Engineered polymerase with improved thermal stability

Abstract

In nucleotide binding and extension reactions by polymerase catalysts, engineered variants of archaeal polymerases are provided herein that exhibit exonuclease-minus activity, improved thermal stability, improved incorporation of 3'-modified nucleotides, improved uracil tolerance, and / or reduced sequence-specific errors compared to wild-type polymerase enzymes. Also provided is the use of the engineered polymerase to form a complex polymerase and to form a binding complex, and the use for performing nucleic acid sequencing reactions.

Description

Technical Field

[0001] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents, and / or patent applications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art to which this disclosure pertains.

[0002] Cross - References to Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 351,294, filed on June 10, 2022, U.S. Provisional Application No. 63 / 422,855, filed on November 4, 2023, and U.S. Provisional Application No. 63 / 491,374, filed on March 21, 2023, the entire disclosures of which are hereby incorporated by reference in their entirety herein.

[0003] Sequence Listing This application contains a sequence listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety herein. The XML copy created on June 7, 2023, is named 55744WO_CRF_sequencelistingand and is 5,651 kilobytes in size.

[0004] Technical Field The present disclosure provides mutant polymerases engineered for improved thermal stability, which exhibit improved binding and / or incorporation of nucleotide reagents, improved uracil resistance, and / or reduced sequence - specific sequencing errors. Exemplary nucleotide reagents include detectably labeled nucleotides, nucleotides containing 3'-chain terminating moieties, nucleotides labeled with phosphate chains, and multivalent molecules. In some embodiments, the engineered polymerase exhibits a decrease in the transition from nucleotide polymerization conformation to exonuclease conformation. The mutant polymerase exhibits an increased incorporation rate compared to the wild - type polymerase.

Background Art

[0005] Background Next-generation sequencing (NGS) technology has become a powerful tool for obtaining sequencing data used in molecular biology techniques, taxonomy, agricultural science, medical diagnosis, and the development of new therapies. The present disclosure provides engineered polymerases useful for performing any nucleic acid sequencing method using labeled or unlabeled chain-terminating nucleotides, where the chain-terminating nucleotides include a 3'-O-azido group (or 3'-O-methylazido group) or any other type of large blocking group at the 3'-position of the sugar. For example, the engineered polymerase can be used to perform sequencing by binding activity (SBA) methods using labeled multivalent molecules and unlabeled chain-terminating nucleotides. Additionally, the engineered polymerase can be used to perform sequencing by synthesis (SBS) methods using labeled chain-terminating nucleotides and to perform sequencing by binding (SBB) methods using unlabeled chain-terminating nucleotides.

[0006] The addition of a single nucleotide alone to a DNA strand does not generate a signal sufficient to be easily detected. Currently available SBS technologies overcome this problem by increasing the signal-to-noise ratio of nucleotide addition, in combination with detection methods having sufficient sensitivity to perform accurate base calling. The most commercially successful platforms use monoclonal template DNA amplification in a spatially constrained matrix to generate individual DNA islands containing multiple copies of the sequence for interrogation. The result of this amplification is a "colony" of DNA copies such that the addition of a single DNA base to all copies concentrates the detection modality in a manner sufficient to overcome the signal-to-noise ratio problem. Sequencing of multiple spatially constrained identical copies of DNA further increases the dependence on a controlled stepwise mechanism to ensure that only one nucleotide base can be added to ensure that all copies within the DNA colony remain at the same position relative to each other (N, N+1, N+2, N+3, etc.).

[0007] The molecular engine necessary to perform SBS is DNA polymerase. In vivo, this class of enzymes is involved in DNA replication and maintaining genomic integrity. Under natural conditions, DNA-dependent DNA polymerases (dDdPs) catalyze the addition of deoxynucleotide triphosphates (dNTPs) to DNA in the 5’ to 3’ direction, creating a phosphodiester bond between the 3’ hydroxyl of the primer DNA end and the 5’ α phosphate of the incoming nucleotide. This chemical reaction occurs with high fidelity for the correct Watson-Crick base pairs due to hydrogen bonding between the correct input dNTP and the template base. This “correct” base pairing induces a conformational change in the enzyme that aligns catalytic amino acids to efficiently perform phosphodiester bond formation. The newly added dNTP also has a 3’OH that is used in the next catalytic round to further extend the DNA strand.

[0008] To ensure that only a single dNTP is added to the growing DNA strand per SBS cycle, reversibly terminated dNTPs are used. These bases contain a modification to the 3’ hydroxyl of the dNTP, blocking subsequent incorporation rounds. The most commercially successful reversible terminator is 3’ methyl azide, although other terminators including 3’-aminoallyl, and 3’ oxyamine have also been used. Each of these reversibly terminated dNTPs functions in the same manner and, once incorporated, the bulky 3’ block inhibits the addition of the next nucleotide since there is no 3’ hydroxyl present. Upon exposure to the catalyst, the 3’ block reacts to regenerate a 3’ hydroxyl that can form a new phosphodiester bond during the next cycle. Although effective, these large 3’ modifications present challenges for the polymerase.

[0009] The evolutionary need for high-fidelity genome replication and stability is 10 4 ~10 7For each incorporation event, it led to a polymerase that incorporated only non-Watson-Crick base pairs. Polymerases also often need to distinguish between vast excesses of nucleotides in the cellular environment. Discrimination between nucleotides is typically done through a steric gate where the presence of the 2’ hydroxyl sterically collides with amino acid side chains at the nucleotide binding site and selects for nucleotide binding and catalysis. Further, damage or modification to the 3’ hydroxyl of nucleotides is also sensed by the enzyme because bases containing a non-viable 3’ hydroxyl can function as chain terminators that inhibit DNA synthesis. Discrimination of these unwanted bases occurs through a kinetic pathway where inappropriate nucleotide substrates bind with a weaker overall binding activity and phosphodiester bond formation occurs more slowly at a rate of 10 2 ~10 4 orders of magnitude. This is due to the lack of an induced fit that properly orients catalytic amino acids for bond formation. As a result, naturally evolved polymerases have poor incorporation of reversible terminator nucleotides.

[0010] The novel advantages and characteristics of the compositions and methods disclosed herein are described in detail in the appended claims. A better understanding of the characteristics and advantages of the compositions and methods of the present disclosure will be obtained by reference to the following detailed description that describes exemplary embodiments and the accompanying drawings.

Brief Description of the Drawings

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[0012] **DETAILED DESCRIPTION** **Definitions**: The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the entire specification.

[0013] Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms employed herein with respect to techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, production of genetically engineered cells, and hybridization are those well known and commonly used in the art. The techniques and procedures described herein are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). Also see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with, and the laboratory procedures and techniques described herein, are those well known and commonly used in the art.

[0014] Unless the context requires otherwise herein, terms in the singular form shall include the plural, and terms in the plural form shall include the singular. The use of the singular forms of "a", "an", and "the", and any word in the singular, is not limited to a single reference and includes plural references, unless clearly and unambiguously limited to one.

[0015] The use of alternative terms (e.g., "or") is understood to mean one or both of the alternative forms, or any combination thereof.

[0016] As used herein, the term "and / or" is to be understood to mean a specific disclosure that each of the particular features or components either has or does not have the other. For example, when used in a phrase such as "A and / or B" herein, the term "and / or" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). In a similar manner, when used in a phrase such as "A, B, and / or C", the term "and / or" is intended to include each of the following aspects: "A, B, and C", "A, B, or C", "A or C", "A or B", "B or C", "A and B", "B and C", "A and C", "A" (alone), "B" (alone), and "C" (alone).

[0017] As used in this specification and the appended claims, the terms "comprising", "including", "having", and "containing", and their grammatical variations as used herein, are intended to be non-limiting such that one or more items in a list are not excluded from other items that may be substituted or added to the listed items. It is understood that any aspect described herein in terms of the term "comprising" also provides a similar aspect described in terms of the terms "consisting of" and / or "consisting essentially of".

[0018] As used herein, the terms "about" or "approximately" refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, and the acceptable error range depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measurement system. For example, "about" or "approximately" can mean within one or two standard deviations per implementation in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Further, especially with respect to biological systems or processes, this term can mean up to one order of magnitude or up to five times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be considered to be within the acceptable error range for this particular value or composition. Also, when ranges and / or sub-ranges of values are provided, the ranges and / or sub-ranges can include the endpoints of the ranges and / or sub-ranges.

[0019] The terms "peptide", "polypeptide", and "protein" and other related terms used herein are used synonymously and refer to polymers of amino acids and are not limited to any particular length. Polypeptides can include natural and non-natural amino acids. Examples of polypeptides include recombinant or chemically synthesized types. Also, polypeptides include precursor molecules that have not yet undergone post-translational modifications such as proteolytic cleavage, cleavage by ribosomal skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass natural and artificial proteins of protein sequences, protein fragments, and polypeptide analogs (such as mutant proteins, variants, chimeric proteins, and fusion proteins), as well as proteins that have been modified post-translationally or otherwise covalently or non-covalently.

[0020] As used herein, the term "polymerase" and its variants include any enzyme capable of catalyzing the polymerization of nucleotides (including analogs thereof) onto a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase includes one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacement activity. Polymerases include naturally occurring polymerases and any of their subunits and truncated, mutant, variant, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments (e.g., catalytically active fragments) thereof that retain the ability to catalyze nucleotide polymerization, but are not limited thereto. In some embodiments, a polymerase may be isolated from a cell or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in a prokaryotic, eukaryotic, viral, or phage organism. In some embodiments, a polymerase can be a post-translationally modified protein or a fragment thereof. A polymerase can be derived from a prokaryote, eukaryote, virus, or phage. Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0021] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization by a template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporating an incorrect nucleotide, i.e., a nucleotide that is not complementary to the template nucleotide). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3' to 5' exonuclease activity of the DNA polymerase.

[0022] As used herein, the term "binding complex" refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and free nucleotides or nucleotide units of a multivalent molecule, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotides or nucleotide units may or may not be bound at the 3'-end of the nucleic acid primer at a position opposite to the complementary nucleotide within the nucleic acid template molecule. A "ternary complex" is an example of a binding complex formed by binding together a nucleic acid duplex, a polymerase, and free nucleotides or nucleotide units of a multivalent molecule, wherein the free nucleotides or nucleotide units are bound at the 3'-end of the nucleic acid primer (as part of the nucleic acid duplex) at a position opposite to the complementary nucleotide within the nucleic acid template molecule.

[0023] The term "duration" and related terms refer to the length of time that a binding complex remains stable without any of its components dissociating, where the components of the binding complex include a nucleic acid template and nucleic acid primer, polymerase, nucleotide units of a multivalent molecule, or free (e.g., unconjugated) nucleotides. The nucleotide units or free nucleotides may be complementary or non-complementary to the nucleotide residues in the template molecule. The nucleotide units or free nucleotides can bind at the 3' end of the nucleic acid primer at a position opposite to the complementary nucleotide residue in the nucleic acid template molecule. The duration indicates the stability of the binding complex and the strength of the binding interaction. The duration can be measured by observing the onset and / or duration of the binding complex, for example, by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent containing one or more nucleotides can be present in the binding complex, thus enabling a signal from the label to be detected during the duration of the binding complex. One exemplary label is a fluorescent label. The binding complex (e.g., a ternary complex) remains stable until it is subjected to conditions that cause dissociation of the interaction between the polymerase, template molecule, primer, and / or any of the nucleotide units or nucleotides. For example, the dissociation conditions include contacting the binding complex with any one or a combination of a detergent, EDTA, and / or water.

[0024] As used herein, the terms "nucleic acid", "polynucleotide", and "oligonucleotide", and other related terms are used interchangeably and refer to polymers of nucleotides, not limited to any particular length. Nucleic acids include recombinant or chemically synthesized types. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric types containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids contain polymers of nucleotides, and nucleotides contain natural or unnatural bases and / or sugars. Nucleic acids contain natural nucleoside linkages, such as phosphodiester linkages. Nucleic acids contain non-natural nucleoside linkages, and non-natural nucleoside linkages include phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, the nucleic acid includes one type of polynucleotide, or a mixture of two or more different types of polynucleotides.

[0025] As used herein, the term "primer" and related terms refer to either natural or synthetic oligonucleotides that can hybridize to a DNA and / or RNA polynucleotide template and form a double-stranded molecule. Primers can have any length, but typically can range from 4 to 50 nucleotides. A typical primer includes a 5' end and a 3' end. The 3' end of the primer can include a 3'OH moiety that functions as a nucleotide polymerization initiation site in a polymerase-mediated primer extension reaction. Alternatively, the 3' end of the primer can lack a 3'OH moiety or can include a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-mediated reaction. Any one nucleotide or two or more nucleotides along the length of the primer can be labeled with a detectable reporter moiety. Primers can be in solution (e.g., soluble primers) or immobilized on a support (e.g., capture primers).

[0026] "Template nucleic acid", "template polynucleotide", "target nucleic acid", "target polynucleotide", "template strand", and other variants refer to nucleic acid strands that function as a basic nucleic acid molecule for generating complementary nucleic acid strands. The sequence of the template nucleic acid can be partially or completely complementary to the sequence of the complementary strand. The template nucleic acid may be obtained from a natural source or recombinant form, or may be chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template nucleic acid can be isolated in any form including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloning, amplification, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, genomic DNA obtained from fresh frozen paraffin-embedded tissues, needle biopsies, cell-free circulating DNA, or any type of nucleic acid library. The template nucleic acid molecule can be isolated from any source including prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, and viruses; cells; tissues; normal or diseased cells or tissues, blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, and body fluids including semen; environmental samples; culture samples; or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The template nucleic acid can be subjected to nucleic acid analysis including sequencing and composition analysis.

[0027] When used in reference to nucleic acid molecules, the terms "hybridize" or "hybridizing" or "hybridization", or other related terms, refer to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a double-stranded region. Hybridization can include Watson-Crick or Hoogsteen bonds to form a double-stranded duplex nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be completely complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other over their entire lengths. Complementary base pairing may be standard A-T or C-G base pairing, or other forms of base pairing interactions. The double-stranded nucleic acid may contain mismatched base-pairing nucleotides.

[0028] The term "nucleotide" and related terms refer to a molecule containing an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of this term. In some embodiments, the phosphate includes monophosphate, diphosphate, or triphosphate, or corresponding phosphate analogs. In some embodiments, the nucleotide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups. The term "nucleoside" refers to a molecule containing an aromatic base and a sugar.

[0029] Nucleotides (and nucleosides) typically contain a heterocyclic base that includes a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, which are commonly found in nucleic acids and include naturally occurring, substituted, modified, or engineered variants, or analogs thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with appropriate complementary bases. Exemplary bases include purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N 6 -Δ 2 -isopentenyladenine (6iA), N 6 -Δ 2 -isopentenyl-2-methylthioadenine (2ms6iA), N 6 -methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine, and O 6 -methylguanine, 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G), pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D), indoles, such as nitroindole and 4-methylindole, pyrroles, such as nitropyrrole, nebularine, inosine, hydroxymethylcytosine, 5-methylcytosine, base (Y), and methylated, glycosylated, and acylated base moieties, among others, are included but not limited to these. Additional exemplary bases can be found in Fasman, 1989, “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0030] Nucleotides (and nucleosides) typically include a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), an acyclic moiety (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274, Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol.7:3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36:2627-2638, Kim, et al., 1993 J. Med. Chem. 36:30-7, Eschenmosser 1999 Science 284:2118-2124, and U.S. Patent No. 5,558,991). The sugar moiety includes ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic, or other modified sugars.

[0031] In some embodiments, the nucleotide includes a chain of 1, 2, or 3 phosphorus atoms, which chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramidate bond. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are joined together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side chain groups containing O, S, or BH3. In some embodiments, the chain includes a phosphate group substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0032] When used with reference to nucleic acids, the terms "extending," "extension," "extends," and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides to the 3'OH terminus of the end of a nucleic acid strand, resulting in the extension of the nucleic acid strand. Nucleotide incorporation can be carried out with natural nucleotides and / or nucleotide analogs. Typically, although not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending a nucleic acid molecule can be used, including primer extension catalyzed by a DNA polymerase or an RNA polymerase.

[0033] The terms "reporter moiety," "reporter moieties," or related terms refer to a compound that produces, or causes the production of, a detectable signal. Reporter moieties are often referred to as "labels." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electrochemiluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophore, radioisotope, electrochemistry, mass spectrometry, Raman, hapten, affinity tag, atom, or enzyme. Reporter moieties produce a detectable signal resulting from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or proximity event). Proximity events include two reporter moieties coming into proximity with each other, associating with each other, or binding to each other. It is well known to those skilled in the art to select reporter moieties such that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from that of other reporter moieties, enabling the monitoring of the presence of different reporter moieties in the same reaction or different reactions. Two or more different reporter moieties having spectrally distinct emission profiles or minimal overlapping spectral emission profiles can be selected. Reporter moieties can be bound (e.g., operably bound) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or supports (e.g., surfaces).

[0034] The reporter moiety (or label) includes a fluorescent label or fluorophore. Exemplary fluorescent moieties that can function as a fluorescent label or fluorophore include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamide fluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives, such as TRITC, TMR, lysamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lysamine rhodamine B sulfonyl chloride, lysamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives, such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives, such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives, such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives, such as Lucifer Yellow iodoacetamide, Lucifer YellowCH, cyanine and derivatives, such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives, such as BCPDA, TBP, TMT,BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE, and their derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and other known ones in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition, Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or any derivatives thereof, or any combinations thereof, but not limited thereto. Cyanine dyes can exist in either sulfonated or non-sulfonated forms and consist of two indolenine, benzoindolium, pyridinium, thiazolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which is 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium, or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which is1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium, or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate (which may be included), and Cy7 (1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-triene-1-yl]-3H-indolium, or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-triene-1-yl]-3H-indolium-5-sulfonate (which may be included), where "Cy" represents "cyanine" and the first number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzoderivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.,

[0035] In some embodiments, the reporter moiety can be a FRET pair, whereby multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET can include Förster (excitation transfer) or Dexter (electron exchange) transfer.,

[0036] Many pH buffers are known to those skilled in the art. The full names of the pH buffers are listed herein. The term "Tris" refers to Tris(hydroxymethyl)-aminomethane, which is a pH buffer. The term "Tris HCl" refers to Tris(hydroxymethyl)-aminomethane hydrochloride, which is a pH buffer. The term "Tris acetate" refers to a pH buffer containing the acetate of Tris(hydroxymethyl)-aminomethane. The term "Tricine" refers to N-[Tris(hydroxymethyl)methyl]glycine, which is a pH buffer. The term "Bicine" refers to N,N-bis(2-hydroxyethyl)glycine, which is a pH buffer. The term "Bis-Tris propane" refers to 1,3-bis[Tris(hydroxymethyl)methylamino]propane, which is a pH buffer. The term "HEPES" refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, which is a pH buffer. The term "MES" refers to 2-(N-morpholino)ethanesulfonic acid), which is a pH buffer. The term "MOPS" refers to 3-(N-morpholino)propanesulfonic acid, which is a pH buffer. The term "MOPSO" refers to 3-(N-morpholino)-2-hydroxypropanesulfonic acid, which is a pH buffer. The term "BES" refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, which is a pH buffer. The term "TES" refers to 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid), which is a pH buffer. The term "CAPS" refers to 3-(cyclohexylamino)-1-propanesulfonic acid, which is a pH buffer. The term "TAPS" refers to N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, which is a pH buffer. The term "TAPSO" refers to N-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid, which is a pH buffer. The term "ACES" refers to N-(2-acetamido)-2-aminoethanesulfonic acid, which is a pH buffer. The term "PIPES" refers to piperazine-1,4-bis(2-ethanesulfonic acid, which is a pH buffer. The term "ethanolamine" refers to a pH buffer also known as 2-aminoethanol.

[0037] The terms "linked," "joined," "attached," and variants thereof include any type of fusion, bonding, adhesion, or association between any combination of compounds or molecules having sufficient stability to withstand use in a particular procedure. The procedure can include, but is not limited to, nucleotide transient bonding, nucleotide incorporation, deblocking, washing, removal, flow, detection, imaging, and / or identification. Such bonding can include, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonding or association including van der Waals forces, and mechanical bonding. In some embodiments, such bonding occurs within a molecule, for example, to join the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonding includes, but is not limited to, bonding between a nucleic acid molecule and a solid surface, bonding between a protein and a detectable reporter moiety, bonding between a nucleotide and a detectable reporter moiety, etc., and can occur between combinations of different molecules, or between a molecule and a non-molecule. Some examples of bonding can be found, for example, in Hermanson, G., "Bioconjugate Techniques", Second Edition (2008), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998).

[0038] As used herein, the terms "linked" and "joined" or related terms, when referring to components in parallel, mean that the components in parallel can be joined together covalently. For example, two nucleic acid components can be ligated together enzymatically, and the bond that joins the two components together includes a phosphodiester bond. The first and second nucleic acid components can be joined together, and the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to the primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) can be ligated to a vector, and the bond enables the expression or function of the transgene sequence contained within the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects the expression of the transgene. In some embodiments, the vector includes at least one host cell regulatory sequence, and the at least one host cell regulatory sequence includes a promoter sequence, an enhancer, a transcription start sequence and / or a translation start sequence, a transcription termination sequence and / or a translation termination sequence, and a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of expression of the transgene.

[0039] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0040] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can have a regular or irregular texture and can include ridges, etching, pores, three-dimensional scaffolds, or any combination thereof.

[0041] In some embodiments, the support includes beads having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0042] The support can be manufactured from any material, including, but not limited to, glass, fused silica, silicon, polymers (such as polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0043] In some embodiments, the surface of the support is coated with one or more compounds to create a passivation layer on the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. Generally, the support can include a low chemically modified layer that is covalently or non-covalently bound, such as a silane layer, a polymer film, and one or more layers of one or more covalently or non-covalently bound oligonucleotides that can be used to immobilize multiple nucleic acid template molecules to the support.

[0044] In some embodiments, the degree of hydrophilicity of the surface coating (or "wettability" with an aqueous solution) can be evaluated, for example, by placing small water droplets on the surface and measuring the contact angle with the surface, such as by measurement of the water contact angle measured using an optical tensiometer. In some embodiments, the static contact angle can be determined. In some embodiments, the advancing or receding contact angle can be determined. In some embodiments, the water contact angle for the surface-treated hydrophilic low-binding support disclosed herein can range from about 0 degrees to about 30 degrees. In some embodiments, the water contact angle for the surface-treated hydrophilic low-binding support disclosed herein can be 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree or less. In many cases, the contact angle is 40 degrees or less. It will be appreciated by those skilled in the art that a given hydrophilic low-binding support surface of the present disclosure can exhibit a water contact angle having a value within any of this range.

[0045] The present disclosure provides a plurality of (e.g., two or more) nucleic acid templates immobilized on a support. In some embodiments, the plurality of immobilized nucleic acid templates have the same sequence or different sequences. In some embodiments, the individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at a site on the support. In some embodiments, the support comprises a plurality of sites arranged in an array. The term "array" refers to a support that includes a plurality of sites located at predetermined positions on the support so as to form an array of sites. The sites may be dispersed and separated by interstitial regions. In some embodiments, the predetermined sites on the support may be arranged in rows or columns in one dimension or in rows or columns in two dimensions. In some embodiments, the plurality of predetermined sites are arranged on the support in an organized manner. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including patterns such as a straight line, a hexagonal pattern, a lattice pattern, a pattern having reflection symmetry, a pattern having rotational symmetry, etc. The pitch between different pairs of sites may be the same or may vary. In some embodiments, the support has a surface density of about 10 2 per 1 mm 2 to 10 15 and may have nucleic acid template molecules immobilized at the plurality of sites. In some embodiments, the support has at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13sites, at least 10 14 sites, or at least 10 15 sites, or more, and these sites are located at predetermined positions on the support. In some embodiments, a plurality of predetermined sites (e.g., 10 2 ~10 15 or more sites) on the support are immobilized with nucleic acid templates to form a nucleic acid template array. In some embodiments, the nucleic acid templates immobilized at a plurality of predetermined sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently bound to the surface capture primers. In some embodiments, the nucleic acid templates are immobilized at a plurality of predetermined sites, e.g., 10 2 ~10 15 or more sites. In some embodiments, the nucleic acid templates immobilized at a plurality of sites on the support include linear or circular nucleic acid template molecules, or a mixture of both linear and circular molecules. In some embodiments, the immobilized nucleic acid templates are clonally amplified to generate immobilized nucleic acid colonies at a plurality of predetermined sites. In some embodiments, individual immobilized nucleic acid template molecules include one copy of a target sequence of interest or a concatemer having two or more tandem copies of the target sequence of interest.

[0046] In some embodiments, a support comprising a plurality of sites located at random positions on the support is herein referred to as a support having sites located at random thereon. The positions of the sites located at random on the support are not predetermined positions. The plurality of randomly located sites are arranged on the support in a disordered and / or unpredictable manner. In some embodiments, the support has at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, or at least 10 15 sites, or more, and these sites are randomly located on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 10 2 ~10 15 or more sites) are immobilized with a nucleic acid template to form a support immobilized with a nucleic acid template. In some embodiments, the nucleic acid template immobilized at a plurality of randomly located sites by hybridization to an immobilized surface capture primer, or the nucleic acid template, is covalently bound to the surface capture primer. In some embodiments, it is immobilized at a plurality of randomly located sites, e.g., 10 2 ~10 15 or more sites. In some embodiments, the nucleic acid template immobilized at a plurality of sites on the support includes linear or circular nucleic acid template molecules, or a mixture of both linear and circular molecules. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate an immobilized nucleic acid colony at a plurality of randomly located sites. In some embodiments, each individual immobilized nucleic acid template molecule contains one copy of a target sequence of interest or a concatemer having two or more tandem copies of the target sequence of interest.

[0047] In some embodiments, with respect to nucleic acid template molecules immobilized at predetermined or random sites on a support, the plurality of immobilized nucleic acid template molecules on the support are in fluid communication with each other such that a solution of reagents (e.g., enzymes including polymerase, multivalent molecules, nucleotides, divalent cations, and / or buffers, etc.) can be flowed over the support so that the plurality of immobilized nucleic acid template molecules on the support can react with the reagents in a superparallel manner. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid template molecules is used to perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) on the plurality of immobilized nucleic acid template molecules, and detection and imaging for superparallel sequencing can be performed. In some embodiments, the term "immobilized" and related terms refer to a nucleic acid molecule or enzyme that is directly bound to the support via covalent or non-covalent interaction, or a nucleic acid molecule or enzyme (e.g., polymerase) that is bound to the support at a predetermined or random position where the nucleic acid molecule or enzyme is bound to a coating on the support.

[0048] As used herein, the term "clonally amplified" and variants thereof refer to nucleic acid template molecules that have been subjected to one or more amplification reactions either in solution or on a support. In the case of template molecules amplified in solution, the resulting amplicons are distributed onto the support. Prior to amplification, the template molecules include a sequence of interest and at least one universal adapter sequence. In some embodiments, clonal amplification includes the use of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding (SSB) protein-dependent amplification, or any combination thereof.

[0049] As used herein, the term "sequencing" and variants thereof typically involve obtaining sequence information from a nucleic acid strand by determining the identity of at least some of the nucleotides (including their nucleobase components) within a nucleic acid template molecule. In some embodiments, "sequencing" a given region of a nucleic acid molecule involves identifying each nucleotide and all nucleotides within the region to be sequenced. However, in some embodiments, "sequencing" includes methods in which the identity of only some of the nucleotides within the region is determined while the identity of some nucleotides remains undetermined or is incorrectly determined. Any suitable sequencing method can be used. In exemplary embodiments, sequencing can include label-free methods or ion-based sequencing methods. In some embodiments, sequencing can include labeled or dye-containing nucleotides, or fluorescence-based nucleotide sequencing methods. In some embodiments, sequencing can include polony-based sequencing methods or bridge sequencing methods. In some embodiments, sequencing includes a massively parallel sequencing platform that uses procedures for sequencing by synthesis, sequencing by hybridization, or sequencing by ligation. Examples of massively parallel sequencing-by-synthesis procedures include polony sequencing, pyrosequencing (e.g., from 454 Life Sciences, U.S. Patent Nos. 7,211,390, 7,244,559, and 7,264,929), chain-terminator sequencing (e.g., from Illumina, U.S. Patent No. 7,566,537, Bentley 2006 Current Opinion Genetics and Development 16:545-552, and Bentley, et al., 2008 Nature 456:53-59), ion-sensitive sequencing (e.g., from Ion Torrent), probe anchor ligation sequencing (e.g., Complete Genomics), DNA nanoball sequencing, nanopore DNA sequencing. Examples of single molecule sequencing include Heliscope single molecule sequencing, and single molecule real time (SMRT) sequencing.Examples of arraying by hybridization include SOLiD sequencing (e.g., from Life Technologies, WO2006 / 084132). Examples of arraying by ligation include Omniome sequencing (e.g., U.S. Patent No. 10,246,744).

[0050] Engineered polymerase showing increased thermal stability The present disclosure provides compositions comprising mutant polymerases having amino acid substitutions and / or truncated amino acid sequences, nucleic acids encoding the mutant polymerases, and systems and kits comprising the mutant polymerases. Methods of using a mutant polymerase, including methods for binding a nucleic acid duplex, methods for binding a complementary nucleotide, or methods for binding a multivalent molecule having complementary nucleotide units, methods for incorporating a complementary nucleotide, methods for extending a primer, and methods for performing nucleic acid sequencing are further provided herein, the methods using any of the mutant polymerases described herein. The mutant polymerase is engineered to exhibit desired properties including exonuclease-minus activity and increased stability, improved uracil tolerance, and / or decreased sequence-specific errors. Further, the mutant polymerase can be engineered to express a higher percentage of soluble expressed enzyme.

[0051] The present disclosure provides mutant polymerases engineered to reduce post-translational modifications to improve protein activity and stability. Post-translational modifications of recombinant proteins can reduce protein activity, cause misfolding, increase heterogeneity, decrease thermal stability, and increase protein aggregation. These modifications pose challenges to achieving consistent manufacturability and maintaining a long shelf life. Many recombinant proteins are subject to post-translational modifications including phosphorylation, acetylation, ubiquitination, succinylation, methylation, glycosylation (e.g., N-linked, O-linked, and C-linked), hydroxylation, oxidation, deamidation, nitrosylation, sulfation, SUMOylation, disulfide bond formation, proteolysis, and / or lipidation (e.g., palmitoylation).

[0052] Examples of the undesirable effects of post-translational modifications include oxidative damage to sulfur-containing amino acids such as cysteine and methionine. Histidine, tryptophan, lysine, and serine are also known to be susceptible to oxidative damage. Deamidation of asparagine and glutamine can lead to proteolysis that decreases the shelf life. Acetylation can involve cleavage of the N-terminal methionine and replacement with an acetyl group. In some proteins, the N-terminal methionine plays an important role in enzyme activity, and removal of this methionine residue can alter the enzyme activity. Acetylation can also involve serine and lysine. Methylation can result in changes in hydrophobicity and side-chain charge. Ubiquitination can lead to proteolysis.

[0053] Analytical techniques such as mass spectrometry and tandem mass spectrometry can be used to detect post-translational modifications, particularly oxidized methionine and lysine residues for which rational mutagenesis can improve protein production and stability.

[0054] The present disclosure provides a mutant polymerase that is engineered to exhibit a reduced editing mode conformation. Many DNA polymerases have 5’→3’ nucleotide polymerization activity and 3’→5’ exonuclease proofreading activity. Generally, the polymerization site in a polymerase is located at a different site from the proofreading site. During the polymerization mode, the growing ends of the template and primer strands are located at the polymerization site. In many family B and C polymerases, the amino acid residues within the finger domain, thumb domain, and palm domain form at least a portion of the polymerization site. In the proofreading mode, the growing end of the primer is physically moved from the polymerization site to the exonuclease site, and the incorrectly incorporated nucleotide at the end of the growing primer strand is excised by the 3’→5’ proofreading activity of the polymerase. The movement of the primer end from the polymerization site to the exonuclease site may involve local mispairing of several base pairs at the primer / template junction that prevents further strand elongation (e.g., polymerization) until the primer end is moved back to the polymerization site and primer / template pairing is restored. The amino acid residues that play a role in the movement of the primer end are independent of the amino acid residues involved in exonuclease activity. The polymerization site and the exonuclease site are located in two different domains. The polymerase may undesirably undergo this polymerization-to-exonuclease conformational switch when the correct nucleotide has been incorporated to stop primer elongation, or when the correct nucleotide unit from a multivalent molecule has bound at the polymerization site. In E. coli DNA polymerase III, the time scale of primer end movement and primer / template mispairing has previously been determined to be approximately 10 milliseconds, an order of magnitude longer than nucleotide incorporation (Dodd, et al., 2020 Nature Communication 11:5379, “Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path”). Thus, it is desirable to engineer a sequencing polymerase that exhibits a reduced editing mode conformation when the correct nucleotide has been incorporated, or when the correct nucleotide unit from a multivalent molecule has bound at the polymerization site.It is assumed that the editing mode mutant polymerase retains 3'→5' exonuclease activity.

[0055] Protein modeling and mass spectrometry can identify key amino acid residues along the pathway that moves the primer terminus from the polymerization site to the exonuclease site without affecting exonuclease activity. Mutations of at least some of these key amino acid residues can reduce the conformational switch from unwanted polymerization to exonuclease. For example, mutations of amino acid residues that play a role in directing the primer terminus to the exonuclease site, or mutations of amino acid residues that stabilize the primer terminus at the exonuclease site, can reduce the transition to the editing mode conformation. In another example, mutations of amino acid residues that interact with and stabilize the template molecule during polymerization can retain the template molecule at the polymerization site.

[0056] The present disclosure provides mutant polymerases that can be used to perform a two-step nucleic acid sequencing method. In some embodiments, the first step generally involves binding a multivalent molecule detectably labeled under conditions suitable to inhibit incorporation of nucleotide units to a complex polymerase to form a multivalent complex polymerase, and detecting the multivalent complex polymerase. The first step can be performed using a trapping polymerase. In some embodiments, the second step generally involves polymerase-catalyzed nucleotide incorporation using a stepping polymerase.

[0057] The present disclosure provides mutant polymerases that can be used to perform a trapping event or a stepping event for nucleic acid sequencing. Some of the mutant polymerases can be used for both trapping events and stepping events.

[0058] The present disclosure provides mutant polymerases that can be used to trap multivalent molecules comprising a complex mutant polymerase that binds to a multivalent molecule having complementary nucleotide units (e.g., exemplary multivalent molecules are shown in FIGS. 2-5). In some embodiments, the multivalent molecule comprises a central core bound to a plurality of polymer arms, each having a nucleotide unit at the end of the arm. The multivalent molecule can be labeled with a detectable reporter moiety. The complex mutant polymerase comprises a mutant polymerase bound to a template / primer duplex. The mutant polymerase is engineered to exhibit reduced sequence-specific errors that occur after a specific motif sequence within the primer strand and / or template strand. The sequence-specific errors of the trapping polymerase can be characterized by base miscalls (e.g., base substitutions) at specific sequencing cycles or a substantial loss of signal intensity leading to no calls. The signal often recovers in the next cycle. The motif sequence leading to the miscall is specific to a given polymerase and can occur on the template strand in either the forward or reverse sequencing direction.

[0059] The present disclosure provides mutant polymerases that can bind to complementary nucleotides (e.g., unconjugated nucleotides) and be used to incorporate nucleotides within the 3' end of a primer, called a stepping event. The mutant polymerase is engineered to exhibit reduced sequence-specific errors characterized by a substantial loss of nucleotide incorporation that occurs after a specific motif sequence within the primer strand and / or template strand. The sequence-specific errors of the stepping enzyme can be characterized by a large-scale phase after the sequence motif. The motif sequence leading to the phase is specific to a given polymerase and can occur on the template strand in either the forward or reverse sequencing direction.

[0060] Although not wishing to be bound by theory, it is hypothesized that mutant polymerases that trap or step array-specific errors with certain array motifs during array determination switch from a nucleotide incorporation conformation to an editing conformation. During a trapping event, the editing conformation prevents the binding of complementary nucleotide units from a multivalent molecule, resulting in a decrease in signal strength. During a stepping event, the editing conformation prevents the binding and incorporation of complementary nucleotides or nucleotide analogs, resulting in a decrease in signal strength. Designing trapping and stepping polymerases with one or more mutant sites that reduce the conformational switch from nucleotide polymerization to editing can reduce trapping array-specific errors.

[0061] In some embodiments, the mutant polymerase comprises a polypeptide or fragment thereof derived from the directed evolution of recently identified novel B-family and A-family polymerases, and the mutant polymerase exhibits improved specificity while maintaining high discrimination for correct Watson-Crick base pairing.

[0062] The present disclosure provides a polymerase engineered to contain substitution mutations including a polymerase having an amino acid sequence backbone of RLF 89458.1 (e.g., Thermococci archaeon, isolate B13_G1) (SEQ ID NO: 1), RLF 78286.1 (e.g., Thermococci archaeon, isolate B89_G9) (SEQ ID NO: 2), NOZ 58130.1 (e.g., Euryarchaeota archaeon, isolate M_BaxBin.100) (SEQ ID NO: 1714), RMF 90817.1 (e.g., Euryarchaeota archaeon, isolate J060) (SEQ ID NO: 2789), MBC 7218772.1 (e.g., Hadesarchaea archaeon, isolate MAG-18) (SEQ ID NO: 2790), WP 175059460.1 (e.g., Thermococcus sp.2319x1) (SEQ ID NO: 2791), KUO 42443.1 (e.g., Candidatus Hadarchaem, yellowstonense, isolate YNP_45) (SEQ ID NO: 2792), and NOZ 77387.1 (e.g., Euryarchaeota archaeon, isolate M_MaxBin.027) (SEQ ID NO: 2793).

[0063] The polypeptides described herein include, but are not limited to, polypeptides having enzymatic activities such as polymerase activity, and are often described as families. In many cases, the polymerase is a DNA polymerase, an RNA polymerase, a template-independent polymerase, a reverse transcriptase, or other enzyme capable of nucleotide binding and nucleotide incorporation (e.g., primer extension). Many DNA polymerases are known in the art, and such enzymes are sometimes mutated to produce the compositions described herein. Members of the DNA polymerase family are often defined in terms of polymerase activity, active site structure, domain homology / function, or sequence homology to other known DNA polymerase family members. For example, DNA polymerases include, but are not limited to, E. coli DNA polymerase I, E. coli DNA polymerase II, or other members of the DNA polymerase family. Known thermostable DNA polymerases include, but are not limited to, Taq polymerase, Pfu polymerase, and 9°N polymerase, or other members of the DNA polymerase family. Wild-type DNA polymerases can be obtained or are obtainable from any number of sources such as eukaryotic, prokaryotic, or viral origins, and in some embodiments, for the purposes of the present disclosure, are obtained or are obtainable from archaeal origins. In some embodiments, a polymerase comprising an amino acid sequence of any of SEQ ID NOs: 1, 2, 1714, 2789-2793, and 2803 is a member of the DNA polymerase family. <>

[0064] The polymerase described in this specification can include one or more amino acid substitutions and / or cleavages that increase the thermal stability of the polymerase. In some embodiments, the thermal stability of the polymerase can be determined by measuring the temperature at which the polymerase unfolds and / or aggregates. For example, a thermal shift assay using differential scanning fluorimetry can be used to determine the thermal stability of the polymerase. Differential scanning fluorimetry can be used to determine the protein unfolding transition (T(m)) at which approximately 50% of the protein is in its native conformation and approximately 50% of the protein is denatured. The protein unfolding transition (T(m)) can be obtained from a melting peak where the increase in temperature is plotted along the x-axis and the first derivative curve of fluorescence over the change in temperature (e.g., dF / dT or dRFU / dT) is plotted along the y-axis. Some proteins exhibit two protein transitions, T(m1) and T(m2). For example, the T(m1) temperature is the temperature at which at least 50% of the protein transitions from a folded state to an unfolded state. In another example, the T(m2) temperature is a temperature higher than the T(m1) temperature, and at the T(m2) temperature, more protein transitions from a folded state to an unfolded state. Completely denatured protein and protein aggregation (T(agg)) can be obtained from a melting curve where the increase in temperature is plotted along the x-axis and fluorescence (e.g., RFU) is plotted along the y-axis. For example, Tables 1-2 (e.g., FIGS. 31-32) list the Tm1 and Tm2 of the engineered polymerase.

[0065] In some embodiments, the engineered polymerase exhibits a protein unfolding transition (e.g., thermal stability) at elevated temperatures compared to a wild-type polymerase or an engineered polymerase having mutations that do not confer increased thermal stability. In some embodiments, the engineered polymerase exhibits increased thermal stability at about 72 - 75 °C, or about 75 - 80 °C, or about 80 - 85 °C, or about 85 - 90 °C, or higher temperatures. Many of the engineered polymerases described herein exhibit nucleotide binding and incorporation activity in a temperature range of about 25 - 50 °C, or about 45 - 75 °C, or about 65 - 80 °C, or about 80 - 90 °C. Thus, these engineered polymerases are thermostable in a moderately high temperature range (e.g., mid-therm polymerases). The engineered polymerases described herein are suitable for performing nucleotide binding, nucleotide unit binding, nucleotide incorporation, and / or nucleic acid sequencing reactions in a temperature range of about 25 - 50 °C, or about 45 - 75 °C, or about 65 - 80 °C, or about 80 - 90 °C, or higher. In some embodiments, the mutant polymerase exhibits increased thermal stability at about 2 - 4 °C, or about 4 - 6 °C, or about 6 - 8 °C, or about 8 - 10 °C.

[0066] In contrast, DNA polymerases that exhibit significantly high thermal stability above 95 °C include 9°N, THERMINATOR, VENT, DEEP VENT, Pfu, and Pyrococcus abyssi. For example, thermostable polymerases such as 9°N, VENT, DEEP VENT, Pfu, and Pyrococcus abyssi polymerases are suitable for use in PCR reactions where typical cycling steps are performed at temperatures above 90 - 95 °C or higher, and may not be suitable for use in nucleotide binding, nucleotide incorporation, and / or nucleic acid sequencing reactions performed in lower temperature ranges. DNA polymerases from Geobacillus stearothermophilus (e.g., Bst DNA polymerase) are typically stable up to a maximum of 65 °C.

[0067] Polymerases include a variety of DNA polymerases, RNA polymerases, template-independent polymerases, reverse transcriptases, or other enzymes capable of catalyzing nucleotide incorporation. Archaeal polymerases are often derived from thermophilic organisms and can thus represent a class of thermostable or heat-resistant enzymes. Accordingly, the polypeptide backbone derived from an archaeal polymerase provides a desirable protein engineering target for further enhancing reversible terminator nucleotide incorporation for uses that can be improved by application of enzymes with improved thermal stability against degradation due to repeated exposure to high temperatures, changes in buffer conditions, etc., or other enzymes with improved resistance.

[0068] The present disclosure provides compositions and methods comprising mutant polymerase enzymes that exhibit an improved shelf life compared to wild-type polymerases or engineered polymerases having mutations that do not confer an improved shelf life. For example, the mutant polymerase can retain enzyme activity after storage for several months or years at a given temperature. In some embodiments, the mutant polymerase can retain about 90 - 100% activity over a storage period of 2 - 12 months at a temperature of about 0 to +27°C, or about -20°C to -45°C, or about -45°C to -80°C. In some embodiments, the mutant polymerase can retain about 80 - 90% activity over a storage period of 2 - 12 months at a temperature of about 0 to +27°C, or about -20°C to -45°C, or about -45°C to -80°C. In some embodiments, the mutant polymerase can retain about 70 - 80% activity over a storage period of 2 - 12 months at a temperature of about 0 to +27°C, or about -20°C to -45°C, or about -45°C to -80°C. In some embodiments, the mutant polymerase can retain about 70 - 100% activity over a storage period of about 12 - 36 months at a temperature of about 0 to +27°C, or about -20°C to -45°C, or about -45°C to -80°C.

[0069] In some embodiments, the wild-type or engineered polymerase can be stored in a storage buffer containing a solvent, at least one pH buffer, at least one salt, at least one chelating agent, at least one viscosity agent, and at least one detergent. In some embodiments, the solvent comprises water.

[0070] In some embodiments, the buffer comprises MES, HEPES, ACES, Tris and / or Tris-HCl, or any other pH buffer known to those skilled in the art. In some embodiments, the pH of the pH buffer is about 6 - 6.5, or the pH of the pH buffer is about 6.5 - 7, or the pH of the pH buffer is about 7 - 7.5, or the pH of the pH buffer is about 7.5 - 8, or the pH of the pH buffer is about 8 - 8.5. In some embodiments, the storage buffer contains at least one pH buffer at a concentration of about 10 - 20 mM, or about 20 - 30 mM, or about 30 - 40 mM, or about 40 - 50 mM.

[0071] In some embodiments, the salt comprises monovalent salts including NaCl, KCl, NH2SO4, and / or potassium glutamate. In some embodiments, the storage buffer contains at least one monovalent salt at a concentration of about 50 - 100 mM, or about 100 - 200 mM, or about 200 - 300 mM, or about 300 - 400 mM, or about 400 - 500 mM, or about 500 - 600 mM, or about 600 - 800 mM.

[0072] In some embodiments, the chelating agent includes EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethyl ethylenediamine triacetic acid), DPTA (diethylenetriamine pentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, ammonium dioxide, citric acid, potassium citrate, and / or magnesium citrate. In some embodiments, the storage buffer includes at least one chelation at a concentration of about 0.0125 to 0.025 mM, or about 0.025 to 0.05 mM, or about 0.05 to 0.1 mM, or about 0.1 to 0.2 mM.

[0073] In some embodiments, the viscosity agent includes sugars such as trehalose, sucrose, cellulose, xylitol, mannitol, sorbitol, and / or inositol. In some embodiments, the viscosity agent includes glycol compounds such as glycerol or ethylene glycol and / or propylene glycol. In some embodiments, the storage buffer includes at least one viscosity agent at about 20 to 30%, or about 30 to 40%, or about 40 to 50%, or about 50 to 60%, or about 60 to 70%, or about 70 to 80%.

[0074] In some embodiments, the detergent comprises an ionic detergent, such as SDS (sodium dodecyl sulfate). In some embodiments, the detergent comprises a non-ionic detergent, such as Triton® X-100, Tween® 20, Tween® 80, or Nonidet P-40. In some embodiments, the detergent comprises an amphoteric ionic detergent, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfate (DetX). In some embodiments, the detergent comprises LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the storage buffer comprises at least one detergent at about 0.025 - 0.5%, or about 0.5 - 0.1%, or about 0.1 - 0.2%, or about 0.2 - 0.4%, or about 0.4 - 0.8%, or about 0.8 - 1.6%.

[0075] The present disclosure provides compositions and methods comprising mutant polymerase enzymes that exhibit an improved ability to bind to complementary nucleotide units of a multivalent molecule, compared to a wild-type polymerase or an engineered polymerase having a mutation that does not confer improved binding to complementary nucleotide units of the multivalent molecule. The multivalent molecule generally comprises a central portion (e.g., a core) attached to a plurality of arms, each arm being attached to a nucleotide unit. The multivalent molecule includes a star, comb, cross-linked, bottlebrush, or dendrimer configuration (e.g., see Figure 2).

[0076] We have made the surprising discovery that many of the engineered polymerases described herein exhibit an improved incorporation rate of nucleotide analogs compared to wild-type polymerases or compared to engineered polymerases having mutations that do not confer an improved incorporation rate of nucleotide analogs. Compared to wild-type polymerases, some of the engineered polymerases exhibit one or more desirable properties including increased binding affinity for nucleotide analogs having a 3'-chain terminating group, improved ability to incorporate dATP nucleotides opposite a uracil-containing template molecule (e.g., uracil-resistant mutant polymerases), improved ability to bind to complementary nucleotide units of a multivalent molecule, increased thermal stability up to approximately 90°C, increased shelf life, and decreased sequence-specific errors.

[0077] The present disclosure provides compositions and methods comprising mutant polypeptides related to polymerase enzymes that exhibit an increased ability to bind and discriminate nucleotide analogs and an improved incorporation of nucleotide analogs compared to the corresponding wild-type polymerase. Nucleotide analogs include, for example, nucleotides containing a chain terminating group attached to the 2' or 3' position of the sugar. The chain terminating group includes an azide, azido, or azidomethyl group, or another type of chain terminating group. The engineered DNA polymerase exhibits an increased incorporation rate of nucleotide analogs compared to the corresponding wild-type polymerase having an amino acid sequence backbone of any of RLF 89458.1 (SEQ ID NO: 1), RLF 78286.1 (SEQ ID NO: 2), NOZ 58130.1 (SEQ ID NO: 1714), RMF 90817.1 (SEQ ID NO: 2789), MBC 7218772.1 (SEQ ID NO: 2790), WP 175059460.1 (SEQ ID NO: 2791), KUO 42443.1 (SEQ ID NO: 2792), and NOZ 77387.1 (SEQ ID NO: 2793).

[0078] The data shown in Tables 1 and 2 provide a number of exemplary mutant polymerases that exhibit increased thermal stability compared to the corresponding wild-type polymerase or compared to engineered polymerases having the same backbone sequence and mutations that do not confer increased thermal stability. Many of these mutant polymerases contain mutations in the LYP motif and / or at other positions. In some embodiments, the mutant polymerase exhibits an increase in the incorporation rate of nucleotide analogs of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 500%, or 1000% compared to the corresponding wild-type enzyme or enzyme variant currently known in the art. Exemplary mutant polymerases that exhibit increased thermal stability are listed in Tables 1 and 2.

[0079] The present disclosure provides compositions and methods comprising mutant polymerases in which at least one amino acid residue is inserted before or after the LYP motif. In some embodiments, at least one amino acid residue inserted before or after the LYP motif can move the LYP motif as a unit in the folded polypeptide, whereby the activity of the LYP motif in the mutant enzyme can be increased or decreased. In some embodiments, inserting at least one amino acid residue before or after the LYP motif can be more effective in modulating the activity of the LYP motif compared to conventional amino acid substitutions designed to change the amino acid side chains within and around the LYP motif. In some embodiments, at least one amino acid residue inserted before or after the LYP motif can modulate (e.g., increase or decrease) the incorporation rate of nucleotide analogs compared to a mutant polymerase lacking the at least one inserted amino acid residue.

[0080] In some embodiments, a polymerase that binds to a nucleotide (or nucleotide analog) or a multivalent molecule under conditions suitable for inhibiting the nucleotide incorporation reaction by the polymerase catalyst includes at least one amino acid residue inserted before or after the LYP motif. In some embodiments, a polymerase that binds to a nucleotide (or nucleotide analog) or a multivalent molecule under conditions suitable for promoting the nucleotide incorporation reaction by the polymerase catalyst includes at least one amino acid residue inserted before or after the LYP motif.

[0081] In some embodiments, at least one amino acid residue inserted before or after the LYP motif includes any of 20 amino acids (e.g., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q). In some embodiments, at least one amino acid residue inserted before or after the LYP motif includes proline or glycine. Exemplary variant polymerases include the amino acid sequence of any of SEQ ID NOs: 1076, 1094, 1197, or 1351.

[0082] In some embodiments, a variant polypeptide having at least one amino acid residue inserted before or after the LYP motif includes a backbone sequence of any of RLF 89458.1 (SEQ ID NO: 1), RLF 78286.1 (SEQ ID NO: 2), NOZ 58130.1 (SEQ ID NO: 1714), RMF 90817.1 (SEQ ID NO: 2789), MBC 7218772.1 (SEQ ID NO: 2790), WP 175059460.1 (SEQ ID NO: 2791), KUO 42443.1 (SEQ ID NO: 2792), and NOZ77387.1 (SEQ ID NO: 2793).

[0083] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif where the LYP motifs of RLF 89458.1 (SEQ ID NO: 1) and RLF 78286 (SEQ ID NO: 2) are located at positions L409, Y410, and P411.

[0084] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif of NOZ 58130 (SEQ ID NO: 1714) where the LYP motif is located at positions L440, Y441, and P442.

[0085] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif of RMF 90817 (SEQ ID NO: 2789) where the LYP motif is located at positions L421, Y422, and P423.

[0086] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif of MBC 7218772 (SEQ ID NO: 2790) where the LYP motif is located at positions L451, Y452, and P453.

[0087] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif of WP 175059460 (SEQ ID NO: 2791) where the LYP motif is located at positions L411, Y412, and P413.

[0088] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif of KUO 42443 (SEQ ID NO: 2792) where the LYP motif is located at positions L448, Y449, and P450.

[0089] In some embodiments, at least one amino acid residue can be inserted before or after the LYP motif of NOZ 77387 (SEQ ID NO: 2793) where the LYP motif is located at positions L432, Y433, and P434.

[0090] In some embodiments, a mutant polypeptide in which at least one amino acid residue is inserted before or after the LYP motif comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803.

[0091] The sites that confer a particular activity to the polypeptide can be conserved and can be positioned by aligning the amino acid sequences of various polymerases. For example, specific residues associated with polymerase activity (e.g., nucleotide incorporation) can be found at residues D405, D539, and / or D541 of a polymerase having the backbone sequence of RLF 89458.1 (SEQ ID NO: 1), or at residues D405, D539, and / or D541 of a polymerase having the backbone sequence of RLF 78286.1 (SEQ ID NO: 2), or at residues D436, D570, and / or D572 of a polymerase having the backbone sequence of NOZ 58130 (SEQ ID NO: 1714), or at residues D417, D551 and / or D553 of a polymerase having the backbone sequence of RMF 90817 (SEQ ID NO: 2789), or at residues D447, D585, and / or D587 of a polymerase having the backbone sequence of MBC 7218772 (SEQ ID NO: 2790), or at residues D407, D543, and / or D545 of a polymerase having the backbone sequence of WP 175059460 (SEQ ID NO: 2791), or at residues D444, D582, and / or D584 of a polymerase having the backbone sequence of KUO 42443 (SEQ ID NO: 2792), or at residues D428, D562, and / or D564 of a polymerase having the backbone sequence of NOZ 77387 (SEQ ID NO: 2793).

[0092] One of ordinary skill in the art can find these sites and other functionally equivalent sites in other polymerases by reviewing the sequence alignments shown in FIG. 33. Such sites are often found at similar positions within other regions and domains, and polypeptides containing such domains are consistent with the methods and compositions described herein.

[0093] Mutations in the polymerases described herein variously include one or more changes to the amino acid residues present within the polypeptide. Additions, substitutions, deletions, and / or cleavages are all examples of mutations that can be used to generate mutant polypeptides. In some embodiments, a substitution includes the exchange of one amino acid for an alternative amino acid, such alternative amino acids being different from the original amino acid with respect to size, shape, conformation, and / or chemical structure. In some embodiments, the mutation is conservative or non-conservative. Conservative mutations include the substitution of an amino acid with an amino acid having similar chemical properties. Additions often include the insertion of one or more amino acids at the N-terminus, C-terminus, or internal position of the polypeptide. In some cases, an addition includes a fusion polypeptide, where one or more additional polypeptides are attached to the polypeptide. In some embodiments, such additional polypeptides include domains having additional activity, or sequences having additional functions (e.g., improved expression, purification aid, improved solubility, binding to a solid support, or other functions). Often, the polypeptides described herein include one or more non-amino acid groups. A fusion polypeptide optionally includes an amino acid or other chemical linker that attaches one or more proteins. Any number of mutations can be introduced into the polypeptides or portions of polypeptides described herein, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more than 50 mutations.

[0094] In some embodiments, an entire domain (a portion of a polypeptide having a defined function) is added to, deleted from, or substituted for a domain from another polypeptide. Exemplary domains include intracellular localization domains such as DNA / RNA binding domains, nucleotide binding domains, nuclease domains, nuclear localization domains, or other domains. In some embodiments, the methods and compositions of the present disclosure include the binding of domains that function as spacers or labels and / or provide for the attachment of linkers such as SNAP tags, avidin moieties, streptavidin moieties, epitope tags, fluorescent proteins, affinity tags, metal binding (i.e., His6 (SEQ ID NO: 2850) or polyhistidine tags). In some embodiments, one or more mutations are present at any position, for example, in an exonuclease domain, nucleic acid binding domain, nucleotide binding domain, and / or catalytic site. The polypeptide includes at least one mutation and can be based on the wild-type backbone sequence of any one of SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, or 2803.

[0095] As used herein, the term "surrounding" an amino acid residue or sequence position includes residues from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more residues away from the named residue, i.e., substitutions, deletions, insertions, or modifications such as post-translational modifications at residues N- or C-terminal to the named residue, and has its ordinary meaning in the art that encompasses it. In some contexts, as will be understood by those skilled in the art, residues or sequence positions N- or C-terminal that are more than 12 residues from the named residue can be considered to "surround" the named residue based on the sequence or structural (i.e., three-dimensional) context.

[0096] Substitutions or modifications of the residues described herein may also incorporate or include non-standard amino acids known in the art, including but not limited to hydroxyproline, N-formylmethionine, selenomethionine, selenocysteine, phosphotyrosine, phosphohistidine, etc. Mutations, modifications, cleavages, substitutions, etc. described herein can be performed by any method known in the art, particularly in the fields of molecular biology and / or protein engineering. Such methods can include site-directed mutagenesis using mutagenic and / or partially denatured primers, in vitro gene assembly, gene editing (e.g., by CRISPR or related methods), etc. Variants or engineered proteins described herein can be further expressed, isolated, and / or purified by any such means known in the art. Related methods are described in Green, M. and Sambrook, J., Molecular Cloning: A Laboratory Manual (4th Edition), which is hereby incorporated by reference in its entirety with respect to the disclosure of methods for modifying, introducing, and expressing recombinant, modified, and engineered gene sequences, as well as methods for extracting, isolating, and / or purifying engineered proteins.

[0097] The polypeptides disclosed herein have been shown to function as nucleotide polymerases that exhibit higher thermal stability, a higher incorporation rate of 3'-O-azidomethylated nucleotides, increased uracil resistance, and / or improved binding to complementary nucleotide units of multivalent molecules, compared to the corresponding wild-type enzymes. The polypeptides disclosed herein can be used for the elongation of nucleic acids during replication or synthesis, or can capture / bind nucleotides at nucleotide addition sites, for example, by the use of non-incorporable or blocked nucleotides, or can be used under conditions where the necessary salts or cofactors are absent. The polypeptides disclosed herein can be utilized, for example, in polynucleotide sequencing applications, such as sequencing by synthesis and sequencing by ligation applications. Variant polymerases are disclosed herein that comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to any one of SEQ ID NO: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, or 2803.

[0098] The present disclosure provides engineered DNA polymerases that typically include a replication polymerase that exhibits high fidelity and includes the amino acid sequence backbone of a Family B or Family A polymerase. Examples of Family B polymerases include Family B archaeal DNA polymerases and Phi29 polymerase. In some embodiments, the engineered DNA polymerase can include a Family B archaeal DNA polymerase selected from Thermococcus, Thermoplasmata, Pyrococcus, Methanococcus, Hadesarchaea, Euryarchaeota, or Candidatus. In some embodiments, the engineered DNA polymerase that is a Family B polymerase includes the amino acid sequence backbone from a 9°N polymerase (including THERMINATOR polymerase), VENT polymerase, DEEP VENT polymerase, Pfu polymerase, or Pyrococcus abyssi polymerase. In some embodiments, the engineered DNA polymerase that is a Family A polymerase includes the amino acid sequence backbone of Geobacillus stearothermophilus (e.g., Bst DNA polymerase).

[0099] The engineered DNA polymerase can be designed and prepared by introducing one or more mutations into the amino acid sequence of the DNA polymerase of interest, and the phenotype of the resulting engineered polymerase can be determined. Any one or any combination of two or more mutation sites can be moved from one type of polymerase to a positionally equivalent site (or functionally equivalent site) in a second type of polymerase. For example, any one of SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, and 2803, or any one of SEQ ID NOs: 3-1713 or 1715-2787, any one or any combination of two or more mutation sites from a DNA polymerase containing the same can be introduced into a positionally equivalent site (or functionally equivalent site) of Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase, SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2801), RB69 polymerase (SEQ ID NO: 2802), or Phi29 polymerase (SEQ ID NO: 2803).

[0100] Exemplary sequence alignments are provided in FIGS. 33-40. The mutations include any one or any combination of substitutions, insertions, deletions, and / or truncations of two or more amino acids.

[0101] Functional equivalents of residues occupy similar positions within a sequence (e.g., sequence alignment) and / or the three-dimensional structure of an enzyme (e.g., DNA polymerase) and include one or more amino acid residues that perform substantially the same function as known amino acid residues within a known enzyme. Functionally equivalent amino acid substitutions include one or more amino acid residues at a specific position of a basic polypeptide that have the same functional role in another polypeptide. Functionally equivalent amino acid substitutions include either one or any combination of conservative and / or non-conservative amino acid substitutions. The sequence alignment has a backbone sequence of any one of SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, and 2803 and provides examples of amino acid residues at sites of DNA polymerases having functionally equivalent amino acid sites of Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N DNA polymerase (relative to SEQ ID NO: 2795 or 2796), THERMINATOR polymerase (relative to SEQ ID NO: 2797), VENT polymerase (relative to SEQ ID NO: 2798), DEEP VENT polymerase (relative to SEQ ID NO: 2799), Pfu DNA polymerase (relative to SEQ ID NO: 2800), Pyrococcus abyssi DNA polymerase (relative to SEQ ID NO: 2801), RB69 polymerase (relative to SEQ ID NO: 2802), or Phi29 polymerase (relative to SEQ ID NO: 2803), as provided in FIGS. 33-40.

[0102] Wild-type polypeptide sequences are often the starting point for protein or enzyme engineering to generate mutant polypeptides. In some embodiments, the mutant polypeptide differs from the wild-type polypeptide by at least one amino acid residue. Often, the mutant polypeptide differs from the closest wild-type polypeptide by at least one amino acid residue. In some embodiments, the mutant polypeptide differs from the wild-type polypeptide by at least two amino acid residues. In some embodiments, the mutant polypeptide differs from the wild-type polypeptide by at least three, four, five, six, or more amino acid residues. Often, the wild-type sequence is the closest wild-type sequence identified by aligning polypeptides that contain at least one mutation within the wild-type sequence. In some embodiments, the wild-type polypeptide sequence includes the sequence of a naturally occurring polypeptide.

[0103] An amino acid substitution refers to replacing an amino acid residue at a selected position within a polypeptide with a different amino acid having similar or different biochemical properties such as similar size, shape, conformation, chemical structure, charge, and / or hydrophobicity. The amino acid substitution can be a conservative or non-conservative amino acid substitution. In some embodiments, the amino acid residue at a selected position within a polypeptide can be replaced with an amino acid having a polar side chain. Examples of amino acids having a polar side chain include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. In some embodiments, the amino acid residue at a selected position within a polypeptide can be replaced with an amino acid having a non-polar side chain. Examples of amino acids having a non-polar side chain include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In some embodiments, the amino acid residue at a selected position within a polypeptide can be replaced with an amino acid having a hydrophobic side chain. Examples of amino acids having a hydrophobic side chain include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, and tryptophan. In some embodiments, the amino acid residue at a selected position within a polypeptide can be replaced with an amino acid having an uncharged side chain. Examples of amino acids having an uncharged side chain include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine. In some embodiments, the amino acid residue at a selected position within a polypeptide can be replaced with an amino acid having a positively charged side chain. Examples of amino acids having a positively charged side chain include arginine, histidine, and lysine. In some embodiments, the amino acid residue at a selected position within a polypeptide can be replaced with an amino acid having a negatively charged side chain. Examples of amino acids having a negatively charged side chain include aspartic acid and glutamic acid.

[0104] The exemplary polypeptide variants described herein are listed in Tables 1-2 (Figures 31-32).

[0105] In some embodiments, the polypeptide comprises the backbone sequence of RLF 89458.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity to either of SEQ ID NO: 1 or 2, and the polypeptide comprises at least one of the mutations listed in Table 1, Figure 31 (e.g., SEQ ID NOs: 3-1713).

[0106] In some embodiments, the polypeptide comprises the backbone sequence of NOZ 58130.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity to either of SEQ ID NO: 1714, and the polypeptide comprises at least one of the mutations listed in Table 2, Figure 32 (e.g., SEQ ID NOs: 1715-2787).

[0107] In some embodiments, the polypeptide comprises the backbone sequence of RMF 90817 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity to SEQ ID NO: 2787, and this polypeptide comprises at least one mutation that is positionally equivalent to the mutations listed in Table 1 and / or 2 (Figures 31 and 32 respectively) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787). In some embodiments, the positionally equivalent amino acid positions are shown in Figures 33 and 36.

[0108] In some embodiments, the polypeptide comprises the backbone sequence of MBC 7218772.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to SEQ ID NO: 2790, and this polypeptide comprises at least one of the mutations listed in Table 1 and / or 2 (FIGS. 31 and 32 respectively) (e.g., SEQ ID NOs: 3 to 1713) (e.g., SEQ ID NOs: 1715 to 2787), and this polypeptide comprises at least one mutation that is positionally equivalent to those mutations. In some embodiments, the positionally equivalent amino acid positions are shown in FIGS. 33 and 37.

[0109] In some embodiments, the polypeptide comprises the backbone sequence of WP 175059460.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to SEQ ID NO: 2791, and this polypeptide comprises at least one mutation that is positionally equivalent to the mutations listed in Table 1 and / or 2 (FIGS. 31 and 32 respectively) (e.g., SEQ ID NOs: 3 to 1713) (e.g., SEQ ID NOs: 1715 to 2787). In some embodiments, the positionally equivalent amino acid positions are shown in FIGS. 33 and 38.

[0110] In some embodiments, the polypeptide comprises the backbone sequence of KUO 42443.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to SEQ ID NO: 2792, and this polypeptide comprises at least one mutation that is positionally equivalent to the mutations listed in Table 1 and / or 2 (FIGS. 31 and 32 respectively) (e.g., SEQ ID NOs: 3 to 1713) (e.g., SEQ ID NOs: 1715 to 2787). In some embodiments, the positionally equivalent amino acid positions are shown in FIGS. 33 and 39.

[0111] In some embodiments, the polypeptide comprises the backbone sequence of NOZ 77387.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to SEQ ID NO: 2793, and this polypeptide comprises at least one mutation that is positionally equivalent to the mutations listed in Table 1 and / or 2 (FIGS. 31 and 32, respectively) (e.g., SEQ ID NOs: 3 to 1713) (e.g., SEQ ID NOs: 1715 to 2787). In some embodiments, the positionally equivalent amino acid positions are shown in FIGS. 33 and 39.

[0112] In some embodiments, the polypeptide comprises the backbone sequence of Phi29 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to SEQ ID NO: 2803, and this polypeptide comprises at least one mutation that is positionally equivalent to the mutations listed in Table 1 and / or 2 (FIGS. 31 and 32, respectively) (e.g., SEQ ID NOs: 3 to 1713) (e.g., SEQ ID NOs: 1715 to 2787).

[0113] Segments or portions of larger polypeptides are further described herein. Optionally, the segment has catalytic activity such as nucleotide incorporation and nucleic acid extension activity, particularly in relation to the nucleotide polymerization or exonuclease domains described herein. A polypeptide comprising any full-length or segment derived from any one of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803, and at least one additional residue (e.g., +1 residue) at the N-terminus or C-terminus is described herein. In some embodiments, both the N-terminus and C-terminus have at least one additional residue, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more than 100 additional residues.

[0114] For example, a polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803, such as adjacent N-terminal aspartic acid, adjacent C-terminal arginine, or a combination thereof (+1 residue), or an additional residue such as a residue identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803, such as adjacent N-terminal glutamine, adjacent C-terminal histidine, or a combination thereof (+1 residue), or an additional residue such as a residue identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803, such as adjacent N-terminal valine, adjacent C-terminal cysteine, or a combination thereof (+1 residue), or an additional residue such as a residue identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803, such as adjacent N-terminal threonine, adjacent C-terminal cysteine, or a combination thereof (+1 residue), or an additional residue such as a residue identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein.A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal threonine, an adjacent C-terminal cysteine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal aspartic acid, an adjacent C-terminal leucine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal aspartic acid, an adjacent C-terminal arginine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal threonine, an adjacent C-terminal threonine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein.A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal threonine, an adjacent C-terminal asparagine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal threonine, an adjacent C-terminal asparagine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein. A polypeptide comprising any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 such as an adjacent N-terminal threonine, an adjacent C-terminal serine, or a combination thereof (+1 residue), or additional residues such as residues identified by alignment of any one of SEQ ID NOs: 1 to 1713, 1714 to 2787, 2789, 2790, 2791, 2792, 2793, and 2803 is described herein.

[0115] Engineered polymerase comprising the RLF 89458.1 or RLF 78286.1 backbone sequence The present disclosure provides one or more mutant polymerases that include the backbone sequence of RLF 89458.1 or RLF 78286.1 and have sequence identity of 100%, at least 99%, at least 98%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% to any of SEQ ID NOs: 1-1713 (Table 1 and FIGS. 11-12). The amino acid sequences of RLF 89458.1 and RLF 78286.1 differ in the amino acid substitution at position 235 where RLF 78286.1 includes D235E.

[0116] In some embodiments, the mutant polymerase having the backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) includes various domains.

[0117] In some embodiments, the N-terminal domain includes amino acid residues 1-134 (SEQ ID NO: 2804) (e.g., FIG. 29).

[0118] In some embodiments, the exonuclease domain (e.g., 3'→5' exonuclease domain) includes amino acid residues 135-356 (SEQ ID NO: 2805) (e.g., FIG. 29).

[0119] In some embodiments, the first palm domain includes amino acid residues 357-454 (SEQ ID NO: 2806) (e.g., FIG. 29).

[0120] In some embodiments, the finger(s) domain includes amino acid residues 455-504 (SEQ ID NO: 2807) (e.g., FIG. 29).

[0121] In some embodiments, the second palm domain includes amino acid residues 505-615 (SEQ ID NO: 2808) (e.g., FIG. 29).

[0122] In some embodiments, the thumb domain comprises amino acid residues 616-765 (SEQ ID NO: 2809) (e.g., FIG. 29).

[0123] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and comprising at least one amino acid substitution mutation that decreases 3'→5' exonuclease activity compared to a polymerase lacking an exon minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D141 and / or position E143. In some embodiments, the mutant polymerase comprises the mutations D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C. In some embodiments, the mutant polymerase comprises the mutations E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143F, E143G, E143H, E143R, E143K, E143D, E143C, or E143Q. In some embodiments, position E143 is not mutated. In some embodiments, the mutant polymerase comprises any combination of mutations at the D141 and E143 sites.

[0124] In some embodiments, the mutant polymerase comprises the non-mutated E143E and the mutations D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C.

[0125] The present disclosure provides compositions and methods comprising mutant polymerase enzymes that can be used for the sequencing of uracil-containing nucleic acid template molecules. The mutant polymerase can exhibit increased uracil tolerance for incorporating dATP at the 3' end of a nucleic acid primer at a position opposite a uracil base within the nucleic acid template molecule. The mutant polymerase can also bind to a nucleotide unit having an adenine of a multivalent molecule at a position opposite a uracil base within the nucleic acid template molecule. The mutant polymerase can exhibit increased uracil tolerance compared to a wild-type polymerase or compared to an engineered polymerase having mutations that do not confer uracil tolerance. A mutant polymerase having the backbone sequence of RLF 89458 or RLF 78286 that is uracil-tolerant (e.g., SEQ ID NO: 1 or 2, respectively) can comprise mutations at positions Y7, T9, H89, Q91, V93, and / or R97. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has an amino acid substitution at position Y7, and this mutation comprises any one of Y7A, Y7F, Y7N, Y7D, Y7R, Y7W, Y7H, or Y7Q. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has an amino acid substitution at position T9, and this mutation comprises any one of T9N, T9E, T9S, T9L, T9I, T9D, T9A, or T9R. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has an amino acid substitution at position H89, and this mutation comprises any one of H89D, H89A, H89Y, H89R, H89N, H89Q, H89K, H89F, H89L, or H89V. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has an amino acid substitution at position Q91, and this mutation comprises any one of Q91L, Q91H, Q91R, Q91W, Q91A, Q91K, Q91N, Q91P, Q91V, or Q91Y.In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), has an amino acid substitution at position V93, and this mutation includes any of V93A, V93M, V93E, V93F, V93Y, V93G, V93S, V93K, V93T, or V93I. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), has an amino acid substitution at position R97, and this mutation includes any of R97C, R97H, R97S, R97P, R97L, R97A, R97N, R97Q, R97E, R97I, R97K, R97M, or R97T.

[0126] Other uracil-resistant mutant polymerases having the backbone sequence of NOZ 58130 (SEQ ID NO: 1714), RMF 90817 (SEQ ID NO: 2789), MBC 7218772 (SEQ ID NO: 2790), WP 175059460 (SEQ ID NO: 2791), KUO 42443 (SEQ ID NO: 2792), or NOZ 77387 (SEQ ID NO: 2793) may include mutations that are positionally equivalent to Y7, T9, H89, Q91, V93, and / or R97 of RLF 89458 (SEQ ID NO: 1). Figure 33 shows the sequence alignment of these various polymerases and their positionally equivalent amino acid residues.

[0127] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and comprising at least one amino acid substitution mutation of the LYP motif, for example, at positions L409, Y410, and P411. In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of nucleotide analogs. In some embodiments, any one or any combination of the first, second, and / or third positions of the LYP motif can be mutated. For example, mutations of the LYP motif include AAG, AAP, AAV, AAI, AGA, AGG, AGI, AGP, AGV, FAA, FAG, FAI, FAP, FAV, FGA, FGG, FGP, FGV, LAG, LAI, LAP, LGG, LGI, LGV, SAA, SAG, SAI, SAV, SGA, SGG, SGI, YAA, YAG, YAI, YAP, YGA, YGG, YGI, YGP, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, SAP, AAA, SGP, LFP, IFP, VFP, LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, SYG, SSG, SSS, CAG, ASG, SSG, MFG, and FTA.

[0128] In some embodiments, a polymerase comprising the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having a substitution mutation at position L409 comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L409 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine. SEQ ID NOs: 1-1713 include exemplary amino acid substitution mutations at position L409.

[0129] In some embodiments, a polymerase that includes the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has a substitution mutation at position Y410 includes a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y410 includes threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine. SEQ ID NOs: 1 to 1713 include exemplary amino acid substitution mutations at position Y410.

[0130] In some embodiments, a polymerase that includes the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has a substitution mutation at position P411 includes a polar uncharged amino acid, a nonpolar amino acid, or a positively charged amino acid. In some embodiments, the amino acid substitution mutation at position P411 includes serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline. SEQ ID NOs: 1 to 1713 include exemplary amino acid substitution mutations at position P411.

[0131] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation for reducing post-translational modifications. In some embodiments, the polymerase comprises at least one mutation of a methionine, lysine, histidine, tryptophan, and / or cysteine residue(s) or any combination thereof. In some embodiments, the polymerase comprises an amino acid substitution at positions M1, M129, M159, M313, M329, M467, and / or M759. In some embodiments, the mutation at position M1 comprises M1F, M1I, M1L, M1S, M1N, M1A, M1V, M1Y, M1Q, M1K, M1V, or M1A. In some embodiments, the mutation at position M129 comprises M129I, M129V, M129K, M129L, M129E, M129F, M129N, M129S, M129R, or M129Y. In some embodiments, the mutation at position M159 comprises M159W, M159F, or M159Y. In some embodiments, the mutation at position M313 comprises M313I, M313K, M313L, M313V, M313D, M313R, M313E, M313A, M313L, or M313N. In some embodiments, the mutation at position M329 comprises M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N, or M329E. In some embodiments, the mutation at position M467 comprises M467V, M467K, M467D, M467T, M467R, M467E, M467Q, or M467L. In some embodiments, the mutation at position M759 comprises M759T, M759S, M759N, M759R, M759E, M759D, or M759A.

[0132] In some embodiments, the polymerase has a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has amino acid substitutions for reducing post-translational modifications including mutations at positions K240, K306, K371, K429, K468, K476, and / or K592. In some embodiments, the mutation at position K240 includes K240S, K240E, K240R, K240D, K240N, K240Q, or K240A. In some embodiments, the mutation at position K306 includes K306R, K306N, K306Q, K306A, K306V, K306I, or K306F. In some embodiments, the mutation at position K371 includes K371R, K371D, K371N, K371Q, K371Y, K371T, K371V, or K371L. In some embodiments, the mutation at position K429 includes K429R, K429S, K429M, K429A, K429N, K429D, K429Q, K429H, K429Y, K429V, K429L, or K429E. In some embodiments, the mutation at position K468 includes K468R, K468E, K468Y, K468T, or K468L. In some embodiments, the mutation at position K476 includes K476R, K476D, K476A, K476F, or K476R. In some embodiments, the mutation at position K592 includes K592Q, K592R, K592W, K592Y, K592A, K592F, K592I, K592T, K592N, or K592S.

[0133] In some embodiments, the polymerase has a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has amino acid substitutions for reducing post-translational modifications including mutations at positions W299 and / or W767. In some embodiments, the mutation at position W299 includes W299F, W299E, W299N, W299Q, W299Y, W299A, or W299F. In some embodiments, the mutation at position W767 includes W767H, W767Y, W767F, W767S, W767R, W767D, W767A, or W767N.

[0134] In some embodiments, the polymerase has a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has an amino acid substitution for reducing post-translational modification including a mutation at position H601. In some embodiments, the mutation at position H601 includes H601R, H601I, H601A, H601T, H601V, H601L, H601N, or H601K.

[0135] In some embodiments, the polymerase has a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has an amino acid substitution for reducing post-translational modification including a mutation at position C428. In some embodiments, the mutation at position C428 includes C428Y.

[0136] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and having at least one mutation for removing unpaired cysteines that can reduce oxidative damage to the polymerase. In some embodiments, the removal of at least one unpaired cysteine increases the thermal stability of the mutant polymerase and / or reduces protein aggregation. In some embodiments, the removal of at least one unpaired cysteine increases the amount of protein in the lysate preparation. In some embodiments, the polymerase includes at least one mutation of cysteines at positions 223 and / or 509 or any combination thereof. In some embodiments, the mutation at position C223 includes the amino acid substitution C223L, C223M, C223A, C223S, C223P, C223K, C223N, C223D, or C223V. In some embodiments, the mutation at position C509 includes the amino acid substitution C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q.

[0137] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions to remove unpaired cysteines including amino acid substitutions at positions C509 and V419. In some embodiments, the mutation at C509 includes any of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q. In some embodiments, the mutation at V419 includes any of V419I, V419L, or V419R.

[0138] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions to remove unpaired cysteines including amino acid substitutions at positions C509 and Q497. In some embodiments, the mutation at C509 includes any of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q. In some embodiments, the mutation at Q497 includes any of Q497H, Q497G, Q497M, Q497N, Q497F, Q497L, Q497R, Q497K, Q497T, Q497E, Q497D, or Q497Y.

[0139] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions to remove unpaired cysteines including amino acid substitutions at positions C509 and S512. In some embodiments, the mutation at C509 includes any of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q. In some embodiments, the mutation at S512 includes any of S512R, S512D, S512E, S512H, S512F, S512K, S512W, or S512D.

[0140] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that increases the thermal stability of the polymerase compared to a wild-type polymerase or compared to an engineered polymerase having mutations that do not confer increased thermal stability. In some embodiments, the polymerase exhibits thermal stability at about 72-75 °C, or about 75-80 °C, or about 80-85 °C, or about 85-90 °C, or higher temperatures. In some embodiments, a thermal shift assay using differential scanning fluorimetry can be used to determine the thermal stability of the polymerase.

[0141] In some embodiments, a polymerase comprising a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having an amino acid substitution to increase thermal stability comprises an amino acid substitution at one or more positions that also confer exonuclease activity, including D141 and / or E143. In some embodiments, a polymerase engineered to exhibit increased thermal stability comprises an amino acid substitution at position M329 alone or in combination with an amino acid substitution at position D141 and / or E143. In some embodiments, a polymerase engineered to exhibit increased thermal stability comprises an amino acid substitution at position D315 alone or in combination with an amino acid substitution at position D141 and / or E143. In some embodiments, a polymerase engineered to exhibit an increased amount of protein (e.g., polymerase enzyme) in a lysate preparation comprises an amino acid substitution at position D141N alone or in combination with an amino acid substitution of either non-mutated E143 or E143. The increase in the amount of protein in the lysate preparation can increase the yield of active polymerase in the manufacturing process.

[0142] In some embodiments, the mutation at D141 includes D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C.

[0143] In some embodiments, the mutation at E143 includes E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143G, E143H, E143R, E143K, E143D, E143C, or E143Q.

[0144] In some embodiments, the mutation at M329 includes M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N, or M329E.

[0145] In some embodiments, the mutation at D315 includes D315A, D315E, D315R, D315W, D315L, D315W, D315F.

[0146] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having a C-terminal truncation to increase the thermal stability of the polymerase compared to a non-truncated polymerase having the same wild-type or engineered backbone sequence. The C-terminal region of the polymerase can be disordered and can contribute to protein aggregation. Thus, truncation in the C-terminal region can reduce protein aggregation and improve stability. In some embodiments, the polymerase exhibits thermal stability at about 72-75 °C, or about 75-80 °C, or about 80-85 °C, or about 85-90 °C, or higher temperatures. In some embodiments, the thermal stability of the polymerase can be determined using a thermal shift assay using differential scanning fluorimetry.

[0147] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and is engineered to exhibit increased thermal stability, and the polymerase comprises cleavage at amino acid positions including K464 (cleaved), R465 (cleaved), E475 (cleaved), Y481 (cleaved), E616 (cleaved), E620 (cleaved), E755 (cleaved), Y756 (cleaved), Q757 (cleaved), R758 (cleaved), M759 (cleaved), T762 (cleaved), W767 (cleaved), or M770 (cleaved). In Tables 1 and 2, cleavage is indicated by "^".

[0148] The present disclosure provides compositions and methods comprising a mutant polymerase having the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and an N-terminal cleavage that increases the thermal stability of the polymerase compared to an uncleaved polymerase having the same wild-type or engineered backbone sequence. In some embodiments, the engineered polymerase comprises a methionine deletion at position 1. Table 1 lists various engineered polymerases having a methionine deletion at position 1, shown as "M1X", and their Tm1 and Tm2 data.

[0149] The present disclosure provides compositions and methods comprising a mutant polymerase having the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and at least one mutation that reduces conformational switching from polymerization to exonuclease compared to a wild-type polymerase or an engineered polymerase having a mutation that does not reduce conformational switching. In some embodiments, protein modeling can identify major amino acid residues that interact with the primer or primer terminus, and these amino acid residues can serve a role in interacting with the primer or in moving the primer terminus from the polymerization site to the exonuclease site. In some embodiments, the polymerase comprises an amino acid substitution at any one or any combination of positions V610, D613, Q664, E668, P677, and / or D671.

[0150] In some embodiments, the mutation at V610 includes V610D, V610A, V610K, V610S, V610T, V610N, V610R, or V610Q.

[0151] In some embodiments, the mutation at D613 includes D613S, D613E, D613R, D613K, D613N, D613Q, D613A, D613V, D613Y, or D613F.

[0152] In some embodiments, the mutation at Q664 includes Q664A, Q664L, Q664V, Q664F, Q664I, Q664R, Q664K, Q664T, Q664N, or Q664M.

[0153] In some embodiments, the mutation at E668 includes E668G, E668K, E668M, E668A, E668P, E668S, E668R, E688N, E688D, E668Y, or E668Q.

[0154] In some embodiments, the mutation at P677 includes P677L, P677R, P677K, or P677A.

[0155] In some embodiments, the mutation at D671 includes D671G, D671R, D671Y, D671S, D671A, D671K, or D671N.

[0156] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that reduces protein aggregation as compared to a wild-type polymerase or as compared to an engineered polymerase having a mutation that does not confer reduced aggregation. In some embodiments, the mutant polymerase aggregates at about 72-75 °C, or about 75-80 °C, or about 80-85 °C, or about 85-90 °C, or at a higher temperature as compared to a wild-type polymerase or as compared to an engineered polymerase having a mutation that does not confer reduced aggregation. In some embodiments, a thermal shift assay using differential scanning fluorimetry can be used to determine the temperature of protein aggregation.

[0157] In some embodiments, a polymerase comprising the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having an amino acid substitution for reducing aggregation comprises an amino acid substitution at one or more of positions N11, K507, K511, and / or K637.

[0158] In some embodiments, the mutation at position N11 comprises N11S, N11A, N11R, N11Q, N11E, N11K, N11T, or N11D.

[0159] In some embodiments, the mutation at position K507 comprises K507L, K507E, K507S, K507A, K507N, K507Q, K507E, K507T, or K507D.

[0160] In some embodiments, the mutation at position K511 comprises E511K, E511S, E511A, E511R, E511N, E511T, E511Q, E511L, E511D, or E511A.

[0161] In some embodiments, the mutation at position K637 comprises K637M, K637A, K637N, K637Q, K637E, K637S, or K637T.

[0162] The present disclosure has a backbone sequence of RLF 89458.1 (SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2), and M1, L3, D4, D6, Y7, I8, T9, E10, N11, G12, K13, P14, V15, I16, R17, I18, F19, K20, K21, E22, K23, G24, E25, F26, K27, I28, E29, Y30, D31, R32, N33, F34, E35, P36, Y37, I38, Y39, A40, L41, L42, E43, D44, D45, E46, S47, I48, E49, D50, I51, K52, K53, I54, T55, R58, G56, E57, R58, H59, G60, K61, K62, V63, I65, I66, R67, V68, E69, K70, V71, K72, K73, K74, F75, L76, G77, E78, P79, I80, E81, V82, W83, K84, L85, V86, F87, E88, H89, P90, Q91, D92, V93, P94, A95, I96, R97, D98, A99, I100, R101, S102, H103, P104, A105, V106, R107, E108, I109, F110, E111, Y112, D113, I114, P115, F116, A117, K118, R119, Y120, L121, I122, D123, K124, L126, V127, P128, M129, E130, G131, G132, E133, L135, K136, L137, L138, A139, F140, D141, I142, E143, T144, F145, Y146, H147, E148, D150, E151, E156, M159, S166, W173, K174, I176, Y180, A190, I191, K192, L195, L198, R199, Q196, P203, V205, L207, Y209, G211, N213, F214, D215, F216, A217, Y218, I219, K220, C223, E224, K225, G227, L228, K229, F230, T231, I232, G233, R234, S237, E238, P239, K240, I241, Q242, R243, M244, G245, D246, R247, A249, E251, L258, Y261, P262, V264, R265, H266, T267, I268, R269, L270, P271, T272, Y273,T274, L275, E276, A277, V278, V282, F283, K285, K286, K287, E288, K289, V290, Y291, A292, I295, E297, A298, W299, K300, S301, E302, L305, K306, R307, V308, A309, Q310, Y311, M313, D315, R317, A318, Y320, E321, P328, V331, M329, E332, L333, A334, I337, G338, Q339, V341, D343, S345, S347, S348, T349, G350, N351, L352, V353, W355, Y356, L357, R359, V360, Y362, N365, E366, L367, K371, P372, G373, G374, E375, E376, Y377, Q378, M381, S383, S384, Y385, I386, G388, Y389, E394, K395, G396, E399, S400, A402, Y403, L404, F406, R407, S408, L409, Y410, P411, S412, I413, V415, H417, V419, P421, D422, T423, L424, E425, E427, C428, K429, N430, Y431, V433, A434, I436, Y439, R440, K443, K446, G447, F448, I449, P450, S451, I452, L453, E454, D455, I457, T459, K462, V463, K464, R465, M467, K468, T470, I471, D472, I474, E475, K476, M478, Y481, R484, A485, L486, K487, I488, N491, S492, Y493, Y494, G495, Q497, G498, Y499, P500, K501, S506, K507, E508, C509, E511, S512, V513, T514, G517, R518, H519, I521, T523, A528, E529, K534, V535, Y537, A538, D539, T540, D541, G542, F543, F544, I547, N549, E550, K551, P552, I555, S557, K558, A559, K560, K561, L563, K564, H565, E568, K569, G572, M573, E575, E577, L583Compositions and methods are provided that include a variant polymerase comprising any one or any combination of positions including G585, F586, V588, T589, K592, Y593, L595, I596, D599, H601, T604, R605, G606, L607, V609, V610, R611, R612, D613, E616, I617, K619, E620, T621, Q622, A623, K624, V625, L626, E627, V628, I629, L630, R631, E632, G633, S634, I635, E636, K637, A638, A639, G640, I641, V642, K644, V645, V646, E647, D648, L649, A650, N651, Y652, R653, V654, V656, E657, K658, I660, H662, E663, Q664, I665, T666, R667, E668, K670, D671, Y672, K673, A674, T675, G676, P677, H678, V679, A680, I681, A682, K683, R684, L685, Q686, A687, R688, G689, I690, K691, V692, K693, P694, T696, I698, S699, Y700, V701, V702, L703, K704, G705, S706, K707, K708, I709, D711, R712, V713, I714, L715, F716, D717, E718, Y719, D720, S721, S722, R723, K725, Y726, P728, Y730, Y731, I732, H733, N734, Q735, V736, P738, A739, V740, L741, R742, I743, L744, E745, A746, F747, G748, Y749, K750, E751, K752, D753, L754, E755, Y756, Q757, R758, M759, K760, Q761, T762, G763, L764, G765, A766, W767, L768, and / or M770.,

[0163] The present disclosure has a backbone sequence of RLF 89458.1 (SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2), and M1F, M1I, M1L, M1S, M1N, M1A, M1V, M1Y, M1Q, M1K, M1V, M1A, L3I, D4R, D4A, D6S, D6R, Y7A, Y7F, Y7N, Y7D, Y7R, Y7W, Y7H, Y7Q, I8S, T9N, T9E, T9S, T9L, T9I, T9D, T9A, T9R, E10V, E10D, E10K, E10R, E10A, E10N, N11S, N11A, N11R, N11Q, N11E, N11K, N11T, N11D, G12S, G12D, G12E, K13E, P14Q, P14N, P14S, V15I, I16T, I16N, I16F, R17H, R17C, I18V, I18L, F19Y, F19S, F19I, K20M, K20E, K21E, E22G, E22V, E22K, K23E, K23M, K23R, K23A, K23N, G24S, E25K, F26L, K27M, I28F, I28N, I28T, I28V, I28F, E29V, E29D, Y30F, Y30N, Y30D, Y30K, D31V, R32C, R32S, N33S, F34S, F34I, E35K, E35G, E35D, P36L, P36A, P36G, P36V, P36M, P36T, P36K, Y37N, Y37F, I38T, I38N, Y39F, A40G, A40V, A40T, L41P, L41F, L41Y, L41D, L41E, L42P, L42Q, E43V, E43K, E43D, D44N, D44G, D45V, E46V, E46S, S47N, S47G, S47R, S47A, I48V, E49G, E49K, D50V, D50G, D50N, D50E, I51K, I51F, I51V, K52I, K52R, K53E, I54T, I54N, I54F, I54K, I54V, T55I, T55S, T55A, G56D, G56S, G56V, G56A, E57G, E57K, R58C, R58L, R58H, H59L, H59Y, G60S, G60D, K61M, K61T, K62N, K62E, K62R, K62T, K62V, V63A, V63I, V63D, I65T, I65V, I65F, I65N, I66V, I66T, I66N, I66K, R67C, V68M, V68A, E69K, K70I, V71I, K72H, K72R, K72V,K72Q, K73E, K74E, K74R, K74N, K74Q, F75C, L76Q, G77D, G77S, E78K, E78G, E78N, E78S, E78R, P79S, I80F, I80N, I80K, I80S, I80R, E81D, E81V, V82A, W83R, K84R, L85V, L85Q, L85A, V86D, V86I, V86A, V86Y, F87I, F87L, F87C, E88K, E88D, E88N, E88T, H89D, H89A, H89Y, H89R, H89N, H89Q, H89K, H89F, H89L, H89V, P90L, P90S, P90D, P90R, P90A, P90G, P90V, P90M, P90T, P90K, Q91L, Q91H, Q91R, Q91W, Q91A, Q91K, Q91N, Q91P, Q91V, Q91Y, D92N, D92V, D92E, D92R, V93A, V93M, V93E, V93F, V93Y, V93G, V93S, V93K, V93T, V93I, V93L, P94L, P94W, P94Y, P94Q, P94F, P94S, A95V, I96T, I96K, I96S, R97C, R97H, R97S, R97P, R97L, R97A, R97N, R97Q, R97E, R97I, R97K, R97M, R97T, D98E, D98N, D98V, A99T, A99K, I100T, R101C, R101H, S102N, S102G, S102E, H103R, H103L, H103Q, H103Y, P104T, P104L, A105S, V106A, V106T, R107C, R107S, R107V, E108V, I109K, I109N, I109F, F110L, F110S, F110Y, E111V, E111G, Y112C, D113G, D113Y, I114T, I114A, I114G, I114V, I114M, I114T, I114K, P115C, P115L, P115S, P115R, P115F, F116L, F116S, F116A, A117T, A117V, A117K, K118M, K118R, K118A, K118Q, K118Y, K118N, R119H, R119S, R119C, R119A, R119G, R119V, R119M, R119T, R119K, R119Y, Y120C, Y120N, L121M, I122V, I122F, I122N, I122D, D123GD123E, D123N, D123V, K124N, K124E, K124R, L126F, L126P, L126Q, V127M, V127I, P128L, P128M, M129I, M129V, M129K, M129L, M129E, M129F, M129N, M129S, M129R, M129Y, E130D, E130G, E130V, E130K, E130T, G131S, G132S, G132D, E133K, L135M, L135P, L135Q, K136E, K136R, K136L, L137F, L137M, L138P, A139E, F140Y, F140L, F140S, D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, D141C, I142V, I142F, I142A, E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143G, E143H, E143R, E143K, E143D, E143C, E143Q, T144F, F145L, Y146C, Y146A, Y146E, Y146S, Y146E, Y146K, Y146T, Y146R, H147E, H147R, H147E, H147D, H147N, H147Q, H147K, H147A, E148S, E148D, E148R, E148A, D150R, D150E, E151R, E156P, M159W, M159F, M159Y, S166E, W173R, W173A, W173Q, W173N, W173H, W173F, K174N, K174D, K174E, K174Q, I176V, Y180F, A190V, A190M, I191L, K192L, L195A, R199H, Q196R, L198I, L198V, P203S, V205A, L207I, L207V, Y209A, Y209E, Y209W, G211S, N213E, N213W, N213Y, F214A, F214E, F214W, F214V, D215A, D215N, D215Q, D215E, D215R, D215K, D215P, F216L, A217N, A217T, A217Q, A217S, Y218H, I219VI219L, K220R, K220N, K220Q, K220H, K220I, K220L, K220M, K220Y, C223V, C223E, C223S, C223L, C223M, C223A, C223P, C223K, C223N, C223D, E224V, E224R, E224K, K225E, G227S, L228P, L228I, L228V, K229R, K229N, K229Q, K229H, F230L, T231I, T231P, I232F, G233D, R234C, S237G, S237C, E238S, E238R, P239S, K240S, K240E, K240R, K240D, K240N, K240Q, K240A, I241T, I241Q, I241E, I241N, I241A, I241S, I241D, I241P, Q242N, Q242S, Q242R, Q242D, Q242E, Q242V, R243E, M244T, M244K, G245D, G245S, G245R, G245A, G245N, G245K, D246R, D246L, D246E, D246V, D246Q, R247E, R247D, R247S, R247H, A249G, A249V, E251S, E251R, E251A, L258I, L258Q, L258F, Y261A, Y261P, Y261T, P262S, P262R, P262L, P262D, P262E, P262Q, V264I, V264A, R265D, R265I, H266F, H266W, H266Y, H266R, H266I, H266L, H266A, H266K, T267A, T267F, T267M, T267V, T267W, T267Y, T267I, T267S, I268A, I268F, I268M, I268V, I268W, I268Y, R269L, R269K, R269S, R269T, R269V, R269N, R269H, R269Y, L270R, P271S, P271F, P271E, P271L, P271Q, P271N, T272A, T272Y, T272V, T272S, T272L, T272E, T272C, T272R, T272W, T272N, T272F, T272H, T272K, Y273A, Y273W, T274E, T274W, T274S, T274D, T274V, T274A, T274R, T274N, L275P, L275M, E276K, A277VV278M, V282L, V282T, V282G, V282I, F283L, K285I, K285Q, K286E, K286P, K287R, E288G, E288K, K289E, K289Q, K289N, K289I, K289R, V290E, Y291F, Y291N, Y291D, Y291K, Y291R, Y291Q, Y291A, A292N, A292T, A292I, I295N, E297G, A298G, W299F, W299E, W299N, W299Q, W299Y, W299A, W299F, K300S, K300E, S301N, S301T, E302G, L305P, K306R, K306N, K306Q, K306A, K306V, K306I, K306F, R307C, V308I, V308A, A309S, Q310R, Y311A, Y311E, Y311W, Y311F, M313I, M313K, M313L, M313V, M313D, M313R, M313E, M313A, M313L, M313N, D315A, D315E, D315R, D315W, D315L, D315W, D315F, R317C, R317K, A318V, Y320F, E321L, P328A, M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N, M329E, V331A, E332K, E332G, E332Q, L333A, L333V, L333I, L333F, A334S, I337V, G338D, Q339N, V341L, D343E, D343N, D343R, D343A, S345C, S345R, S347N, S347T, S347R, S347A, S348C, T349A, T349E, T349F, T349I, T349L, T349N, T349S, T349Y, G350S, N351S, N351Q, N351R, N351I, N351Y, N351K, N351A, N351E, L352M, V353Q, V353E, W355R, W355F, Y356N, Y356C, Y356L, Y356F, L357P, R359H, V360A, V360D, V360I, V360K, Y362I, Y362E, Y362F, Y362N, Y362D, Y362K, Y362R, Y362T, N365S, E366A, E366D, E366N, E366Q, E366R, L367P, K371R, K371DK371N, K371Q, K371Y, K371T, K371V, K371L, P372S, P372M, G373S, G373D, G374E, E375R, E375K, E376K, Y377L, Q378R, Q378A, Q378E, Q378K, M381I, M381R, M381V, M38, 1D, M381L, S383G, S383Q, S383E, S383T, S384A, S384R, S384D, S384Q, S384E, S384T, Y385R, Y385S, Y385F, Y385K, Y385H, Y385W, Y385A, Y385M, Y385F, Y385D, I386A, I386N, I386D, I386E, I386K, I386T, I386L, G388S, G388R, Y389R, Y389S, Y389F, Y389N, Y389D, Y389K, Y389A, Y389Q, Y389E, Y389I, E394G, K395R, G396S, E399D, S400N, S400D, A402T, A402V, Y403H, Y403L, Y403D, Y403Q, Y403E, Y403H, Y403K, Y403F, Y403W, Y403R, L404Q, F406Y, F406R, F406I, R407N, R407K, R407A, R407L, R407V, R407I, S408A, S408G, L409S, L409F, L409A, L409Y, L409I, L409V, L409T, L409N, L409C, L409M, Y410A, Y410G, Y410F, Y410M, Y410L, Y410D, Y410T, Y410I, Y410N, Y410V, Y410E, Y410S, Y410L, P411G, P411A, P411I, P411V, P411S, P411T, P411L, S412N, S412A, S412G, I413F, I413V, V415M, V415K, V415R, V415N, V415T, V415I, H417I, H417R, H417F, H417Y, H417V, V419I, V419L, V419R, P421S, D422V, T423I, T423L, L424Q, E425N, E427G, E427R, C428Y, K429R, K429S, K429M, K429A, K429N, K429D, K429Q, K429H, K429Y, K429V, K429L, K429E, N430E, Y431A, Y431D, V433A, A434V, A434D, A434P, I436T, I436F, I436A, I436R, I436N, I436D, I436Q, I436E, I436H, I436K, I436S, Y439H, R440H, K443R, K446P, G447D, F448I, F448L, I449NI449F, P450L, S451N, S451T, S451A, S451D, I452L, L453Q, E454D, E454N, E454T, E454G, D455N, I457L, T459E, K462D, V463M, V463I, K464C, R465C, R465T, M467V, M467K, M467D, M467T, M467R, M467E, M467Q, M467L, K468R, K468E, K468Y, K468T, K468L, T470S, T470A, I471K, I471Q, I471S, I471V, I471K, D472V, D472E, D472N, I474C, I474F, I474V, I474L, E475C, K476R, K476D, K476A, K476F, K476R, M478L, Y481C, Y481A, Y481F Y481T, Y481V, Y481W, R484D, R484N, R484K, R484S, R484T, R484V, R484L, A485S, A485T, A485L, A485V, A485G, A485R, L486I, K487M, K487R, K487N, K487A, K487Q, K487Y, I488A, I488V, I488S, I488T, I488M, I488R, I488N, I488Q, I488E, I488K, I488L, N491T, N491S, N491A, N491I, S492G, S492Y, S492D, S492K, S492T, S492N, S492E, Y493T, Y493S, Y493I, Y493F, Y493W, Y494A, Y494N, Y494G, Y494F, Y494W, G495S, Q497H, Q497G, Q497M, Q497N, Q497F, Q497L, Q497R, Q497K, Q497T, Q497E, Q497D, Q497Y, G498R, G498D, G498E, G498F, G498I, G498S, Y499F, P500A, K501R, K501A, K501Q, K501Y, K501N, S506C, S506R, S506A, S506L, S506T, S506N, S506D, S506H, S506V, K507L, K507E, K507S, K507A, K507N, K507Q, K507E, K507T, K507D, E508Q, E508C, C509V, C509Y, C509S, C509M, C509A, C509N, C509DC509H, C509Q, E511K, E511S, E511A, E511R, E511N, E511T, E511Q, E511L, E511D, E511A, S512R, S512D, S512E, S512H, S512F, S512K, S512W, S512D, V513T, V513I, V513L, V513M, V513F, V513A, V513S, T514A, T514G, T514S, T514V, T514I, T514S, G517A, G517S, G517V, G517T, R518C, H519N, H519Y, H519E, H519Q, I521N, I521T, I521E, I521H, T523I, T523A, A528L, A528I, E529N, K534N, K534S, K534R, V535N, V535K, V535S, V535R, Y537H, A538V, A538G, D539A, D539G, D539E, D539V, D539L, D539S, D539N, D539I, T540I, D541A, D541G, D541E, G542S, G542E, G542D, G542N, G542R, G542T, F543L, F544H, F544Y, I547F, I547T, I547P, N549G, E550A, K551D, P552L, I555V, S557C, S557K, K558A, K558V, K558Q, K558R, A559K, K561N, K561E, L653M, K564S, K564Q, H565Y, H565S, H565N, E568K, K569E, G572S, M573I, M573V, M573K, M573D, M573A, M573R, M573L, E575K, E577D, L583P, G585D, G585A, G585I, G585V, G585Y, G585F, G585T, F586I, V588E, V588T, T589K, K592Q, K592R, K592W, K592Y, K592A, K592F, K592I, K592T, K592N, K592S, Y593R, Y593N, Y593E, Y593H, Y593K, Y593F, Y593A, L595V, I596T, D599E, H601R, H601I, H601A, H601T, H601V, H601L, H601N, H601K, T604S, R605K, R605N, R605Q, R605H, R605D, R605E, R605S, R605TR605Y, R605A, G606S, G606R, G606Y, G606Q, G606N, G606E, G606D, L607F, V609I, V609L, V610D, V610A, V610K, V610S, V610T, V610N, V610R, V610Q, R611M, R611E, R612E, R612H, R612F, R612W, R612M, R612S, R612N, R612G, R612L, R612I, D613S, D613E, D613R, D613K, D613N, D613Q, D613A, D613V, D613Y, D613F, E616C, E616G, I617V, K619R, K619A, K619S, K619T, K619V, E620D, E620K, E620C, E620V, T621I, T621S, Q622L, A623T, A623C, A623K, K624I, K624R, V625F, L626I, E627K, V628L, V628I, V628A, I629F, I629C, L630Q, L630M, R631H, R631C, R631D, R631K, E632G, E632C, E632D, E632H, G633S, G633D, S634C, S634D, I635V, I635N, I635T, E636G, E636K, E636D, K637M, K637A, K637N, K637Q, K637E, K637S, K637T, A638E, A638V, A638T, A639T, A639V, G640D, G640R, I641F, I641V, I641A, V642I, V642A, K644E, V645E, V645I, V645M, V646A, V646D, E647G, E647D, E647K, D648V, D648C, D648L, D648G, L649Q, A650E, A650V, A650T, A650N, A650S, N651S, N651K, Y652H, Y652C, Y652M, Y652L, Y652F, R653C, R653H, R653Y, R653E, V654M, V656I, V656I, E657V, K658R, K658E, K658I, K658L, I660V, H662V, E663K, E663R, E663S, E663M, E663Q, E663V, Q664A, Q664L, Q664V, Q664F, Q664I, Q664R, Q664K, Q664T, Q664N, Q664M, I665VI665F, I665P, I665M, I665F, T666A, R667E, E668G, E668K, E668M, E668A, E668P, E668S, E668R, E688N, E688D, E668Y, E668Q, K670E, K670I, K670R, K670S, D671G, D671R, D671Y, D671S, D671A, D671K, D671N, Y672F, K673I, K673Y, K673R, K673S, K673E, A674T, A674V, A674S, T675S, T675I, T675A, G676S, G676F, G676E, G676Y, G676R, G676L, G676Q, P677L, P677R, P677K, P677A, H678R, H678K, H678Q, H678F, H678W, V679S, V679M, A680V, A680I, A680D, I681T, I681L, I681F, I681V, A682T, A682S, K683R, R684H, L685E, Q686R, Q686C, Q686L, Q686A, A687C, A687T, A687S, R688S, G689S, G689D, I690V, I690F, I690R, I690N, I690Q, I690K, I690T, I690Y, K691R, K691V, K691T, K691D, K691E, K691Q, K691N, V692I, K693M, K693V, K693Q, K693R, K693A, K693T, K693N, K693Y, K693S, K693E, K693D, P694R, T696S, T696I, T696L, T696M, I698K, I698M, I698F, I698Q, S699I, S699G, Y700W, Y700S, V701I, V702A, V702I, L703P, K704E, K704I, K704N, G705D, S706N, S706C, S706G, K707I, K707G, K707N, K7P728A, Y730H, Y731H, I732T, I732F, I732N, H733R, H733E, N734Y, N734R, N734P, N734D, N734K, N734T, Q735H, Q735R, V736A, P738L, A739V, V740I, L741A, L741Q, L741E, R742K, R742L, R742C, I743V, I743E, L744A, E745V, E745F, E745R, A746V, A746G, F747L, F747Y, G748V, G748K, Y749F, Y749E, K750N, K750R, E751K, E751D, E751M, K752E, K752L, D753V, D753E, D753G, L754Y, L754S, E755G, E755Q, E755D, E755K, E755Y, E755R, Y756C, Y756F, Y756I, Y756R, Y756Q, Y756K, Q757L, Q757H, Q757S, Q757M, R758H, R758A, R758K, M759T, M759S, Mx759N, M759R, M759E, M759D, M759A, Q761L, T762N, G765S, G765R, G765L, G765F, G765Y, G765I, G765T, G765D, G765E, W767H, W767Y, W767F, W767S, W767R, W767D, W767A, W767N, M770S, M770T, and / or M770N, and compositions and methods comprising a variant polymerase comprising any one or any combination of positions so included are provided.

[0164] In some embodiments, the variant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and comprises an amino acid deletion at any one or any combination of positions including M1 (deletion), R58 (deletion), V93 (deletion), and / or E755 (deletion).

[0165] Note: There was a typo in the original text where "Mx759N" was written. It was assumed to be "M759N" for the translation.In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position M1. In some embodiments, the amino acid substitution at position M1 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0166] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position Y7. In some embodiments, the amino acid substitution at position Y7 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0167] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position K74. In some embodiments, the amino acid substitution at position K74 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0168] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position E88. In some embodiments, the amino acid substitution at position E88 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0169] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position V93. In some embodiments, the amino acid substitution at position V93 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0170] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position A217. In some embodiments, the amino acid substitution at position A217 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0171] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position Y261. In some embodiments, the amino acid substitution at position Y261 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0172] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position T267. In some embodiments, the amino acid substitution at position T267 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, or Q), or has a non-natural amino acid known to those skilled in the art.

[0173] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position I268. In some embodiments, the amino acid substitution at position I268 includes any of the 20 natural amino acids (i.e., W, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0174] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position E366. In some embodiments, the amino acid substitution at position E366 includes any of the 20 natural amino acids (i.e., W, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0175] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position I436. In some embodiments, the amino acid substitution at position I436 includes any of the 20 natural amino acids (i.e., W, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0176] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position A485. In some embodiments, the amino acid substitution at position A485 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, V, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0177] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position T514. In some embodiments, the amino acid substitution at position T514 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, or Q), or has a non-natural amino acid known to those skilled in the art.

[0178] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and has a substitution mutation at position D671. In some embodiments, the amino acid substitution at position D671 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0179] Engineered polymerase comprising the NOZ 58130.1 backbone sequence The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of NOZ 58130.1 and having a sequence identity of 100%, at least 99%, at least 98%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% to any of SEQ ID NOs: 1714 - 2787 (Table 2 of FIG. 32 and FIG. 13).

[0180] In some embodiments, the mutant polymerase having a backbone sequence of NOZ 58130.1 (e.g., SEQ ID NO: 1714) includes various domains.

[0181] In some embodiments, the N-terminal domain includes amino acid residues 1 - 162 (SEQ ID NO: 2810) (e.g., FIG. 30).

[0182] In some embodiments, the exonuclease domain (e.g., 3’→5’ exonuclease domain) comprises amino acid residues 163 to 405 (SEQ ID NO: 2811) (e.g., FIG. 30).

[0183] In some embodiments, the palm domain comprises amino acid residues 406 to 640 (SEQ ID NO: 2812) (e.g., FIG. 30).

[0184] In some embodiments, the finger(s) subdomain within the palm domain comprises amino acid residues 480 to 527 (SEQ ID NO: 2813) (e.g., FIG. 30).

[0185] In some embodiments, the thumb domain comprises amino acid residues 641 to 792 (SEQ ID NO: 2814) (e.g., FIG. 30).

[0186] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of NOZ 58130.1 (e.g., SEQ ID NO: 1714) and comprising at least one amino acid substitution mutation that reduces 3’→5’ exonuclease activity compared to a polymerase lacking an exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D168 and / or position E170. In some embodiments, the mutant polymerase comprises the mutation D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168K, D168T, D168S, D168W, D168M, D168P, D168G, D168H, D168R, D168E, D168C, or D168Q. In some embodiments, the mutant polymerase comprises the mutation E170A, E170V, E170L, E170I, E170F, E170Y, E170N, E170K, E170T, E170S, E170W, E170M, E170P, E170G, E170H, E170R, E170D, E170C, or E170Q. In some embodiments, the mutant polymerase comprises any combination of mutations at the D168 and E170 sites. SEQ ID NOs: 1714 to 2787 comprise exemplary amino acid substitution mutations at positions D168 and E170.

[0187] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and comprising at least one amino acid substitution mutation of the LYP motif at positions L440, Y441, and P442, for example. In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of nucleotide analogs. In some embodiments, any one or any combination of the first, second, and / or third positions of the LYP motif can be mutated. For example, mutations of the LYP motif include YAG, FAG, YGP, YAP, FGP, SAP, AAA, YGA, YAA, FGA, FTA, AAG, AAP, AAV, AAI, AGA, AGG, AGI, AGP, AGV, FAA, FAI, FAP, FAV, FGG, FGV, LAG, LAI, LAP, LGG, LGI, LGV, SAA, SAG, SAI, SAV, SGA, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, SGP, LFP, IFP, VFP, LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, MFG, and SYG.

[0188] In some embodiments, a polymerase comprising the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and having a substitution mutation at position L440 comprises a non-polar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L440 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine. SEQ ID NOs: 1714-2787 include exemplary amino acid substitution mutations at position L440.

[0189] In some embodiments, a polymerase that includes the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position Y441 includes a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y441 includes threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine. SEQ ID NOs: 1715-2787 include exemplary amino acid substitution mutations at position Y441.

[0190] In some embodiments, a polymerase that includes the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position P442 includes a polar uncharged amino acid, a nonpolar amino acid, or a positively charged amino acid. In some embodiments, the amino acid substitution mutation at position P442 includes serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline. SEQ ID NOs: 1715-2787 include exemplary amino acid substitution mutations at position P442.

[0191] The present disclosure provides compositions and methods that include a mutant polymerase having the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and having at least one mutation for reducing post-translational modification as compared to a wild-type polymerase or an engineered polymerase lacking mutations for reducing post-translational modification. In some embodiments, the polymerase includes at least one mutation of a methionine, lysine, tryptophan, and / or serine residue(s) or any combination thereof. In some embodiments, the polymerase is engineered to include any one or any combination of amino acid substitution mutations at W135, M187, W329, K335, M389, S473, M527, K552, M629, W641, K650, K711, M723, and / or W791.

[0192] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W135 including W135S, W135L, W135R, W135Y, W135F, W135D, W135A, W135V, or W135G.

[0193] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M187 including M187S, M187L, M187R, M187Y, M187I, M187T, M187A, or M187V.

[0194] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W329 including W329Y, W329F, W329L, W329D, W329A, or W329V.

[0195] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K335 including K335R, K335L, K335S, or K335A.

[0196] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M389 including M389D, M389E, M389L, M389Y, M389S, M389A, or M389V.

[0197] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position S473 including S473K, S473R, S473T, S473Q, or S473A.

[0198] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M527 including M527H, M527G, M527Q, M527L, M527D, M527A, or M527V.

[0199] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M549 including M549N, M549Y, M549H, M549T, M549D, M549R, M549A, or M549V.

[0200] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K552 including K552R, K552T, K552N, K552Q, or K552A.

[0201] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M629 including M629L, M629A, M629D, M629R, or M629V.

[0202] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W641 including W641R, W641A, W641L, W641F, W641Y, or W641V.

[0203] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K650 including K650T, K650C, K650A, K650R, or K650S.

[0204] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K711 including K711R, K711L, K711T, or K711D.

[0205] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M723 including M723S, M723I, M723T, M723N, M723R, M723L, M723A, or M723C.

[0206] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W791 including W791R, W791Y, W791D, W791S, W791L, W791A, or W791V.

[0207] The present disclosure provides compositions and methods comprising a mutant polymerase having the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and having at least one mutation to remove unpaired cysteines that can reduce oxidative damage to the polymerase. In some embodiments, removal of at least one unpaired cysteine increases the thermal stability of the mutant polymerase and / or reduces protein aggregation. In some embodiments, removal of at least one unpaired cysteine increases the amount of protein in the lysate preparation. In some embodiments, the polymerase comprises at least one mutation of cysteine at position 362 and / or position 539 or any combination thereof. In some embodiments, the mutation at position C362 comprises the amino acid substitution C362A, C362L, C362I, C362S, C362F, C362Y, C362V, C362P, C362K, C362N, or C362D. In some embodiments, the mutation at position C539 comprises the amino acid substitution C539A, C539V, C539L, C539S, C539Y, C539D, C539K, C539N, or C539P.

[0208] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714), has non-mutated or mutated C362, and has the amino acid substitution R536C.

[0209] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714), has non-mutated or mutated C539, and has the amino acid substitution R536C.

[0210] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has non-mutated or mutated C362 and the amino acid substitution D451C.

[0211] The present disclosure provides compositions and methods comprising a mutant polymerase having a C-terminal truncation that increases the thermal stability of the polymerase compared to a non-cleaved polymerase having the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and the same wild-type or engineered backbone sequence. The C-terminal region of the polymerase can be disordered and can contribute to protein aggregation. Thus, truncation in the C-terminal region can reduce protein aggregation and improve stability. In some embodiments, the polymerase exhibits thermal stability at about 72-75 °C, or about 75-80 °C, or about 80-85 °C, or about 85-90 °C, or higher temperatures. In some embodiments, the thermal stability of the polymerase can be determined using a thermal shift assay using differential scanning fluorimetry.

[0212] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a C-terminal truncation for increasing thermal stability that includes M723 (cleavage), G773 (cleavage), D777 (cleavage), E781 (cleavage), T784 (cleavage), Q785 (cleavage), R790 (cleavage), W791 (cleavage), or F792 (cleavage). In Table 2, cleavage is indicated by "^".

[0213] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and having at least one mutation that reduces the conformational switch from polymerization to exonuclease compared to wild-type polymerase or compared to an engineered polymerase having a mutation that does not reduce conformational switching. In some embodiments, protein modeling can identify major amino acid residues that interact with the primer or primer terminus, and these amino acid residues can serve a role in interacting with the primer or in moving the primer terminus from the polymerization site to the exonuclease site. In some embodiments, the polymerase comprises an amino acid substitution at any one or any combination of positions Q95, I186, V304, L313, and / or E318.

[0214] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position Q95 comprising Q95L, Q95H, Q95R, Q95W, Q95A, Q95K, Q95N, or Q95P.

[0215] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position I186 comprising I186R or I186N.

[0216] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position V304 comprising V304D.

[0217] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position L313 comprising L313M, L313D, L313F, L313K, L313R, L313A, or L313E.

[0218] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position E318 including E318V.

[0219] The present disclosure provides a composition and method comprising a mutant polymerase having at least one mutation that reduces protein aggregation, having the backbone sequence of NOZ 58130 (SEQ ID NO: 1714) and compared to wild-type polymerase or an engineered polymerase having a mutation that does not confer reduced aggregation. In some embodiments, the mutant polymerase aggregates at about 72-75 °C, or about 75-80 °C, or about 80-85 °C, or about 85-90 °C, or at a higher temperature compared to wild-type polymerase or an engineered polymerase having a mutation that does not confer reduced aggregation. In some embodiments, a thermal shift assay using differential scanning fluorimetry can be used to determine the temperature of protein aggregation. In some embodiments, the mutant polymerase comprises a sequence having at least 70%, or 75%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or more than 99% sequence identity to any of SEQ ID NOs: 1714-2787 and comprises at least one mutation that reduces protein aggregation by reducing intermolecular interactions.

[0220] In some embodiments, a polymerase comprising the backbone sequence of NOZ 58130 (SEQ ID NO: 1714) and having an amino acid substitution for reducing aggregation comprises an amino acid substitution at one or more of positions E76, K254, R537, E541, and / or K664.

[0221] In some embodiments, the mutation at position E76 includes E76N, E76A, E76R, E76K, E76T, E76V, E76S, E76Q, E76D, or E76I.

[0222] In some embodiments, the mutation at position K254 includes K254E, K254D, K254A, K254N, K254T, K254V, K254S, K254Q, K254G, K254I, or K254R.

[0223] In some embodiments, the mutation at position R537 includes R537S, R537T, R537N, R537Q, R537A, R537V, R537D, R537I, R537E, R537G, R537K, or R537L.

[0224] In some embodiments, the mutation at position E541 includes E541A, E541R, E541N, E541K, E541T, E541V, E541S, E541Q, E541D, or E541I.

[0225] In some embodiments, the mutation at position K664 includes K664R, K664A, K664N, K664Q, K664E, K664S, K664T, K664D, K664G, K664V, or K664I.

[0226] In some embodiments, the mutant polymerase includes a deletion in any of the domains including the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714), and the N-terminal domain (SEQ ID NO: 2810), the exonuclease domain (SEQ ID NO: 2811), the first palm domain (SEQ ID NO: 2812), the finger domain (SEQ ID NO: 2813), and the thumb domain (SEQ ID NO: 2814).

[0227] In some embodiments, the mutant deletion polymerase includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to any of SEQ ID NOs: 1714 to 2787, and includes a deletion in any of these domains.

[0228] In some embodiments, the mutant deletion polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714), and the deleted portion comprises I130 - P160 or a partial region thereof. In some embodiments, the deleted portion comprises the N-terminus of any one of I130, T134, L136, L138, R143, G151, or E156. In some embodiments, the deleted portion comprises the N-terminus of any one of P160, E156, V152, A147, G145, or L136.

[0229] In some embodiments, the deleted portion comprises any one of I130 - P160, L136 - P160, L138 - P160, R143 - P160, I130 - V152, L136 - V152, L138 - V152, R143 - V152, I130 - A147, L136 - A147, L138 - A147, R143 - A147, I130 - G145, L136 - G145, L138 - G145, R143 - G145, I130 - L136, G151 - P160, G151 - V152, E156 - P160, or T134 - E156.

[0230] In some embodiments, the portion deleted in the mutant polymerase (e.g., having the backbone sequence of NOZ 58130.1, SEQ ID NO: 1714) is a sequence or region that is not present in other archaeal family B polymerases including RLF 89458 (SEQ ID NO: 1), RLF 78286 (SEQ ID NO: 2), WP 175059460 (SEQ ID NO: 2791), 9°N (SEQ ID NOs: 2795 and 2796), THERMINATOR (SEQ ID NO: 2797), VENT (SEQ ID NO: 2798), DEEP VENT (SEQ ID NO: 2799), Pfu (SEQ ID NO: 2800), and P. abyssi (SEQ ID NO: 2801). See, for example, the sequence alignments of FIGS. 33 and 35, which show regions that are present in NOZ 58130 but not in other archaeal family B polymerases. In some embodiments, the portion deleted in the mutant polymerase comprises the 23 - amino - acid - long amino - acid sequence TWLRLEVEERDGRALLRGVEQLE (SEQ ID NO: 2788). In some embodiments, the deleted portion is larger or smaller than SEQ ID NO: 2788.

[0231] This disclosure has a backbone sequence of NOZ 58130.1 (e.g., SEQ ID NO: 1714), and Y14, E15, V17, E18, R20, F26, L25, L27, G29, F34, V35, V36, F41, S42, P43, F45, L48, P49, G50, R55, E58, L61, A62, S63, A65, E67, A68, I69, K71, V72, I74, E76, K77, L79, F80, T82, P83, R84, V85, A86, L87, T90, V91, S92, H93, P94, Q95, D96, V97, P98, R99, I100, R101, E102, R103, R105, E108, D111, L112, I113, N114, E115, H116, D117, I118, V121, R122, R123, Y124, I126, R128, I130, K131, P132, L133, T134, W135, L136, E139, G145, R150, E153, E156, E157, E158, R163, V164, A165, V167, D168, I169, E170, V171, Y172, N173T, P174, E184, I186, M187, V190, T192, S193, E197, L200, V205, G207, E209, Q210, Q215, D216, M220, L222, E226, K229, G231, Y233, I236, V237, G238, N240, T241, S243, F244, Y248, R252, L253, K254, L260, L265, D266, L269, S272, G275, A276, L277, I282, A286, V288, L290, Y291, P292, I293, V294, R295, H297, V298, K299, N301, S302, Y303, V304, S307, V309, L312, L313, G314, E318, K319, L320, D321, G322, R324, L325, F326, T327, W329, D330, E331, K335, R336, L338, L339, A343, Y342, L344, D346, A352, A354, K356, L360, C362, I367, A376, M378, T379, V384, L387, M389, R390, T393, L398, I399, P400, E407, Y408, A409, R413, Y416, R422, V429, V434, F435,Compositions and methods are provided that include a mutant polymerase having an amino acid substitution mutation in any one or any combination of positions including D436, F437, S439, L440, Y441, P442, S443, I444, I445, V446, T454, A465, S473, F479, I480, R496, D504, F511, A515, S522, F523, Y524, Y526, M527, R536, R537, E538, C539, E541, V543, A544, F546, A547, M549, I551, K552, M555, A558, E559, F562, L564, E565, V566, D570, D572, V576, V577, I578, P580, L585, A586, Q587, K588, K592, V593, E595, M597, I601, F608, L613, V615, T616, R619, L622, L623, K628, M629, V631, F636, V637, R638, R639, D640, W641, A642, K650, I655, L656, A660, K664, A665, L668, I673, E674, R675, R677, S682, D685, T687, Y689, T690, Q691, R695, S698, S701, E703, V707, A708, K711, E718, V719, M723, I724, I729, T730, K734, G735, S737, Q738, T741, D752, D758, N759, I761, I765, R767, I772, Y774, L779, K780, E781, G782, I783, T784, Q785, T786, S787, L788, S789, R790, W791, and / or F792.,

[0232] The present disclosure has a backbone sequence of NOZ 58130.1 (e.g., SEQ ID NO: 1714), and Y14F, Y14D, Y14I, Y14N, E15I, V17E, E18S, E18N, E18D, R20K, L25F, F26Y, F26S, F26I, L27Q, L27K, G29E, G29K, F34K, V35M, V35K, V36F, V36N, V36T, V36I, V36E, F41S, F41I, F41Y, S42K, S42G, S42D, S42E, P43L, P43A, P43G, P43V, P43M, P43T, P43K, F45T, F45N, F45I, L48D, P49V, P49K, P49D, P49E, P49L, G50K, R55G, R55K, R55E, R55I, E58V, L61I, L61S, L61A, L61T, A62D, A62S, A62V, A62G, S63G, S63K, S63E, A65L, A65Y, A65H, E67M, E67K, E67V, A68R, I69A, I69D, I69V, K71T, K71V, K71F, K71N, K71I, V72T, V72N, V72I, I74K, E76Q, E76N, E76A, E76R, E76K, E76T, E76V, E76S, E76D, E76I, K77E, L79F, F80L, T82K, TL114T, E115V, E115G, H116C, H116Y, H116L, D117G, D117Y, I118T, I118A, I118G, I118V, I118M, I118T, I118K, V121T, V121K, V121A, R122S, R122M, R122K, R123H, R123S, R123C, R123A, R123G, R123V, R123M, R123T, R123K, R123Y, Y124R, Y124C, I126V, I126F, I126N, I126D, R128K, I130L, K131I, P132L, P132M, L133I, L133V, L133K, L133L, L133E, L133M, T134E, W135S, W135L, W135R, W135Y, W135F, W135D, W135A, W135V, W135G, L136D, E139V, G145S, R150A, R150V, R150L, R150K, R150F, E153A, E153V, E153L, E153K, E153R, E153F, E156N, E156R, E157A, E157V, E157L, E157K, E157R, E157F, E157D, E157G, E157T, E158S, E158G, R163E, R163L, R163K, R163H, VI236L, V237I, G238T, N240G, N240T, N240S, T241G, S243N, F244S, Y248D, Y248R, R252A, R252S, L253V, L253E, L253C, K254E, K254R, K254A, K254N, K254Q, K254S, K254T, K254D, K254G, K254V, K254I, L260F, L265D, D266G, L269P, S272Q, G275N, G275K, G275S, G275R, A276M, A276N, A276Q, A276D, L277R, L277M, L277D, I282V, A286I, V288F, L290I, Y291A, Y291P, Y291G, Y291D, Y291N, P292R, I293V, V294M, V294I, R295A, H297F, H297Y, V298I, K299N, N301R, N301P, S302N, S302T, Y303G, Y303D, Y303A, V304D, V304A, V304E, V304H, V304I, V304L, V304M, V304P, V304R, V304T, V304W, V304Y, V304F, V304G, V304K, V304N, V304Q, V304S, S307A, V309Y, L312V, L312I, L313M, L313D, L313F, L313K, L313R, L313A, L313E, G314S, G314D, G314K, G314R, G314E, E318V, K319V, K319R, L320V, D321F, G322D, G322S, R324E, L325R, F326N, F326T, F326A, T327Q, W329Y, W329F, W329L, W329D, W329A, W329V, D330N, D330E, E331N, E331R, K335R, K335L, K335S, K335A, R336L, L338E, L339V, Y342N, Y342A, Y342R, A343S, L344M, D346G, D346A, D346R, A352L, A352E, A352D, A352Q, A354G, K356R, K356D, K356E, L360I, L360Q, L360V, L360M, C362A, C362L, C362I, C362S, C362F, C362Y, C362V, C362P, C362K, C362N, C362D, I367L, A376C, A376R,A376S, M378R, M378T, M378A, T379D, T379K, T379N, T379S, V384Q, V384E, L387N, L387C, L387Y, L387F, M389D, M389E, M389L, M389Y, M389S, M389A, M389V, R390M, L398D, I399A, I399N, I399R, I399F, P400H, P400N, P400S, E407R, Y408R, A409R, A409Q, R413Q, R413T, Y416H, R422V, R422T, R422D, V429W, V434H, V434L, V434Y, F435L, D436T, F437Y, F437R, F437I, S439A, S439G, S439R, L440, L440Y, L440F, L440S, L440A, Y441, Y441A, Y441G, Y441T, P442, P442G, P442A, S443R, S443N, S443A, S443G, I444F, I445L, I445F, V446M, V446K, V446R, V446N, V446T, D451C, T454I, T454L, T454R, A465V, A465D, A465P, S473K, S473R, S473T, S473Q, S473A, F479I, F479L, I480F, I480Y, R496T, R496A, R496G, R496C, D504E, F511Y, F511L, F511V, A515L, A515S, A515T, A515V, A515G, A515R, S522D, S522K, S522T, S522N, S522E, S522G, S522Y, F523A, F523S, F523T, F523V, F523I, F523Y, Y524A, Y524N, Y524G, Y524F, Y524L, Y526C, M527H, M527G, M527Q, M527L, M527D, M527A, M527V, R536C, R537K, R537E, R537G, R537S, R537L, R537A, R537N, R537Q, R537T, R537D, R537V, R537I, E538A, E538C, C539A, C539V, C539L, C539S, C539Y, C539D, C539K, C539N, C539P, E541K, E541S, E541A, E541R, E541N, E541T, E541V, E541Q, E541D, E541I, V543TV543I, V543A, V543S, V543G, A544G, A544S, A544T, F546W, A547G, M549N, M549Y, M549H, M549T, M549D, M549R, M549A, M549V, I551N, I551T, I551E, I551H, I551L, I551V, I551A, K552R, K552T, K552N, K552Q, K552A, M555Y, M555I, A558I, E559N, E559K, E559D, F562E, F562N, F562Q, F562R, L564F, E565N, E565K, E565S, E565R, V566N, V566K, V566S, V566R, D570A, D570G, D570E, D570V, D570L, D570S, D572A, D572G, D572E, V576A, V577T, I578F, I578T, I578P, I578R, I578T, I578E, I578N, P580G, L585K, L585S, L585E, L585Q, L585R, L585T, L585V, A586K, K588N, K588Q, K588R, K588S, K592A, K592G, K592R, K592T, K592N, K592S, V593I, V593A, E595K, M597L, I601L, F608Y, L613F, V615E, V615T, T616K, R619E, L622V, L623I, K628R, K628I, K628H, M629L, M629A, M629D, M629R, M629V, M629I, V631T, F636I, V637D, V637N, V637R, R638D, R639D, R639N, D640R, D640K, W641R, W641A, W641L, W641F, W641Y, W641V, A642S, K650T, K650C, K650A, K650R, K650S, I655L, I655V, I655A, L656I, A660G, A665E, A665V, A665T, K664R, K664A, K664N, K664Q, K664E, K664S, K664T, K664D, K664G, K664V, K664I, L668I, I673T, E674G, E674D, E674K, R675V, R675C, R675L, R675D, R677E, R677V, R677T, R677N, R677A, S682P, D685R, D685E, D685I,D685L, D685K, T687V, Y689D, Y689N, T690K, T690R, T690S, T690M, T690Q, T690V, T690E, T690N, Q691S, Q691T, R695K, S698D, S698K, S698R, S698G, S698Y, S698D, S701T, , S701V, S701A, S701R, S701E, E703R, E703S, E703K, V707I, V707D, V707A, A708V, K711R, K711L, K711T, K711D, E718R, E718V, E718K, V719I, M723S, M723I, M723T, M723N, M723R, M723L, M723A, M723C, I724D, I724V, I729V, T730L, K734I, K734G, K734N, G735M, G735R, G735K, G735S, G735P, G735T, G735E, S737R, S737E, Q738D, Q738S, Q738E, Q738, Q738R, T741I, D752Q, D752T, D758N, N759P, N759D, N759K, N759T, N759Y, N759R, I761V, I765V, R767E, I772L, I772Y, I772F, Y774F, Y774E, L779G, L779Q, L779D, L779K, L779Y, L779E, K780C, K780F, K780I, K780R, K780Q, K780Y, E781L, E781H, E781S, E781M, E781Q, G782H, G782A, G782K, G782R, Q785L, T786N, S789G, W791R, W791Y, W791D, W791S, W791L, W791A, W791V, and / or F792R, and compositions and methods comprising a mutant polymerase comprising an amino acid substitution mutation at any one or any combination of the positions containing the same are provided.

[0233] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position Y14. In some embodiments, the amino acid substitution at position Y14 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0234] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position L48. In some embodiments, the amino acid substitution at position L48 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0235] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position E76. In some embodiments, the amino acid substitution at position E76 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0236] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position V97. In some embodiments, the amino acid substitution at position V97 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0237] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position R122. In some embodiments, the amino acid substitution at position R122 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0238] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position Y124. In some embodiments, the amino acid substitution at position Y124 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0239] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position R150. In some embodiments, the amino acid substitution at position R150 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0240] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position K254. In some embodiments, the amino acid substitution at position K254 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0241] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position V304. In some embodiments, the amino acid substitution at position V304 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0242] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position C362. In some embodiments, the amino acid substitution at position C362 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0243] [[ID=?]]In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position R496. In some embodiments, the amino acid substitution at position R496 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0244] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position A515. In some embodiments, the amino acid substitution at position A515 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, V, L, H, R, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0245] It seems there is a typo in ID=8 where it should be "R496" instead of "?". The above translation is based on the corrected content.In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position R537. In some embodiments, the amino acid substitution at position R537 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0246] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position E559. In some embodiments, the amino acid substitution at position E559 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0247] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position K664. In some embodiments, the amino acid substitution at position K664 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, N, Y, C, S, T, or Q), or has a non-natural amino acid known to those skilled in the art.

[0248] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and includes an amino acid deletion at any position including D117 (deletion).

[0249] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and contains a cleavage at an amino acid position including Q587 (cleavage), M723 (cleavage), G773 (cleavage), Y774 (cleavage), D777 (cleavage), E781 (cleavage), G782 (cleavage), T784 (cleavage), Q785 (cleavage), R790 (cleavage), W791 (cleavage), or F792 (cleavage). The cleaved polymerase can exhibit increased thermal stability as compared to an uncleaved polymerase having the same backbone sequence. In Table 2, the cleavage is indicated by the symbol "^".

[0250] In some embodiments, the mutant polymerase has the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and contains a cleaved portion at the C-terminus. In some embodiments, the cleaved mutant polymerase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to any of SEQ ID NOs: 1714 to 2787 and contains a cleaved portion at the C-terminus. In some embodiments, the cleaved portion can be 2 - 5, 5 - 10, 10 - 15, or 15 - 20 amino acids in length. In some embodiments, the cleaved portion includes G782 - F792 and is 11 amino acids in length. The cleaved polymerase can exhibit increased thermal stability as compared to an uncleaved polymerase having the same backbone sequence. In Table 2, the cleaved portion is indicated by the letter "X".

[0251] A composition comprising an engineered polymerase The present disclosure provides a polymerase mutated at two or more positions to increase the thermal stability of the enzyme, which exhibits improved binding of nucleotide reagents and / or improved binding and incorporation of nucleotide reagents, an improved incorporation rate of nucleotide analogs, improved uracil resistance, and / or reduced sequence-specific errors as compared to a wild-type polymerase comprising any of the amino acid sequences of SEQ ID NOs: 1, 2, 1714, 2789-2793, and 2803. For example, the mutant polymerase exhibits increased thermal stability in a temperature range of about 25-50 °C, or about 45-75 °C, or about 65-90 °C. In another example, the mutant polymerase exhibits an increased incorporation rate of nucleotide analogs comprising a chain-terminating moiety (e.g., a blocking moiety) at the 2'- and / or 3'-sugar positions. The mutant polymerase may exhibit increased uracil resistance. The mutant polymerase may exhibit improved binding to complementary nucleotide units of a multivalent molecule. In some embodiments, the mutant polymerase has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical, or has a higher level of sequence identity, to any of SEQ ID NOs: 1, 2, 1714, 2789-2793, and 2803.

[0252] In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458.1 or RLF 78286.1, comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 1713, and comprises an amino acid substitution that can confer exonuclease minus activity including any one of D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458.1 or RLF 78286.1, comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 1713, and comprises an amino acid substitution that can confer exonuclease minus activity including non-mutated E143, or any one of mutated E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143F, E143G, E143H, E143R, E143K, E143D, E143C, or E143Q.

[0253] In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ 58130.1, comprises any of the amino acid sequences of SEQ ID NOs: 1714-2787 and 2249-2479, and comprises an amino acid substitution capable of conferring exonuclease minus activity comprising any of D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168K, D168T, D168S, D168W, D168M, D168P, D168G, D168H, D168R, D168E, D168C, or D168Q. In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ 58130, comprises any of the amino acid sequences of SEQ ID NOs: 1714-2787, and comprises an amino acid substitution capable of conferring exonuclease minus activity comprising any of E170A, E170V, E170L, E170I, E170F, E170Y, E170N, E170K, E170T, E170S, E170W, E170M, E170P, E170G, E170H, E170R, E170D, E170C, or E170Q.

[0254] The present disclosure provides engineered archaeal family B DNA or family A polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including SEQ ID NO: 2797, THERMINATOR polymerase), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2801), and also RB69 polymerase (SEQ ID NO: 2802), which are M1, L3, D4, D6, Y7, I8, T9, E10, N11, G12, K13, P14, V15, I16, R17, I18, F19, K20, K21, E22, K23, G24, E25, F26, K27, I28, E29, Y30, D31,R32, N33, F34, E35, P36, Y37, I38, Y39, A40, L41, L42, E43, D44, D45, E46, S47, I48, E49, D50, I51, K52, K53, I54, T55, R58, G56, E57, R58, H59, G60, K61, K62, V63, I65, I66, R67, V68, E69, K70, V71, K72, K73, K74, F75, L76, G77, E78, P79, I80, E81, V82, W83, K84, L85, V86, F87, E88, H89, P90, Q91, D92, V93, P94, A95, I96, R97, D98, A99, I100, R101, S102, H103, P104, A105, V106, R107, E108, I109, F110, E111, Y112, D113, I114, P115, F116, A117, K118, R119, Y120, L121, I122, D123, K124, L126, V127, P128, M129, E130, G131, G132, E133, L135, K136, L137, L138, A139, F140, D141, I142, E143, T144, F145, Y146, H147, D150, E151, M159, W173, K174, I176, Y180, A190, I191, K192, L195, L198, R199, Q196, P203, V205, L207, Y209,G211, N213, F214, F216, A217, Y218, I219, C223, E224, G227, L228, F230, T231, I232, G233, R234, S237, E238, P239, K240, I241, Q242, R243, M244, G245, D246, R247, A249, E251, L258, Y261, P262, V264, R265, H266, T267, I268, R269, L270, P271, T272, Y273, T274, L275, E276, A277, V278, V282, F283, K285, K286, K287, E288, K289, V290, Y291, A292, I295, E297, A298, W299, K300, S301, L305, K306, R307, V308, A309, Y311, M313, D315, R317, A318, Y320, E321, P328, M329, V331, E332, L333, A334, I337, G338, Q339, V341, D343, S345, S347, S348, T349, G350, N351, L352, V353, W355, Y356, L357, R359, V360, Y362, N365, E366, L367, K371, P372, G373, E376, Q378, M381, S384, Y385, I386, G388, Y389, E394, G396, A402, Y403, L404, F406, R407, S408, L409, Y410, P411, S412, I413, V415, H417, V419, P421, D422, T423, L424, E427, C428, K429, A434, I436, R440, K443, G447, F448, I449, P450, S451, I452, L453, E454, D455, I457, V463, K464, R465, M467, K468, D472, I474, E475, K476, Y481, R484, A485, L486, K487, I488, N491, S492, Y493, Y494, G495, Q497, G498, Y499, P500, K501, S506, K507, E508, C509, E511, S512, V513, T514, G517, R518, H519, I521, T523, A528, E529, K534, V535, A538, E539, D541, G542, I547, I555, P552S557, K558, A559, K560, K561, L563, K564, H565, E568, K569, G572, M573, E575, E577, L583, G585, F586, V588, T589, K592, L595, I596, H601, T604, G606, V609, V610, R611, R612, D613, E616, I617, K619, E620, T621, Q622, A623, K624, V625, L626, E627, V628, I629, L630, R631, E632, G633, S634, I635, E636, K637, A638, A639, G640, I641, V642, V645, V646, E647, D648, L649, A650, N651, Y652, R653, V654, V656, E657, K658, I660, H662, E663, Q664, I665, T666, R667, E668, K670, D671, Y672, K673, A674, T675, G676, P677, H678, V679, A680, I681, A682, K683, R684, L685, Q686, A687, R688, G689, I690, K691, V692, K693, P694, T696, I698, S699, Y700, V701, V702, L703, K704, G705, S706, K707, K708, I709, D711, R712, V713, I714, L715, F716, D717, E718, D720, S721, S722, R723, K725, Y726, P728, Y730, Y731, I732, H733, N734, Q735, V736, P738, A739, V740, L741, R742, I743, L744, E745, A746, F747, G748, Y749, K750, E751, K752, D753, L754, E755, Y756, Q757, R758, M759, K760, Q761, T762, G763, L764, G765, A766, W767, L768 and / or M770, including RLF 89458.1 (e.g.,It is mutated at any one or any combination of positions of amino acid substitutions that are positionally equivalent (or functionally equivalent sites) to one or more positions among the backbone sequences of polymerase having SEQ ID NO: 1 or any one of SEQ ID NOs: 3 to 1713) or RLF 78286.1 (SEQ ID NO: 2). From the sequence alignment shown in FIG. 34, those skilled in the art can identify positions that are positionally equivalent (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NOs: 2795 or 2796) (including THERMINATOR polymerase, SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2801), as well as RB 69 polymerase (SEQ ID NO: 2802).

[0255] The present disclosure provides engineered archaeal family B DNA or family A polymerases comprising Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including SEQ ID NO: 2797, THERMINATOR polymerase), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802), which are Y14, E18, F26, L27, G29, F34, V35, V36, F41, S42, P43, F45, L48, P49, R55, L61, A62, S63, A65, E67, I69, K71, V72, E76, K77, T82, P83, R84, V85, T90, V91, S92, H93, P94, Q95, D96, V97, P98, R99, I100, R101, E102, R103, R105, E108, D111, L112, I113, N114, E115, H116, D117, I118, V121, R122, R123, Y124, I126, I130, P132, L133, W135, G145, R150, E153, E156, E157, E158, R163, V164, A165, V167, D168, I169, E170, V171, Y172, N173T, P174, E184, I186, M187, V190, L200, V205, M220, K229, Y233, I236, V237, G238, N240, F244, Y248, R252, L253, K254, L260, L269, G275, A276, L277, I282, A286, V288, L290, Y291, P292, I293, V294, R295, H297, V298, N301, S302, Y303, V304, S307, V309, L312, L313, G314, E318, K319, L320, D321, G322, L325, F326, T327, W329, D330, E331, K335, L338, L339, A343, Y342, L344, D346, A352, A354, K356, L360, C362, I367, A376, M378,One or any combination of amino acid substitutions at any one of the positions of a polymerase having a backbone sequence of NOZ 58130.1 (for example, any one of SEQ ID NOs: 1714 to 2787) containing T379, V384, L387, M389, R390, T393, L398, I399, P400, E407, Y408, A409, R413, Y416, R422, V429, V434, F435, D436, F437, S439, L440, Y441, P442, S443, I444, I445, V446, T454, A465, S473, F479, I480, R496, D504, F511, A515, S522, F523, Y524, Y526, M527, R536, R537, E538, C539, E541, V543, A544, F546, A547, M549, I551, K552, M555, A558, E559, F562, L564, E565, V566, D570, D572, V576, V577, I578, P580, L585, A586, Q587, K588, K592, V593, E595, M597, I601, F608, L613, V615, T616, R619, L622, L623, K628, M629, V631, F636, V637, R638, R639, D640, W641, A642, K650, I655, L656, A660, K664, A665, L668, I673, E674, R675, R677, S682, D685, T687, Y689, T690, Q691, R695, S698, S701, E703, V707, A708, K711, E718, V719, M723, I724, I729, T730, K734, G735, S737, Q738, T741, D752, D758, N759, I761, I765, R767, I772, Y774, L779, K780, E781, G782, I783, T784, Q785, T786, S787, L788, S789, R790, W791, and / or F792 is mutated at one or more positions that are positionally equivalent (functionally equivalent sites). From the sequence alignment shown in FIG. 35, one of ordinary skill in the art can identify Geobacillus stearothermophilus (for example, Bst DNA polymerase) (SEQ ID NO: 2794),It is possible to identify positions that are positionally equivalent (or functionally equivalent sites) in 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase, SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2801), and also in RB 69 polymerase (SEQ ID NO: 2802).

[0256] The present disclosure provides engineered archaeal family B DNA or family A polymerases that include Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including SEQ ID NO: 2797, THERMINATOR polymerase), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802), which include Y11, D15, F23, K25, I28, L29, F34, Q35, P36, F38, H43, E49, G55, A56, V57, R62, R67, I75, L76, S77, H78, P79, S80, E81, V82, P83, K84, I85, R86, E87, E88, R90, E96, I98, E100, H101, D102, I103, A106, R108, I111, P117, L118, E138, G139, R144, V145, M146, D149, I150, E151, T152, A234, Y272, C307, R312, E333, A35...

Claims

1. A manipulated polymerase comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, and ●Having a first amino acid substitution selected from the group consisting of D141N, D141V, D141A, D141L, D141I, D141F, D141Y, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, and D141C, ● The manipulated polymerase having a second amino acid substitution selected from the group consisting of E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143F, E143G, E143L, E143H, E143R, E143K, E143D, E143C, and E143Q.

2. A manipulated polymerase having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, and having a non-mutation position E143E, and an amino acid substitution selected from the group consisting of D141N, D141V, D141A, D141L, D141I, D141F, D141Y, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, and D141C.

3. (a) an amino acid substitution selected from the group consisting of V93A, V93M, V93E, V93F, V93Y, V93G, V93S, V93K, V93T, and V93I; (b) Amino acid substitutions L409S, Y410A, and P411G; (c) Amino acid substitutions selected from the group consisting of M1F, M1I, M1L, M1S, M1N, M1A, M1V, M1Y, M1Q, M1K, M1V, and M1A; (d) Amino acid substitutions selected from the group consisting of M129I, M129V, M129K, M129L, M129E, M129F, M129N, M129S, M129R, and M129Y; (e) Amino acid substitutions selected from the group consisting of M159W, M159F, and M159Y; (f) Amino acid substitutions selected from the group consisting of M313I, M313K, M313L, M313V, M313D, M313R, M313E, M313A, M313L, and M313N; (g) Amino acid substitutions selected from the group consisting of M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N, and M329E; (h) Amino acid substitutions selected from the group consisting of M467V, M467K, M467D, M467T, M467R, M467E, M467Q, and M467L; (i) Amino acid substitutions selected from the group consisting of M759T, M759S, M759N, M759R, M759E, M759D, and M759A; (j) Amino acid substitutions selected from the group consisting of K240R, K240D, K240N, K240Q, and K240A; (k) Amino acid substitution selected from the group consisting of K306R, K306N, K306Q, K306A, K306V, K306I, and K306F; (l) Amino acid substitutions selected from the group consisting of K371R, K371D, K371N, K371Q, K371Y, K371T, K371V, and K371L; (m) Amino acid substitutions selected from the group consisting of K429R, K429S, K429M, K429A, K429N, K429D, K429Q, K429H, K429Y, K429V, and K429L; (n) Amino acid substitution selected from the group consisting of K468R, K468E, K468Y, K468T, and K468L; (o) Amino acid substitutions selected from the group consisting of K476R, K476D, K476A, and K476F; (p) Amino acid substitutions selected from the group consisting of K592Q, K592R, K592W, K592Y, K592A, K592F, K592I, K592T, K592N, and K592S; (q) Amino acid substitution selected from the group consisting of W299F, W299E, W299N, W299Q, W299Y, W299A, and W299F; (r) Amino acid substitution selected from the group consisting of H601R, H601I, H601A, H601T, H601V, H601L, and H601N; (s) Amino acid substitutions selected from the group consisting of C428Y; (t) Methionine deficiency at position 1; (u) Amino acid substitutions selected from the group consisting of C223V, C223E, C223S, C223L, C223M, C223A, C223P, C223K, C223N, and C223D; (v) Amino acid substitution selected from the group consisting of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, and C509Q; Cutting at (w) K464, R465, E475, Y481, E616, E620, E755, Y756, Q757, R758, M759, T762, W767, or M770; (x) Amino acid substitution selected from the group consisting of V610D, V610A, V610K, V610S, V610T, V610N, V610R, and V610Q; (y) Amino acid substitution selected from the group consisting of D613S, D613E, D613R, D613K, D613N, D613Q, D613A, D613V, D613Y, and D613F; (z) Amino acid substitutions selected from the group consisting of Q664A, Q664L, Q664V, Q664F, Q664I, Q664R, Q664K, Q664T, Q664N, and Q664M; (aa) Amino acid substitutions selected from the group consisting of E668G, E668K, E668M, E668A, E668P, E668S, E668R, E688N, E688D, E668Y, and E668Q; (bb) Amino acid substitution selected from the group consisting of P677L, P677R, P677K, and P677A; (cc) Amino acid substitutions selected from the group consisting of D671G, D671R, D671Y, D671S, D671A, D671K, and D671N; (dd) Amino acid substitution selected from the group consisting of N11S, N11A, N11R, N11Q, N11E, N11K, and N11T; (ee) Amino acid substitution selected from the group consisting of K507L, K507E, K507S, K507A, K507N, K507Q, K507E, and K507T; (ff) Amino acid substitution selected from the group consisting of E511K, E511S, E511A, E511R, E511N, and E511T; (gg) Amino acid substitution selected from the group consisting of K637M, K637A, K637N, K637Q, K637E, K637S, and K637T The manipulated polymerase according to claim 1 or 2, further comprising:

4. A manipulated polymerase comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 1714, and ●Having a first amino acid substitution selected from the group consisting of D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168T, D168S, D168W, D168M, D168P, D168G, D168H, D168R, D168E, D168C, D168K, or D168Q, ● The manipulated polymerase having a second amino acid substitution selected from the group consisting of E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143G, E143H, E143R, E143K, E143D, E143C, or E143Q.

5. The L440, Y441, and P442 positions further contain amino acid substitutions of the LYP motif, and the mutation of the LYP motif is ●L440F, Y441G, and P442P, or ●The manipulated polymerase according to claim 4, comprising L440S, Y441G, and P442P.

6. (a) an amino acid substitution selected from the group consisting of Y14F, Y14D, Y14I, and Y14N; (b) Amino acid substitutions selected from the group consisting of W135S, W135L, W135R, W135Y, W135F, W135D, W135A, and W135V; (c) Amino acid substitutions selected from the group consisting of M187S, M187L, M187R, M187Y, M187I, M187T, M187A, and M187V; (d) Amino acid substitutions selected from the group consisting of W329Y, W329F, W329L, W329D, W329A, and W329V; (e) Amino acid substitutions selected from the group consisting of K335R, K335L, K335S, and K335A; (f) Amino acid substitutions selected from the group consisting of S473K, S473R, S473T, S473Q, and S473A; (g) Amino acid substitutions selected from the group consisting of M527H, M527G, M527Q, M527L, M527D, M527A, and M527V; (h) Amino acid substitutions selected from the group consisting of M549N, M549Y, M549H, M549T, M549D, M549R, M549A, and M549V; (i) Amino acid substitutions selected from the group consisting of K552R, K552T, K552N, K552Q, and K552A; (j) Amino acid substitutions selected from the group consisting of M629L, M629A, M629D, M629R, and M629V; (k) Amino acid substitution selected from the group consisting of W641R, W641A, W641L, W641F, W641Y, and W641V; (l) Amino acid substitutions selected from the group consisting of K650T, K650C, K650A, K650R, and K650S; (m) Amino acid substitution selected from the group consisting of K711R, K711L, K711T, and K711D; (n) Amino acid substitution selected from the group consisting of M723S, M723I, M723T, M723N, M723R, M723L, M723A, and M723C; (o) Amino acid substitutions selected from the group consisting of W791R, W791Y, W791D, W791S, W791L, W791A, and W791V; (p) Amino acid substitution selected from the group consisting of C362A, C362L, C362I, C362S, C362F, C362Y, C362V, C362P, C362K, C362N, and C362D; (q) Amino acid substitution selected from the group consisting of C539A, C539V, C539L, C539S, C539Y, C539D, C539K, C539N, and C539P; (r) Cutting at positions M723, G773, D777, E781, T784, Q785, R790, W791, or F792; (s) Amino acid substitution selected from the group consisting of Q95L, Q95H, Q95R, Q95W, Q95A, Q95K, Q95N, and Q95P; (t) Amino acid substitution I186R; (u) Amino acid substitution V304D; or (v) Amino acid substitution selected from the group consisting of L313M, L313D, L313F, L313K, L313R, L313A, and L313E The manipulated polymerase according to claim 4, further comprising:

7. (a) an amino acid substitution selected from the group consisting of M389D, M389E, M389L, M389Y, M389S, M389A, and M389V; or (b) Amino acid substitution E318V The manipulated polymerase according to claim 5, further comprising:

8. The manipulated polymerase according to any one of claims 1, 2, 4, or 5, further comprising a plurality of manipulated polymerases, a plurality of nucleic acid template molecules, and a plurality of nucleotide polymerization initiation sites having a 3'-extendable end.

9. (i) The plurality of nucleic acid template molecules include linear nucleic acid molecules, cyclic nucleic acid molecules, or a mixture of linear nucleic acid molecules and cyclic nucleic acid molecules; (ii) The plurality of nucleic acid template molecules include cloned amplified template molecules; (iii) At least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules contains one copy of the target sequence of interest, or contains a concatemer having two or more tandem copies of the target sequence of interest; or (iv) The manipulated polymerase according to claim 8, wherein each of the plurality of nucleotide polymerization initiation sites comprises a nucleic acid primer that hybridizes to at least one portion of the nucleic acid template molecule, or each of the plurality of nucleotide polymerization initiation sites comprises at least one self-priming terminal portion of the nucleic acid template molecule.

10. The manipulated polymerase according to claim 8, wherein the plurality of manipulated polymerases, the plurality of nucleic acid template molecules, and the plurality of nucleotide polymerization initiation sites form a plurality of complex polymerases, each of which contains a polymerase bound to a nucleic acid double strand, and the double strand contains a nucleic acid template molecule hybridized to a nucleic acid primer.

11. The manipulated polymerase according to claim 10, wherein the plurality of nucleic acid template molecules include target sequences of the same interest or target sequences of different interests.

12. The manipulated polymerase according to claim 10, wherein the plurality of complex polymerases further comprises a plurality of polyvalent molecules, each of which comprises (a) a core and (b) a plurality of nucleotide arms, each comprising (i) a core-binding portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is bound to the plurality of nucleotide arms via their core-binding portions, the spacer is bound to the linker, and the linker is bound to the nucleotide unit.

13. (i) The linker comprises an aliphatic chain having 2 to 6 subunits, or an oligoethylene glycol chain having 2 to 6 subunits; (ii) The plurality of nucleotide arms attached to the core have nucleotide units of the same type, the nucleotide units include dATP, dGTP, dCTP, dTTP, or dUTP; (iii) The plurality of polyvalent molecules include one type of polyvalent molecule, and each polyvalent molecule in the plurality of polyvalent molecules has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP; (iv) The plurality of polyvalent molecules comprises a mixture of any combination of two or more types of polyvalent molecules, each type having a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP; (v) At least one of the plurality of polyvalent molecules comprises a core labeled with a fluorophore; or (vi) The manipulated polymerase according to claim 12, wherein at least one of the plurality of polyvalent molecules comprises a nucleotide unit labeled with a fluorophore.

14. The manipulated polymerase according to claim 10, wherein the plurality of complex polymerases further comprises a plurality of nucleotides, and each of the plurality of nucleotides comprises an aromatic base, a pentacose, and 1 to 10 phosphate groups.

15. (i) The plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP; (ii) The plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP; (iii) At least one nucleotide in the plurality of nucleotides is labeled with a fluorophore; (iv) The plurality of nucleotides lack fluorophore labeling; or (v) The manipulated polymerase according to claim 14, wherein at least one of the nucleotides in the plurality of nucleotides comprises a removable chain termination portion bonded to the 3' carbon position of a sugar group, the removable chain termination portion comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, and the removable chain termination portion is cleavable with a chemical compound to produce an extendable 3'OH portion on the sugar group.

16. (i) The plurality of complex polymerases further comprises a plurality of non-catalyzed divalent cations that inhibit polymerase-catalyzed nucleotide incorporation, wherein the non-catalyzed divalent cations include strontium or barium; (ii) The plurality of composite polymerases further comprises a plurality of catalytic divalent cations that promote polymerase-catalyzed nucleotide incorporation, wherein the catalytic divalent cations include magnesium or manganese; or (iii) The manipulated polymerase according to claim 10, wherein the plurality of composite polymerases are immobilized on a support or immobilized on a coating on the support.

17. (a) The density of the plurality of composite polymerases immobilized on the support is 1 mm ２ 10 hits ２ ~10 １２ Constitutes, (b) The plurality of immobilized complex polymerases are immobilized at predetermined sites on the support, or at random sites on the support, (c) The coating comprises at least one hydrophilic polymer coating layer comprising unbranched polyethylene glycol (PEG), or the coating comprises at least one hydrophilic polymer coating layer comprising branched polyethylene glycol (PEG) having at least four branches, or (d) The manipulated polymerase according to claim 16, wherein the plurality of immobilized complex polymerases are in fluid communication with one another, allowing the reagent solution to flow over the support so that the plurality of immobilized complex polymerases on the support react with the reagent solution in a massively parallel manner.

18. The manipulated polymerase according to claim 17, wherein the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

19. A method for forming a plurality of complex polymerases, comprising contacting a plurality of manipulated polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid primers under conditions suitable for forming a plurality of complex polymerases, each comprising a polymerase bound to a nucleic acid double strand, wherein the nucleic acid double strand comprises a nucleic acid template molecule hybridized to a nucleic acid primer, and the plurality of manipulated polymerases comprises the amino acid sequence described in any one of claims 1, 2, 4, or 5.

20. A method for determining the sequence of a nucleic acid template, a) Contacting a plurality of first polymerases with (i) a plurality of nucleic acid template molecules, each containing a target sequence of interest, and (ii) a plurality of nucleic acid primers, wherein the contact is carried out under conditions suitable for binding the plurality of first polymerases to the plurality of nucleic acid template molecules and the plurality of nucleic acid primers, thereby forming a plurality of first complex polymerases, each containing a first polymerase bound to a nucleic acid double strand, wherein the nucleic acid double strand contains a nucleic acid template molecule hybridized to a nucleic acid primer, and the plurality of first polymerases contain the amino acid sequence described in any one of claims 1, 2, 4, or 5. b) Forming a plurality of polyvalent complexes by contacting a plurality of first complex polymerases with a plurality of polyvalent molecules, wherein each polyvalent molecule in the plurality of polyvalent molecules contains a core bound to a plurality of nucleotide arms, each nucleotide arm being bound to a nucleotide unit, and the contact is carried out under conditions suitable for binding the complementary nucleotide unit of the polyvalent molecule to at least two of the plurality of first complex polymerases, thereby forming a plurality of polyvalent complexes, wherein the conditions are suitable for inhibiting the incorporation of the complementary nucleotide unit of the plurality of polyvalent complexes into the primers, c) To detect the multiple polyvalent binding complexes, d) A method comprising identifying the bases of the complementary nucleotide units within the plurality of polyvalent bond complexes, thereby determining the sequence of the nucleic acid template molecule.

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