Polymerases engineered with reduced sequence-specific errors

Abstract

Provided herein are engineered variants of archaeal polymerases that exhibit exonuclease-minus activity, enhanced thermostability, enhanced incorporation of 3' modified nucleotides, improved uracil tolerance, and / or reduced sequence-specific errors in polymerase-catalyzed nucleotide binding and extension reactions compared to wild-type polymerase enzymes. Uses of the engineered polymerases to form conjugated polymerases, form binding complexes, and perform nucleic acid sequencing reactions are also provided.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. The XML copy created on February 17, 2023 is named 52268WO_CRF_SL.xml and is 4,685,344 bytes in size.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application Nos. 63 / 311,939, filed February 18, 2022, 63 / 343,036, filed May 17, 2022, and 63 / 479,490, filed January 11, 2023, and U.S. Nonprovisional Application Nos. 18 / 160,947, filed January 27, 2023, 18 / 160,951, filed January 27, 2023, and 18 / 160,952, filed January 27, 2023, each of which is incorporated by reference herein in its entirety for all purposes.

[0003] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents, and / or patent applications are incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0004] The present disclosure provides mutant polymerases that are engineered for improved thermostability and that exhibit improved binding and / or improved binding and incorporation of nucleotide reagents, and / or improved uracil resistance and / or reduced sequence-specific sequencing errors. Exemplary nucleotide reagents include detectably labeled nucleotides, nucleotides that include 3' chain terminating moieties, phosphate-labeled nucleotides, and multivalent molecules. The mutant polymerases exhibit increased incorporation rates compared to wild-type polymerases. [Background technology]

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

[0006] The addition of a single nucleotide alone to a strand of DNA does not generate enough signal to be easily detected. Currently available SBS technologies overcome this problem by increasing the signal-to-noise ratio of nucleotide addition coupled with a detection method sensitive enough to make accurate base calls. The most commercially successful platforms use monoclonal template DNA amplification in a spatially constrained matrix to generate discrete DNA islands containing multiple copies of a sequence for matching. 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 relies on a controlled step mechanism to ensure that only one nucleotide base can be added to ensure that all copies in a DNA colony remain in the same position relative to each other (N, N+1, N+2, N+3, etc.).

[0007] The molecular engine required to carry out SBS is DNA polymerase. In vivo, this class of enzymes is involved in DNA replication and maintaining genome integrity. Under native conditions, DNA-dependent DNA polymerase (dDdP) catalyzes the addition of deoxynucleotide triphosphates (dNTPs) to DNA in a 5' to 3' direction, creating a phosphodiester bond between the 3' hydroxyl of the primer DNA end and the 5' alpha phosphate of the input nucleotide. This chemical reaction occurs with high fidelity to the correct Watson-Crick base pairing, with hydrogen bonding between the correct input dNTP and the template base. This "correct" base pairing induces a conformational change in the enzyme that aligns the catalytic amino acid to efficiently perform phosphodiester bond formation. The newly added dNTP also has a 3'OH that is used in the next round of catalysis to further extend the DNA strand.

[0008] To ensure that only a single dNTP is added to the growing strand of DNA per SBS cycle, reversibly terminated dNTPs are used. These bases contain a modification to the 3' hydroxyl of the dNTP, blocking subsequent rounds of incorporation. The most commercially successful reversible terminator is the 3' methyl azide, but other terminators, including 3'-aminoallyl and 3' oxyamine, have also been used. Each of these reversibly terminated dNTPs functions in the same way: once incorporated, the bulky 3' block inhibits the addition of the next nucleotide because the 3' hydroxyl is absent. Upon exposure to catalyst, the 3' block reacts to regenerate a 3' hydroxyl that can form a new phosphodiester bond during the next cycle. Although effective, these bulky 3' modifications present a challenge for the polymerase.

[0009] The evolutionary requirement for high-fidelity genome replication and stability has been 4 ~10 7This resulted in polymerases that only incorporate non-Watson-Crick base pairs per incorporation event. Polymerases also often need to discriminate between the vast excess of nucleotides in the cellular environment. Discrimination between nucleotides is typically achieved through a steric gate where the presence of the 2' hydroxyl sterically clashes with the amino acid side chain at the nucleotide binding site, selecting for nucleotide binding and catalysis. In addition, damage or modification to the 3' hydroxyl of a nucleotide is also sensed by the enzyme, as bases containing non-viable 3' hydroxyls can function as chain terminators that inhibit DNA synthesis. Discrimination of these undesirable bases results in inappropriate nucleotide substrates being bound with weaker overall binding activity, and phosphodiester bond formation being delayed by 10 2 ~10 4 This occurs via a kinetic pathway that occurs much more slowly, at rates of several orders of magnitude. This is due to a lack of induced fit to properly align the catalytic amino acids for bond formation. As a result, naturally evolved polymerases are poor at incorporating reversible chain terminator nucleotides.

[0010] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0011] The novel advantages and properties of the compositions and methods disclosed herein are set forth with particularity in the appended claims. A better understanding of the properties and advantages of the compositions and methods of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments and the accompanying drawings. [Brief description of the drawings]

[0012] [Figure 1]1 is a schematic diagram of an exemplary low binding support comprising alternating layers of a glass substrate and a hydrophilic coating covalently or non-covalently adhered to the glass, and further comprising chemically reactive functional groups that serve as binding sites for oligonucleotide primers (e.g., capture oligonucleotides). In alternative embodiments, the support can be made from any material, such as glass, plastic, or polymeric materials. [Diagram 2] Schematic diagrams of various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of a multivalent molecule with a "starburst" or "helter skelter" configuration. Center (Class II): Schematic diagram of a multivalent molecule with a dendrimer configuration. Right (Class III) Schematic diagram of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS bearing biotin and dNTPs. Nucleotide units are represented as "N", biotin is represented as "B", and streptavidin is represented as "SA". [Diagram 3] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a generic core attached to multiple nucleotide arms. [Figure 4] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms. [Diagram 5] 1 shows a schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, the nucleotide arms comprising biotin, a spacer, a linker, and a nucleotide unit. [Figure 6] FIG. 1 is a schematic diagram of an exemplary nucleotide arm comprising a core binding moiety, a spacer, a linker, and a nucleotide unit. [Figure 7] The chemical structures of an exemplary spacer (top) and various exemplary linkers (bottom) are shown, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker. [Figure 8] 1 shows the chemical structures of various exemplary linkers, including linkers 1-9. [Figure 9A]1 shows the chemical structures of various exemplary linkers that link / bond nucleotide units. [Figure 9B] 1 shows the chemical structures of various exemplary linkers that link / bond nucleotide units. [Figure 9C] 1 shows the chemical structures of various exemplary linkers that link / bond nucleotide units. [Figure 9D] 1 shows the chemical structures of various exemplary linkers that link / bond nucleotide units. [Figure 10] 1 shows the chemical structure of an exemplary biotinylated nucleotide arm, in which the nucleotide unit is connected to the linker via a propargylamine bond at position 5 of the pyrimidine base or position 7 of the purine base. [Figure 11] 1 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from RLF89458.1 (SEQ ID NO:1). [Figure 12] 1 is the amino acid sequence of a wild-type DNA polymerase having a framework sequence from RLF78286.1 (SEQ ID NO:2). [Figure 13] 1 is the amino acid sequence of a wild-type DNA polymerase having a framework sequence from NOZ58130.1 (SEQ ID NO: 1316). [Figure 14] 22 is the amino acid sequence of a wild-type DNA polymerase having the backbone sequence from RMF90817.1 (SEQ ID NO: 2215). [Figure 15]

[0036] Figure 2 is the amino acid sequence of a wild type DNA polymerase having the backbone sequence from MBC7218772.1 (SEQ ID NO: 2367). [Figure 16] 1 is the amino acid sequence of a wild-type DNA polymerase having the backbone sequence from WP175059460.1 (SEQ ID NO: 2393). [Figure 17] 2 is the amino acid sequence of a wild-type DNA polymerase having the backbone sequence from KUO42443.1 (SEQ ID NO: 2408). [Figure 18] 2 is the amino acid sequence of a wild-type DNA polymerase having a framework sequence from NOZ77387.1 (SEQ ID NO: 2436). [Figure 19]

[0033] Figure 2 is the amino acid sequence of a wild-type DNA polymerase having the backbone sequence from Geobacillus stearthermophilus (Bst polymerase) (SEQ ID NO: 2502). [Figure 20]

[0043] Figure 2 is the amino acid sequence of 9°N polymerase (SEQ ID NO: 2503). [Figure 21]

[0046] Figure 2 is the amino acid sequence of 9°N polymerase UniProt Q56366 (SEQ ID NO: 2504). [Figure 22]

[0046] Figure 2 is the amino acid sequence of Therminator polymerase (SEQ ID NO: 2505). [Figure 23]

[0043] Figure 2 is the amino acid sequence of Vent polymerase UniProt P30317 (SEQ ID NO: 2506). [Figure 24]

[0043] Figure 2 is the amino acid sequence of deep vent polymerase UniProt Q51334 (SEQ ID NO: 2507). [Diagram 25]

[0046] Figure 2 is the amino acid sequence of Pfu polymerase UniProt P61875 (SEQ ID NO: 2508). [Figure 26]

[0046] Figure 2 is the amino acid sequence of Pyrococcus abyssi polymerase UniProt P0CL77 (SEQ ID NO: 2509). [Figure 27]

[0041] Figure 2 is the amino acid sequence of RB69 polymerase (SEQ ID NO:2510). [Figure 28]

[0041] Figure 2 is the amino acid sequence of Phi29 polymerase (SEQ ID NO:2455). [Figure 29A] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29B]Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29C] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29D] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29E] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29F]Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29G] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Fig. 29H] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29I] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29J]Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29K] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29L] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29M] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29N]Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29O] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29P] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 29Q] Table 1, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 3-293) with various mutation substitution sites. Table 1 lists the incorporation activity of 3' methyl azido nucleotide at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild type DNA polymerase with the backbone sequence of RLF89458.1. [Figure 30A]Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30B] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30C] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30D] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30E] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30F] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30G] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30H]Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30I] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30J] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30K] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30L] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30M] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30N] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30O]Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30P] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30Q] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30R] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30S] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30T] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 30U] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30V]Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 30W] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 30X] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 30Y] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig.30Z] Table 2 lists the fluorescent signal intensities of mutant variants (SEQ ID NOs: 294 to 725) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1 of the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31A] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31B] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31C] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31D] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31E] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31F] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31G] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31H] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31I]Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31J] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31K] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31L] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31M] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31N] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31O]Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31P] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31Q] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31R] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31S] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31T] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31U]Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31V] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31W] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31X] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31Y] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig.31Z] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31AA]Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Diagram 31BB] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31CC] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig.31DD] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Fig. 31EE] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Diagram 31FF] Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 31GG]Table 3 lists mutant DNA polymerases (SEQ ID NOs: 726-1315) having the backbone sequence of RLF89458.1 and various mutation substitution sites relative to the numbering in SEQ ID NO: 1 for the wild-type DNA polymerase having the backbone sequence of RLF89458.1. [Figure 32A] Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Figure 32B] Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Figure 32C] Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Fig. 32D]Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Figure 32E] Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Fig. 32F] Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Fig. 32G] Table 4, which lists the relative incorporation activity of mutant variants (SEQ ID NO: 1317-1447) with various mutation substitution sites. Table 4 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase with the backbone sequence of NOZ58130.1. [Diagram 33]Table 5 shows the fluorescent signal intensities of mutant variants (SEQ ID NOs: 1448-1450) with various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34A] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34B] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34C] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34D] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34E] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34F]Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34G] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34H] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34I] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34J] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34K] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34L]Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34M] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34N] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34O] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34P] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34Q] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34R]Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34S] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34T] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34U] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34V] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34W] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34X]Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34Y] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig.34Z] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34AA] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Diagram 34BB] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34CC] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig.34DD]Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig.34EE] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Diagram 34FF] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34GG] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34HH] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34II] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34JJ]Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Diagram 34KK] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34LL] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Fig. 34MM] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Figure 34NN] Table 6 lists mutant DNA polymerases (SEQ ID NOs: 1451-2214) having the backbone sequence of NOZ58130.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 1316 of the wild-type DNA polymerase having the backbone sequence of NOZ58130.1. [Diagram 35]Table 7, which lists the relative incorporation activity of mutant variants (SEQ ID NOs: 2216-2236) with various mutation substitution sites. Table 7 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide strand at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild type DNA polymerase with the backbone sequence of RMF90817.1. [Figure 36A] Table 8 lists mutant DNA polymerases (SEQ ID NOs: 2237-2366) having the backbone sequence of RMF90817.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild-type DNA polymerase having the backbone sequence of RMF90817.1. [Figure 36B] Table 8 lists mutant DNA polymerases (SEQ ID NOs: 2237-2366) having the backbone sequence of RMF90817.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild-type DNA polymerase having the backbone sequence of RMF90817.1. [Figure 36C] Table 8 lists mutant DNA polymerases (SEQ ID NOs: 2237-2366) having the backbone sequence of RMF90817.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild-type DNA polymerase having the backbone sequence of RMF90817.1. [Figure 36D] Table 8 lists mutant DNA polymerases (SEQ ID NOs: 2237-2366) having the backbone sequence of RMF90817.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild-type DNA polymerase having the backbone sequence of RMF90817.1. [Figure 36E]Table 8 lists mutant DNA polymerases (SEQ ID NOs: 2237-2366) having the backbone sequence of RMF90817.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild-type DNA polymerase having the backbone sequence of RMF90817.1. [Figure 36F] Table 8 lists mutant DNA polymerases (SEQ ID NOs: 2237-2366) having the backbone sequence of RMF90817.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2215 of the wild-type DNA polymerase having the backbone sequence of RMF90817.1. [Figure 37] Table 9 lists the relative incorporation activity of mutant variants (SEQ ID NO: 2368-2387) with various mutation substitution sites. Table 9 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide strand at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2367 of the wild type DNA polymerase having the backbone sequence of MBC7218772.1. [Figure 38] Table 10 lists mutant DNA polymerases (SEQ ID NOs: 2388-2392) having the backbone sequence of MBC7218772.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2367 of the wild-type DNA polymerase having the backbone sequence of MBC7218772.1. [Figure 39A] Table 11 lists mutant DNA polymerases (SEQ ID NOs: 2394-2407 and 2511-2523) having the backbone sequence of WP175059460.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2393 of the wild type DNA polymerase having the backbone sequence of WP175059460.1. [Figure 39B]Table 11 lists mutant DNA polymerases (SEQ ID NOs: 2394-2407 and 2511-2523) having the backbone sequence of WP175059460.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2393 of the wild type DNA polymerase having the backbone sequence of WP175059460.1. [Diagram 40] Table 12 lists the relative incorporation activity of mutant variants (SEQ ID NOs: 2409-2424) with various mutation substitution sites. Table 12 lists the incorporation activity of 3' methyl azido nucleotides at the N+1 position of the elongated polynucleotide chain at 42°C. The mutant variants are present in lysates removed from the expression strain. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2408 of the wild type DNA polymerase having the backbone sequence of KUO42443.1. [Diagram 41] Table 13 lists mutant DNA polymerases (SEQ ID NOs: 2425-2435) having the backbone sequence of KUO42443.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2408 of the wild-type DNA polymerase having the backbone sequence of KUO42443.1. [Diagram 42] Table 14 lists mutant DNA polymerases (SEQ ID NOs: 2437-2454) having the backbone sequence of NOZ77387.1 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2436 of the wild-type DNA polymerase having the backbone sequence of NOZ77387.1. [Figure 43A] Table 15 lists mutant DNA polymerases (SEQ ID NOs: 2456-2501) having the backbone sequence of Phi29 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2455 of the wild-type DNA polymerase having the backbone sequence of Phi29. [Figure 43B]Table 15 lists mutant DNA polymerases (SEQ ID NOs: 2456-2501) having the backbone sequence of Phi29 and various mutation substitution sites. The mutation substitution sites are relative to the numbering in SEQ ID NO: 2455 of the wild-type DNA polymerase having the backbone sequence of Phi29. [Figure 44A] 1 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (sequence number 1), NOZ58130 (sequence number 1316), RMF90817 (sequence number 2215), MBC7218772 (sequence number 2367), WP175059460 (sequence number 2393), KUO42443 (sequence number 2408), and NOZ77387 (sequence number 2436). [Figure 44B] 1 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (sequence number 1), NOZ58130 (sequence number 1316), RMF90817 (sequence number 2215), MBC7218772 (sequence number 2367), WP175059460 (sequence number 2393), KUO42443 (sequence number 2408), and NOZ77387 (sequence number 2436). [Figure 44C] 1 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (sequence number 1), NOZ58130 (sequence number 1316), RMF90817 (sequence number 2215), MBC7218772 (sequence number 2367), WP175059460 (sequence number 2393), KUO42443 (sequence number 2408), and NOZ77387 (sequence number 2436). [Fig.44D] 1 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (sequence number 1), NOZ58130 (sequence number 1316), RMF90817 (sequence number 2215), MBC7218772 (sequence number 2367), WP175059460 (sequence number 2393), KUO42443 (sequence number 2408), and NOZ77387 (sequence number 2436). [Figure 44E]1 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (sequence number 1), NOZ58130 (sequence number 1316), RMF90817 (sequence number 2215), MBC7218772 (sequence number 2367), WP175059460 (sequence number 2393), KUO42443 (sequence number 2408), and NOZ77387 (sequence number 2436). [Figure 45A] 25 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (SEQ ID NO: 1), Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2502), 9°N (SEQ ID NO: 2503), Pfu polymerase (SEQ ID NO: 2508), and Pyrococcus abyssi polymerase (SEQ ID NO: 2509). [Figure 45B] 25 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (SEQ ID NO: 1), Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2502), 9°N (SEQ ID NO: 2503), Pfu polymerase (SEQ ID NO: 2508), and Pyrococcus abyssi polymerase (SEQ ID NO: 2509). [Figure 45C] 25 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (SEQ ID NO: 1), Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2502), 9°N (SEQ ID NO: 2503), Pfu polymerase (SEQ ID NO: 2508), and Pyrococcus abyssi polymerase (SEQ ID NO: 2509). [Figure 45D] 25 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (SEQ ID NO: 1), Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2502), 9°N (SEQ ID NO: 2503), Pfu polymerase (SEQ ID NO: 2508), and Pyrococcus abyssi polymerase (SEQ ID NO: 2509). [Figure 45E]25 shows an amino acid sequence alignment of DNA polymerases from: RLF89458.1 (SEQ ID NO: 1), Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2502), 9°N (SEQ ID NO: 2503), Pfu polymerase (SEQ ID NO: 2508), and Pyrococcus abyssi polymerase (SEQ ID NO: 2509). [Figure 46A] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ58130 (sequence number 1316), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 46B] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ58130 (sequence number 1316), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 46C] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ58130 (sequence number 1316), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 46D] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ58130 (sequence number 1316), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 46E]2 shows an amino acid sequence alignment of DNA polymerases from: NOZ58130 (sequence number 1316), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 47A] 2 shows an amino acid sequence alignment of DNA polymerases from: RMF90817 (sequence number 2215), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 47B] 2 shows an amino acid sequence alignment of DNA polymerases from: RMF90817 (sequence number 2215), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 47C] 2 shows an amino acid sequence alignment of DNA polymerases from: RMF90817 (sequence number 2215), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 47D] 2 shows an amino acid sequence alignment of DNA polymerases from: RMF90817 (sequence number 2215), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 47E]2 shows an amino acid sequence alignment of DNA polymerases from: RMF90817 (sequence number 2215), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 48A] 2 shows an amino acid sequence alignment of DNA polymerases from: MBC7218772 (sequence number 2367), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 48B] 2 shows an amino acid sequence alignment of DNA polymerases from: MBC7218772 (sequence number 2367), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 48C] 2 shows an amino acid sequence alignment of DNA polymerases from: MBC7218772 (sequence number 2367), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 48D] 2 shows an amino acid sequence alignment of DNA polymerases from: MBC7218772 (sequence number 2367), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 48E]2 shows an amino acid sequence alignment of DNA polymerases from: MBC7218772 (sequence number 2367), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 49A] 2 shows an amino acid sequence alignment of DNA polymerases from: WP175059460 (sequence number 2393), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 49B] 2 shows an amino acid sequence alignment of DNA polymerases from: WP175059460 (sequence number 2393), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 49C] 2 shows an amino acid sequence alignment of DNA polymerases from: WP175059460 (sequence number 2393), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 49D] 2 shows an amino acid sequence alignment of DNA polymerases from: WP175059460 (sequence number 2393), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 49E] 2 shows an amino acid sequence alignment of DNA polymerases from: WP175059460 (sequence number 2393), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 50A] 2 shows an amino acid sequence alignment of DNA polymerases from: KUO42443 (sequence number 2408), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 50B] 2 shows an amino acid sequence alignment of DNA polymerases from: KUO42443 (sequence number 2408), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 50C] 2 shows an amino acid sequence alignment of DNA polymerases from: KUO42443 (sequence number 2408), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 50D] 2 shows an amino acid sequence alignment of DNA polymerases from: KUO42443 (sequence number 2408), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 50E] 2 shows an amino acid sequence alignment of DNA polymerases from: KUO42443 (sequence number 2408), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 51A] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ77387 (sequence number 2436), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 51B] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ77387 (sequence number 2436), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 51C] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ77387 (sequence number 2436), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Fig. 51D] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ77387 (sequence number 2436), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 51E] 2 shows an amino acid sequence alignment of DNA polymerases from: NOZ77387 (sequence number 2436), Geobacillus stearothermophilus (Bst polymerase) (sequence number 2502), 9°N (sequence number 2503), Pfu polymerase (sequence number 2508), and Pyrococcus abyssi polymerase (sequence number 2509). [Figure 52] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation L253S. [Figure 53] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation Y254A. [Figure 54] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation Y254G. [Figure 55] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation L381A. [Figure 56] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation L381S. [Figure 57] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation L381V. [Figure 58] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation M102K. [Figure 59] Ribbon model based on the crystal structure of the ternary complex of Phi29 DNA polymerase containing the amino acid substitution mutation V425K. [Figure 60] 1 is a graph showing the % error of a 150 cycle sequencing run of a nucleic acid library prepared from PhiX. [Figure 61]61 is a schematic diagram of an exemplary immobilized nucleic acid template molecule hybridized to a first and a second nucleic acid primer. The nucleic acid template molecule shown in Figure 61 comprises a concatemer hybridized to a plurality of nucleic acid primers. [Figure 62] FIG. 1 is a schematic diagram of exemplary polymerase composites, indicated by dashed circles; each polymerase composite comprises a DNA polymerase that binds to nucleic acid duplexes, each duplex comprising a nucleic acid template hybridized to a nucleic acid primer. [Figure 63] FIG. 63 is a schematic diagram of an exemplary first binding complex (e.g., shown as a dashed circle) including a first nucleic acid primer, a first DNA polymerase, and a first multivalent molecule that are bound to a first portion of a concatemer template molecule, thereby forming a first binding complex. FIG. 63 also shows a plurality of multivalent molecules that are not part of the first binding complex. [Figure 64] FIG. 1 is a schematic diagram of an exemplary avidity complex (e.g., indicated by dashed circles) including: (i) a first binding complex comprising a first nucleic acid primer, a first DNA polymerase, and a first multivalent molecule bound to a first portion of a concatemer template molecule, thereby forming a first binding complex, where a first nucleotide unit of the multivalent molecule binds to the first DNA polymerase; and (ii) a second binding complex comprising a second nucleic acid primer, a second DNA polymerase, and the same first multivalent molecule bound to a second portion of the same concatemer template molecule, thereby forming a second binding complex, where a second nucleotide unit of the multivalent molecule binds to the second DNA polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex. Summary of the Invention

[0013] An embodiment of the present disclosure is a polymerase that is at least 85% identical to any one of SEQ ID NOs: 1-2501 and 2511-2523, and has the amino acid substitution mutations Asp141Ala and Glu143Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogs compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 1 or 2 (RLF89458.1 or RLF78286.1). amino acid substitution mutations Asp168Ala and Glu170Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogs compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 1316 (NOZ58130.1); amino acid substitution mutations Asp149Ala and Glu151Ala, wherein the engineered polymerase has the amino acid sequence of SEQ ID NO: 2215 (RMF90817.1); and amino acid substitution mutations Asp173Ala and Glu175Ala, which have increased incorporation of a chain-terminating nucleotide analog compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2367 (MBC7218772.1). Ala, amino acid substitution mutations Asp141Ala and Glu143Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogs compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2393 (WP175059460.1), amino acid substitution mutations Asp170Ala and Glu172Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogs compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2408 (KUO42443.1), wherein the engineered polymerase comprises an amino acid sequence having the amino acid substitution mutations Asp161Ala and Glu163Ala, which has increased incorporation of a chain-terminating nucleotide analog compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2436 (NOZ77387.1).

[0014] In some embodiments, the method further comprises a plurality of engineered polymerases, a plurality of nucleic acid template molecules, and a plurality of nucleotide polymerization initiation sites having 3' extendable ends.

[0015] In some embodiments, the plurality of nucleic acid template molecules comprises linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear and circular nucleic acid molecules.

[0016] In some embodiments, the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

[0017] In some embodiments, at least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules comprises one copy of a target sequence of interest or comprises a concatemer having two or more tandem copies of a target sequence of interest.

[0018] In some embodiments, an individual nucleotide polymerization initiation site within 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 an individual nucleotide polymerization initiation site within the plurality of nucleotide polymerization initiation sites comprises a self-priming end portion of at least one of the nucleic acid template molecules.

[0019] In some embodiments, the multiple polymerases, the multiple nucleic acid template molecules, and the multiple nucleotide polymerization initiation sites form multiple polymerase multiplexes, each multiple polymerase multiplex comprising a polymerase bound to a nucleic acid duplex, the duplex comprising a nucleic acid template molecule hybridized to a nucleic acid primer.

[0020] In some embodiments, the multiple nucleic acid template molecules comprise the same target sequence of interest or different target sequences of interest.

[0021] In some embodiments, the plurality of composite polymerases further comprises a plurality of multivalent molecules, each multivalent molecule in the plurality of multivalent molecules comprising (a) a core and (b) a plurality of nucleotide arms comprising (i) a core-binding moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core-binding moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0022] In some embodiments, the linker comprises an aliphatic chain having 2 to 6 subunits or an oligoethylene glycol chain having 2 to 6 subunits.

[0023] In some embodiments, the multiple nucleotide arms attached to a given core have the same type of nucleotide unit, which nucleotide unit comprises dATP, dGTP, dCTP, dTTP, or dUTP.

[0024] In some embodiments, the plurality of multivalent molecules comprises one type of multivalent molecule, and each multivalent molecule of the plurality has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0025] In some embodiments, the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0026] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a core labeled with a fluorophore.

[0027] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a nucleotide unit labeled with a fluorophore.

[0028] In some embodiments, the plurality of composite polymerases further comprises a plurality of nucleotides, wherein each nucleotide in the plurality of nucleotides comprises an aromatic base, a 5-carbon sugar, and from 1 to 10 phosphate groups.

[0029] In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0030] In some embodiments, 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.

[0031] In some embodiments, at least one nucleotide in the plurality of nucleotides is labeled with a fluorophore.

[0032] In some embodiments, a plurality of nucleotides lacks a fluorophore label.

[0033] In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a removable chain terminating moiety attached to the 3' carbon position of the sugar group, the removable chain terminating moiety comprising an acetal group, 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, the removable chain terminating moiety being cleavable with a chemical compound to generate a 3' OH moiety that can be extended on the sugar group.

[0034] In some embodiments, the multiple polymerases further comprise multiple non-catalytic divalent cations that inhibit polymerase-catalyzed incorporation of nucleotides, wherein the non-catalytic divalent cations comprise strontium or barium.

[0035] In some embodiments, the multiple composite polymerases further comprise multiple catalytic divalent cations that facilitate polymerase-catalyzed incorporation of nucleotides, the catalytic divalent cations comprising magnesium or manganese, hi some embodiments, the multiple composite polymerases are immobilized on a support or in a coating on a support.

[0036] In some embodiments, the density of the multiple polymerases immobilized on the support is greater than or equal to 1 mm 2 10 per 2 ~10 12 include.

[0037] In some embodiments, the multiple immobilized composite polymerases are immobilized at predetermined sites on the support or at random sites on the support.

[0038] In some embodiments, 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.

[0039] In some embodiments, the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

[0040] In some embodiments, multiple immobilized composite polymerases are in fluid communication with each other, allowing a solution of reagents to be flowed over the support so as to react with the solution in a massively parallel manner.

[0041] In some embodiments, the plurality of composite polymerases further comprises first and second binding complexes, the first binding complex comprising a first nucleic acid primer, a first polymerase, and a first multivalent molecule bound to a first portion of the concatemeric template molecule, thereby forming a first binding complex, the first nucleotide unit of the multivalent molecule binds to the first polymerase, the second binding complex comprising a second nucleic acid primer, a second polymerase, and the first multivalent molecule bound to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, the second nucleotide unit of the multivalent molecule binds to the second polymerase, and the first and second binding complexes comprising the same multivalent molecule form a binding activity complex.

[0042] Another aspect of the present disclosure includes a method for forming a plurality of composite polymerases comprising contacting a plurality of engineered polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid primers under suitable conditions to form a plurality of composite polymerases, each comprising a polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer, and the plurality of engineered polymerases comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:1-2501 and 2511-2523.

[0043] In some embodiments, the plurality of nucleic acid template molecules comprises linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear and circular nucleic acid molecules.

[0044] In some embodiments, the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

[0045] In some embodiments, each nucleic acid template molecule in the plurality of nucleic acid molecules comprises one copy of a target sequence of interest, or each nucleic acid template molecule in the plurality of nucleic acid molecules comprises a concatemer having two or more tandem copies of a target sequence of interest.

[0046] In some embodiments, the multiple nucleic acid molecules comprise the same target sequence of interest or different target sequences of interest.

[0047] In some embodiments, the method includes contacting a plurality of composite polymerases with a plurality of multivalent molecules, where each multivalent molecule in the plurality of multivalent molecules comprises (a) a core and (b) a plurality of nucleotide arms comprising (i) a core-binding moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, where the core is attached to the plurality of nucleotide arms via their core-binding moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0048] In some embodiments, the linker comprises an aliphatic chain having 2 to 6 subunits or an oligoethylene glycol chain having 2 to 6 subunits.

[0049] In some embodiments, multiple nucleotide arms attached to a given core have the same type of nucleotide unit, where the types of nucleotide units include dATP, dGTP, dCTP, dTTP, or dUTP.

[0050] In some embodiments, the plurality of multivalent molecules comprises one type of multivalent molecule, and each multivalent molecule of the plurality has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0051] In some embodiments, the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0052] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore.

[0053] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a core labeled with a fluorophore.

[0054] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide units that are labeled with a fluorophore.

[0055] In some embodiments, the contacting is performed under conditions suitable for binding of at least one complementary nucleotide unit of the multivalent molecule to at least one of the multiple polymerases.

[0056] In some embodiments, the method further comprises contacting the multiple composite polymerases with multiple non-catalytic divalent cations that inhibit polymerase-catalyzed incorporation of nucleotides, wherein the non-catalytic divalent cations comprise strontium or barium.

[0057] In some embodiments, the method further comprises contacting a plurality of multiplex polymerases with a plurality of nucleotides, wherein each nucleotide in the plurality of nucleotides comprises an aromatic base, a 5-carbon sugar, and from 1 to 10 phosphate groups.

[0058] In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0059] In some embodiments, 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.

[0060] In some embodiments, at least one nucleotide of the plurality of nucleotides is labeled with a fluorophore. In some embodiments, the plurality of nucleotides lacks a fluorophore label.

[0061] In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a removable chain terminating moiety attached to the 3' carbon position of the sugar group, the removable chain terminating moiety comprising an acetal group, 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, the removable chain terminating moiety being cleavable with a chemical compound to generate a 3' OH moiety that can be extended on the sugar group.

[0062] In some embodiments, the contacting is performed under conditions suitable for binding at least one complementary nucleotide from the plurality of nucleotides to the at least one composite polymerase. In some embodiments, the method further comprises contacting the plurality of composite polymerases with a plurality of catalytic divalent cations that facilitate polymerase-catalyzed incorporation of nucleotides, wherein the catalytic divalent cation comprises magnesium or manganese.

[0063] In some embodiments, the multiple polymerases are immobilized on a support or in a coating on a support. In some embodiments, the density of the multiple polymerases immobilized on a support is greater than 1 mm 2 10 per 2 ~10 12 In some embodiments, the multiple immobilized composite polymerases are immobilized at predetermined sites on the support, or the multiple immobilized composite polymerases are immobilized at random sites on the support.

[0064] In some embodiments, 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.

[0065] In some embodiments, the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

[0066] In some embodiments, multiple immobilized composite polymerases are in fluid communication with each other, allowing solutions of reagents to be flowed over the support so as to react with the solutions of reagents in a massively parallel manner.

[0067] In some embodiments, forming the multiple binding complexes comprises binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of a concatemeric template molecule, thereby forming a first binding complex, where a first nucleotide unit of the first multivalent molecule binds to the first polymerase, and binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, where a second nucleotide unit of the first multivalent molecule binds to the second polymerase, and the first and second binding complexes comprising the same multivalent molecule form a binding activity complex.

[0068] Another aspect of the present disclosure includes a method for determining a sequence of a nucleic acid template, comprising contacting a plurality of first polymerases with (i) a plurality of nucleic acid templates, each comprising a target sequence of interest, and (ii) a plurality of nucleic acid primers, wherein the contacting is performed under conditions suitable to bind 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 composite polymerases, each comprising a first polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer, and wherein the plurality of first polymerases comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 1-2501 and 2511-2523, and contacting a plurality of first composite polymerases with a plurality of multivalent molecules to form binding complexes, where each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a number of nucleotide arms, each nucleotide arm binding to a nucleotide unit, the contacting being performed under conditions suitable for binding a complementary nucleotide unit of the multivalent molecule to at least two of the plurality of first composite polymerases, thereby forming a plurality of multivalent binding complexes, the conditions being suitable for inhibiting incorporation of the complementary nucleotide unit into a primer of the plurality of multivalent binding complexes; detecting the plurality of multivalent binding complexes; and identifying bases of the complementary nucleotide units in the plurality of multivalent binding complexes, thereby determining a sequence of the nucleic acid template molecule.

[0069] In some embodiments, the method includes dissociating the plurality of multivalent binding complexes by removing the plurality of first polymerases and their bound multivalent molecules, retaining the plurality of nucleic acid duplexes, and contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second polymerases under conditions suitable for binding the plurality of second polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second polymerase complexes, each comprising a second polymerase bound to a nucleic acid duplex, and wherein the plurality of second polymerases bind to a nucleic acid duplex comprising a nucleic acid sequence similar to SEQ ID NO:1. and contacting a plurality of second composite polymerases with a plurality of nucleotides, the contacting being performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second composite polymerases, thereby forming a plurality of nucleotide binding complexes, the conditions being suitable for promoting nucleotide incorporation of the bound complementary nucleotides into a primer of the nucleotide binding complex.

[0070] In some embodiments, the method further comprises (h) detecting a complementary nucleotide incorporated into the primer of the nucleotide complex polymerase.

[0071] In some embodiments, the method further comprises detecting a complementary nucleotide incorporated into the primer for the nucleotide complex polymerase and identifying a base of the complementary nucleotide incorporated into the primer for the nucleotide complex polymerase.

[0072] In some embodiments, contacting the plurality of first composite polymerases with the plurality of multivalent molecules of step (b) is performed in the presence of a non-catalytic divalent cation that inhibits polymerase-catalyzed incorporation of nucleotides, wherein the non-catalytic divalent cation comprises strontium or barium.

[0073] In some embodiments, contacting the plurality of second composite polymerases with the plurality of nucleotides in step (g) is performed in the presence of a catalytic divalent cation that promotes polymerase-catalyzed incorporation of nucleotides, wherein the catalytic divalent cation comprises magnesium or manganese.

[0074] In some embodiments, the plurality of nucleic acid template molecules of step (a) comprises clonally amplified template molecules.

[0075] In some embodiments, each nucleic acid template molecule in the plurality of nucleic acid molecules of step (a) comprises one copy of a target sequence of interest or comprises a concatemer having two or more tandem copies of a target sequence of interest. In some embodiments, the nucleic acid template molecules in the plurality of nucleic acid molecules of step (a) comprise the same target sequence of interest or different target sequences of interest.

[0076] In some embodiments, each multivalent molecule in the plurality of multivalent molecules comprises (a) a core and (b) a plurality of nucleotide arms comprising (i) a core binding moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core binding moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0077] In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligoethylene glycol chain having 2-6 subunits. In some embodiments, the nucleotide arms attached to a given core have the same type of nucleotide unit, the type of nucleotide unit comprising dATP, dGTP, dCTP, dTTP, or dUTP. In some embodiments, the multivalent molecules comprise one type of multivalent molecule, each of the plurality of multivalent molecules having the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0078] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore.

[0079] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a core labeled with a fluorophore.

[0080] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide units that are labeled with a fluorophore.

[0081] In some embodiments, each nucleotide in the plurality of nucleotides of step (g) comprises an aromatic base, a 5-carbon sugar, and from 1 to 10 phosphate groups.

[0082] In some embodiments, the plurality of nucleotides in step (g) comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, or 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.

[0083] In some embodiments, at least one of the nucleotides in the plurality of nucleotides of step (g) is labeled with a fluorophore. In some embodiments, the plurality of nucleotides of step (g) lacks a fluorophore label. In some embodiments, at least one of the nucleotides in the plurality of nucleotides of step (g) comprises a removable chain terminating moiety attached to a 3' carbon position of a sugar group, the removable chain terminating moiety comprising an acetal group, 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, the removable chain terminating moiety being cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

[0084] In some embodiments, the plurality of first composite polymerases in step (a) is immobilized on a support or in a coating on a support. In some embodiments, the density of the plurality of first composite polymerases immobilized on a support is greater than 1 mm 2 10 per 2 ~10 12 In some embodiments, the plurality of first composite polymerases are immobilized at predetermined sites on the support or at random sites on the support.

[0085] In some embodiments, 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.

[0086] In some embodiments, the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

[0087] In some embodiments, the multiple immobilized first composite polymerases are in fluid communication with each other, allowing a solution of reagents to be flowed over the support such that the multiple immobilized first composite polymerases on the support react with the solution of reagents in a massively parallel manner.

[0088] In some embodiments, the method comprises forming a plurality of binding complexes comprising: binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of a concatemeric template molecule, thereby forming a first binding complex, where a first nucleotide unit of the first multivalent molecule binds to the first polymerase; and binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, where a second nucleotide unit of the first multivalent molecule binds to the second polymerase, and the first and second binding complexes comprising the same multivalent molecule form a binding activity complex.

[0089] In some embodiments, the method includes contacting a plurality of polymerases and a plurality of nucleic acid primers with different portions of a concatemeric nucleic acid template molecule to form at least a first and a second composite polymerase on the same concatemeric template molecule; contacting a plurality of multivalent molecules with at least a first and a second composite polymerase on the same concatemeric template molecule under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first and the second composite polymerases, wherein at least a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase comprising a first primer hybridized to a first portion of the concatemeric template molecule, thereby forming a first binding complex, and at least a second nucleotide unit of the single multivalent molecule binds to the first composite polymerase comprising a first primer hybridized to a first portion of the concatemeric template molecule, thereby forming a first binding complex. and contacting the first and second binding complexes with a second composite polymerase comprising a second primer hybridized to a second portion of the template molecule, thereby forming a second binding complex, under conditions suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes, whereby the first and second binding complexes bound to the same multivalent molecule form a binding activity complex; detecting the first and second binding complexes on the same concatemer template molecule; identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] Definition: The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. Generally, the terms related to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization techniques described herein are 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 the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with the experimental procedures and techniques described herein, as well as the experimental procedures and techniques, are well known and commonly used in the art.

[0092] Unless otherwise required by context herein, singular terms include plurals and plural terms include the singular. Use of the singular forms "a," "an," and "the," as well as the singular form of any word, includes plural referents unless clearly and unambiguously limited to one referent.

[0093] The use of alternative terms (eg, "or") is understood to mean either one or both of the alternatives, or any combination thereof.

[0094] As used herein, the term "and / or" should be understood to mean a specific disclosure that each of the specified features or components has or does not have the other. For example, when used herein in phrases such as "A and / or B", the term "and / or" is intended to include "A and B", "A or B", "A" (A only), and "B" (B only). In a similar manner, when used in phrases such as "A, B, and / or C", the term "and / or" is intended to include each of the following embodiments: "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" (A only), "B" (B only), and "C" (C only).

[0095] As used in this specification and the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be open-ended so that one item or more items in a list do not exclude other items that may be substituted for or added to the listed items. Wherever an embodiment is described herein with the term "comprising," it is understood that alternatively similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0096] As used herein, the term "about" or "approximately" refers 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, the acceptable error range depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) or more, 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. Furthermore, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to 5 times. 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 an acceptable error range for this particular value or composition. Also, when a range and / or subrange of values ​​is provided, the range and / or subrange can include the endpoints of the range and / or subrange.

[0097] The terms "peptide", "polypeptide" and "protein" as well as other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides can include natural and unnatural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also 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, protein fragments and polypeptide analogs of protein sequences, such as muteins, variants, chimeric proteins, and fusion proteins, as well as proteins that have been post-translationally or otherwise covalently or non-covalently modified.

[0098] The term "polymerase" and variants thereof, as used herein, includes any enzyme capable of catalyzing the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase comprises one or more active sites, at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase can bind to a nucleotide, a nucleotide analog, or a multivalent molecule, and may or may not incorporate a nucleotide, a nucleotide analog, or a nucleotide unit of a multivalent molecule. In some embodiments, a polymerase comprises other enzymatic activities, such as a 3' to 5' exonuclease activity or a 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacement activity. Polymerases can include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. In some embodiments, the polymerase may be isolated from a cell or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, the polymerase may be expressed in a prokaryotic, eukaryotic, viral, or phage organism. In some embodiments, the polymerase can be a post-translationally modified protein or fragment thereof. The polymerase can be derived from a prokaryotic, eukaryotic, viral, or phage. Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0099] 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.

[0100] As used herein, the term "binding complex" refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotide or nucleotide unit may or may not be bound to the 3' end of the nucleic acid primer at a position opposite the complementary nucleotide in 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 a free nucleotide or nucleotide unit of a multivalent molecule, where the free nucleotide or nucleotide unit is bound to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) at a position opposite the complementary nucleotide in the nucleic acid template molecule.

[0101] The term "duration" and related terms refer to the length of time during which the binding complex remains stable without any of the components dissociating, the components of the binding complex including the nucleic acid template and nucleic acid primer, the polymerase, the nucleotide unit of the multivalent molecule, or the free (e.g., unconjugated) nucleotide. The nucleotide unit or free nucleotide may be complementary or non-complementary to the nucleotide residue in the template molecule. The nucleotide unit or free nucleotide may be bound to the 3' end of the nucleic acid primer at a position opposite 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 labeling reagent including one or more nucleotides, can be present in the binding complex, thus allowing the signal from the label to be detected during the duration of the binding complex. One exemplary label is a fluorescent label. The bound complex (e.g., ternary complex) remains stable until it is subjected to conditions that cause dissociation of interactions between the polymerase, the template molecule, the primer, and / or any of the nucleotide units or nucleotides. For example, dissociation conditions include contacting the bound complex with any one of detergent, EDTA, and / or water, or any combination thereof.

[0102] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside linkages, e.g., phosphodiester linkages. Nucleic acids include non-natural internucleoside linkages, where the non-natural internucleoside linkages include phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids include one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0103] The term "primer" and related terms as used herein refers to an oligonucleotide, either natural or synthetic, that can hybridize to a DNA and / or RNA polynucleotide template and form a double-stranded molecule. A primer can be of any length, but typically ranges 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 or more nucleotides along the length of the primer can be labeled with a detectable reporter moiety. The primer can be in solution (e.g., a soluble primer) or immobilized on a support (e.g., a capture primer).

[0104] "Template nucleic acid", "template polynucleotide", "target nucleic acid", "target polynucleotide", "template strand", and other variations refer to a nucleic acid strand that serves as a base nucleic acid molecule for generating a complementary nucleic acid strand. The sequence of the template nucleic acid can be partially or completely complementary to the sequence of the complementary strand. The template nucleic acid can be obtained from naturally occurring sources or recombinant forms, or can be chemically synthesized to contain any type of nucleic acid analog. The template nucleic acid can be linear, circular, or in other forms. The template nucleic acid can be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloned, amplified, RNA such as cDNA, precursor mRNA or mRNA, oligonucleotides, total genomic DNA obtained from fresh frozen paraffin-embedded tissue, needle biopsy, cell-free circulating DNA, or any type of nucleic acid library. Template nucleic acid molecules can be isolated from any source, including prokaryotes, eukaryotes (e.g., human, plant, and animal), fungi, and viruses; cells; tissues; bodily fluids, including normal or diseased cells or tissues, blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, and semen; environmental samples; culture samples; or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. Template nucleic acids can be subjected to nucleic acid analysis, including sequencing and compositional analysis.

[0105] When used in reference to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization", or other related terms, refers 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 with a double-stranded region. Hybridization can include Watson-Crick or Hoogstein binding to form a double-stranded double-stranded 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, can be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other over their entire length. Complementary base pairing can be standard AT or CG base pairing, or other forms of base pairing interactions. Double-stranded nucleic acids can include mismatched base pairing nucleotides.

[0106] The term "nucleotide" and related terms refer to a molecule that includes an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or nonstandard nucleotides are consistent with the use of this term. In some embodiments, the phosphate includes a monophosphate, diphosphate, or triphosphate, or the corresponding phosphate analogs. In some embodiments, a nucleotide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups. The term "nucleoside" refers to a molecule that includes an aromatic base and a sugar.

[0107] Nucleotides (and nucleosides) typically contain a heterocyclic base that contains a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, which are commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants, or analogs thereof. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include 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-deazadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); cytosine (C), 5-propynylcytosine, isocytosine, thymosine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 Examples of bases include, but are not limited to, purines and pyrimidines such as -methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydracyl; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methylcytosine (methycytosine); base (Y); and methylated, glycylated, and acylated base moieties. Additional exemplary bases can be found in Fasman, 1989, in "Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, CRC Press, Boca Raton, Fla.

[0108] Nucleotides (and nucleosides) typically include a sugar moiety, e.g., 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 another sugar moiety (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). Sugar moieties include ribosyl, 2'-deoxyribosyl, 3'-deoxyribosyl, 2',3'-dideoxyribosyl, 2',3'-didehydrodideoxyribosyl, 2'-alkoxyribosyl, 2'-azidoribosyl, 2'-aminoribosyl, 2'-fluororibosyl, 2'-mercaptoriboxyl, 2'-alkylribosyl, 3'-alkoxyribosyl, 3'-azidoribosyl, 3'-aminoribosyl, 3'-fluororibosyl, 3'-mercaptoriboxyl, 3'-alkylribosylcarbosyl, acyclic, or other modified sugars.

[0109] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, typically linked to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain are O, S, or BH. 3 In some embodiments, the tethers include phosphate groups replaced with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methyl phosphoramidate groups.

[0110] When used in reference to nucleic acids, the terms "extend", "extending", "extension" and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves polymerization of one or more nucleotides to the terminal 3'OH terminus of a nucleic acid chain, resulting in the extension of the nucleic acid chain. Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method of extending a nucleic acid molecule can be used, and suitable methods include primer extension catalyzed by DNA polymerase or RNA polymerase.

[0111] The terms "reporter moiety", "reporter moieties" or related terms refer to a compound that produces or can be caused to produce a detectable signal. Reporter moieties are often referred to as "labels". Any suitable reporter moiety can be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or enzyme. The reporter moiety produces a detectable signal due to a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or a proximity event). A proximity event includes two reporter moieties coming into close 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 wavelengths distinguishable from other reporter moieties, allowing the presence of different reporter moieties in the same or different reactions to be monitored. Two or more different reporter moieties can be selected that have spectrally distinct emission profiles or that have minimal overlapping spectral emission profiles. The reporter moiety can be attached (e.g., operably attached) to a nucleotide, a nucleoside, a nucleic acid, an enzyme (e.g., a polymerase or a reverse transcriptase), or a support (e.g., a surface).

[0112] Reporter moieties (or labels) include fluorescent labels or fluorophores. Exemplary fluorescent moieties that can function as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and rhodamine derivatives. Conductors such as TRITC, TMR, Lissamine rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-Rhodamine, TMR-Iodoacetamide, Lissamine rhodamine B sulfonyl chloride, Lissamine rhodamine B sulfonyl hydrazine, 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, cyanines and derivatives such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT,Examples of suitable dyes include, but are not limited to, 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, stilbenes, DEG dyes, NR dyes, near infrared dyes, and others known 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 combination thereof. Cyanine dyes can exist in either sulfonated or non-sulfonated form and consist of two indolenine, benzoindolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the 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 can include 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 can include1-(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-yne 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) Cy5 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), and Cy6 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for "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 benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0113] In some embodiments, the reporter moieties can be FRET pairs, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET can include excitation exchange (Förster) transfer, or electron exchange (Dexter) transfer.

[0114] The terms "linked," "joined," "attached," and variants thereof include any type of fusion, binding, adhesion, or association between any combination of compounds or molecules that have sufficient stability to withstand use in a particular procedure. Procedures can include, but are not limited to, nucleotide transient binding, nucleotide incorporation, deblocking, washing, removal, flow, detection, imaging, and / or identification. Such binding can include, for example, covalent bonds, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity binding, binding or association involving van der Waals forces, and mechanical binding, and the like. In some embodiments, such binding occurs intramolecularly, for example, by joining the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such binding can occur between different molecular combinations or between a molecule and a non-molecular entity, including, but not limited to, a nucleic acid molecule and a solid surface, a protein and a detectable reporter moiety, a nucleotide and a detectable reporter moiety, and the like. Some examples of conjugation 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).

[0115] As used herein, the terms "operably linked" and "operably linked" or related terms refer to the juxtaposition of components. The juxtaposed components may be covalently linked together. For example, two nucleic acid components may be enzymatically ligated together, and the bond linking the two components together comprises a phosphodiester bond. A first and a second nucleic acid component may be linked together, and the first nucleic acid component may 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 capable of binding to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) may be ligated into a vector, and the bond allows for 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 expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, 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.

[0116] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is any combination of porous, semi-porous, non-porous, or 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.

[0117] In some embodiments, the surface of the support can be substantially smooth, hi some embodiments, the support can be textured, regular or irregular, including bumps, etchings, pores, three-dimensional scaffolds, or any combination thereof.

[0118] In some embodiments, the support comprises a bead having any shape, including spherical, hemispherical, cylindrical, barrel, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0119] The support may be made of any material, including, but not limited to, glass, fused silica, silicon, polymer (e.g., 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.

[0120] In some embodiments, the surface of the support is coated with one or more compounds to generate a passivation layer on the support.In some embodiments, the support comprises a low non-specific binding surface, which allows improved nucleic acid hybridization and amplification performance on the support.Generally, the support can comprise one or more layers of covalently or non-covalently bound low chemical modification layers, such as silane layers, polymer films, and one or more layers of covalently or non-covalently bound oligonucleotides, which can be used to immobilize multiple nucleic acid template molecules on the support.

[0121] In some embodiments, the degree of hydrophilicity (or "wettability" with aqueous solutions) of a surface coating can be assessed, for example, by measuring water contact angles, where a small drop of water is placed on the surface and the contact angle with the surface is measured, for example, using an optical tensiometer. In some embodiments, a static contact angle can be determined. In some embodiments, an advancing or receding contact angle can be determined. In some embodiments, the water contact angle for a 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 a 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. One of skill in the art will recognize that a given hydrophilic low-binding support surface of the present disclosure can exhibit a water contact angle having a value anywhere within this range.

[0122] The present disclosure provides a plurality (e.g., two or more) of nucleic acid templates immobilized on a support. In some embodiments, the immobilized plurality of nucleic acid templates have the same sequence or different sequences. In some embodiments, 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 sites on the support. In some embodiments, the support comprises a plurality of sites arranged in an array. The term "array" refers to a support comprising a plurality of sites located at predetermined locations on the support 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 arranged in rows and columns in two dimensions. In some embodiments, the plurality of predetermined sites are arranged in an organized manner on the support. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including linear, hexagonal, lattice, patterns with reflection symmetry, patterns with rotational symmetry, and the like. The pitch between different pairs of sites may be the same or may vary. In some embodiments, the support is 1 mm thick to form a nucleic acid template array. 2 About 10 per 2 ~10 15 In some embodiments, the support may have immobilized nucleic acid template molecules at a surface density of at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12at least 10 sites 13 at least 10 sites 14 at least 10 sites 15 In some embodiments, the substrate comprises a plurality of predetermined sites (e.g., 10 sites, 20 sites, 30 sites, 40 sites, 50 sites, 60 sites, 70 sites, 80 sites, 90 sites, 10 ... 2 ~10 15 In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites by hybridization to the immobilized surface capture primer, or the nucleic acid template is covalently linked to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites, e.g., 10 2 ~10 15 Nucleic acid templates immobilized at a plurality of sites or more. In some embodiments, the nucleic acid templates immobilized at a plurality of sites on a support comprise 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 polonies at a plurality of predetermined sites. In some embodiments, each immobilized nucleic acid template molecule comprises one copy of the target sequence of interest, or comprises a concatemer having two or more tandem copies of the target sequence of interest.

[0123] In some embodiments, a support comprising a plurality of sites located at random positions on the support is referred to herein as a support having randomly located sites thereon. The positions of the randomly located sites on the support are not predetermined. The plurality of randomly located sites are arranged on the support in a non-ordered and / or unpredictable manner. In some embodiments, the support is at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 at least 10 sites 15 In some embodiments, the substrate comprises a plurality of randomly arranged sites (e.g., 10 sites, 20 sites, 30 sites, 40 sites, 50 sites, 60 sites, 70 sites, 80 sites, 90 sites, 1 ...10 sites, 20 sites, 30 sites, 40 sites, 50 sites, 60 sites, 70 sites, 80 sites, 90 sites, 10 sites, 10 sites, 20 sites, 2 ~10 15 A plurality of randomly located sites (e.g., 10 or more sites) are immobilized with the nucleic acid template to form a nucleic acid template-immobilized support. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites by hybridization to the immobilized surface capture primer, or the nucleic acid template is covalently attached to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites, e.g., 10 or more sites. 2 ~10 15 Nucleic acid templates immobilized at multiple sites or more. In some embodiments, the nucleic acid templates immobilized at multiple sites on the support comprise 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 polonies at multiple randomly located sites. In some embodiments, each immobilized nucleic acid template molecule comprises one copy of the target sequence of interest or comprises a concatemer having two or more tandem copies of the target sequence of interest.

[0124] In some embodiments, with respect to nucleic acid template molecules immobilized at predetermined or random sites on a support, the multiple immobilized nucleic acid template molecules on the support are in fluid communication with each other to allow solutions of reagents (e.g., enzymes including polymerases, multivalent molecules, nucleotides, divalent cations, and / or buffers, etc.) to be flowed over the support so that the multiple immobilized nucleic acid template molecules on the support can react with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the multiple immobilized nucleic acid template molecules can be used to perform nucleotide binding assays and / or perform nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​the multiple immobilized nucleic acid template molecules, as well as detection and imaging for massively parallel sequencing. In some embodiments, the term "immobilized" and related terms refer to a nucleic acid molecule or enzyme (e.g., a polymerase) that is bound to a support at a predetermined or random location, where the nucleic acid molecule or enzyme is directly bound to the support via covalent or non-covalent interactions, or the nucleic acid molecule or enzyme is bound to a coating on the support.

[0125] As used herein, the term "clonal amplified" and its variants refer to a nucleic acid template molecule that has been subjected to one or more amplification reactions, either in solution or on a support. In the case of a template molecule amplified in solution, the resulting amplicons are distributed on a support. Prior to amplification, the template molecule comprises a sequence of interest and at least one universal adaptor 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-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof.

[0126] As used herein, the term "sequencing" and its variants typically involves obtaining sequence information from a nucleic acid strand by determining the identity of at least some nucleotides (including their nucleobase components) in a nucleic acid template molecule. In some embodiments, "sequencing" a given region of a nucleic acid molecule involves identifying each and every nucleotide in the region to be sequenced, although in some embodiments, "sequencing" includes methods in which the identity of only some nucleotides in a region is determined, while the identity of some nucleotides is undetermined or erroneously determined. Any suitable sequencing method may be used. In exemplary embodiments, the sequencing method may use label-free or ion-based sequencing methods. In some embodiments, the sequencing method may use labeled or dye-containing nucleotide or fluorescent-based nucleotide sequencing methods. In some embodiments, the sequencing method may use labeled or unlabeled multivalent molecules. In some embodiments, the sequencing may include polony-based sequencing or bridge sequencing methods. In some embodiments, the sequencing may include massively parallel sequencing platforms using sequence-by-synthesis, sequence-by-hybridization, or sequence-by-binding procedures. Examples of massively parallel sequence-by-synthesis procedures include polony sequencing, pyrosequencing (e.g., from 454 Life Sciences, U.S. Pat. Nos. 7,211,390, 7,244,559, and 7,264,929), chain terminator sequencing (e.g., from Illumina, U.S. Pat. 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 sequencing by hybridization include SOLiD sequencing (e.g., from Life Technologies, WO2006 / 084132). Examples of sequencing by binding include Omniome sequencing (e.g., U.S. Patent No. 10,246,744).

[0127] Engineered polymerases exhibiting reduced sequence-specific errors The present disclosure provides compositions including mutant polymerases with amino acid substitutions and / or truncated amino acid sequences, nucleic acids encoding the mutant polymerases, and systems and kits that include the mutant polymerases. Further provided herein are methods of using the mutant polymerases for binding nucleic acid duplexes, binding and / or incorporating nucleotide reagents, binding complementary nucleotides or binding multivalent molecules with complementary nucleotide units, incorporating complementary nucleotides, extending primers, and performing nucleic acid sequencing, which methods use any of the mutant polymerases described herein. The mutant polymerases are engineered to exhibit desired properties including exonuclease-minus activity and increased thermostability, improved uracil resistance, and / or reduced sequence-specific errors compared to the corresponding wild-type polymerase. Additionally, the mutant polymerases can be engineered to express a higher percentage of soluble expressed enzyme.

[0128] The present disclosure provides mutant polymerases that can be used to perform two-step nucleic acid sequencing methods. In some embodiments, the first step generally comprises: binding detectably labeled multivalent molecules to a composite polymerase to form a multivalent composite polymerase under conditions suitable for inhibiting incorporation of nucleotide units; and detecting the multivalent composite polymerase. The first step can be performed using a trapping polymerase. In some embodiments, the second step generally comprises polymerase-catalyzed incorporation of nucleotides using a stepping polymerase.

[0129] The present disclosure provides mutant polymerases that can be used to perform trapping or stepping events for nucleic acid sequencing. Some of the mutant polymerases can be used for both trapping and stepping events.

[0130] The present disclosure provides mutant polymerases that can be used to trap multivalent molecules, including composite mutant polymerases that bind to multivalent molecules with complementary nucleotide units (e.g., exemplary multivalent molecules are shown in Figures 2-5). In some embodiments, the multivalent molecule includes a central core that binds to multiple polymer arms, each with a nucleotide unit at the end of the arm. The multivalent molecule can be labeled with a detectable reporter moiety. The composite mutant polymerase includes a mutant polymerase bound to a template / primer duplex. The mutant polymerase is engineered to exhibit reduced sequence-specific errors that occur after specific motif sequences in the primer strand and / or template strand. The trapping polymerase sequence-specific errors can be characterized by a substantial loss of signal intensity that leads to a base miscall (e.g., base substitution) or no call at a particular sequencing cycle. The signal often recovers in the next cycle. The motif sequence that leads to a miscall is specific to a given polymerase and can occur on the template strand in either the forward or reverse sequencing direction.

[0131] The present disclosure provides mutant polymerases that can be used to bind complementary nucleotides (e.g., unconjugated nucleotides) and incorporate nucleotides into the 3' end of a primer, referred to as stepping events. The mutant polymerases are engineered to exhibit reduced sequence-specific errors, characterized by a substantial loss of nucleotide incorporation that occurs after a specific motif sequence in the primer strand and / or template strand. The sequence-specific errors of stepping enzymes can be characterized by extensive phasing after the sequence motif. The motif sequence that leads to the phasing is specific to a given polymerase and can occur on the template strand in either the forward or reverse sequencing direction.

[0132] Without wishing to be bound by theory, it is hypothesized that mutant polymerases that exhibit trapping sequence-specific errors at certain sequence motifs during sequencing switch from nucleotide incorporation conformation to editing conformation.The editing conformation blocks the binding of complementary nucleotide units from multivalent molecules during trapping events, which leads to a decrease in signal intensity.Designing polymerases that carry one or more mutation sites that reduce the switch conformation from nucleotide incorporation to editing can reduce trapping sequence-specific errors.

[0133] In some embodiments, the mutant polymerases comprise polypeptides or fragments thereof derived from directed evolution of recently identified novel B-family and A-family polymerases, where the mutant polymerases exhibit improved specificity while maintaining high discrimination for correct Watson-Crick base pairing.

[0134] The present disclosure relates to RLF89458.1 (e.g., Thermococci archaeon, isolate B13_G1) (SEQ ID NO: 1), RLF78286.1 (e.g., Thermococci archaeon, isolate B89_G9) (SEQ ID NO: 2), NOZ58130.1 (e.g., Euryarchaeota archaeon, isolate M_BaxBin.100) (SEQ ID NO: 1316), RMF90817.1 (e.g., Euryarchaeota archaeon, isolate J060) (SEQ ID NO: 2215), MBC7218772.1 (e.g., Hadesarchaea archaeon, isolate MAG-18) (SEQ ID NO: 2367), WP175059460.1 (e.g., Thermococcus sp.2319x1) (SEQ ID NO: 2393), KUO42443.1 (e.g., Candidatus Hadarchaem, yellowstonense, isolate YNP_45) (SEQ ID NO: 2408), and NOZ 77387.1 (e.g., Euryarchaeota archaeon, isolate M_MaxBin.027) (SEQ ID NO: 2436).

[0135] The polypeptides described herein include, but are not limited to, polypeptides having enzymatic activity, such as polymerase activity, and are often described as families. Often, 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 optionally mutated to generate 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 Taq polymerase, Pfu polymerase, and 9°N polymerase, or other members of the DNA polymerase family. Wild-type DNA polymerases may be obtained or derived from any number of origins, such as eukaryotic, prokaryotic, or viral origins, and in some embodiments, for purposes of this disclosure, may be obtained or derived from an archaeal origin. In some embodiments, a polymerase comprising any of the amino acid sequences of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, and 2511-2523 is a member of the DNA polymerase family.

[0136] The polymerases described herein can include mutations in the LYP motif, which can increase the thermostability of the polymerase to about 72°C, or about 73°C, or about 74°C, or about 75°C, or about 76°C, or about 77°C, or about 78°C, or about 79°C, or about 80°C, or higher. Many of the engineered polymerases described herein exhibit nucleotide binding and incorporation activity at temperatures ranging from about 25-50°C, or about 45-75°C, or about 65-80°C. Thus, these engineered polymerases are thermostable at moderately high temperature ranges (e.g., moderate heat polymerases). The engineered polymerases described herein are suitable for performing nucleotide binding, nucleotide unit binding, nucleotide incorporation, and / or nucleic acid sequencing reactions at temperatures ranging from about 25-50°C, or about 45-75°C, or about 65-80°C, or higher. In some embodiments, the mutant polymerases exhibit increased thermostability of about 2-4°C, or about 4-6°C, or about 6-8°C, or about 8-10°C.

[0137] In contrast, DNA polymerases that exhibit significantly higher thermostability 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 may be suitable for use in PCR reactions in which the typical cycling step is performed at temperatures above 90-95°C or higher, but may not be suitable for use in nucleotide binding, nucleotide incorporation, and / or nucleic acid sequencing reactions that are performed at lower temperature ranges. DNA polymerases from Geobacillus stearothermophilus (e.g., Bst DNA polymerase) are typically stable up to 65°C.

[0138] Polymerases include a variety of DNA polymerases, RNA polymerases, template-independent polymerases, reverse transcriptases, or other enzymes that can catalyze the incorporation of nucleotides. Archaeal polymerases are often derived from thermophilic organisms and therefore may represent a class of thermostable or thermotolerant enzymes. Thus, polypeptide backbones derived from archaeal polymerases provide desirable protein engineering targets to further enhance the incorporation of reversible terminator nucleotides for applications that may be improved by application of enzymes with increased thermostability or other increased resistance to degradation due to repeated exposure to high temperatures, changes in buffer conditions, etc.

[0139] The present disclosure provides compositions and methods comprising mutant polymerase enzymes that exhibit improved ability to bind complementary nucleotide units of multivalent molecules. Multivalent molecules generally include a central portion (e.g., core) bound to multiple arms, each arm binding to a nucleotide unit. Multivalent molecules include star, comb, bridge, bottle-brush, or dendrimer configurations (see, e.g., FIG. 2).

[0140] It has been surprisingly discovered that many of the engineered polymerases described herein exhibit increased rates of incorporation of nucleotide analogs compared to wild-type polymerases. Compared to wild-type polymerases, some of the engineered polymerases exhibit one or more desirable properties, including increased binding affinity to nucleotide analogs having a 3' chain terminator, 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 about 80°C, and reduced sequence-specific errors.

[0141] The present disclosure provides compositions and methods comprising mutant polypeptides related to polymerase enzymes that exhibit increased ability to bind and discriminate between nucleotide analogs and improved incorporation of nucleotide analogs compared to the corresponding wild-type polymerase. Nucleotide analogs include, for example, nucleotides that include a chain terminating group attached to the 2' or 3' sugar position. The chain terminating group can include an azide, azido, or azidomethyl group, or another type of chain terminating group. The engineered DNA polymerases exhibit increased incorporation rates of nucleotide analogs compared to the corresponding wild-type polymerases having the amino acid sequence backbone of any of RLF78286.1 (SEQ ID NO: 1), RLF78286 (SEQ ID NO: 2), NOZ58130 (SEQ ID NO: 1316), RMF90817 (SEQ ID NO: 2215), MBC7218772 (SEQ ID NO: 2367), WP175059460 (SEQ ID NO: 2393), KUO42443 (SEQ ID NO: 2408), or NOZ77387 (SEQ ID NO: 2436). The data presented in Tables 1, 4, 7, 9, and 12 provide a number of exemplary mutant polymerases that exhibit increased incorporation rates of nucleotide analogs compared to the corresponding wild-type polymerases. Many of these mutant polymerases contain mutations in the LYP motif. In some embodiments, the mutant polymerase exhibits about a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 500%, or 1000% increased rate of incorporation of nucleotide analogs compared to the corresponding wild-type enzyme or enzyme variant currently known in the art. Exemplary mutant polymerases that exhibit increased rates of incorporation of nucleotide analogs are listed in Tables 1, 4, 7, 9, and 12.

[0142] The present disclosure provides compositions and methods including mutant polymerase enzymes that can be used for sequencing uracil-containing nucleic acid template molecules. The mutant polymerases can exhibit uracil resistance with increased ability to incorporate dATP into the 3' end of a nucleic acid primer at a position opposite the uracil base in the nucleic acid template molecule. The mutant polymerases can also bind to adenine-bearing nucleotide units of multivalent molecules at a position opposite the uracil base in the nucleic acid template molecule. The mutant polymerases having the backbone sequence of RLF89458 or RLF78286 (e.g., SEQ ID NO: 1 or 2, respectively) that are uracil resistant can include a mutation at V93. Other uracil resistant mutant polymerases having the backbone sequence of NOZ58130 (SEQ ID NO:1316), RMF90817 (SEQ ID NO:2215), MBC7218772 (SEQ ID NO:2367), WP175059460 (SEQ ID NO:2393), KUO42443 (SEQ ID NO:2408), or NOZ77387 (SEQ ID NO:2436) may contain a positionally equivalent mutation to V93 in RLF89458 (SEQ ID NO:1). Figure 44 shows a sequence alignment of these various polymerases and their positionally equivalent amino acid residues.

[0143] Sites that confer a particular activity to a polypeptide may be conserved and may be located by aligning the amino acid sequences of various polymerases. For example, particular residues associated with polymerase activity (e.g., nucleotide incorporation) may be residues D405, D539, and / or D541 of a polymerase having a backbone sequence of RLF89458.1 (SEQ ID NO:1), or residues D405, D539, and / or D541 of a polymerase having a backbone sequence of RLF78286.1 (SEQ ID NO:2), or residues D436, D570, and / or D572 of a polymerase having a backbone sequence of NOZ58130 (SEQ ID NO:1316), or residue D417 of a polymerase having a backbone sequence of RMF90817 (SEQ ID NO:2215). , D551 and / or D553, residues D447, D585 and / or D587 of a polymerase having a backbone sequence of MBC7218772 (SEQ ID NO: 2367), residues D407, D543 and / or D545 of a polymerase having a backbone sequence of WP175059460 (SEQ ID NO: 2393), residues D444, D582 and / or D584 of a polymerase having a backbone sequence of KUO42443 (SEQ ID NO: 2408), or residues D428, D562, and / or D564 of a polymerase having a backbone sequence of NOZ77387 (SEQ ID NO: 2436).

[0144] One of skill in the art can find these and other functionally equivalent sites in other polymerases by reviewing the sequence alignment shown in Figure 44. Such sites are often found in analogous locations in other regions and domains, and polypeptides containing such domains are consistent with the methods and compositions described herein.

[0145] Mutations in the polymerases described herein variously include one or more changes to amino acid residues present in the polypeptide. Additions, substitutions, deletions, and / or truncations are all examples of mutations used to generate mutant polypeptides. In some embodiments, a substitution involves the exchange of one amino acid with an alternative amino acid, which differs from the original amino acid in terms of size, shape, conformation, and / or chemical structure. In some embodiments, the mutations are conservative or non-conservative. Conservative mutations involve the replacement of an amino acid with an amino acid having similar chemical properties. Additions often involve the insertion of one or more amino acids at the N-terminus, C-terminus, or internal positions of the polypeptide. In some cases, additions include fusion polypeptides, in which one or more additional polypeptides are attached to the polypeptide. In some embodiments, such additional polypeptides include domains with additional activities or sequences with additional functions (e.g., improving expression, aiding in purification, improving solubility, binding to solid supports, or other functions). In many cases, the polypeptides described herein include one or more non-amino acid groups. Fusion polypeptides optionally include amino acid or other chemical linkers connecting one or more proteins. Any number of mutations can be introduced into a polypeptide or portion of a polypeptide described herein, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more than 50 mutations.

[0146] In some embodiments, entire domains (portions of a polypeptide with a defined function) are added, deleted, or replaced with domains from other polypeptides. Exemplary domains include DNA / RNA binding domains, nucleotide binding domains, nuclease domains, subcellular localization domains such as nuclear localization domains, or other domains. In some embodiments, the disclosed methods and compositions include linking of domains that act 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: 2524) or polyhistidine tags). In some embodiments, one or more mutations are present at any position, for example, in the exonuclease domain, nucleic acid binding domain, nucleotide binding domain, and / or catalytic site. The polypeptides may contain at least one mutation and may be based on the wild-type backbone sequence of any of SEQ ID NOs: 1, 2, 1316, 2215, 2367, 2393, 2408, or 2436.

[0147] As used herein, the term "surrounding" an amino acid residue or sequence position has its normal meaning in the art to include and incorporate modifications such as substitutions, deletions, insertions, or post-translational modifications at 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., residues N-terminal or C-terminal to the named residue. In some contexts, as will be understood by those of skill in the art, more than 12 residues or sequence positions N- or C-terminal from the named residue can be considered to "surround" the named residue based on sequence or structural (i.e., three-dimensional) context.

[0148] It is understood that the substitutions or modifications of 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, and the like. The mutations, modifications, truncations, substitutions, and the like described herein may be made by any method as known in the art, particularly in the art of molecular biology and / or protein engineering. Such methods may include site-directed mutagenesis using mutagenesis and / or partially degenerate primers, in vitro gene assembly, gene editing (e.g., by CRISPR or related methods), and the like. The mutant or engineered proteins described herein may be further expressed, isolated, and / or purified by any such means as known in the art. Relevant methods are described in Green, M. and Sambrook, J., Molecular Cloning: A Laboratory Manual (4th Edition), which is incorporated by reference in its entirety, particularly with respect to its disclosure of methods for modifying, introducing and expressing recombinant, modified, altered and engineered genetic sequences, and methods for extracting, isolating and / or purifying engineered proteins.

[0149] The polypeptides disclosed herein have been shown to function as nucleotide polymerases that exhibit higher thermal stability and higher incorporation rates of 3'-O-azidomethyl derivatized nucleotides, increased uracil resistance, and / or improved binding of multivalent molecules to complementary nucleotide units compared to the corresponding wild-type enzymes. The polypeptides disclosed herein can be used for the extension of nucleic acids during replication or synthesis, or can capture / bind nucleotides to nucleotide addition sites, for example, by using non-incorporable or blocked nucleotides, or can be used under conditions in which required 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 binding applications. Disclosed herein are mutant polymerases that comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2435, 2437-2454, 2456-2501, or 2511-2523.

[0150] The present disclosure provides engineered DNA polymerases that include an amino acid sequence backbone of a Family B or Family A polymerase, typically including replicative polymerases that exhibit high fidelity. Examples of Family B-type polymerases include Family B archaeal DNA polymerases and Phi29 polymerase. In some embodiments, the engineered DNA polymerase includes a Family B archaeal DNA polymerase that may be selected from Thermococcus, Thermoplasmata, Pyrococcus, Methanococcus, Hadesarchaea, Euryarchaeota, or Candidatus. In some embodiments, the engineered DNA polymerase that is a Family B polymerase includes an amino acid sequence backbone from 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 comprises an amino acid sequence backbone of Geobacillus stearothermophilus (eg, Bst DNA polymerase).

[0151] Engineered DNA polymerases can be designed and prepared by introducing one or more mutations into the amino acid sequence of a 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 transferred from one type of polymerase to a positionally equivalent site (or functionally equivalent site) in a second type of polymerase. For example, any one or any combination of two or more mutation sites from a DNA polymerase comprising any one of SEQ ID NOs: 3 to 1315, 1317 to 2214, 2216 to 2366, 2368 to 2392, 2394 to 2407, 2409 to 2435, 2437 to 2454, 2456 to 2501, or 2511 to 2523, can be used in combination with ... abyssi polymerase (SEQ ID NO: 2509), RB69 polymerase (SEQ ID NO: 2510), or Phi29 (SEQ ID NO: 2455) at a positionally equivalent site (or functionally equivalent site). Exemplary sequence alignments are provided in Figures 44-51. Mutations include any one or any combination of two or more amino acid substitutions, insertions, deletions, and / or truncations.

[0152] Functional equivalents of residues include one or more amino acid residues that occupy a similar position in a sequence (e.g., sequence alignment) and / or the three-dimensional structure of an enzyme (e.g., a DNA polymerase) and perform substantially the same function as a known amino acid residue in a known enzyme. Functionally equivalent amino acid substitutions include one or more amino acid residues at a particular position of a basic polypeptide that have the same functional role in another polypeptide. Functionally equivalent amino acid substitutions include any one or any combination of conservative and / or non-conservative amino acid substitutions. Sequence alignments are provided in Figures 44-50, which list examples of amino acid residues at sites in DNA polymerases having the backbone sequence of any of SEQ ID NOs:1, 2, 1316, 2215, 2367, 2393, 2408, or 2436, as well as functionally equivalent amino acid sites in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N DNA polymerase (for SEQ ID NOs: 2372 or 2373), Pfu DNA polymerase (for SEQ ID NO: 2508), or Pyrococcus abysssi DNA polymerase (for SEQ ID NO: 2509).

[0153] A wild-type polypeptide sequence is often the starting point for protein or enzyme engineering to generate mutant polypeptides. In some embodiments, a mutant polypeptide differs from a wild-type polypeptide by at least one amino acid residue. Often, a mutant polypeptide differs from the closest wild-type polypeptide by at least one amino acid residue. In some embodiments, a mutant polypeptide differs from a wild-type polypeptide by at least two amino acid residues. In some embodiments, a mutant polypeptide differs from a 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 in the wild-type sequence. In some embodiments, the wild-type polypeptide sequence comprises the sequence of a naturally occurring polypeptide.

[0154] Amino acid substitution refers to replacing an amino acid residue at a selected position in 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. Amino acid substitutions can be conservative or non-conservative amino acid substitutions. In some embodiments, an amino acid residue at a selected position in a polypeptide can be replaced with an amino acid having a polar side chain. Examples of amino acids having polar side chains include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. In some embodiments, an amino acid residue at a selected position in a polypeptide can be replaced with an amino acid having a non-polar side chain. Examples of amino acids having non-polar side chains include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, prolific, tryptophan, tyrosine, and valine. In some embodiments, an amino acid residue at a selected position in a polypeptide can be replaced with an amino acid having a hydrophobic side chain. Examples of amino acids having hydrophobic side chains include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, and tryptophan. In some embodiments, amino acid residues at selected positions in a polypeptide can be replaced with amino acids having uncharged side chains. Examples of amino acids having uncharged side chains include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine. In some embodiments, amino acid residues at selected positions in a polypeptide can be replaced with amino acids having positively charged side chains. Examples of amino acids having positively charged side chains include arginine, histidine, and lysine. In some embodiments, amino acid residues at selected positions in a polypeptide can be replaced with amino acids having negatively charged side chains. Examples of amino acids having negatively charged side chains include aspartic acid and glutamic acid.

[0155] Exemplary polypeptide variants described herein are listed in Tables 1-15 (Figures 29-43, respectively).

[0156] In some embodiments, the polypeptide comprises the backbone sequence of RLF89458.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs:1-1315, and the polypeptide comprises at least one of the mutations listed in Tables 1, 2, and 3 (Figures 29, 30, and 31, respectively).

[0157] In some embodiments, the polypeptide comprises the backbone sequence of NOZ58130.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs: 1316-2214, and the polypeptide comprises at least one of the mutations listed in Tables 4-6 (Figures 32-33, respectively).

[0158] In some embodiments, the polypeptide comprises the backbone sequence of RMF90817 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs:2215-2366, and the polypeptide comprises at least one of the mutations listed in Tables 7 and 8 (Figures 35 and 36, respectively).

[0159] In some embodiments, the polypeptide comprises the backbone sequence of MBC7218772.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs: 23672392, and the polypeptide comprises at least one of the mutations listed in Tables 9 and 10 (Figures 37 and 38, respectively).

[0160] In some embodiments, the polypeptide comprises the backbone sequence of WP175059460.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs:2393-2407 and 2511-2523, and the polypeptide comprises at least one of the mutations listed in Table 11 (Figure 39).

[0161] In some embodiments, the polypeptide comprises the backbone sequence of KUO42443.1 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 any of SEQ ID NOs:2408-2435, and the polypeptide comprises at least one of the mutations listed in Tables 12 and 13 (Figures 40 and 41, respectively).

[0162] In some embodiments, the polypeptide comprises the backbone sequence of NOZ77387.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to any of SEQ ID NOs:2436-2454, and the polypeptide comprises at least one of the mutations listed in Table 14 (Figure 42).

[0163] In some embodiments, the polypeptide comprises a 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 greater than 99% sequence identity to any of SEQ ID NOs:2455-2501, and the polypeptide comprises at least one of the mutations listed in Table 15 (Figure 43).

[0164] Additional polypeptides contemplated and disclosed herein include a DNA polymerase domain having at least one mutation at a position similar to at least one of the positions of any of Tables 1-15, and optionally achieving a polypeptide having one or more of the mutations shown in Tables 1-15 at the homologous position.

[0165] Segments or portions of larger polypeptides are further described herein. Optionally, the segments have catalytic activity, such as nucleotide incorporation and nucleic acid extension activity, particularly in the context of a reverse transcriptase domain or polymerase domain as described herein. Described herein are polypeptides that include any full length or segment from any one of SEQ ID NOs: 1-2501 and at least one additional residue (e.g., +1 residue) at the N-terminus or C-terminus. 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.

[0166] Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal aspartic acid, an adjacent C-terminal arginine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal glutamine, an adjacent C-terminal histidine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal valine, an adjacent C-terminal cysteine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal cysteine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal cysteine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal aspartic acid, an adjacent C-terminal leucine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. For example, polypeptides that include additional residues, such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal aspartic acid, an adjacent C-terminal arginine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501, are described herein.Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal threonine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal asparagine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. Described herein are polypeptides that include additional residues such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal asparagine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501. For example, polypeptides that include additional residues, such as any one of SEQ ID NOs: 1-2501 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal serine, or a combination thereof, or a residue identified by alignment of any one of SEQ ID NOs: 1-2501, are described herein.

[0167] Engineered polymerases containing RLF89458.1 or RLF78286.1 backbone sequences The disclosure provides one or more mutant polymerases that comprise the backbone sequence of RLF89458.1 or RLF78286.1 and have 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% sequence identity to any of SEQ ID NO:11315 (Tables 1-3 and Figures 11-12 and Figures 29-31). The amino acid sequences of RLF89458.1 and RLF78286.1 differ by an amino acid substitution at position 235, where RLF78286.1 contains D235E.

[0168] In some embodiments, the mutant polymerase has a backbone sequence of RLF89458.1 (e.g., SEQ ID NO:1) or RLF78286.1 (SEQ ID NO:2) and comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D141 and / or E143. In some embodiments, the mutant polymerase comprises a mutation D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, or D141S. In some embodiments, the mutant polymerase comprises a mutation E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, or E143S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D141 and E143 sites.

[0169] In some embodiments, the mutant polymerase has a backbone sequence of RLF89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L409, Y410, and P411. In some embodiments, the at least one mutation in the LYP motif can increase the rate of incorporation of a nucleotide analog. 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 in 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, Examples include 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, and FTA.

[0170] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 1-1315, and has an amino acid substitution mutation at position L409 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L416 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine.

[0171] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 1-1315, and has an amino acid substitution mutation at position Y410 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y410 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0172] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 1-1315, and the amino acid substitution mutation at position P411 comprises 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 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0173] The present disclosure relates to a method for the preparation of a polypeptide having the backbone sequence of RLF89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2), and including the steps of D4, D6, Y7, I8, E10, N11, G12, K13, P14, I16, R17, F19, K20, K21, E22, K23, G24, E25, F26, K27, I28, E29, 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, E 57, 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, H89, P 90, Q91, D92, V93, P94, A95, I96, R97, D98, A99, I100, R101, S102, H103, P10 4, A105, V106, R107, E108, I109, F110, E111, Y112, D113, I114, P115, F116, A 117, K118, R119, Y120, L121, I122, D123, K124, L126, V127, P128, M129, E13 0, G131, G132, E133, L135, K136, L137, L138, A139, F140, D141, I142, E143, T144, Y146, H147, Y180, A190, K192, R199, Q196, P203, V205, Y209, G211, N2 13, F214, F216, Y218, I219, C223, E224, G227, L228, F230, T231, I232, G233, R234, S237, E238, P239, K240, Q242, R243, M244, G245, D246, R247, A249, E2 51, L258, Y261, P262, V264, R265, T267, I268, R269, P271, T272, Y273, T274, L275, E276, A277, V278, V282, F283, K285, K286, K287, E288, K289, V290, A2 92, I295, E297, A298, K300, S301, L305, R307, V308, A309, Y311, M313, D315,<h2 style=";text-align:left;direction:ltr">R317, Y320, E321, P328, M329, E332, L333, G338, Q339, D343, S345, S347, S348, G350, N351, L352, V353, W355, Y356, L357, R359, V360, Y362, N365, L367 P372、G373、E376、Q378、M381、Y385、G388、Y389、E394、G396、A402、Y403、L4 04、F406、R407、S408、L409、Y410、P411、S412、I413、V415、V419、P421、D422、 T423、L424、E427、C428、K429、A434、I436、R440、K443、G447、F448、I449、P4 50、S451、L453、E454、D455、V463、K464、R465、E475、K468、D472、I474、Y481、 A485, K487, I488, N491, S492, Y493, Y494, G495, Q497, Y499, S506, K507, E508, C509, E511, S512, V513, T514, G517, R518, H519, I521, T523, E529, K534 V535, A538, E539, D541, I547, P552, S557, K558, A559, K560, K561, L563, H565, E568, K569, G572, M573, E575, E577, L583, G585, F586, V588, T589, K592 I596、H601、T604、G606、V610、R611、R612、D613、E616、I617、K619、E620、T6 21、Q622、A623、K624、V625、L626、E627、V628、I629、L630、R631、E632、G633、 S634、I635、E636、K637、A638、A639、G640、I641、V642、V645、V646、E647、D6 48、L649、A650、N651、Y652、R653、V654、V656、E657、K658、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, I690K691, V692, K693, P694, T696, I698, S699, 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, Mutant polymerases are provided that comprise amino acid substitution mutations at any one or any combination of positions including: 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. In some embodiments, the amino acid substitution mutations comprise D141A and E143A.

[0174] The present disclosure relates to a method for the preparation of a polypeptide having a framework sequence of RLF89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2), comprising: D4R, D4A, D6S, D6R, Y7A, Y7F, I8S, E10V, E10D, E10K, N11S, G12S, G12D, G12E, K13E, P14Q, I16T, I16N, I16F, R17H, R17C, F19Y, F19S, F19I, K20M, K20E, K21E, E22G, E22V, E22K, K23E, K23M, G24S, E25K, F26L, K27M, I28F, I28N, I28T, E29V, E2 9D, D31V, R32C, R32S, N33S, F34S, F34I, E35K, E35G, E35D, P36L, P36A, P36G , P36V, P36M, P36T, P36K, Y37N, Y37F, I38T, I38N, Y39F, A40G, A40V, A40T, L 41P, L42P, L42Q, E43V, E43K, E43D, D44N, D44G, D45V, E46V, S47N, S47G, S47 R, I48V, E49G, E49K, D50V, D50G, D50N, I51K, I51F, K52I, K52R, K53E, I54T, I 54N, I54F, I54K, T55I, T55S, T55A, G56D, G56S, G56V, E57G, E57K, R58C, R58 L, R58H, H59L, H59Y, G60S, G60D, K61M, K62N, K62E, K62R, V63A, V63I, V63D, I65T, I65V, I65F, I65N, I66V, I66T, I66N, R67C, V68M, E69K, K70I, V71I, K7 2H, K72R, K72V, K73E, K74E, K74R, F75C, L76Q, G77D, G77S, E78K, E78G, E78N, E78S, P79S, I80F, I80N, I80K, I80S, I80R, E81D, E81V, V82A, W83R, K84R, L8 5V, L85Q, L85A, V86D, V86I, V86A, F87I, F87L, F87C, H89D, H89A, H89Y, P90L, P90S, P90D, P90R, P90A, P90G, P90V, P90M, P90T, P90K, Q91L, Q91H, Q91R, Q9 1W, Q91A, Q91K, Q91N, Q91P, D92N, D92V, V93A, V93M, V93E, V93F, V93Y, V93G,V93S、V93K、V93T、P94L、P94W、P94Y、P94Q、P94F、P94S、A95V、I96T、I96K、I96S、R97C、R97H、R97S、R97P、R97L、D98E、D98N、D98V、A99T、I100T、R101C、R101H、S102N、S102G、H103R、H103L、H103Q、H103Y、P104T、P104L、A105S、V106A、V106T、R107C、R107S、E108V、I109K、I109N、I109F、F110L、F110S、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、R119H、R119S、R119C、R119A、R119G、R119V、R119M、R119T、R119K、R119Y、Y120C、Y120N、L121M、I122V、I122F、I122N、I122D、D123G、D123E、D123N、D123V、K124N、K124E、K124R、L126F、L126P、L126Q、V127M、P128L、P128M、M129I、M129V、M129K、M129L、M129E、E130D、E130G、E130V、E130K、E130T、G131S、G132S、E133K、L135M、L135P、L135Q、K136E、K136R、K136L、L137F、L138P、A139E、F140Y、F140L、F140S、D141A、D141V、D141L、D141I、D141F、D141Y、D141N、D141T、D141S、I142V、I142F、I142A、E143A、E143V、E143L、E143I、E143F、E143Y、E143N、E143T、E143S、T144F、Y146C、Y146A、Y146E、Y146S、H147E、Y180F、A190V、K192L、R199H、Q196R、P203S、V205A、Y209A、Y209E、Y209W、G211S、N213E、N213W、N213Y、F214A、F214E、F214W、F214V、F216L、<h2 style=";text-align:left;direction:ltr">Y218H, I219V, C223V, C223E, C223S, C223L, C223M, C223A, C223P, C223K, C223N, C223D, E224V, G227S, L228P, F230L, T231I, I232F, G233D, R234C, S237 G、S237C、E238S、E238R、P239S、K240S、K240E、Q242N、Q242S、R243E、M244T、 M244K、G245D、G245S、G245R、G245A、G245N、G245K、D246R、D246L、D246E、D24 6V、R247E、R247D、R247S、R247H、A249G、A249V、E251S、E251R、E251A、L258I 、L258Q、Y261A、Y261P、Y261T、P262S、P262R、P262L、V264I、V264A、R265D、R 265I, T267A, T267F, T267M, T267V, T267W, T267Y, T267I, T267S, I268A, I268F, I268M, I268V, I268W, I268Y, R269L, R269K, R269S, R269T, R269V, R269N R269H, P271S, T272A, T272Y, T272V, T272S, T272L, T272E, T272C, T272R, T272W, T272N, T272F, T272H, T272K, Y273A, Y273W, T274E, T274W, T274S, L275 P、L275M、E276K、A277V、V278M、V282L、V282T、V282G、F283L、K285I、K286E、 K287R、E288G、E288K、K289E、K289Q、K289N、V290E、A292N、A292T、A292I、I29 5N, E297G, A298G, K300S, S301N, L305P, R307C, V308I, V308A, A309S, Y311A, Y311E, Y311W, Y311F, M313I, M313K, M313L, D315A, D315E, D315R, D315W, R 317C, Y320F, E321L, P328A, M329L, M329S, M329W, M329A, E332K, E332G, L333A, L333V, L333I, G338D, Q339N, D343E, D343N, D343R, D343A, S345C, S345RS347N、S347T、S347R、S348C、G350S、N351S、N351Q、L352M、V353Q、V353E、W355R、W355F、Y356N、Y356C、Y356L、L357P、R359H、V360A、V360D、Y362I、Y362E、N365S、L367P、P372S、P372M、G373S、E376K、Q378R、M381I、M381R、Y385R、Y385S、G388S、G388R、Y389R、Y389S、E394G、G396S、A402T、Y403H、Y403L、L404Q、F406Y、F406R、F406I、R407N、R407K、S408A、S408G、L409S、L409F、L409A、L409Y、L409I、L409V、L409T、L409N、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、V419I、P421S、D422V、T423I、T423L、L424Q、E427G、C428Y、K429R、A434V、A434D、A434P、I436T、I436F、R440H、K443R、G447D、F448I、F448L、I449N、I449F、P450L、S451N、L453Q、E454D、E454N、E454T、D455N、V463M、K464C、R465C、R465T、E475C、K468R、D472V、D472E、I474C、I474F、I474V、Y481C、Y481A、Y481F Y481T、Y481V、Y481W、A485S、A485T、A485L、A485V、A485G、A485R、K487M、K487R、K487N、I488A、I488V、I488S、I488T、I488M、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、Y499F、S506C、S506R、S506A、S506L、S506T、K507L、K507E、K507S、E508Q、E508C、C509V、C509Y、C509S、C509M、C509A、C509N、C509D、C509H、C509Q、E511K、E511S、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、I521N、I521T、I521E、I521H、T523I、T523A、E529N、K534N、K534S、K534R、V535N、V535K、V535S、V535R、A538V、D539A、D539G、D539E、D539V、D539L、D539S、D541A、D541G、D541E、I547F、I547T、I547P、P552L、S557C、K558A、A559K、K561N、L653M、H565Y、E568K、K569E、G572S、M573I、E575K、E577D、L583P、G585D、G585A、F586I、V588E、V588T、T589K、K592Q、I596T、H601R、H601I、T604S、G606S、V610D、V610A、V610K、V610S、V610T、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、V625F、L626I、E627K、V628L、V628I、I629F、I629C、L630Q、L630M、R631H、R631C、E632G、E632C、G633S、G633D、S634C、I635V、I635N、I635T、E636G、E636K、K637M、A638E、A638V、A638T、A639T、G640D、I641F、I641V、I641A,V642I、V642A、V645E、V645I、 、V645M、V646A、V646D、E647G、E647D、E647K、D648V、D648C、D648L、D648G、L649Q、A650E、A650V、A650T、A650N、N651S、Y652H、Y652C、Y652M、Y652L、Y652F、R653C、R653H、R653Y、V654M、V656I、E657V、K658R、K658E、K658I、K658L、H662V、E663K、E663R、E663S、E663M、E663Q、E663V、Q664A、Q664L、I665V、I665F、I665P、T666A、R667E、E668G、E668K、E668M、E668A、E668P、E668S、E668R、E688N、E688D、K670E、K670I、K670R、K670S、D671G、D671R、D671Y、D671S、D671A、D671K、D671N、Y672F、K673I、K673Y、K673R、K673S、K673E、A674T、A674V、A674S、T675S、T675I、T675A、G676S、P677L、H678R、H678K、H678Q、V679S、V679M,A680V, ​​A680I, A680D, I681T, A682T, K683R, R684H, L685E, Q686R, Q686 C, Q686L, A687C, A687T, A687S, R688S, G689S, G689D, I690V, I690F, K69 1R, K691V, V692I, K693M, K693V, P694R, T696S, T696I, I698K, S699I, S6 99G, V702A, V702I, L703P, K704E, K704I, K704N, G705D, S706N, S706C, S 706G, K707I, K707G, K707N, K708M, K708R, I709F, I709V, I709L, D711G, R712C, V713I, I714F, L715P, L715Q, F716L, D717N, E718K, E718V, D720V , D720Y, D720E, S721N, S721C, S721G, S722G, R723H, K725E, K725L, K725 R, Y726F, P728S, P728L, Y730H, Y731H, I732T, I732F, I732N, H733R, N73 4Y, N734R, N734P, N734D, N734K, N734T, Q735H, Q735R, V736A, P738L, A 739V, V740I, L741A, L741Q, R742K, R742L, R742C, I743V, I743E, L744A, E745V, E745F, A746V, A746G, F747L, F747Y, G748V, G748K, Y749F, Y749E , K750N, E751K, E751D, E751M, K752E, K752L, D753V, D753E, D753G, L754 and / or M770N.

[0175] In some embodiments, the mutant polymerase has a backbone sequence of RLF89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and includes amino acid deletions at any one or any combination of positions including R58 (deletion), V93 (deletion), and / or E755 (deletion).

[0176] In some embodiments, the mutant polymerase has a backbone sequence of RLFRLF89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and includes a truncation at an amino acid position including K464 (truncated), R465 (truncated), E475 (truncated), Y481 (truncated), E616 (truncated), E620 (truncated), E755 (truncated), Y756 (truncated), Q757 (truncated), R758 (truncated), M759 (truncated), T762 (truncated), W767 (truncated), or M770 (truncated). The truncated polymerase can exhibit increased thermostability compared to a non-truncated polymerase having the same backbone sequence. In Tables 1-15, truncations are indicated with a "^".

[0177] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position Y7. In some embodiments, the amino acid substitution at position Y7 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0178] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position V93. In some embodiments, the amino acid substitution at position V93 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0179] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position Y261. In some embodiments, the amino acid substitution at position Y261 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0180] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position T267. In some embodiments, the amino acid substitution at position T267 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0181] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position I268. In some embodiments, the amino acid substitution at position I268 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0182] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position A485. In some embodiments, the amino acid substitution at position A485 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0183] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position T514. In some embodiments, the amino acid substitution at position T514 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0184] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1-1315 and has an amino acid substitution mutation at position D671. In some embodiments, the amino acid substitution at position D671 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0185] Engineered polymerase containing the NOZ58130.1 scaffold sequence The disclosure provides one or more mutant polymerases comprising the backbone sequence of NOZ58130.1 and having 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% sequence identity to any of SEQ ID NOs:1316-2214 (Tables 4-6, and Figures 13 and 32-34).

[0186] In some embodiments, the mutant polymerase has a backbone sequence of NOZ58130.1 (e.g., any of SEQ ID NOs: 1316-2214) and includes at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase includes at least one amino acid substitution at positions D168 and / or E170. In some embodiments, the mutant polymerase includes a mutation D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168T, or D168S. In some embodiments, the mutant polymerase includes a mutation E170A, E170V, E170L, E170I, E170F, E170Y, E170N, E170T, or E170S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D168 and E170 sites.

[0187] In some embodiments, the mutant polymerase has a backbone sequence of NOZ58130.1 (e.g., any of SEQ ID NOs: 1316-2214) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L440, Y441, and P442. In some embodiments, the at least one mutation in the LYP motif can increase the rate of incorporation of a nucleotide analog. 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 in 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, Examples include LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, and SYG.

[0188] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 1316-2214, and has an amino acid substitution mutation at position L440 that comprises a nonpolar 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.

[0189] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 1316-2214, and has an amino acid substitution mutation at position Y441 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y441 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0190] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 1316-2214, and the amino acid substitution mutation at position P442 comprises 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 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0191] The present disclosure relates to a polypeptide having a backbone sequence of NOZ58130.1 (e.g., SEQ ID NO: 1316) and comprising: Y14, E18, F26, G29, F34, V35, V36, F41, S42, P43, F45, 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, D111, I113, E115, H116, D117, I118, V121, R122, R123, I126, P132, L133, W135, R150, E153, E157, E158 , R163, V164, A165, D168, I169, E170, V171, L253, G275, A276, L277, V288 , L290, Y291, P292, V298, L313, K319, D321, F326, T327, D330, E331, L338 , A352, C362, A376, M378, V384, L387, T393, E407, Y408, A409, R413, R422 , V434, D436, F437, S439, L440, Y441, P442, S443, I444, I445, V446, T45 4, A465, F479, I480, R496, F511, A515, S522, F523, Y524, M527, R537, E53 8, C539, E541, V543, A544, A547, M549, I551, M555, E559, E565, V566, D5 70, D572, I578, L585, A586, Q587, E595, V615, T616, R619, K628, K650, I6 In some embodiments, the mutant polymerases include amino acid substitution mutations at any one or any combination of positions including: 55, A665, E674, R675, R677, D685, T690, S698, S701, E703, V707, E718, M723, K734, G735, S737, Q738, D752, D758, N759, R767, I772, Y774, L779, K780, E781, G782, I783, T784, Q785, T786, S787, L788, S789, R790, W791, and / or F792. In some embodiments, the amino acid substitution mutations include D168A and E170A.

[0192] In some embodiments, the mutant polymerase has a backbone sequence of NOZ58130.1 (e.g., SEQ ID NO: 1316) and is selected from the group consisting of Y14F, Y14D, Y14I, Y14N, E18S, E18N, F26Y, F26S, F26I, G29E, G29K, V35M, V35K, V36F, V36N, V36T, V36I, F41S, F41I, S42K, S42G, S42D, S42E, P43L, P43A, P43G, P43V, P43M, P43T, P43K, F45T, F45N, F45I, P49V, P49K, P49D, P49E, R55G, R 55K, R55E, L61I, L61S, L61A, L61T, A62D, A62S, A62V, A62G, S63G, S63K, S63 E, A65L, A65Y, A65H, E67M, E67K, I69A, I69D, I69V, K71T, K71V, K71F, K71N, K71I, V72T, V72N, V72I, E76Q, E76N, K77E, T82K, T82G, T82N, T82S, T82E, P8 3R, R84N, R84K, R84S, V85R, T90D, T90I, T90A, T90V, V91I, V91L, V91C, V91F, H93D, H93A, H93Y, P94L, P94S, P94D, P94R, P94A, P94G, P94V, P94M, P94T, P9 4K, Q95L, Q95H, Q95R, Q95W, Q95A, Q95K, Q95N, Q95P, D96N, D96V, V97S, V97A , V97F, V97Y, P98L, P98W, P98Y, P98Q, P98F, P98S, R99V, R99A, I100T, I100K , 100S, R101C, R101H, R101S, R101P, R101L, E102N, E102V, E102D, R103T, R10 3A, R105C, R105H, D111C, D111S, D111R, I113K, I113N, I113F, E115V, E115G , H116C, H116Y, D117G, D117Y, I118T, I118A, I118G, I118V, I118M, I118T, I 118K, V121T, V121K, V121A, R122S, R122M, R122K, R123H, R123S, R123C, R12 3A, R123G, R123V, R123M, R123T, R123K, R123Y, I126V, I126F, I126N, I126D,P132L、P132M、L133I、L133V、L133K、L133L、L133E、L133M、W135S、W135L、W135R、R150A、R150V、R150L、R150K、R150F、E153A、E153V、E153L、E153K、E153R、E153F、E157A、E157V、E157L、E157K、E157R、E157F、E157D、E157G、E157T、E158S、E158G、R163E、R163L、R163K、V164F、V164L、A165P、A165L、D168A、D168V、D168L、D168I、D168F、D168Y、D168N、D168T、D168S、I169V、I169F、I169A、E170A、E170V、E170L、E170I、E170F、E170Y、E170N、E170T、E170S、V171F、V171T、L253V、L253E、L253C、G275N、G275K、G275S、G275R、A276M、A276N、A276Q、L277R、L277M、V288F、L290I、Y291A、Y291P、P292R、V298I、L313M、K319V、K319R、D321F、F326N、F326T、F326A、T327Q、D330N、D330E、E331N、L338E、A352L、A352E、A352D、A352Q、C362A、C362L、C362I、C362S、C362F、C362Y、C362V、C362P、C362K、C362N、C362D、A376C、A376R、A376S、M378R、M378T、M378A、V384Q、V384E、L387N、L387C、L387Y、E407R、Y408R、A409R、A409Q、R413Q、R413T、R422V、R422T、R422D、V434H、V434L、V434Y、F437Y、F437R、F437I、S439A、S439G、L440、L440Y、L440F、L440S、L440A、Y441、Y441A、Y441G、Y441T、P442、P442G、P442A、S443R、S443N、S443A、S443G、I444F、I445L、I445F、V446M、V446K、V446R、V446N、V446T、T454I、T454L、A465V、A465D、A465P、F479I、F479L、I480F、I480Y、R496T、R496A、R496G、R496C、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、M527H、M527G、M527Q、R537K、R537E、R537G、R537S、R537L、R537S、E538Q、E538C、C539、C539A、C539V、C539L、C539S、C539Y、C539S、E541K、E541S、V543T、V543I、V543A、V543S、V543G、A544G、A544S、A544T、A547G、M549N、M549Y、M549H、I551N、I551T、I551E、I551H、I551L、I551V、I551A、M555Y、M555I、E559N、E559K、E559D、E565N、E565K、E565S、E565R、V566N、V566K、V566S、V566R、D570A、D570G、D570E、D570V、D570L、D570S、D572A、D572G、D572E、I578F、I578T、I578P、L585K、A586K、E595K、V615E、V615T、T616K、R619E、K628R、K628I、K628H、K650T、K650C、K650A、I655L、I655V、A655E、A655V、A655T、E674G、E674D、E674K、R675V、R675C、R675L、R675D、R677E、R677V、R677T、R677N、R677A、D685R、D685E、D685I、D685L、D685K、T690K、T690R、T690S、T690M、T690Q、T690V、T690E、S698D、S698K、S698R、S698G、S698Y、S698D、S701T、S701V、S701A、S701R、S701E、E703R、E703S、V707I、V707D、V707A、E718R、E718V、E718K、M723S、M723I、M723T、M723N、K734I、K734G、K734N、G735M、G735R、G735K, G735S, G735P, G735T, G735E, S737R, S737E, Q738D, Q738S, Q738E, D752Q, D752T, D758N, N759P, N75 9D, N759K, N759T, N759Y, N759R, R767E, I772L, I772Y, I772F, Y774F, Y774E, L779G, L779Q, L779D, L779K, The present invention further comprises an amino acid substitution mutation at any one or any combination of positions including: L779Y, L779E, K780C, K780F, K780I, K780R, K780Q, K780Y, E781L, E781H, E781S, E781M, E781Q, G782H, G782A, G782K, G782R, Q785L, T786N, S789G, W791R, and / or F792R.

[0193] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214, and has an amino acid substitution mutation at position Y14. In some embodiments, the amino acid substitution at position Y14 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0194] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position V97. In some embodiments, the amino acid substitution at position V97 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0195] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position R122. In some embodiments, the amino acid substitution at position R122 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0196] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position R150. In some embodiments, the amino acid substitution at position R150 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0197] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position C362. In some embodiments, the amino acid substitution at position C362 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0198] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position R496. In some embodiments, the amino acid substitution at position R496 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0199] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position A515. In some embodiments, the amino acid substitution at position A515 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0200] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position R537. In some embodiments, the amino acid substitution at position R537 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0201] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 1316-2214 and has an amino acid substitution mutation at position E559. In some embodiments, the amino acid substitution at position E559 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0202] In some embodiments, the mutant polymerase has the backbone sequence of NOZ58130.1 (eg, any of SEQ ID NOs: 1316-2214) and contains an amino acid deletion at any position, including D117 (the deletion).

[0203] In some embodiments, the mutant polymerase has a backbone sequence of NOZ58130.1 (e.g., any of SEQ ID NOs: 1316-2214) and includes a truncation at an amino acid position including M723 (truncated), G773 (truncated), Y774 (truncated), D777 (truncated), G782 (truncated), Q785 (truncated), R790 (truncated), or F792 (truncated). The truncated polymerase can exhibit increased thermostability compared to a non-truncated polymerase having the same backbone sequence. In Tables 1-15, truncations are indicated with a "^".

[0204] Engineered polymerases containing the RMF90817.1 scaffold sequence The disclosure provides one or more mutant polymerases that comprise the backbone sequence of RMF90817.1 and have 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% sequence identity to any of SEQ ID NOs:2215-2366 (Tables 7-8, and Figures 14 and 35-36).

[0205] In some embodiments, the mutant polymerase has a backbone sequence of RMF90817.1 (e.g., SEQ ID NO: 2215) and comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D149 and / or E151. In some embodiments, the mutant polymerase comprises a mutation D149A, D149V, D149L, D149I, D149F, D149Y, D149N, D149T, or D149S. In some embodiments, the mutant polymerase comprises a mutation E151A, E151V, E151L, E151I, E151F, E151Y, E151N, E151T, or E151S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D149 and E151 sites.

[0206] In some embodiments, the mutant polymerase has a backbone sequence of RMF90817.1 (e.g., SEQ ID NO: 2215) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L421, Y422, and P423. In some embodiments, at least one mutation in the LYP motif can increase the rate of incorporation of a nucleotide analog. 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 in the LYP motif include AAA, AAG, AGA, AGP, FAA, FAG, FGA, FGP, SAA, SAG, SGA, SGP, YAA, YAG, YGA, YGP, FAP, SAP, YAP, AAP, AGV, AGG, AGI, AAV, AAI, FAI, FTA, FAV, FGG, FGV, LAG, LAI, LAP, LGG, LGI, LGV, SAI, SAV, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, LFP, IFP, VFP, Examples include LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, and SYG.

[0207] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2215-2366, and has an amino acid substitution mutation at position L421 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L421 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine.

[0208] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2215-2366, and has an amino acid substitution mutation at position Y422 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y422 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0209] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2215-2366, and the amino acid substitution mutation at position P423 comprises 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 P423 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0210] The present disclosure relates to a polypeptide having the backbone sequence of RMF90817.1 (e.g., SEQ ID NO: 2215) and including the following amino acids: 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, T1 52, A234, Y272, C307, R312, E333, A357, V365, L368, F374, L390, V415, D417, F418, S420, L421, Y422, P423, I425, V427, T435, P445, F459, A496, S503, F504, Y505, M508, K518, E519, C520, S523, V524, T525, M530, T5 32, D551, D553, V559, R566, A567, M568, R576, I596, T597, N609, Q631, V636, A646, N655, R656, K658, D666, Mutant polymerases are provided that comprise an amino acid substitution mutation at any one or any combination of positions including: T671, R679, N682, K688, E699, M704, G715, L716, N740, L753, Y755, K761, E762, E763, M764, V765, Q766, G767, S768, L769, Q770, R771, W772, and / or F773. In some embodiments, the amino acid substitution mutations comprise D149A and E151A.

[0211] In some embodiments, the mutant polymerase comprises the backbone sequence of RMFRMF90817.1 (e.g., SEQ ID NO: 2215) and is selected from the group consisting of Y11F, Y11A, D15S, F23Y, F23S, F23I, K25E, I28F, I28N, I28T, L29V, L29D, F34S, F34I, Q35K, Q35G, Q35D, P36L, P36A, P36G, P36V, P36M, P36T, P36K, F38T, F38N, H43V, H43K, H43D, E49G, E49K, G55I, G55S, G55A, G55T, A56D, A56S, A56T ... V, A56G, V57A, V57I, V57D, V57M, R62E, R62K, R67K, R67G, R67N, R67S, R67E, I75D, I75A, I75V, L76I, L76C, L76F, S77E, S77N, H78D, H78A, H78Y, P79L, P7 9S, P79D, P79R, P79A, P79G, P79V, P79M, P79T, P79K, S80L, S80H, S80R, S80W , S80A, S80K, S80N, S80P, S80Q, E81N, E81V, E81D, V82A, V82M, V82E, V82F, V8 2Y, V82G, V82S, V82K, V82T, P83L, P83W, P83Y, P83Q, P83F, P83S, K84V, K84A , I85T, I85K, I85S, R86C, R86H, R86S, R86P, R86L, E87N, E87V, E87D, E88T, E 88A, R90C, R90H, E96C, E96S, E96R, I98K, I98N, I98F, E100V, E100G, H101C, H101Y, D102G, D102Y, I103T, I103A, I103G, I103V, I103M, I103T, I103K, A10 6T, A106V, A106K, R108H, R108S, R108C, R108A, R108G, R108V, R108M, R108T , R108K, R108Y, I111V, I111F, I111N, I111D, P117L, P117M, L118I, L118V, L 118K, L118E, I118M, E138D, E138G, E138V, E138K, E138T, G139S, R144E, R14 4L, R144K, V145F, V145L, M146P, M146L, D149A, D149V, D149L, D149I, D149F,D149Y、D149N、D149T、D149S、I150V、I150F、I150A、E151A、E151V、E151L、E151I、E151F、E151Y、E151N、E151T、E151S、T152F、A234V、A234E、A234C、Y272A、Y272P、C307A、C307V、R312S、R312K、E333L、A357C、A357R、A357S、V365Q、V365E、L368N、L368C、L368Y、F374I、F374E、F374Y、L390R、L390Q、V415H、V415L、V415Y、F418Y、F418R、F418I、L421A、L421F、L421S、L421Y、L421I、L421V、L421T、L421N、Y422A、Y422G、Y422F、Y422M、Y422L、Y422D、Y422T、Y422I、Y422N、P423A、P423G、P423I、P423V、I425F、V427M、V427K、V427R、V427N、V427T、T435I、T435L、P445V、P445D、P445A、F459I、F459L、A496L、A496S、A496T、A496V、A496G、A496R、S503G、S503Y、S503D、S503K、S503T、S503N、S503E、F504T、F504S、F504I、F504Y、Y505A、Y505N、Y505G、M508H、M508G、M508Q、K518L、K518E、E519Q、E519C、C520A、C520V、C520Y、C520S、S523A、S523R、V524T、V524I、T525A、T525S、T525G、M530N、M530Y、M530H、T532N、T532E、T532H、D551A、D551G、D551E、D551V、D551L、D551S、D553A、D553G、D553E、V559F、V559T、V559P、V559I、R566A、R566K、A567K、M568N、R576K、R576E、I596E、I596T、I596V、T597K、N609R、N609I、N609H、Q631T、Q631C、Q631K、Q631A、V636L、V636I、A646E、A646V、A646T、N655G、N655D、N655K、N655E、R656V、R656C、R656L、R656D, K658E, K658V, K658T, K658N, K658A, D666R, D666E, D666I, D666L, D666K, T671K, T671R, T671S, T671M, T671Q, T671V, T671E, R679G, R679Y, R679D, N682T, N682V, N682S, N682A, K688V, K688I, K688D, K688A, E699R, E699V, E699K, M704S, M704I, M704T, G715I, G715N, G715K, L716M, L716R, L716K, N740P, N740D, N740K, N740T, N740Y, N740R, L753Y, L753F, Y755F, Y755E, K761G, K761Q, K761D, K761Y, K761E, E762L, E762H, E762S, E762M, E762Q, E763H, E763A, E763K, E763R, M764I, M764V, M764L, M764A, M764K, V765K, V765T, V765S, V765N, V765Q, V765A, V765I, V765L, and / or F773I.

[0212] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366, and has an amino acid substitution mutation at position Y11. In some embodiments, the amino acid substitution at position Y11 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0213] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366 and has an amino acid substitution mutation at position Y272. In some embodiments, the amino acid substitution at position Y272 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0214] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366 and has an amino acid substitution mutation at position C307. In some embodiments, the amino acid substitution at position C307 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0215] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366 and has an amino acid substitution mutation at position A496. In some embodiments, the amino acid substitution at position A496 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0216] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366 and has an amino acid substitution mutation at position C520. In some embodiments, the amino acid substitution at position C520 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0217] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366 and has an amino acid substitution mutation at position S523. In some embodiments, the amino acid substitution at position S523 comprises any of the 20 naturally occurring amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, T, or Q) or an unnatural amino acid known to one of skill in the art.

[0218] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2215-2366 and has an amino acid substitution mutation at position T525. In some embodiments, the amino acid substitution at position T525 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0219] Engineered polymerases containing the MBC7218772.1 scaffold sequence The disclosure provides one or more mutant polymerases that comprise the backbone sequence of MBC7218772.1 and have 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% sequence identity to any of SEQ ID NOs:2367-2392 (Tables 9-10 and Figures 15 and 37-38).

[0220] In some embodiments, the mutant polymerase has a backbone sequence of MBC7218772.1 (e.g., SEQ ID NO: 2367) and comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D173 and / or E175. In some embodiments, the mutant polymerase comprises a mutation D173A, D173V, D173L, D173I, D173F, D173Y, D173N, D173T, or D173S. In some embodiments, the mutant polymerase comprises a mutation E175A, E175V, E175L, E175I, E175F, E175Y, E175N, E175T, or E175S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D173 and E175 sites.

[0221] In some embodiments, the mutant polymerase has a backbone sequence of MBC7218772.1 (e.g., SEQ ID NO: 2367) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L451, Y452, and P453. In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of a nucleotide analog. 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 in the LYP motif include AAA, AAG, AGA, AGP, FAA, FAG, FGA, FGP, SAA, SAG, SGA, SGP, YAA, YAG, YGA, YGP, FAP, SAP, YAP, AAP, LAP, AGV, AGG, AGI, AAV, AAI, FAI, FTA, FAV, FGG, FGV, LAG, LAI, LGG, LGI, LGV, SAI, SAV, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, LFP, IFP, VFP, Examples include LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, and SYG.

[0222] In some embodiments, the polymerase comprises an amino acid sequence of any of SEQ ID NOs: 2367-2392, and has an amino acid substitution mutation at position L451 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L451 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine.

[0223] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2367-2392, and has an amino acid substitution mutation at position Y452 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y452 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0224] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2367-2392, and the amino acid substitution mutation at position P453 comprises 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 P453 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0225] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2367-2392 and has an amino acid substitution mutation at position I10. In some embodiments, the amino acid substitution at position I10 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0226] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2367-2392 and has an amino acid substitution mutation at position C468. In some embodiments, the amino acid substitution at position C468 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0227] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2367-2392 and has an amino acid substitution mutation at position T560. In some embodiments, the amino acid substitution at position T560 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0228] WP175059460.1 Engineered polymerases containing scaffold sequences The disclosure provides one or more mutant polymerases comprising the backbone sequence of WP175059460.1 and having 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% sequence identity to any of SEQ ID NOs:2393-2407 and 2511-2523 (Table 11 and Figures 16 and 39).

[0229] In some embodiments, the mutant polymerase has a backbone sequence of WP175059460.1 (e.g., SEQ ID NO: 2393) and comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D141 and / or E143. In some embodiments, the mutant polymerase comprises a mutation D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, or D141S. In some embodiments, the mutant polymerase comprises a mutation E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, or E143S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D141 and E143 sites.

[0230] In some embodiments, the mutant polymerase has a backbone sequence of WP175059460.1 (e.g., SEQ ID NO: 2393) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L451, Y452, and P453. In some embodiments, at least one mutation in the LYP motif can increase the rate of incorporation of a nucleotide analog. 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 in the LYP motif include AAA, AAG, AGA, AGP, FAA, FAG, FGA, FGP, SAA, SAG, SGA, SGP, YAA, YAG, YGA, YGP, FAP, SAP, YAP, AAP, LAP, AGV, AGG, AGI, AAV, AAI, FAI, FTA, FAV, FGG, FGV, LAG, LAI, LGG, LGI, LGV, SAI, SAV, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, LFP, IFP, VFP, Examples include LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, and SYG.

[0231] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position L451 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L451 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine.

[0232] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position Y452 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y452 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0233] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and the amino acid substitution mutation at position P453 comprises 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 P453 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0234] The present disclosure relates to a polypeptide having the framework sequence of WP175059460.1 (e.g., SEQ ID NO: 2393) and including the following amino acids: Y7, D11, I51, K61, V93, A117, M129, D141, I142, E143, T144, A223, E302, E323, D407, F408, S410, L411, Y412, P413, R487, A488, S495, Y4 In some embodiments, the mutant polymerases include amino acid substitution mutations at one or more positions, including: 96, K510, T517, I524, K562, A563, R564, S572, T593, R605, K652, D675, K695, T700, R712, R759, Y760, Q761, S762, S763, K764, Q765, and / or T766. In some embodiments, the amino acid substitution mutations include D141A and E143A.

[0235] In some embodiments, the mutant polymerase has a backbone sequence of WP175059460.1 (e.g., SEQ ID NO: 2393) and is selected from the group consisting of Y7F, Y7A, Y7V, Y7I, Y7L, Y7M, Y7W, D11K, D11G, D11E, D11N, I51R, I51K, I51H, K61M, K61R, V93Q, V93A, V93I, V93L, V93M, V93F, V93Y, V93W, V93S, V93T, V93Y, V93T, V93S, V93T ... , V93T, V93N, V93R, V93E, V93K, V93D, A117V, M129A, D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141 T, D141S, I142A, I142G, I142M, I142V, I142L, E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E14 3S, T144A, T144K, T144R, A223S, A223C, E302N, E302D, E302K, E323Q, S410A, S410G, S410M, S410V, S410L, S4 10I, L411A, L411S, L411Q, L411H, L411F, L411Y, Y412A, Y412G, P413G, P413V, P413A, P413I, P413S, P413T, P 413L, P413I, A488V, A488S, A488L, S495G, Y496I, Y496V, Y496M, K510I, K510L, K510V, K510R, T517S, T517N, T517Q, T517A, I524H, I524T, I524V, I524S, I524G, I524A, I524L, I524F, K562D, K562N, A563V, R564G, R564E and / or Q765W.

[0236] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position I228. In some embodiments, the amino acid substitution at position I228 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0237] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position G441. In some embodiments, the amino acid substitution at position G441 comprises any of the 20 naturally occurring amino acids (i.e., W, I, M, P, F, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to one of skill in the art.

[0238] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position K443. In some embodiments, the amino acid substitution at position K443 comprises any of the 20 naturally occurring amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to one of skill in the art.

[0239] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position A488. In some embodiments, the amino acid substitution at position A488 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0240] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position K510. In some embodiments, the amino acid substitution at position K510 comprises any of the 20 naturally occurring amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to one of skill in the art.

[0241] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position I524. In some embodiments, the amino acid substitution at position I524 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0242] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position G710. In some embodiments, the amino acid substitution at position G710 comprises any of the 20 naturally occurring amino acids (i.e., W, I, M, P, F, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to one of skill in the art.

[0243] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position R712. In some embodiments, the amino acid substitution at position R712 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0244] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2393-2407 and 2511-2523, and has an amino acid substitution mutation at position A770. In some embodiments, the amino acid substitution at position A770 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0245] Engineered polymerase containing the KUO42443.1 scaffold sequence The disclosure provides one or more mutant polymerases that comprise the backbone sequence of KUO42443.1 and have 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% sequence identity to any of SEQ ID NOs:2408-2435 (Tables 12-13, and Figures 17, 40-41).

[0246] In some embodiments, the mutant polymerase has a backbone sequence of KUO42443.1 (e.g., SEQ ID NO: 2408) and comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D170 and / or E172. In some embodiments, the mutant polymerase comprises a mutation D170A, D170V, D170L, D170I, D170F, D170Y, D170N, D170T, or D170S. In some embodiments, the mutant polymerase comprises a mutation E172A, E172V, E172L, E172I, E172F, E172Y, E172N, E172T, or E172S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D170 and E172 sites.

[0247] In some embodiments, the mutant polymerase has a backbone sequence of KUO42443.1 (e.g., SEQ ID NO: 2408) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L448, Y449, and P450. In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of a nucleotide analog. 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 in the LYP motif include AAA, SAG, SGP, SAP, LAP, AAG, AGA, AGP, FAA, FAG, FGA, FGP, SAA, SGA, YAG, YGA, YGP, FAP, YAP, AAP, YAA, AGV, AGG, AGI, AAV, AAI, FAI, FTA, FAV, FGG, FGV, LAG, LAI, LGG, LGI, LGV, SAI, SAV, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, LFP, IFP, VFP, Examples include LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, and SYG.

[0248] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2408-2435, and has an amino acid substitution mutation at position L448 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L448 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine.

[0249] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2408-2435, and has an amino acid substitution mutation at position Y449 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y449 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0250] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2408-2435, and the amino acid substitution mutation at position P450 comprises 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 P450 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0251] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2408-2435 and has an amino acid substitution mutation at position Y7. In some embodiments, the amino acid substitution at position Y7 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0252] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2408-2435 and has an amino acid substitution mutation at position D170. In some embodiments, the amino acid substitution at position D170 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0253] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2408-2435 and has an amino acid substitution mutation at position E172. In some embodiments, the amino acid substitution at position E172 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0254] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2408-2435 and has an amino acid substitution mutation at position T557. In some embodiments, the amino acid substitution at position T557 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0255] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2408-2435 and has an amino acid substitution mutation at position S558. In some embodiments, the amino acid substitution at position S558 comprises any of the 20 naturally occurring amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, T, or Q) or an unnatural amino acid known to one of skill in the art.

[0256] Engineered polymerases containing the NOZ77387.1 scaffold sequence The disclosure provides one or more mutant polymerases comprising the backbone sequence of NOZ77387.1 and having 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% sequence identity to any of SEQ ID NOs:2436-2454 (Table 14 and Figures 18 and 42).

[0257] In some embodiments, the mutant polymerase has a backbone sequence of NOZ77387.1 (e.g., SEQ ID NO: 2436) and comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D161 and / or E163. In some embodiments, the mutant polymerase comprises a mutation D161A, D161V, D161L, D161I, D161F, D161Y, D161N, D161T, or D161S. In some embodiments, the mutant polymerase comprises a mutation E163A, E163V, E163L, E163I, E163F, E163Y, E163N, E163T, or E163S. In some embodiments, the mutant polymerase comprises any combination of mutations at the D161 and E163 sites.

[0258] In some embodiments, the mutant polymerase has a backbone sequence of NOZ77387.1 (e.g., SEQ ID NO: 2436) and includes at least one amino acid substitution mutation in the LYP motif, e.g., at positions L448, Y449, and P450. In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of a nucleotide analog. 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 in the LYP motif include AAA, SAP, FAG, FGP, YAG, YGP, FAP, YAP, SAG, SGP, LAP, AAG, AGA, AGP, FAA, FGA, SAA, SGA, YGA, AAP, YAA, AGV, AGG, AGI, AAV, AAI, FAI, FTA, FAV, FGG, FGV, LAG, LAI, LGG, LGI, LGV, SAI, SAV, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, LFP, IFP, VFP, Examples include LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, and SYG.

[0259] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2436-2454, and has an amino acid substitution mutation at position L432 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L432 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine.

[0260] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2436-2454, and has an amino acid substitution mutation at position Y433 that comprises a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y433 comprises a threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine.

[0261] In some embodiments, the polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 2436-2454, and the amino acid substitution mutation at position P434 comprises 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 P434 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline.

[0262] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2436-2454 and has an amino acid substitution mutation at position Y10. In some embodiments, the amino acid substitution at position Y10 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0263] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2436-2454 and has an amino acid substitution mutation at position C41. In some embodiments, the amino acid substitution at position C41 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0264] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2436-2454 and has an amino acid substitution mutation at position C531. In some embodiments, the amino acid substitution at position C531 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0265] In some embodiments, the polymerase comprises the amino acid sequence of any one of SEQ ID NOs: 2436-2454 and has an amino acid substitution mutation at position T536. In some embodiments, the amino acid substitution at position T536 comprises any of the 20 naturally occurring 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 an unnatural amino acid known to one of skill in the art.

[0266] Engineered polymerase containing the Phi29 backbone sequence The present disclosure provides mutant polymerases having the backbone sequence of Phi29. An exemplary wild-type Phi29 backbone sequence includes SEQ ID NO: 2455 (e.g., FIG. 28). In some embodiments, the mutant Phi29 polymerase includes amino acid substitutions at sites that improve binding to nucleotide analogs carrying chain-terminating moieties. For example, the mutant Phi29 polymerase can be mutated at sites positionally equivalent to the LYP motif in RLF89458, such as positions L409, Y410, and P411 in RLF89458 (see, e.g., SEQ ID NOs: 1 and 2). The mutant Phi29 polymerase can be mutated at sites positionally equivalent to A485 in RLF89458 (see, e.g., SEQ ID NOs: 1 and 2). Positionally equivalent amino acid residues in Phi29 and RL89458 can be identified by comparing ribbon models based on the crystal structure of the ternary complex of mutant Phi29 polymerase complexed with a DNA template and a primer (see, e.g., Figures 52-59). Exemplary mutant polymerases include SEQ ID NOs: 2456-2501 (see, Table 15). In some embodiments, the mutant Phi29 polymerase comprises an amino acid substitution at L253 (e.g., any of SEQ ID NOs: 2472-2476), which may be positionally equivalent to L409 in RLF89458. In some embodiments, the mutant Phi29 polymerase comprises an amino acid substitution at Y254 (e.g., any of SEQ ID NOs: 2477-2479), which may be positionally equivalent to Y410 in RLF89458. In some embodiments, the mutant Phi29 polymerase comprises an amino acid substitution at L381 (eg, any of SEQ ID NOs: 2498-2501), which may be positionally equivalent to A485 in RLF89458.

[0267] In some embodiments, a mutant polymerase having a backbone sequence of Phi29 (e.g., SEQ ID NO: 2455, FIG. 28) comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase lacking the exo-minus mutation. In some embodiments, the mutant polymerase comprises a mutation D12A, D12S, D12N, D12Q, or D12K. In some embodiments, the mutant polymerase comprises a mutation E14A. In some embodiments, the mutant polymerase comprises a mutation D66A. In some embodiments, the mutant polymerase comprises a mutation Y165F or Y165C. In some embodiments, the mutant polymerase comprises a mutation D169A. In some embodiments, the mutant polymerase comprises any one or any combination of two or more mutations at sites D12, E14, D66, Y165, and / or D169.

[0268] In some embodiments, any of the mutant Phi29 polymerases can bind and catalyze the incorporation of a nucleotide analog having a nucleobase that is complementary to a base in a template molecule. The nucleotide analog can be 3'-O-azido, 3'-O-methyl-azido, 3'-deoxynucleotide, 2,3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-aminomethyl, 3'-ethyl, 3'butyl, 3'-tertbutyl, 3'-fluorenylmethyloxycarbonyl, 3'-amino ... Contains chain terminating moieties including tert-butyloxycarbonyl, 3'-O-alkynylhydroxylamino groups, 3'-phosphorothioate, 3'-O-benzyl, 3-acetal moieties, or derivatives thereof.

[0269] The disclosure provides one or more mutant polymerases that comprise a backbone sequence of Phi29 and have 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% sequence identity to any of SEQ ID NOs:2455-2501 (Table 15 and Figures 28 and 43).

[0270] Compositions Comprising Engineered Polymerases The present disclosure provides polymerases mutated at two or more positions to increase the thermostability of the enzyme, exhibit improved binding of nucleotide reagents and / or improved incorporation rate of nucleotide analogs, improved uracil tolerance and / or reduced sequence-specific sequencing errors, as compared to a wild-type polymerase comprising any of the amino acid sequences of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523. For example, the mutant polymerase exhibits increased thermostability in a temperature range of about 25-50° C., or about 45-75° C., or about 65-80° C. In another example, the mutant polymerase exhibits an increased incorporation rate of nucleotide analogs that include chain terminating moieties (e.g., blocking moieties) at the sugar 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 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523.

[0271] In some embodiments, the mutant polymerase comprises the backbone sequence of RLF89458.1 or RLF78286.1, and comprises the amino acid sequence of any of SEQ ID NOs:1-1315, and comprises amino acid substitutions capable of conferring exonuclease-minus activity, including any of D141A and E143A, D141V and E143A, D141L and E143A, D141I and E143A, D141F and E143A, or D141Y and E143A.

[0272] In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ58130 and comprises the amino acid sequence of any of SEQ ID NOs: 1316-2214, and comprises amino acid substitutions capable of conferring exonuclease-minus activity, including any of D168A and E170A, D168V and E170A, D168L and E170A, D168I and E170A, D168F and E170A, or D168Y and E170A.

[0273] In some embodiments, the mutant polymerase comprises the backbone sequence of RMF90817.1 and comprises the amino acid sequence of any of SEQ ID NOs:2215-2366, and comprises amino acid substitutions capable of conferring exonuclease-minus activity, including any of D149A and E151A, D149V and E151A, D149L and E151A, D149I and E151A, D149F and E151A, or D149Y and E151A.

[0274] In some embodiments, the mutant polymerase comprises the backbone sequence of MBC7218772.1 and comprises the amino acid sequence of any of SEQ ID NOs:2367-2392, and comprises amino acid substitutions capable of conferring exonuclease-minus activity, including any of D173A and E175A, D173V and E175A, D173L and E175A, D173I and E175A, D173F and E175A, or D173Y and E175A.

[0275] In some embodiments, the mutant polymerase comprises the backbone sequence of WP175059460.1 and comprises the amino acid sequence of any of SEQ ID NOs:2393-2407 and 2511-2523, and comprises amino acid substitutions capable of conferring exonuclease-minus activity, including any of D173A and E175A, D173V and E175A, D173L and E175A, D173I and E175A, D173F and E175A, or D173Y and E175A.

[0276] In some embodiments, the mutant polymerase comprises the backbone sequence of KUO42443.1 and comprises the amino acid sequence of any of SEQ ID NOs:2408-2435, and contains amino acid substitutions capable of conferring exonuclease-minus activity, including any of D170A and E172A, D170V and E172A, D170L and E172A, D170I and E172A, D170F and E172A, or D170Y and E172A.

[0277] In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ77387.1 and comprises the amino acid sequence of any of SEQ ID NOs:2436-2454, and comprises amino acid substitutions capable of conferring exonuclease-minus activity, including any of D161A and E163A, D161V and E163A, D161L and E163A, D161I and E163A, D161F and E163A, or D161Y and E163A.

[0278] The present disclosure relates to engineered archaeal family B polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509), and RB69 polymerase (SEQ ID NO: 2510). Provided are DNA or Family A polymerases, including D4, D6, Y7, I8, E10, N11, G12, K13, P14, I16, R17, F19, K20, K21, E22, K23, G24, E25, F26, K27, I28, E29, 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, G56, E57, R58, H59, G60, K61, K62, V63, I65, I66, R67, V68, E69, K70, V71, K7 2, K73, K74, F75, L76, G77, E78, P79, I80, E81, V82, W83, K84, L85, V86, F87, H89, P90, Q91, D92, V93, P94, A95, I 96, R97, D98, A99, I100, R101, S102, H103, P104, A105, V106, R107, E108, I109, F110, E111, Y112, D113, I114, P 115, F116, A117, K118, R119, Y120, L121, I122, D123, K124, L126, V127, P128, M129, E130, G131, G132, E133, L1 35, K136, L137, L138, A139, F140, D141, I142, E143, T144, Y146, H147, Y180, A190, K192, R199, Q196, P203, V20 5, Y209, G211, N213, F214, F216, Y218, I219, C223, E224, G227, L228, F230, T231, I232, G233, R234, S237, E238,<h2 style=";text-align:left;direction:ltr">P239, K240, Q242, R243, M244, G245, D246, R247, A249, E251, L258, Y261, P262, V264, R265, T267, I268, R269, P271, T272, Y273, T274, L275, E276, A277 V278, V282, F283, K285, K286, K287, E288, K289, V290, A292, I295, E297, A298, K300, S301, L305, R307, V308, Y311, M313, D315, R317, Y320, E321, P328 M329, E332, L333, G338, Q339, D343, S345, S347, S348, G350, N351, L352, V353, W355, Y356, L357, R359, V360, Y362, N365, L367, P372, G373, E376, Q378 M381、Y385、G388、Y389、E394、G396、A402、Y403、L404、F406、R407、S408、L4 09、Y410、P411、S412、I413、V415、V419、P421、D422、T423、L424、E427、C428、 K429、A434、I436、R440、K443、G447、F448、I449、P450、S451、L453、E454、D4 55、V463、K464、R465、E475、K468、D472、I474、Y481、A485、K487、I488、N491、 S492, Y493, Y494, G495, Q497, Y499, S506, K507, E508, C509, E511, S512, V513, T514, G517, R518, H519, I521, T523, E529, K534, V535, E539, D541, I547 P552, S557, K558, A559, K560, K561, H565, E568, K569, G572, M573, E575, E577, L583, G585, F586, V588, T589, K592, I596, H601, T604, G606, V610, R611 R612, D613, E616, I617, K619, E620, T621, Q622, A623, K624, L626, E627, V628, I629, L630, R631, E632, G633, S634, I635, E636, K637, A638, A639, G640I641, V642, V645, V646, E647, D648, L649, A650, N651, Y652, R653, V654, V656, E657, K658, H662, E663, Q6 64, 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, V702, L703, K 704, 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, E 745, A746, F747, G748, Y749, K750, E751, K752, D753, L754, E755, Y756, Q757, R758, M759, K760, Q761, T762 45, one of skill in the art can ascertain positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509). ,

[0279] The present disclosure relates to engineered archaeal family B polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509) and RB69 polymerase (SEQ ID NO: 2510). DNA or Family A polymerases are provided, including Y14, E18, F26, G29, F34, V35, V36, F41, S42, P43, F45, 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, R1 01, E102, R103, R105, D111, I113, E115, H116, D117, I118, V121, R122, R123, I126, P132, L133, W135, R150, E15 3, E157, E158, R163, V164, A165, D168, I169, E170, V171, L253, G275, A276, L277, V288, L290, Y291, P292, V298, L313, K319, D321, F326, T327, D330, E331, L338, A352, C362, A376, M378, V384, L387, T393, E407, Y408, A409, R 413, R422, V434, D436, F437, S439, L440, Y441, P442, S443, I444, I445, V446, T454, A465, F479, I480, R496, F5 11, A515, S522, F523, Y524, M527, R537, E538, C539, E541, V543, A544, A547, M549, I551, M555, E559, E565, V56 6, D570, D572, I578, L585, A586, Q587, E595, V615, T616, R619, K628, K650, I655, A665, E674, R675, R677, D685,The polymerase is mutated at one or more positions that are positionally equivalent (or functionally equivalent sites) to the amino acid substitutions at any one or any combination of positions of the polymerase having the backbone sequence of NOZ58130.1 (SEQ ID NOs: 1316-2214), including T690, S698, S701, E703, V707, E718, M723, K734, G735, S737, Q738, D752, D758, N759, R767, I772, Y774, L779, K780, E781, G782, I783, T784, Q785, T786, S787, L788, S789, R790, W791, and / or F792. From the sequence alignment shown in Figure 46, one of skill in the art can identify positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509).

[0280] The present disclosure relates to engineered archaeal family B polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509) and RB69 polymerase (SEQ ID NO: 2510). DNA or Family A polymerases are provided, including 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, A357, V365, L368, F374, L390, V415, D417, F418, S420, L 421, Y422, P423, I425, V427, T435, P445, F459, A496, S503, F504, Y505, M508, K518, E519, C520, S5 23, V524, T525, M530, T532, D551, D553, V559, R566, A567, M568, R576, I596, T597, N609, Q631, V63 6, RMF90817 including A646, N655, R656, K658, D666, T671, R679, N682, K688, E699, M704, G715, L716, N740, L753, Y755, K761, E762, E763, M764, V765, Q766, G767, S768, L769, Q770, R771, W772, and / or F773.47, one of skill in the art can ascertain positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509).

[0281] The disclosure provides engineered archaeal family B DNA or family A polymerases including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509) and RB69 polymerase (SEQ ID NO: 2510), which are mutated at one or more positions that are positionally equivalent (or functionally equivalent sites) to the amino acid substitutions at any one or any combination of positions of a polymerase having a backbone sequence of MBC7218772.1 (SEQ ID NOs: 2215-2366), including I10, C468, and / or F773. From the sequence alignment shown in Figure 48, one of skill in the art can identify positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509).

[0282] The present disclosure relates to engineered archaeal family B polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509) and RB69 polymerase (SEQ ID NO: 2510). DNA or Family A polymerases are provided, including Y7, D11, I51, K61, V93, A117, M129, D141, I142, E143, T144, A223, E302, E323, D407, F408, S410, L411, Y412, P413, R487, A488, S495, Y496, K510, T517, I524, K562, A563, R564, S572, T593, R605, K652, The polymerase is mutated at one or more positions that are positionally equivalent (or functionally equivalent sites) to the amino acid substitution at any one or any combination of positions in a polymerase having the backbone sequence of WP175059460.1 (SEQ ID NOs: 2393-2407 and 2511-2523), including D675, K695, T700, R712, R759, Y760, Q761, S762, S763, K764, Q765, and / or T766. From the sequence alignment shown in Figure 49, one of skill in the art can identify positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509).

[0283] The present disclosure relates to engineered archaeal family B polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509) and RB69 polymerase (SEQ ID NO: 2510). Provided are DNA or Family A polymerases that are mutated at one or more positions that are positionally equivalent (or functionally equivalent sites) to the amino acid substitutions at any one or any combination of positions of a polymerase having the backbone sequence of KUO42443.1 (SEQ ID NOs: 2408-2435), including Y7, D170, E172, T557, and / or S558. From the sequence alignment shown in Figure 50, one of skill in the art can ascertain positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509).

[0284] The disclosure provides engineered archaeal family B DNA or family A polymerases, including Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509) and RB69 polymerase (SEQ ID NO: 2510), which are mutated at one or more positions that are positionally equivalent (or functionally equivalent sites) to the amino acid substitutions at any one or any combination of positions of a polymerase having a backbone sequence of NOZ77387.1 (SEQ ID NOs: 2436-2454), including Y10, C41, C531, and / or T536. From the sequence alignment shown in Figure 51, one of skill in the art can identify positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NO: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus abyssi polymerase (SEQ ID NO: 2509).

[0285] The present disclosure provides a polymerase operably linked to a detectable reporter moiety. Any of the polymerases described herein may be selected from the group consisting of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523, Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NOs: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus Any of the polymerases described herein can be labeled with a detectable reporter moiety, including polymerases having a mutant amino acid sequence backbone of any of the polymerases described herein, including any of abyssi polymerase (SEQ ID NO: 2509), RB69 polymerase (SEQ ID NO: 2510), and Phi29 (SEQ ID NO: 2455).

[0286] In some embodiments, the detectable reporter moiety generates a detectable signal due to a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity, or a proximity event such as FRET). In some embodiments, the detectable reporter moiety comprises a luminescent moiety, a fluorescent moiety, or a quencher. In some embodiments, the detectable moiety comprises a fluorescent moiety that acts as a FRET donor or acceptor. The detectable reporter moiety can be attached to the polymerase at the N-terminus, C-terminus, or any internal location. The detectable reporter moiety is attached to the polymerase in a manner that does not interfere with the ability of the polymerase to bind to a nucleic acid template molecule, a nucleic acid primer, or a nucleotide. The detectable reporter moiety is attached to the polymerase in a manner that does not interfere with the catalytic activity of the polymerase, including the incorporation of nucleotides.

[0287] The present disclosure provides recombinant fusion polypeptides comprising any of the DNA polymerases described herein operably linked to any one or any combination of two or more exogenous amino acid sequences for affinity purification, cleavage, or solubilization. In some embodiments, the recombinant fusion polypeptide is selected from the group consisting of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523, Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2502), 9°N polymerase (SEQ ID NOs: 2503 or 2504) (including THERMINATOR polymerase; SEQ ID NO: 2505), VENT polymerase (SEQ ID NO: 2506), DEEP VENT polymerase (SEQ ID NO: 2507), Pfu polymerase (SEQ ID NO: 2508), and / or Pyrococcus These include polymerases having a variant amino acid sequence backbone of any polymerase described herein, including any of: abyssi polymerase (SEQ ID NO: 2509), RB69 polymerase (SEQ ID NO: 2510), and Phi29 (SEQ ID NO: 2455).

[0288] In some embodiments, the recombinant fusion polypeptide comprises any of the wild-type and mutant polymerases described herein operably linked at their N-terminus and / or C-terminus(s) to at least one affinity purification tag sequence(s), wherein the affinity purification tag sequence(s) comprises a histidine tag (e.g., a hexa-histidine tag (SEQ ID NO: 2524)), a FLAG tag, a T7 tag, a Strep II tag, an S tag (e.g., from pancreatic ribonuclease A), an HA tag (e.g., from human influenza hemagglutinin protein), and / or a c-Myc tag.

[0289] In some embodiments, the recombinant fusion polypeptide comprises any of the wild-type and mutant polymerases described herein operably linked at their N-terminus and / or C-terminus(s) to at least one polypeptide cleavage sequence, or the polypeptide cleavage sequence can be located between the affinity tag sequence and the N-terminus or C-terminus of the polymerase sequence. In some embodiments, the polypeptide cleavage sequence can be recognized and cleaved by a protease or reducing conditions. In some embodiments, the polypeptide cleavage sequence comprises a thrombin cleavage sequence, a TEV cleavage sequence (e.g., from tobacco etch virus, including AcTEV and ProTEV), a factor Xa cleavage sequence, an enterokinase cleavage sequence, and a SUMO cleavage sequence (e.g., small ubiquitin-like modification, including Ulp1, Senp2, and SUMOstar). In some embodiments, the recombinant fusion polypeptide comprises any of the wild-type and mutant polymerases described herein operably linked at the N-terminus and / or C-terminus(s) to at least one exogenous amino acid sequence for improving solubilization, including maltose binding protein (MBP), small ubiquitin-like modifier (SUMO), and glutathione S-transferase (GST).

[0290] The present disclosure provides compositions comprising one or more mutant polymerases, at least one nucleic acid template molecule, and at least one nucleic acid primer. In some embodiments, the one or more mutant polymerases may or may not bind to the at least one nucleic acid template molecule and the at least one nucleic acid primer. In some embodiments, the primer provides an initiation site for nucleotide polymerization. In some embodiments, the primer comprises a 3' extendable end for a polymerase-catalyzed nucleotide incorporation reaction, or the primer comprises a 3' non-extendable end. In some embodiments, the nucleic acid template molecule comprises at least one uridine nucleotide, or is devoid of uridine nucleotides. In some embodiments, the mutant polymerase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523. In some embodiments, the mutant polymerase comprises an amino acid substitution that confers exonuclease-minus activity. In some embodiments, the polymerase comprises at least one mutation that increases the thermostability of the enzyme, improves binding and / or improved binding and incorporation of nucleotide reagents, improves the rate of incorporation of nucleotide analogs, improves uracil tolerance, and / or reduces sequence-specific sequencing errors, as compared to the corresponding wild-type polymerase.

[0291] The present disclosure provides compositions comprising one or more mutant polymerases and at least one nucleic acid template molecule having a self-priming 3' end. In some embodiments, the one or more mutant polymerases may or may not bind to at least one nucleic acid template molecule having a self-priming 3' end. In some embodiments, the self-priming 3' end of the template molecule provides an initiation site for nucleotide polymerization. In some embodiments, the nucleic acid template molecule comprises at least one uridine nucleotide or lacks uridine nucleotides. In some embodiments, the mutant polymerase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523. In some embodiments, the mutant polymerase comprises an amino acid substitution that confers exonuclease-minus activity, hi some embodiments, the polymerase comprises at least one mutation that increases the thermostability of the enzyme, improves the rate of incorporation of nucleotide analogs, and / or improves uracil tolerance compared to the corresponding wild-type polymerase.

[0292] In some embodiments, the compositions include one or more mutant polymerases bound to a nucleic acid duplex, each comprising a nucleic acid template hybridized to a nucleic acid primer, thereby forming a composite polymerase. In some embodiments, the primer provides an initiation site for nucleotide polymerization. In some embodiments, the mutant polymerase binds to a nucleic acid template molecule having a self-priming 3' end to form a composite polymerase lacking a separate primer molecule. In some embodiments, the nucleic acid template molecule includes at least one uridine nucleotide or lacks uridine nucleotides. In some embodiments, the mutant polymerase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523. In some embodiments, the mutant polymerase is a recombinant polymerase.

[0293] In some embodiments, the composition comprises one or more mutant polymerases, at least one nucleic acid template molecule, and an initiation site for nucleotide polymerization, where the mutant polymerase is in solution, the nucleic acid template molecule is in solution, and the initiation site (e.g., a primer) is in solution. In some embodiments, the composition comprises one or more mutant polymerases, at least one nucleic acid template molecule, and an initiation site for nucleotide polymerization, where the composition comprises any combination of mutant polymerases in solution, nucleic acid template molecules in solution or immobilized on a support, and initiation sites (e.g., a primer) in solution or immobilized on a support. In some embodiments, the composition comprises one or more mutant polymerases, at least one nucleic acid template molecule, and an initiation site for nucleotide polymerization, where the composition comprises any combination of mutant polymerases in solution or immobilized on a support, nucleic acid template molecules in solution or immobilized on a support, and initiation sites (e.g., a primer) in solution or immobilized on a support.

[0294] In some embodiments, the mutant polymerase exhibits increased thermostability as compared to a wild-type polymerase having the amino acid sequence of any of SEQ ID NOs: 1, 2, 1316, 2215, 2367, 2393, 2408, or 2436. For example, the mutant polymerase exhibits increased thermostability in a temperature range of about 25-50°C or about 45-80°C.

[0295] In some embodiments, the mutant polymerases exhibit increased rates of incorporation of nucleotide analogs compared to wild-type polymerases comprising any of SEQ ID NOs:1, 2, 1316, 2215, 2367, 2393, 2408, or 2436, and the nucleotide analogs comprise chain terminating moieties (e.g., blocking moieties) at the 2' and / or 3' sugar positions.

[0296] In some embodiments, the mutant polymerase exhibits increased uracil resistance compared to a wild-type polymerase comprising any of SEQ ID NOs:1, 2, 1316, 2215, 2367, 2393, 2408, or 2436.

[0297] In some embodiments, the mutant polymerase exhibits an increased ability to bind complementary nucleotide units of a multivalent molecule compared to a wild-type polymerase comprising any of SEQ ID NOs: 1, 2, 1316, 2215, 2367, 2393, 2408, or 2436.

[0298] In some embodiments, the composition comprises one or more mutant polymerases and a plurality of nucleic acid duplexes, each comprising a nucleic acid template hybridized to a nucleic acid primer. In some embodiments, the one or more polymerases and the nucleic acid duplexes further comprise a nucleotide reagent. The one or more mutant polymerases may or may not bind to the nucleic acid duplex. The one or more mutant polymerases may or may not bind to the nucleotide reagent. In some embodiments, the one or mutant polymerases bind to the nucleic acid duplex comprising the nucleic acid template hybridized to the nucleic acid primer, thereby forming a multiplex polymerase. In some embodiments, the multiplex polymerase further comprises a nucleotide reagent. In some embodiments, the mutant polymerase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523. In some embodiments, the mutant polymerase is a recombinant polymerase.

[0299] In some embodiments, the nucleotide reagent comprises any one or any combination of nucleotides and / or multivalent molecules. In some embodiments, the nucleotide comprises a canonical nucleotide. In some embodiments, the nucleotide comprises a detectably labeled nucleotide, each comprising a detectable reporter moiety linked to one of the nucleobases or the phosphate moieties of the phosphate chain. In some embodiments, the nucleotide comprises a nucleotide bearing a removable or non-removable chain terminating moiety. In some embodiments, the reversible chain terminating nucleotide may be detectably labeled or unlabeled. In some embodiments, each multivalent molecule comprises a central core attached to multiple polymer arms, each having a nucleotide unit at the end of the arm.

[0300] In some embodiments, the composite polymerase further comprises a nucleotide reagent comprising a nucleotide. In some embodiments, the nucleotide can bind to the composite polymerase without being incorporated. In some embodiments, a complementary nucleotide can bind to the composite polymerase without undergoing polymerase-catalyzed incorporation and form a ternary complex in which the complementary nucleotide is attached to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand.

[0301] In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a base, a sugar, and at least one phosphate group. In some embodiments, at least one nucleotide of the plurality of nucleotides comprises an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1-10 phosphate groups). The plurality of nucleotides can comprise at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides can comprise in any combination mixture of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0302] In some embodiments, at least one nucleotide in the plurality of nucleotides comprises a chain of one, two, or three phosphorus atoms, typically linked to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide in the plurality of nucleotides is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain are O, S, or BH. 3 In some embodiments, the tethers include phosphate groups replaced with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methyl phosphoramidate groups.

[0303] In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a nucleotide analog having a chain terminating moiety (e.g., a blocking moiety) at the sugar 2' position, the sugar 3' position, or the sugar 2' and 3' positions. In some embodiments, the chain terminating moiety can inhibit polymerase-catalyzed incorporation of a subsequent nucleotide unit or free nucleotide in the nascent chain during a primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3' sugar hydroxyl position, where the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain terminating moiety is removable / cleavable from the 3' sugar hydroxyl position to generate a nucleotide having a 3'OH sugar group that is extendable with a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide 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, a silyl group, or an acetal group. In some embodiments, the chain terminating moiety is cleavable / removable from the nucleotide, for example, by reacting the chain terminating moiety with a chemical agent, a pH change, light, or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl, and allyl are cleavable / removable from the nucleotide using a method such as tetrakis(triphenylphosphine)palladium(0) (Pd(PPh 3 ) 4 ), or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, chain terminating moieties aryl and benzyl can be cleaved using HPd / C. In some embodiments, chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide can be cleaved using phosphines, or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). In some embodiments, chain terminating moieties carbonate can be cleaved using potassium carbonate (K 2 CO 3 ) in MeOH. 2 CO 3), triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the chain terminating moieties can be cleaved / removable using nitrous acid. In some embodiments, the chain terminating moieties can be cleaved / removable using a solution containing nitrite, for example, a combination of nitrite and an acid, for example, acetic acid, sulfuric acid, or nitric acid. In some further embodiments, the solution can include an organic acid.

[0304] In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a terminator nucleotide analog having a chain terminating moiety (e.g., a blocking moiety) at the sugar 2' position, the sugar 3' position, or the sugar 2' and 3' positions. In some embodiments, the chain terminating moiety comprises an azide, azido, or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3'-O-azido or 3'-O-azidomethyl group. In some embodiments, the chain terminating azide, azido, and azidomethyl groups are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP). In some embodiments, a chain terminating moiety comprising one or more of a 3'-O-amino group, a 3'-O-aminomethyl group, a 3'-O-methylamino group, or derivatives thereof may be cleaved with nitrous acid via a mechanism utilizing nitrous acid or using a solution comprising nitrous acid. In some embodiments, a chain terminating moiety comprising one or more of a 3'-O-amino group, a 3'-O-aminomethyl group, a 3'-O-methylamino group, or derivatives thereof may be cleaved using a solution comprising a nitrite. In some embodiments, for example, the nitrite may be combined with or contacted with an acid, such as acetic acid, sulfuric acid, or nitric acid. In some further embodiments, for example, the nitrite may be combined with or contacted with an organic acid, such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, etc. In some embodiments, the chain terminating moiety comprises a 3'-acetal moiety that may be cleaved with a palladium deblocking reagent (e.g., Pd(0)).

[0305] In some embodiments, the nucleotide analog comprises a chain terminating moiety selected from the group consisting of 3'-deoxynucleotides, 2',3'-dideoxynucleotides, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'butyl, 3'-tertbutyl, 3'-fluorenylmethyloxycarbonyl, 3' tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino groups, 3'-phosphorothioates, and 3-O-benzyl, or derivatives thereof.

[0306] In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides lacking a detectable reporter moiety, e.g., a fluorophore. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. The detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base by a linker that is cleavable / removable from the base.

[0307] In some embodiments, the cleavable linker on the base comprises a cleavable moiety comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide 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. In some embodiments, the cleavable linker on the base is cleavable / removable from the base by reacting the cleavable moiety with a chemical agent, a pH change, light, or heat. In some embodiments, the cleavable moieties alkyl, alkenyl, alkynyl, and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh)) with piperidine.3 ) 4 ), or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the cleavable moieties aryl and benzyl can be cleaved using HPd / C. In some embodiments, the cleavable moieties amine, amide, keto, isocyanate, phosphate, thio, and disulfide can be cleaved using phosphines, or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). In some embodiments, the cleavable moieties carbonate can be cleaved using potassium carbonate (K 2 CO 3 ) in MeOH. 2 CO 3 ), triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the cleavable moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0308] In some embodiments, the cleavable linker on the base comprises a cleavable moiety comprising an azide, azido, or azidomethyl group. In some embodiments, the cleavable moieties azide, azido, and azidomethyl groups are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0309] In some embodiments, the chain terminating moiety (e.g., at the sugar 2' and / or sugar 3' positions) and the cleavable linker on the base have the same or different cleavable moieties. In some embodiments, the chain terminating moiety (e.g., at the sugar 2' and / or sugar 3' positions) and the detectable reporter moiety attached to the base are chemically cleavable / removable with the same chemical agent. In some embodiments, the chain terminating moiety (e.g., at the sugar 2' and / or sugar 3' positions) and the detectable reporter moiety attached to the base are chemically cleavable / removable with different chemical agents.

[0310] In some embodiments, the composition comprises one or more mutant polymerases and a plurality of nucleic acid duplexes, each comprising a nucleic acid template hybridized to a nucleic acid primer. In some embodiments, the one or more polymerases and the nucleic acid duplexes further comprise a plurality of nucleotide reagents. In some embodiments, the one or more polymerases and the nucleic acid duplexes further comprise a plurality of multivalent molecules. The one or more mutant polymerases may or may not bind to the nucleic acid duplex. The one or more mutant polymerases may or may not bind to one or more of the multivalent molecules. In some embodiments, the one or mutant polymerases bind to a nucleic acid duplex comprising a nucleic acid template hybridized to a nucleic acid primer, thereby forming a multiplex polymerase. In some embodiments, the multiplex polymerase further comprises at least one nucleotide reagent (e.g., a plurality of multivalent molecules). In some embodiments, the mutant polymerase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any of SEQ ID NOs: 3-1315, 1317-2214, 2216-2366, 2368-2392, 2394-2407, 2409-2435, 2437-2454, 2456-2501, or 2511-2523. In some embodiments, the mutant polymerase is a recombinant polymerase.

[0311] In some embodiments, the nucleotide reagent comprises any one or any combination of nucleotides and / or multivalent molecules. In some embodiments, the nucleotide comprises a canonical nucleotide. In some embodiments, the nucleotide comprises a nucleotide analog, including a detectably labeled nucleotide and / or a nucleotide carrying a removable or non-removable chain terminating moiety. In some embodiments, each multivalent molecule comprises a central core that is linked to multiple polymer arms, each having a nucleotide unit at the end of the arm.

[0312] In some embodiments, a multivalent molecule generally comprises a central portion (e.g., core) bound to multiple arms, each arm being bound to a nucleotide unit. The multivalent molecule comprises a star, comb, bridge, bottle-brush, or dendrimer configuration. In some embodiments, the multivalent molecule may comprise 2-4, 4-10, 10-20, or up to 64 arms. In some embodiments, the arms may radiate out from the central portion.

[0313] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises (a) a core and (b) a plurality of nucleotide arms comprising (i) a core-binding moiety, (ii) a spacer (e.g., comprising a PEG moiety), (iii) a linker, and (iv) a nucleotide unit, where the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, and the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, and both linker chains have 2-6 subunits. In some embodiments, the linker also comprises an aromatic moiety. Exemplary multivalent molecules are shown in Figures 2-5. An exemplary nucleotide arm is shown in Figure 6. An exemplary spacer is shown in Figure 7 (top). Various exemplary linkers are shown in Figures 7 (bottom) and 8. Examples of various linkers linking / bonding nucleotide units are shown in Figures 9A-D, where the 5-position of the pyrimidine base or the 7-position of the purine base is bonded to the linker via a propargylamine bond (see also Figure 10).

[0314] In some embodiments, the nucleotide arms are designed such that the nucleotide units of the nucleotide arms can interact with a polymerase enzyme in a manner similar to free nucleotides. The nucleotide units of the nucleotide arms can bind to a polymerase complexed with a nucleic acid template and a nucleic acid primer (e.g., nucleotide association). The nucleotide units can also dissociate from the polymerase complex and rebind to the same polymerase complex or bind to a different polymerase complex that is in close proximity to the multivalent molecule. Because the multivalent molecule contains multiple nucleotide arms, the nucleotide units of a single multivalent molecule can bind to multiple polymerase complexes simultaneously. The multivalent molecule effectively increases the local concentration of nucleotides, which can enhance the signal in the nucleotide binding reaction.

[0315] In some embodiments, a nucleotide unit of a multivalent molecule can bind to a composite polymerase without being incorporated. In some embodiments, a complementary nucleotide unit of a multivalent molecule can bind to a composite polymerase without undergoing polymerase-catalyzed incorporation, with the complementary nucleotide attached to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand.

[0316] In some embodiments, a nucleotide unit of a multivalent molecule can undergo primer extension by binding to a composite polymerase and incorporating into the 3' end of an extendable primer (e.g., complexed with a polymerase), resulting in primer extension. If the nucleotide unit contains a sugar 3'OH, it can incorporate a subsequent nucleotide into the nascent extended primer. If the nucleotide unit contains a sugar 3'OH substituted with a blocking group, it blocks the subsequent nucleotide from being incorporated into the nascent extended primer strand. A nucleotide unit (of a multivalent molecule) can be attached to the 3' end of a primer at a position opposite the complementary nucleotide in the template strand. The nucleotide unit can undergo nucleotide incorporation in a polymerase-catalyzed reaction, thereby extending the primer by one nucleotide.

[0317] In some embodiments, the core, linker, and / or nucleotide units of the multivalent molecule can be labeled with a detectable reporter moiety (e.g., a fluorophore) in a manner that allows for differentiation between different multivalent molecules carrying different types of nucleotide units. For example, the core unit of a first multivalent molecule is labeled with a first fluorophore, and the first multivalent molecule comprises a plurality of nucleotide arms with dGTP nucleotide units. The core unit of a second multivalent molecule is labeled with a second fluorophore (different from the first fluorophore), and the second multivalent molecule comprises a plurality of nucleotide arms with dATP nucleotide units. The binding and incorporation events of the nucleotide units can be detected, and the specific bases of the nucleotide units (as part of the multivalent molecule) can be identified based on the detection and identification of the detectable reporter moiety on the core. In another example, the linker and / or nucleotide units of the first multivalent molecule are labeled with a first fluorophore, and the first multivalent molecule comprises a plurality of nucleot...

Claims

1. An engineered polymerase that is at least 85% identical to any one of SEQ ID NOs: 1-2501 and 2511-2523; and the amino acid substitution mutations Asp141Ala and Glu143Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 1 or 2 (RLF89458.1 or RLF78286.1); - the amino acid substitution mutations Asp168Ala and Glu170Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 1316 (NOZ58130.1); - the amino acid substitution mutations Asp149Ala and Glu151Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2215 (RMF90817.1); - the amino acid substitution mutations Asp173Ala and Glu175Ala, wherein said engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2367 (MBC7218772.1); - the amino acid substitution mutations Asp141Ala and Glu143Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2393 (WP175059460.1); - the amino acid substitution mutations Asp170Ala and Glu172Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2408 (KUO42443.1), or - An engineered polymerase comprising an amino acid sequence having the amino acid substitution mutations Asp161Ala and Glu163Ala, wherein the engineered polymerase has increased incorporation of chain-terminating nucleotide analogues compared to a wild-type polymerase having the amino acid sequence of SEQ ID NO: 2436 (NOZ77387.1).

2. 2. The engineered polymerase of claim 1, further comprising a plurality of said engineered polymerases, a plurality of nucleic acid template molecules, and a plurality of nucleotide polymerization initiation sites having 3' extendable ends.

3. 3. The engineered polymerase of claim 2, wherein the plurality of nucleic acid template molecules comprises linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear and circular nucleic acid molecules.

4. 3. The engineered polymerase of claim 2, wherein the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

5. 3. The engineered polymerase of claim 2, wherein at least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules comprises one copy of a target sequence of interest or comprises a concatemer having two or more tandem copies of a target sequence of interest.

6. 3. The engineered polymerase of Claim 2, wherein an individual nucleotide polymerization initiation site within the plurality of nucleotide polymerization initiation sites comprises a nucleic acid primer that hybridizes to a portion of at least one of the nucleic acid template molecules, or an individual nucleotide polymerization initiation site within the plurality of nucleotide polymerization initiation sites comprises a self-priming end portion of at least one of the nucleic acid template molecules.

7. 3. The engineered polymerase of Claim 2, wherein the plurality of polymerases, the plurality of nucleic acid template molecules, and the plurality of nucleotide polymerization initiation sites form a plurality of polymerase composites, each comprising a polymerase that binds to a nucleic acid duplex, the duplex comprising a nucleic acid template molecule hybridized to a nucleic acid primer.

8. 8. The engineered polymerase of claim 7, wherein the multiple nucleic acid template molecules comprise the same target sequence of interest or different target sequences of interest.

9. 8. The engineered polymerase of Claim 7, wherein the plurality of composite polymerases further comprises a plurality of multivalent molecules, each multivalent molecule in the plurality of multivalent molecules comprising: (a) a core; and (b) a plurality of nucleotide arms comprising: (i) a core binding moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core binding moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

10. 10. The engineered polymerase of claim 9, wherein the linker comprises an aliphatic chain having 2 to 6 subunits or an oligoethylene glycol chain having 2 to 6 subunits.

11. 10. The engineered polymerase of claim 9, wherein the multiple nucleotide arms bound to a given core have the same type of nucleotide unit, the nucleotide unit comprising dATP, dGTP, dCTP, dTTP, or dUTP.

12. 10. The engineered polymerase of Claim 9, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, and each multivalent molecule in the plurality of multivalent molecules has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

13. 10. The engineered polymerase of claim 9, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

14. 10. The engineered polymerase of claim 9, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore.

15. 10. The engineered polymerase of claim 9, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises a nucleotide unit that is labeled with a fluorophore.

16. 8. The engineered polymerase of claim 7, wherein the plurality of composite polymerases further comprises a plurality of nucleotides, each nucleotide in the plurality of nucleotides comprising an aromatic base, a 5-carbon sugar, and from 1 to 10 phosphate groups.

17. 17. The engineered polymerase of claim 16, wherein the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

18. 17. The engineered polymerase of Claim 16, wherein 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.

19. 17. The engineered polymerase of claim 16, wherein at least one nucleotide of the plurality of nucleotides is labeled with a fluorophore.

20. 17. The engineered polymerase of claim 16, wherein the plurality of nucleotides lacks a fluorophore label.

21. 17. The engineered polymerase of claim 16, wherein at least one of the nucleotides in the plurality of nucleotides comprises a removable chain terminating moiety attached to a 3' carbon position of a sugar group, the removable chain terminating moiety comprising an acetal group, 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 terminating moiety is cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

22. 8. The engineered polymerase of claim 7, wherein the plurality of composite polymerases further comprises a plurality of non-catalytic divalent cations that inhibit polymerase-catalyzed incorporation of nucleotides, the non-catalytic divalent cations comprising strontium or barium.

23. 8. The engineered polymerase of claim 7, wherein the plurality of composite polymerases further comprises a plurality of catalytic divalent cations that facilitate polymerase-catalyzed incorporation of nucleotides, the catalytic divalent cations comprising magnesium or manganese.

24. 8. The engineered polymerase of claim 7, wherein the multiple composite polymerases are immobilized on a support or in a coating on the support.

25. The density of the multiple polymerases immobilized on the support is 1 mm ２ 10 per ２ ~10 １２ 25. The engineered polymerase of claim 24, comprising

26. 25. The engineered polymerase of claim 24, wherein the multiple immobilized composite polymerases are immobilized at predetermined sites on the support or are immobilized at random sites on the support.

27. 25. The engineered polymerase of claim 24, wherein 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 a branched polyethylene glycol (PEG) having at least four branches.

28. 28. The engineered polymerase of claim 27, wherein the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

29. 25. The engineered polymerase of claim 24, wherein the multiple immobilized composite polymerases are in fluid communication with one another to allow a solution of reagents to be flowed over a support such that the multiple immobilized composite polymerases on the support react with the solution of reagents in a massively parallel manner.

30. the plurality of complex polymerases further comprising first and second binding complexes; (i) the first binding complex comprises a first nucleic acid primer, a first polymerase, and a first multivalent molecule that is bound to a first portion of a concatemeric template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the multivalent molecule binds to the first polymerase; (ii) the second binding complex comprises a second nucleic acid primer, a second polymerase, and the first multivalent molecule bound to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the multivalent molecule binds to the second polymerase; 10. The engineered polymerase of claim 9, wherein the first and second binding complexes comprising the same multivalent molecule form a binding activity complex.

31. 10. A method for forming a plurality of composite polymerases, comprising contacting a plurality of engineered polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid primers under conditions suitable to form a plurality of composite polymerases, each comprising a polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer, and wherein the plurality of engineered polymerases comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-2501 and 2511-2523 of claim 1.

32. 32. The method of claim 31 , wherein the plurality of nucleic acid template molecules comprises linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear and circular nucleic acid molecules.

33. 32. The method of claim 31 , wherein the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

34. 32. The method of claim 31 , wherein each nucleic acid template molecule in the plurality of nucleic acid molecules comprises one copy of a target sequence of interest, or each nucleic acid template molecule in the plurality of nucleic acid molecules comprises a concatemer having two or more tandem copies of a target sequence of interest.

35. 32. The method of claim 31 , wherein the multiple nucleic acid molecules comprise the same target sequence of interest or different target sequences of interest.

36. 32. The method of claim 31 , further comprising contacting the plurality of composite polymerases with a plurality of multivalent molecules, wherein each multivalent molecule in the plurality of multivalent molecules comprises: (a) a core; and (b) a plurality of nucleotide arms comprising: (i) a core-binding moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core-binding moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

37. 37. The method of claim 36, wherein the linker comprises an aliphatic chain having 2 to 6 subunits or an oligoethylene glycol chain having 2 to 6 subunits.

38. 37. The method of claim 36, wherein the multiple nucleotide arms attached to a given core have the same type of nucleotide unit, the type of nucleotide unit comprising dATP, dGTP, dCTP, dTTP, or dUTP.

39. 37. The method of claim 36, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, and each multivalent molecule in the plurality of multivalent molecules has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

40. 37. The method of claim 36, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

41. 37. The method of claim 36, wherein at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore.

42. 37. The method of claim 36, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore.

43. 37. The method of claim 36, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide units that are labeled with a fluorophore.

44. 37. The method of claim 36, wherein said contacting is performed under conditions suitable for binding of at least one complementary nucleotide unit of said multivalent molecule to at least one of said multiplex polymerases.

45. 37. The method of claim 36, further comprising contacting the multiple composite polymerases with a multiple non-catalytic divalent cations that inhibit polymerase-catalyzed incorporation of nucleotides, wherein the non-catalytic divalent cations comprise strontium or barium.

46. 32. The method of claim 31, further comprising contacting said plurality of composite polymerases with a plurality of nucleotides, wherein each nucleotide in said plurality of nucleotides comprises an aromatic base, a 5-carbon sugar, and from 1 to 10 phosphate groups.

47. 47. The method of claim 46, wherein the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

48. 47. The method of claim 46, wherein 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.

49. 47. The method of claim 46, wherein the at least one nucleotide in the plurality of nucleotides is labeled with a fluorophore.

50. 47. The method of claim 46, wherein the plurality of nucleotides lacks a fluorophore label.

51. 47. The method of claim 46, wherein at least one of the nucleotides in the plurality of nucleotides comprises a removable chain terminating moiety attached to a 3' carbon position of a sugar group, the removable chain terminating moiety comprising an acetal group, 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, the removable chain terminating moiety being cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

52. 47. The method of claim 46, wherein said contacting is performed under conditions suitable for binding of at least one complementary nucleotide from said plurality of nucleotides to at least one multiplex polymerase.

53. 47. The method of claim 46, further comprising contacting the multiple composite polymerases with a multiple catalytic divalent cations that facilitate polymerase-catalyzed incorporation of nucleotides, wherein the catalytic divalent cations comprise magnesium or manganese.

54. 32. The method of claim 31 , wherein the multiple composite polymerases are immobilized on a support or in a coating on the support.

55. The density of the multiple polymerases immobilized on the support is 1 mm ２ 10 per ２ ~10 １２ 55. The method of claim 54, comprising:

56. 55. The method of claim 54, wherein the plurality of immobilized composite polymerases are immobilized at predetermined sites on the support, or the plurality of immobilized composite polymerases are immobilized at random sites on the support.

57. 55. The method of claim 54, wherein 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 a branched polyethylene glycol (PEG) having at least four branches.

58. 58. The method of claim 57, wherein the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

59. 55. The method of claim 54, wherein the multiple immobilized composite polymerases are in fluid communication with one another to allow a solution of reagents to be flowed over the support such that the multiple immobilized composite polymerases on the support react with the solution of reagents in a massively parallel manner.

60. forming a plurality of binding complexes; a) binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of a concatemeric template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first polymerase; b) binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second polymerase; 37. The method of claim 36, wherein the first and second binding complexes comprising the same multivalent molecule form an avidity complex.

61. 1. A method for determining the sequence of a nucleic acid template, comprising: a) contacting a plurality of first polymerases with (i) a plurality of nucleic acid templates, each comprising a target sequence of interest, and (ii) a plurality of nucleic acid primers, said contacting being performed under conditions suitable for binding of said plurality of first polymerases to said plurality of nucleic acid template molecules and said plurality of nucleic acid primers, thereby forming a plurality of first composite polymerases, each of said first composite polymerases comprising a first polymerase bound to a nucleic acid duplex, said nucleic acid duplex comprising a nucleic acid template molecule hybridized to a nucleic acid primer, said plurality of first polymerases comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 1-2501 and 2511-2523 as set forth in claim 1; b) contacting said plurality of first composite polymerases with a plurality of multivalent molecules to form a plurality of multivalent binding complexes, each multivalent molecule in said plurality of multivalent molecules comprising a core attached to a plurality of nucleotide arms, each nucleotide arm being bound to a nucleotide unit, said contacting being performed under conditions suitable for binding a complementary nucleotide unit of said multivalent molecule to at least two of said plurality of first composite polymerases, thereby forming a plurality of multivalent binding complexes, said conditions suitable for inhibiting incorporation of said complementary nucleotide unit into said primer of said plurality of multivalent binding complexes; c) detecting said plurality of multivalent binding complexes; and d) identifying bases of the complementary nucleotide units in the plurality of multivalent binding complexes, thereby determining the sequence of the nucleic acid template molecule.

62. e) dissociating the plurality of multivalent binding complexes by removing the plurality of first polymerases and their bound multivalent molecules, retaining the plurality of nucleic acid duplexes; f) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second polymerases under conditions suitable for binding the plurality of second polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second composite polymerases, each of which comprises a second polymerase bound to a nucleic acid duplex, wherein the plurality of second polymerases comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 1-2501 and 2511-2523 as set forth in claim 1; 62. The method of claim 61, further comprising: g) contacting said plurality of second composite polymerases with a plurality of nucleotides, said contacting being performed under conditions suitable for binding complementary nucleotides from said plurality of nucleotides to at least two of said second composite polymerases, thereby forming a plurality of nucleotide binding complexes, said conditions suitable for promoting nucleotide incorporation of said bound complementary nucleotides into said primers of said nucleotide binding complexes.

63. 63. The method of claim 62, further comprising (h) detecting the complementary nucleotide incorporated into the primer of the nucleotide complex polymerase.

64. h) detecting the complementary nucleotide incorporated into the primer by the nucleotide complex polymerase; 63. The method of claim 62, further comprising: i) identifying the base of the complementary nucleotide incorporated into the primer of the nucleotide complex polymerase.

65. 62. The method of Claim 61, wherein the contacting of the plurality of first composite polymerases with the plurality of multivalent molecules of step (b) is performed in the presence of a non-catalytic divalent cation that inhibits polymerase-catalyzed incorporation of nucleotides, the non-catalytic divalent cation comprising strontium or barium.

66. 63. The method of Claim 62, wherein the contacting of the plurality of second composite polymerases with the plurality of nucleotides of step (g) is performed in the presence of a catalytic divalent cation that promotes polymerase-catalyzed incorporation of nucleotides, wherein the catalytic divalent cation comprises magnesium or manganese.

67. 62. The method of Claim 61, wherein the plurality of nucleic acid template molecules of step (a) comprises clonally amplified template molecules.

68. 62. The method of Claim 61, wherein each nucleic acid template molecule in the plurality of nucleic acid molecules of step (a) comprises one copy of a target sequence of interest or comprises a concatemer having two or more tandem copies of a target sequence of interest.

69. 62. The method of Claim 61, wherein the nucleic acid template molecules in the plurality of nucleic acid molecules of step (a) comprise the same target sequence of interest or different target sequences of interest.

70. 62. The method of claim 61 , wherein each multivalent molecule in the plurality of multivalent molecules comprises: (a) a core; and (b) a plurality of nucleotide arms comprising: (i) a core binding moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core binding moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

71. 71. The method of claim 70, wherein the linker comprises an aliphatic chain having 2 to 6 subunits or an oligoethylene glycol chain having 2 to 6 subunits.

72. 71. The method of claim 70, wherein the multiple nucleotide arms attached to a given core have the same type of nucleotide unit, the type of nucleotide unit comprising dATP, dGTP, dCTP, dTTP, or dUTP.

73. 71. The method of claim 70, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, and each multivalent molecule in the plurality of multivalent molecules has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

74. 71. The method of claim 70, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

75. 71. The method of claim 70, wherein at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore.

76. 71. The method of claim 70, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore.

77. 71. The method of claim 70, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide units that are labeled with a fluorophore.

78. 63. The method of claim 62, wherein each nucleotide in the plurality of nucleotides of step (g) comprises an aromatic base, a 5-carbon sugar, and from 1 to 10 phosphate groups.

79. 79. The method of claim 78, wherein the plurality of nucleotides in step (g) comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, or 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.

80. 80. The method of claim 78, wherein at least one of the nucleotides in the plurality of nucleotides of step (g) is labeled with a fluorophore.

81. 80. The method of claim 78, wherein said plurality of nucleotides of step (g) lacks a fluorophore label.

82. 80. The method of claim 78, wherein at least one of the nucleotides in the plurality of nucleotides of step (g) comprises a removable chain terminating moiety attached to a 3' carbon position of a sugar group, wherein the removable chain terminating moiety comprises an acetal group, 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, wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

83. 62. The method of claim 61 , wherein the plurality of first composite polymerases of step (a) are immobilized to a support or to a coating on the support.

84. The density of the plurality of first composite polymerases immobilized on the support is 1 mm ２ 10 per ２ ~10 １２ 84. The method of claim 83, comprising:

85. 84. The method of claim 83, wherein the plurality of first composite polymerases are immobilized at predetermined sites on the support or are immobilized at random sites on the support.

86. 84. The method of claim 83, wherein 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 a branched polyethylene glycol (PEG) having at least four branches.

87. 87. The method of claim 86, wherein the hydrophilic polymer coating has a water contact angle of 45 degrees or less.

88. 84. The method of claim 83, wherein the plurality of immobilized first composite polymerases are in fluid communication with one another to allow a solution of reagents to be flowed over the support such that the plurality of immobilized first composite polymerases on the support react with the solution of reagents in a massively parallel manner.

89. forming a plurality of binding complexes; a) binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of a concatemeric template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first polymerase; b) binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second polymerase; 71. The method of claim 70, wherein the first and second binding complexes comprising the same multivalent molecule form an avidity complex.

90. a) contacting said plurality of polymerases and said plurality of nucleic acid primers with different portions of a concatemeric nucleic acid template molecule to form at least first and second composite polymerases on the same concatemeric template molecule; b) contacting a plurality of multivalent molecules with said at least a first and a second composite polymerase on the same concatemeric template molecule under conditions suitable for binding of a single multivalent molecule from said plurality of multivalent molecules to said first and second composite polymerases, wherein at least a first nucleotide unit of said single multivalent molecule binds to said first composite polymerase comprising a first primer hybridized to a first portion of said concatemeric template molecule, thereby forming a first binding complex, and at least a second nucleotide unit of said single multivalent molecule binds to said second composite polymerase comprising a second primer hybridized to a second portion of said concatemeric template molecule, thereby forming a second binding complex; said contacting is performed under conditions suitable to inhibit polymerase-catalyzed incorporation of said linked first and second nucleotide units in said first and second binding complexes; contacting said first and second binding complexes that bind to the same multivalent molecule to form an avidity complex; c) detecting said first and second binding complexes on the same concatemeric template molecule; 71. The method of claim 70, further comprising: d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule; and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule.

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