi29-XT, and vent DNA polymerase.

[0123] In certain embodiments, a variant of any suitable nucleic acid polymerase may be used according to the present invention. For example, a suitable variant may be a commercially available variant of wildtype Bst or Bst large fragment DNA polymerase, such as “Neobolt Bst” (available from Varizymes, Middleton, WI). In other embodiments, a suitable commercially available variant of Bst or Bst large fragment DNA polymerase may be Bst 2.0 or Bst 3.0, (available from NEB) or Thermo Lyo Ready Bst DNA polymerase, commercially available from ThermoFisher Scientific.

[0124] In other embodiments, a suitable variant polymerase may be a variant of any of the DNA polymerase disclosed herein. In certain embodiments, the suitable variant may have from around 95% to around 99% identity, from around 90% to around 95% identity, from around 85% to around 90% identity, from around 80% to around 85% or less than around 80% identity to any of the DNA polymerases set forth herein. Of course, a suitable variant will retain the ability to synthesize a complementary copy of a duplex template construct during the linear amplification methods disclosed herein.

[0125] Other reactive materials are needed in the linear amplification reaction, such as a suitable buffer, dNTPs or analogs thereof, and other application-dependent additives. In certain embodiments, the nicking enzyme is functional in the same reaction conditions as the polymerase, so balancing the conditions to favor the appropriate activities for each enzyme is necessary. In certain embodiments, for example, it may be desirable to optimize the activity of the DNA polymerase while minimizing the activity of the nickase enzyme. In other embodiments, it may be desirable to optimize the activity of the nickase enzyme while minimizing the activity of the DNA polymerase.

[0126] In certain non-limiting embodiments, a linear amplification protocol may include an extension reaction including template DNA, a primer that hybridizes to an end region ofAttorney Docket P39649-WO the template DNA, a mixture of dNTPs, a pyrophosphatase, a single stranded binding protein and a Bst DNA polymerase and an amplification reaction that further includes a nicking endonuclease added to the extension reaction. In some embodiments, the mixture of dNTPs may include 7-deaza dGTP and / or N4-methyl dCTP. In certain embodiments, the mixture of dNTPs may include from around 25% to around 100% 7-deaza dGTP and / or N4-methyl dCTP in place of native dGTP and dCTP.

[0127] In certain embodiments, the template DNA will be provided by any of the DNA libraries disclosed herein. For example, the template DNA may include a double stranded insert (e.g., a library or target fragment) joined on both ends to a Y adapter. In some embodiments, the Y adapter may be a “YS” or a “YSU” adapter that provides a nickase cleavage site in one of the single stranded arm regions of the adapter. An initial DNA replication reaction will provide two double stranded template products that each include a double stranded nickase site that can be used to initiate linear amplification of the template product.

[0128] As used herein, the terms “isothermal conditions” and “constant temperature” may be used interchangeably and refer to a set of reaction conditions where the temperature of the reaction is kept essentially or substantially constant during the course of the amplification reaction. An advantage of isothermal amplification is that the temperature does not need to be cycled between an upper temperature and a lower temperature. The nicking and extension reaction will work at the same temperature or within the same narrow temperature range. However, it is not necessary that the temperature be maintained at precisely one temperature.

[0129] In certain embodiments, the isothermal conditions may include incubation of an amplification reaction at a temperature from around 25 degrees Celsius to around 80 degrees Celsius, from around 35 degrees Celsius to around 70 degrees Celsius, from around 40 degrees Celsius to around 65 degrees Celsius, or around 45 degrees Celsius to around 60 degrees Celsius. In some embodiments, the isothermal conditions may include incubation of an amplification reaction at a temperature from around 50 degrees Celsius to around 55 degrees Celsius. In one embodiment, the isothermal conditions may include incubation of an amplification reaction at a temperature around 52 degrees Celsius.

[0130] In certain embodiments, the isothermal conditions may include incubation for around 5 hours to around 24 hours. (V) Mitigation of Repetitive Sequences in Genomic DNA TemplatesAttorney Docket P39649-WO

[0131] It is known in the art that genomic DNA includes many repetitive sequences, such as SINE-ALU sequences, which make up approximately 10% of the human genome. One potentially deleterious consequence of repetitive sequences is that a pool of amplified copies of genomic DNA fragments may cross-hybridize with each other to form higher order nucleic acid structures. This template cross-hybridization may compromise subsequent steps of the sequencing workflow and ultimately impact the quality of the sequence data (for example, by creating coverage gaps). To address this problem in the art, the methods of the present invention include strategies to mitigate the propensity of single strands of genomic DNA to cross-hybridize following linear amplification.

[0132] One such method is depicted in simplified form in FIG.4. In this embodiment, single stranded amplicons in an amplified pool of library fragments are converted into a partially double stranded form prior to Xpandomer synthesis. Here, in step 1, double stranded genomic library fragment 400 is contacted with YSU adapter 405 under DNA ligation conditions. YSU adapters are described in greater detail with reference to FIG.3. Briefly, YSU adapter 405 includes arm portion 407 and stem portion 409, here depicted in their precursor form prior to the ligation reaction that provides the full YSU adapter structure. Arm portion 407 includes 3’ single stranded arm region that includes nickase cleavage (antisense) site 411. In certain embodiments, the 3’ single stranded arm region may also include an amplification primer hybridization sequence and a SID sequence. Arm portion 407 includes a 5’ second single stranded arm region that may include an extension oligonucleotide hybridization sequence. In certain embodiments, YSU stem portion includes a UMI sequence. In other embodiments, an alternative adapter may be a YS adapter that includes arm portion 405 and lacks stem portion 409. Ligation of the YSU adapter to the genomic library fragment produces adapter-ligated library fragment 420.

[0133] In step 2, adapter ligated library fragment 420 functions as a template for a linear amplification step, which provides a pool of single stranded linear amplicons 425. As described in further detail herein, linear amplification includes a first DNA synthesis reaction in which the adapter-ligated library fragment is contacted with amplification primer 423 and a first nucleic acid polymerase under nucleic acid hybridization and synthesis conditions. The amplification primer is designed to include a sequence complementary to the amplification primer hybridization sequence in the 3’ single stranded arm of the YSU adapter. The first DNA synthesis reaction generates two double stranded nucleic acid products that each include a newly synthesized complementary copy strand hybridized to oneAttorney Docket P39649-WO strand of the adapter-ligated library fragment (not shown). The newly synthesized stands of the double stranded nucleic acid products will include a (sense) copy of nickase cleavage site 411.

[0134] A second DNA synthesis reaction (i.e. the amplification reaction) is then conducted in which the double stranded nucleic acid products are contacted with a nicking endonuclease and a nucleic acid polymerase under nucleic acid nicking and synthesis conditions. The nicking endonuclease generates a single stranded nick in the newly synthesized strands, which creates a free 3’ end that is used as an initiation site for nucleic acid synthesis by the nucleic acid polymerase. As a new complementary copy of the library fragment template is synthesized, the prior complementary copy is displaced from the template strand and is finally released into solution. In this manner, repeated cycles of nucleic acid nicking, synthesis, and strand displacement produce the pool of single stranded amplicons 425.

[0135] In step 3, the pool of single stranded amplicons is contacted with blocker primer 427 under nucleic acid hybridization conditions. The blocker primer is designed to include a sequence that hybridizes to a sequence near the 3’ end of the single stranded amplicons (in one embodiment, with the sequence being partially derived from an arm region and partially derived from the stem region of the YSU adapter). In certain embodiments, the blocker primer is only capable of hybridizing to the single stranded copies of the double stranded nucleic acid product. In certain embodiments, the blocker primer may provide an initiation site for nucleic acid synthesis. In certain embodiments, the blocker primer may be included in the linear amplification reaction. As such, it will hybridize near the 3’ end of the newly synthesized copies of the template strand and enable synthesis of complementary copies 429 of the newly synthesized copies to produce a pool of partial double stranded nucleic acid products 430. The partial double stranded nucleic acid products include 3’ single stranded regions 435 that include a hybridization site for an extension oligonucleotide. Advantageously, the double stranded form of the nucleic acid products significantly reduces amplicon cross-hybridization.

[0136] In step 4, solid support 440 is provided that is covalently joined to extension oligonucleotide 445. The extension oligonucleotide provides a hybridization site for the partially double stranded nucleic acid products, which is mediated by the 3’ single stranded regions of the nucleic acid products. The partially double stranded nucleic acid products areAttorney Docket P39649-WO contacted with the support under nucleic acid hybridization conditions to produce hybridized nucleic acid products 450.

[0137] In step 5, the hybridized nucleic acid products are contacted with an Xpandomer synthetase (e.g., a variant of DPO4 polymerase) under Xpandomer synthesis conditions. The Xpandomer synthetase uses the free 3’ end of the extension oligonucleotide to initiate Xpandomer synthesis to produce full length Xpandomer copies 460 of the library fragments. In certain embodiments, the Xpandomer synthesis conditions may be suitable for displacement of the non-template strand of the hybridized nucleic acid products by the Xpandomer synthetase. Suitable Xpandomer synthesis conditions are described in further detail herein. Solid-Phase Synthesis

[0138] In certain embodiments, one or more steps of generating the library constructs, single stranded templates 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. 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.

[0139] The invention encompasses solid-phase synthesis methods in which a capture moiety is immobilized on a solid support. In certain instances, the capture moiety includes a first end covalently bound to the solid support and a second end that provides a functional group capable of binding to the 5’ end of a single stranded target sequence. As used herein, the term "immobilized", refers to the association, attachment, or binding between a molecule (e.g., linker, adapter, or oligonucleotide) and a support in a manner that provides a stable association under the conditions of elongation, amplification, ligation, and other processes as described herein. Such binding can be covalent or non-covalent. Non-covalent binding includes electrostatic, hydrophilic and hydrophobic interactions. Covalent binding is the formation of covalent bonds that are characterized by sharing of pairs of electrons betweenAttorney Docket P39649-WO atoms. Such covalent binding can be directly between the molecule and the support or can be formed by a cross linker or by inclusion of a specific reactive group on either the support or the molecule or both. Covalent attachment of a molecule can be achieved using a binding partner, such as avidin or streptavidin, immobilized to the support and the non-covalent binding of the biotinylated molecule to the avidin or streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions.

[0140] Any suitable covalent attachment means known in the art may be used for these purposes. The chosen attachment chemistry will depend on the nature of the solid support and any derivatization or functionalities applied thereto. The extension oligonucleotide may include a moiety, which may be a non-nucleotide chemical modification, to facilitate attachment. Certain exemplary embodiments of suitable surface chemistries include conventional streptavidin / biotin interaction chemistry and involve functionalization of a solid support, e.g., with a linker moiety that includes terminal a biotin moiety. In this embodiment, the 5’ end of single stranded DNA fragment (or oligonucleotide) is bound to the linker moiety. Attachment is mediated by a streptavidin moiety provided by the 5’ end of the single stranded DNA fragment. The linker moieties disclosed herein may be of sufficient length to connect the single stranded DNA fragment to the support such that the support does not significantly interfere with primer extension reaction.

[0141] 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. An alkyne moiety provided by the end of the linker distal to the substrate is capable of reacting with an azide group provided by the 5’ end of the capture oligonucleotide. Methods of functionalizing a solid support with maleimide-linker polymers is provided in Applicant’s published Patent Application No. WO2020 / 172479, which is herein incorporated by reference in its entirety.

[0142] In certain instances, the linkage between the capture moiety and the solid support is cleavable, enabling primer extension products to be released from the support following synthesis. Cleavable linkers and methods of cleaving such linkers are known and can be employed in the provided methods using the knowledge of those of skill in the art. For example, the cleavable linker can be cleaved by an enzyme, a catalyst, a chemical compound, temperature, electromagnetic radiation or light. Optionally, the cleavable linker includes aAttorney Docket P39649-WO 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. The Xpandomer Synthesis Reaction

[0143] The Xpandomer synthesis reaction represent a critical step in SBX , as it isresponsible for accurately transcribing the sequence of the DNA template of interest into the sequence of the Xpandomer, which is the polymer directly read by the nanopore sensor. Through trial and error, the inventors have developed a complex reaction mixture for Xpandomer synthesis, which includes a DNA polymerase and many additives that enable incorporation of the bulky XNTP substrates by the polymerase into the very large Xpandomer structure.

[0144] In certain embodiments, a non-limiting Xpandomer synthesis reaction mixture may include the following reagents: a buffer / salt system, polymerase cofactors, polymerase enhancing moieties (PEMs), a DNA polymerase, XNTP substrates, a phosphate shield molecule, a solvent, a crowding agent, and optionally, additional additives. In some embodiments, the buffer / salt system may include TrisCl and NaCl; the polymerase cofactors may include MnCl2 formulated in MES; the PEMs may include molecules disclosed in Applicant’s published PCT applications, WO2019 / 135975 and WO2020 / 263703 and PCT application no. PCT / US2024 / 061051, which are herein incorporated by reference in their entireties; the DNA polymerase may include a variant of DPO4 polymerase (e.g., an Xpandomer synthase) as disclosed in Applicant’s U.S. patent no.s 11,299,725, 11,708,566, 11,530,392 and published PCT application no. WO / 2025 / 082960, each of which is herein incorporated by reference in their entireties; the phosphate shield molecule may include hexametaphosphate (HMP); the solvent may include NMP and DMSO; the crowding agent may include PEG8k; and the additional additives may include imidazole and betaine.

[0145] In optimizing Xpandomer synthesis conditions, e.g., when a double stranded DNA fragment provides the template, the inventors have tested numerous biological additives and other physical forces or manipulations to increase the percentage of full-length Xpandomers synthesized. The following classes of additives were observed to increase the efficiency of Xpandomer synthesis: 1) single stranded binding proteins (SSBs), which are proteins that bind to and help stabilize single stranded regions of DNA and prevent formation of more stable secondary structures; 2) proteins, or enzymes, known to participate in DNAAttorney Docket P39649-WO recombination processes, or to otherwise manipulate regions of single stranded DNA; 3) non- protein additives known to beneficially impact Xpanodmer synthesis (e.g., PEMs or polyphosphate analogs); 4) the biochemistry conditions of the Xpandomer synthesis reaction, including order of addition (e.g., pre-treatment of the template with an additive such as SSB prior to the Xpandomer synthesis reaction) ; 4) stretching forces proposed to enhance solid- state replication of duplexed template constructs.

[0146] In certain embodiments, an Xpandomer synthesis reaction may include a SSB selected from the group consisting of TTH, KOD, Gp32, RPA, E. coli SSB, NCp7, RecA, E. coli Helicase. Xpandomer synthesis conditions are further disclosed in Applicant’s PCT application no. PCT / US25 / 43277, which is herein incorporated by reference in its entirety.Attorney Docket P39649-WO EXAMPLES Example 1 DNA Library and Template Preparation with Linear Amplification for Xpandomer Synthesis

[0147] This example describes an exemplary workflow for library and template preparation for Xpandomer synthesis. Xpandomer copies of DNA library constructs may be used for nanopore sequence determination. This workflow includes the following steps: 1) fragmentation and A-tailing of genomic DNA to provide double stranded library fragment inserts; 2) Y adapter preparation; 3) ligation of Y adapters to DNA library fragments to generate adapter-ligated library constructs; and 4) linear amplification of adapter-ligated library constructs to provide single stranded DNA templates for Xpandomer synthesis.

[0148] Fragmentation and A-tailing. A “Fragmentation ReadyMix” is provided by a commercial kit, e.g., the KAPA EvoPrep kit, commercially available from Roche Sequencing Solutions, Inc. The ReadyMix is diluted to an appropriate amount with water, e.g., to 45%v / v. A 50ng sample of unsheared genomic DNA is brought to a volume of 35 L with 10mMTris HCl, pH 8. A 25 L sample of diluted Fragmentation ReadyMix is added to the sampleof DNA and mixed by pipetting. The mixed sample is then placed in a thermal cycler and incubated at 37 degrees C. for 12 minutes, followed by 55 degrees C. for 10 minutes, and held at 4 degrees C.

[0149] The sample of fragmented and A-tailed genomic DNA is purified by SPRI bead clean-up (using, e.g., KAPA Hyper Pure beads commercially available from Roche SequenceSolutions, Inc.) as follows: a 19 L sample of resuspended SPRI beads is added to the sampleof fragmented DNA, mixed well and incubated at room temperature for 10 minutes; the beadsare captured with a magnet and the supernatant is removed; the beads are washed with 100 Lof 80% ethanol for 30 seconds at room temperature and the supernatant is discarded; the beads are then washed two more times as described above; the beads are allowed to air dryfor seven minutes and then resuspended in 30 L of water; the beads are mixed and incubatedfor 5 minutes; and the beads are captured against a magnet and the supernatant containing the eluted DNA is collected and stored on ice.

[0150] Preparation of Y adapters. Suitable Y adapters for this workflow include the “YSU” and “YS” adapters described with reference to FIG.3. The two oligonucleotidesAttorney Docket P39649-WO comprising the Y adapter may include a “5’ oligo” and a “3’ oligo”. In one example, the 5’ oligo may include the following features, from the 5’ to the 3’ direction: a 25 base extension oligonucleotide hybridization sequence, a six base runway sequence, and a 12 base SID sequence.; and the 3’ oligo may include the following features, from the 3’ to the 5’ direction: an 18 base extension oligonucleotide hybridization sequence, an eight base nickase site, a 10 base blocker oligonucleotide hybridization sequence, a three base runway sequence, and a 12 base SID sequence. The 12 base SID sequences of the two oligonucleotides are designed to be complementary to each other and mediate hybridization of the two oligonucleotides to form the “stem” region of the Y adapter, while the other sequences form the single stranded “arm” regions of the Y adapter.

[0151] The 3’ oligonucleotide is treated with polynucleotide kinase (PNK) by adding a 50μL sample including 10nmol of the 3’ oligonucleotide to a 50μL PNK mix and incubating the sample at 37 degrees C. for 60 minutes, followed by 75 degrees C. for ten minutes. A 25μL sample of the PNK reaction is then added to a 37μL sample including 7.5nmol of the 5’ oligonucleotide in hybridization buffer (125mM NaCl in 5mM Tris pH 8) and the two oligonucleotides are hybridized by incubating the sample at 85 degrees C. for one minute, followed by 20 degrees C. for one minute.

[0152] Ligation of YSU adapters. A 10 L (0.75pmol) sample of Y adapter is added tothe 30 L sample of fragmented and A-tailed DNA. A 20 L sample of Ligation MasterMix(including 0.67U / L deadenylase and 0.03U / DNA ligase in 1X ligation buffer) is added tothe sample of adapter and insert DNA and incubated at 23 degrees C. for 30 minutes. A 5 Lsample of Ligation Digest (including proteinase K) is then added to the sample of ligated DNA and incubated at 55 degrees C. for 10 minutes followed by 95 degrees C. for 3 minutes. The ligated DNA is then purified by SPRI bead clean up, as described above.

[0153] Linear Amplification. An amplification mix (40 L) is prepared that contains thefollowing reagents: 4pmol extension primer, 1.6mM each dNTP (with 100% 7-deaza dGTP),0.04U pyrophosphatase, 20 g single stranded binding protein, and 40U Thermo BSTpolymerase in 1X isothermal amplification buffer. For the initial extension, the 40 L sampleof amplification mix is added to the 40 L sample of adapter-ligated DNA library constructsand incubated at 37 degrees C. for 60 minutes.

[0154] To amplify the library construct, an amplification initiator is prepared thatincludes 3U / L of Nt.BspQ1 nickase and added to the initial extension reaction; the sample isincubated at 52 degrees C. for three hours, followed by 95 degrees C. for four minutes. TheAttorney Docket P39649-WO resulting single stranded amplicons are then subjected to proteinase K treatment and SPRI bead clean up, as described above. The amplified library may then be quantitated and used directly for Xpandomer synthesis.

Claims

Attorney Docket P39649-WO CLAIMS 1. A method for preparing a sample enriched for single stranded nucleic acid templates, comprising the steps of: a) providing a nucleic acid library construct, wherein the nucleic acid library construct comprises a double stranded nucleic acid library fragment joined at both ends to a Y adapter; b) providing an asymmetric PCR reaction mixture, wherein the asymmetric PCR reaction mixture comprises a forward primer and a reverse primer, wherein the forward and reverse primers are capable of hybridizing to the Y adapter, and wherein one of the forward primer or the reverse primer is present in molar excess relative to the other primer; c) performing a PCR reaction using the nucleic acid library construct as a template; and d) collecting a sample enriched for single stranded nucleic acid templates, wherein the single stranded acid templates comprise the sequence of the primer present in molar excess in the asymmetric PCR reaction.

2. The method of claim 1, wherein the molar ratio of one of the forward or the reverse primer to the other of the forward or the reverse primer is from 1:20 to 1:

40.

3. The method of any one of claims 1 or 2, wherein the asymmetric PCR reaction comprises from 15 to 40 PCR cycles.

4. The method of any one of claims 1-3, wherein the asymmetric PCR reaction mixture comprises 7-deaza-dGTP.

5. A method for preparing a sample enriched for single stranded nucleic acid templates, comprising the steps of: a) providing a nucleic acid library construct, wherein the nucleic acid library construct comprises a double stranded nucleic acid library fragment joined at both ends to a Y adapter; b) providing a PCR reaction mixture, wherein the PCR reaction mixture comprises a forward primer and a reverse primer, wherein the forward and reverse primers are capable of hybridizing to the Y adapter, and wherein one of the forward primer or the reverse primer comprises a 5’ biotin moiety;Attorney Docket P39649-WO c) performing a PCR reaction using the nucleic acid library construct as a template to provide a sample of double stranded nucleic acid amplicons, wherein one of the strands of the double stranded nucleic acid amplicons comprises the 5’ biotin moiety; d) contacting the double stranded nucleic acid amplicons with streptavidin-coated beads to provide a sample of double stranded nucleic acid amplicons bound to streptavidin-coated beads; e) contacting the double stranded nucleic acid amplicons with denaturing conditions, wherein the denaturing conditions separate the two strands of the double stranded nucleic acid amplicons; and f) collecting a sample enriched for single stranded nucleic acid templates, wherein the single stranded nucleic acid templates lack the 5’ biotin moiety.

6. The method of claim 5, wherein the primer comprising the 5’ biotin moiety comprises two biotin moieties and one or more PEG6 spacer moieties.

7. The method of claim 5 or 6, wherein the denaturing conditions comprise around 200mM NaOH.

8. A method for preparing a sample enriched for single stranded nucleic acid templates, comprising the steps of: a) providing a nucleic acid library construct, wherein the nucleic acid library construct comprises a double stranded nucleic acid library fragment joined at both ends to a Y adapter; b) providing a PCR reaction mixture, wherein the PCR reaction mixture comprises a forward primer and a reverse primer, wherein the forward and reverse primers are capable of hybridizing to the Y adapter, and wherein one or both of the forward primer and the reverse primer comprises a phosphorthioate bond; c) performing a PCR reaction using the nucleic acid library construct as a template to provide a samples of double stranded nucleic acid amplicons, wherein one or both of the strands of the double stranded nucleic acid amplicons comprises a phosphorthioate bond; and d) contacting the sample of double stranded nucleic acid amplicons with a 5’ to 3’ exonuclease enzyme, wherein the 5’ to 3’ exonuclease enzyme digests the 5’ end of a stand of the nucleic acid amplicons up to the position of theAttorney Docket P39649-WO phosphothioate bond to produce a region of single stranded nucleic acid at the 3’ end of the opposite strand of the nucleic acid amplicons.

9. The method of claim 8, wherein both the forward and the reverse primer comprise a phosphothioate bond.

10. The method of claim 8 or 9, wherein the region of single stranded nucleic acid at the 3’ end of the opposite strand comprises a binding site for an extension oligonucleotide.

11. The method of any one of claims 8-10 wherein the 5’ to 3’ exonuclease is a lambda exonuclease.

12. The method of claim 8, wherein one of the forward and reverse primers comprises the phosphorthioate bond, and wherein the strand lacking the phosphorthioate bond is completely digested by the 5’ to 3’ exonuclease enzyme to produce a sample enriched for the single stranded nucleic acid templates.

13. A method for preparing a sample enriched for single stranded nucleic acid templates, comprising the steps of: a) providing a nucleic acid library construct, wherein the nucleic acid library construct comprises a double stranded nucleic acid library fragment joined at both ends to a Y adapter, wherein the Y adapter comprises a nickase endonuclease cleavage site; b) providing an extension reaction mixture, wherein the extension reaction mixture comprises an extension oligonucleotide and a strand displacing nucleic acid polymerase, wherein the extension oligonucleotide is capable of hybridizing to the Y adapter; c) performing an extension reaction using the nucleic acid library construct as a template, wherein the extension reaction produces a double stranded nucleic acid product, wherein the double stranded nucleic acid product comprises a complementary copy of the nucleic acid library construct; d) contacting the double stranded nucleic acid product with a nicking endonuclease under endonuclease conditions, wherein the nicking endonuclease cleaves the nickase endonuclease cleavage site to produce a free 3’ end in the complementary copy of the nucleic acid library construct; e) contacting the free 3’ end with the strand displacing nucleic acid polymerase under nucleic acid synthesis conditions, wherein the strand displacing nucleicAttorney Docket P39649-WO acid polymerase synthesizes a new complementary copy of the nucleic acid library construct, wherein the new complementary copy of the nucleic acid library construct displaces the complementary copy of step c) from the double stranded nucleic acid product; and f) repeating steps d) and e) to provide an amplified population of single stranded nucleic acid templates, wherein the single stranded nucleic acid templates comprise the sequence of the complementary copy of the nucleic acid library construct.

14. The method of claim 13, wherein the nickase endonuclease is selected from the group consisting of Nb. BbvCl, Nb. Bsml, Nt. BstNBI, Nt. BspQI, Nt. BspD61, Nt. Bst9I, Nt. BstSEI, Nt. BsmAI, Nt. AIwI, Nb. BsrD1, and Nt. CviPII, or variants thereof.

15. The method of claim 13or 14, wherein the strand displacing nucleic acid polymerase is a strand displacing DNA polymerase selected from the group consisting of Bst wildtype, Bst 2.0, Bst 3.0, Bsu, and Klenow fragment, or variants thereof.

16. The method of any one of claims 13-15, wherein the nickase endonuclease conditions and the DNA synthesis conditions are provided in the same reaction mixture.

17. The method of any one of claims 13-16, wherein the reaction mixture comprises a nucleotide analog, wherein the nucleotide analog comprises N4-Me dCTP or 7-deaza dGTP.

18. The method of any one of claims 13-17, wherein the reaction mixture comprises one or more of a single stranded binding protein, a pyrophosphatase, a DPO4 polymerase or a variant thereof, and a translesion repair enzyme.

19. The method of any one of claims 13-18, wherein the extension reaction further comprises a blocker primer, wherein the blocker primer is capable of hybridizing to the single stranded copies of the nucleic acid library construct, and wherein the blocker primer provides an initiation site for nucleic acid synthesis.

20. The method of claim 19, wherein the blocker primer is capable of hybridizing to a sequence derived from the heterologous adapter.

21. The method of any one of claims 1-20, wherein the Y adapter comprises one or more of an SID and a UMI sequence.

22. The method of any one of claims 1-22, wherein the double stranded nucleic acid library fragment comprises a cDNA copy of single cell RNA (scRNA).

23. A method of synthesizing an Xpandomer, comprising,Attorney Docket P39649-WO a) providing the sample enriched for single stranded nucleic acid templates according to any one of claims 1-22; b) providing an Xpandomer synthesis reaction mixture; and c) providing Xpandomer synthesis conditions.

24. The method of claim 24, wherein the Xpandomer synthesis conditions comprise providing an extension oligonucleotide bound to a solid support, wherein the extension oligonucleotide comprises a sequence complementary to a sequence in the heterologous adapter of the double stranded nucleic acid constructs, and wherein the single stranded template is capable of hybridizing to the extension oligonucleotide bound to the solid support.

25. The method of claim 23 or 24, wherein the Xpandomer synthesis reaction mixture comprises one or more of XNTP substrates, a variant of DPO4 polymerase, a manganese salt, a polymerase enhancing molecule (PEM), and a single stranded binding protein.

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