Highly accurate de novo polynucleotide synthesis

Abstract

The invention relates to highly accurate de novo polynucleotide synthesis. Described herein are methods, systems, compositions, and devices for preparing high quality building blocks, such as polynucleotides. The processes described herein provide for efficient washing of residual reagents, solvents, or by-products from previous synthesis steps to allow for the production of polynucleotides with low error rates. The processes described herein also provide for a reduction in deletion rate during chemical nucleic acid synthesis. In addition, the methods and devices described herein allow for rapid construction and assembly of large libraries of highly precise polynucleotides.

Description

[0001] This application is a divisional application of an application with a filing date of December 23, 2019, application number 201980093128.8, and invention name “Highly Accurate De Novo Polynucleotide Synthesis”.

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 785,015, filed on December 26, 2018, which is incorporated herein by reference in its entirety. Background Art

[0004] De novo gene synthesis is a powerful tool for both basic biological research and biotechnological applications. Although various methods are known for designing and synthesizing relatively short fragments on a small scale, these techniques are generally unsatisfactory in terms of predictability, scalability, automation, speed, precision, and cost. Summary of the invention

[0005] Provided herein are systems, methods, and compositions for the efficient de novo synthesis of highly accurate and uniform polynucleotide libraries.

[0006] Provided herein is a method for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; d) depositing a washing solvent on the surface, wherein the washing solvent comprises a ketone, an ester, an ether, a hydrocarbon, or a functional equivalent thereof; and e) repeating steps bd to synthesize a plurality of polynucleotides. Further provided herein is that the washing solvent comprises acetone, tetrahydrofuran, ethyl acetate, toluene, benzene, ethanol, or a combination thereof. Further provided herein is that the washing solvent comprises a ketone, an ether, or a functional equivalent thereof. Further provided herein is that the washing solvent comprises acetone or a functional equivalent thereof. Further provided herein is that the washing solvent is functionally equivalent to the major component by volume of the reagent solution previously contacted. Further provided herein is that the surface is washed with the washing solvent after one or more steps in steps b) to d). Further provided herein is that the surface is washed with acetonitrile after one or more steps in steps b) to d). Further provided herein is that the surface is washed with the washing solvent after each step after the coupling step. It is further provided herein that the surface is washed with the washing solvent after each step after the deblocking step. It is further provided herein that the method further comprises depositing a capping solution on the surface, wherein capping prevents coupling of unblocked nucleosides. It is further provided herein that the capping solution comprises an acyl halide or anhydride. It is further provided herein that the capping solution comprises acetyl chloride or acetic anhydride. It is further provided herein that the capping solution comprises an amine base. It is further provided herein that the surface is washed with the washing solvent after each step after the capping step. It is further provided herein that the method further comprises depositing a deblocking solution on the surface, wherein deblocking allows coupling of the polynucleotide to the nucleoside. It is further provided herein that the at least one nucleoside comprises a phosphoramidite. It is further provided herein that the at least one nucleoside comprises a 5' blocking group. It is further provided herein that the at least one nucleoside comprises a 3' blocking group. It is further provided herein that the structure is a plate, a tape, a belt or a bead. It is further provided herein that the method further comprises depositing a deblocking solution on the surface, wherein deblocking allows coupling of the polynucleotide to the nucleoside. Further provided herein, the method further comprises depositing a capping solution on the surface before or after depositing the oxidizing solution on the surface. Methods are provided herein, further comprising depositing the capping solution on the surface before and after depositing the oxidizing solution on the surface. Methods are further provided herein, further comprising depositing a washing solvent after the steps of: (i) coupling the at least one nucleoside to the polynucleotide attached to the surface; (ii) depositing the capping solution; and (iii) depositing the oxidizing solution. Further provided herein, the oxidizing solution comprises iodine.Further provided herein, the oxidizing solution comprises I2 or an iodine salt, and the I2 or iodine salt has a greater solubility or an increased dissolution rate in the washing solvent compared to acetonitrile. Further provided herein is a method, wherein the oxidizing solution further comprises an amine base. Further provided herein, the amine base is selected from pyridine, lutidine, collidine, N-methylmorpholine or a functional equivalent thereof. Provided herein is a method, further comprising: providing a predetermined sequence for a plurality of preselected polynucleotides before step (a); assembling the plurality of preselected polynucleotides after step (e), wherein the washing solvent dissolves the active ingredients or byproducts of the oxidizing solution. Further provided herein, the method further comprises additional washing before or after depositing the oxidizing solution on the surface, wherein washing comprises depositing the washing solvent on the surface. Further provided herein, the polynucleotide or nucleoside comprises DNA or RNA.

[0007] Provided herein is a composition for polynucleotide synthesis comprising: a) at least one base; b) at least one O-nucleophile; and c) at least one solvent. Further provided herein, the at least one base is selected from pyridine, lutidine and collidine. Further provided herein, the at least one nucleophile is an O-nucleophile selected from acetic acid, formic acid, propionic acid, methoxyacetic acid, phenoxyacetic acid and water. Further provided herein, the O-nucleophile is selected from acetic acid and water. Further provided herein, the concentration of the O-nucleophile is 0.01-3M. Further provided herein, the concentration of the O-nucleophile is 0.1-0.5M. Further provided herein, the at least one solvent is acetonitrile, acetone or THF. Further provided herein, the concentration of the at least one base is 0.01-3M. Further provided herein, the concentration of the at least one base is 0.1-0.5M.

[0008] Provided herein are methods for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; and d) repeating steps (b)-(c) to synthesize a plurality of polynucleotides; wherein the method comprises depositing a composition of any one of the compositions described herein. Further provided herein, the depositing is performed during any one of steps (a)-(d). Further provided herein, the depositing is performed after step (b). Provided herein are methods, further comprising at least one washing step with a washing solvent, wherein the washing solvent comprises acetone or THF.

[0009] Provided herein is a composition for polynucleotide synthesis comprising: a) at least one base; b) at least one O-nucleophile; c) at least one electrophile; and d) at least one solvent. Further provided herein, the at least one base is selected from pyridine, lutidine and collidine. Further provided herein, the O-nucleophile is selected from acetic acid, formic acid, propionic acid, methoxyacetic acid, phenoxyacetic acid and water. Further provided herein, the O-nucleophile is selected from acetic acid, methoxyacetic acid, phenoxyacetic acid and water. Further provided herein, the concentration of the O-nucleophile is 0.01-3M. Further provided herein, the concentration of the O-nucleophile is 0.1-0.5M. Further provided herein, the electrophile is an anhydride, NHS ester or acyl halide. Further provided herein, the at least one electrophile is an anhydride or acyl halide. Further provided herein, the anhydride is acetic anhydride. Further provided herein, the composition further comprises an activator. Further provided herein, the activator is N-methylimidazole or DMAP. It is further provided herein that the concentration of the activator is 0.001-0.05M. It is further provided herein that the at least one solvent is acetonitrile, acetone or THF. It is further provided herein that the concentration of the at least one base is 0.01-3M. It is further provided herein that the concentration of the at least one base is 0.1-0.5M. It is provided herein that a method for synthesizing polynucleotides comprises: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; d) depositing a capping solution on the surface, wherein the capping solution comprises a composition as described herein; and e) repeating steps (b)-(d) to synthesize a plurality of polynucleotides. It is further provided herein that the method further comprises at least one washing step comprising acetone or THF. It is further provided herein that the nucleoside comprises a protected nucleoside phosphoramidite. It is further provided herein that the nucleoside comprises a protected base. It is further provided herein that the protected nucleoside phosphoramidite comprises a protected guanine or thymine. It is further provided herein that the protected nucleoside phosphoramidite is selected from:

[0010] Where X is -N=CHNR 2 , and R is CH3 or CH2CH3; and wherein R is H, OCH3, F or tert-butyl; wherein DMT is dimethoxytrityl; and CE is cyanoethyl.

[0011] Incorporation by reference

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0014] Figure 1A A workflow for nucleoside phosphoramidite-based polynucleotide synthesis is depicted.

[0015] Figure 1B Depicted are sequential loop arrangements of polynucleotide synthetic structures.

[0016] Figure 1C Depicted is the roll-to-roll arrangement of a polynucleotide synthetic structure.

[0017] Figure 1D A plate configured for polynucleotide synthesis is shown, comprising 24 regions or subdomains, each having an array of 256 clusters.

[0018] Figure 1E Shows Figure 1D A close-up view of the subfield in , which has 16×16 clusters, each with 121 individual seats.

[0019] Figure 1F Shows Figure 1D Detailed view of a cluster in , where the cluster has 121 seats.

[0020] Figure 2 A schematic diagram of the generation of a polynucleotide library from cluster amplification is depicted.

[0021] Figure 3 A polynucleotide synthetic material deposition apparatus is depicted.

[0022] Figure 4 The polynucleotide synthesis workflow is depicted.

[0023] Figure 5 Depicts a computer system.

[0024] Figure 6 A block diagram illustrating the architecture of a computer system is depicted.

[0025] Figure 7 A network is depicted that is configured to incorporate multiple computer systems, multiple cell phones and personal data assistants, and network attached storage (NAS).

[0026] Figure 8 A multiprocessor computer system using a shared virtual address storage space is shown.

[0027] Fig. 9A Depicted are the block deletion rates and single base deletion rates between clusters on the surface for polynucleotides synthesized using acetone wash condition A.

[0028] Fig. 9B Depicted are the block deletion rates and single base deletion rates between clusters on the surface for polynucleotides synthesized using wash condition B with acetonitrile.

[0029] Fig. 9C Depicted are the single base deletion rates between clusters on the surface for polynucleotides synthesized using acetone wash condition A. Darker shaded circles indicate higher deletion rates.

[0030] Fig.9D Depicted are the single base deletion rates between clusters on the surface for polynucleotides synthesized using acetonitrile wash condition B. Darker shaded circles indicate higher deletion rates.

[0031] Fig.10 Depicted are the overall error rates for three different sets of samples determined using Sanger sequencing.

[0032] Fig.11A Polynucleotide error rates by error class (deletion, insertion, or mismatch) determined using next generation sequencing (NGS) are depicted.

[0033] Fig. 11B Depicted are polynucleotide error rates for various substitutions determined using next generation sequencing (NGS).

[0034] Fig. 11C Depicted are the polynucleotide error rates of deletions determined using NGS.

[0035] Fig.11D Depicted are the polynucleotide error rates of insertions determined using NGS.

[0036] Fig.11E Plotted are the error rates for various error categories (insertions, mismatches, single deletions, and block deletions) at different base positions on a synthetic polynucleotide as determined using NGS. The base at the first position is farthest from the synthesis surface and is therefore added last to the synthetic polynucleotide chain.

[0037] Fig. 12A Depicted are the representation uniformity, dropout rate, and total reads of synthetic polynucleotides at different locations on the synthetic surface. Lighter shaded squares on the surface represent lower average read counts, and darker shaded squares represent higher average read counts.

[0038] Fig. 12B The representation uniformity of the synthetic polynucleotide library is depicted.

[0039] Fig. 12C Depicted are the representation uniformity of synthetic polynucleotide libraries as a function of clusters (upper panel) and devices (lower panel). Different levels of shading represent individual clusters (upper panel) or devices (lower panel). DETAILED DESCRIPTION

[0040] Provided herein are systems, methods and compositions for efficient de novo synthesis of highly accurate and uniform polynucleotide libraries. Further provided herein are methods comprising washing with a solvent after an oxidation step, wherein the solvent dissolves a major reagent or a reagent byproduct of the oxidation step.

[0041] definition

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these inventions belong.

[0043] Throughout the present disclosure, numerical features are given in range format. It should be understood that the description of the range format is only for convenience and simplicity, and should not be interpreted as a hard limit to the scope of any embodiment. Therefore, unless the context clearly stipulates otherwise, the description of the range should be considered to clearly disclose all possible sub-ranges and each numerical value accurate to one-tenth of the lower limit unit in the range. For example, the description of the range such as from 1 to 6 should be considered to have clearly disclosed such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and each value in the range, for example, 1.1, 2, 2.3, 5 and 5.9. Regardless of the width of the range, this is applicable. The upper and lower limits of these intermediate ranges can be independently included in a smaller range, and are also included in the present invention, but are subject to any clearly excluded limit in the range. Unless the context clearly stipulates otherwise, in the case where the range includes one or both of the limits, the scope of excluding any one or both of these included limits is also included in the present invention.

[0044] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit any embodiment. Unless the context clearly stipulates otherwise, the singular forms "one", "a kind of" and "the" as used herein are also intended to include plural forms. It should be further understood that the terms "include" and / or "comprising" refer to the presence of the features, integers, steps, operations, elements and / or components when used in this specification, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or their groups. As used herein, the term "and / or" includes any and all combinations of one or more related listed items.

[0045] Unless otherwise specified or apparent from the context, as used herein, the term "about" with respect to a number or numerical range should be understood to mean the stated number and + / - 10% thereof, or, for values ​​listed in a range, 10% below the listed lower limit to 10% above the listed upper limit.

[0046] As used herein, the terms "preselected sequence", "predefined sequence" or "predetermined sequence" are used interchangeably. These terms mean that the sequence of a polymer is known and selected prior to the synthesis or assembly of the polymer. In particular, aspects of the invention are described herein primarily with respect to the preparation of polynucleotides, and the sequence of the oligonucleotides or polynucleotides is known and selected prior to the synthesis or assembly of the polynucleotides.

[0047] The methods, systems, devices, and compositions described herein in various aspects include contacting a surface with a solvent or solvent mixture, which is variously described as a "wash," "wash solvent," "wash buffer," "bath," "cleaning solvent," or "rinse." In some cases, a wash step is used to push, rinse, purge, remove, exchange, or replace other reagent solutions (including unreacted reagents (active ingredients), solvents, or reagent byproducts (chemical products resulting from reagent reactions) or previous wash solvents that are in contact with the surface. In some cases, residual reagent solution or solvent is used in the step immediately preceding the wash step.

[0048] The term "functional equivalent" used herein with respect to solvents describes alternative solvents or solvent mixtures with similar properties. In some cases, these properties are physical properties (including, for example, boiling point, melting point, heat of vaporization, viscosity, miscibility, solubility, density, purity or other physical properties). Similar properties also optionally include performance metrics, for example, when used together with the methods, systems, compositions and devices described herein, solvents and functional equivalents all provide similar results (reduced error rates, increase in error rate uniformity, dissolution of reagents or reagent byproducts, or other results).

[0049] Provided herein are methods and compositions for producing synthetic (i.e., de novo synthesized or chemically synthesized) polynucleotides. Throughout the text, the terms oligonucleotide, oligonucleotide, and polynucleotide are defined as synonyms. The library of synthetic polynucleotides described herein may include multiple polynucleotides that jointly encode one or more genes or gene fragments. In some cases, the polynucleotide library includes coding sequences or non-coding sequences. In some cases, the polynucleotide library encodes multiple cDNA sequences. The reference gene sequence based on the cDNA sequence may contain introns, while the cDNA sequence does not contain introns. The polynucleotides described herein may encode genes or gene fragments from organisms. Exemplary organisms include, but are not limited to, prokaryotes (e.g., bacteria) and eukaryotes (e.g., mice, rabbits, humans, and non-human primates). In some cases, the polynucleotide library includes one or more polynucleotides, each of which encodes the sequence of multiple exons. Each polynucleotide in the library described herein may encode different sequences, i.e., non-identical sequences. In some cases, each polynucleotide in the library described herein includes at least one portion complementary to the sequence of another polynucleotide in the library. Unless otherwise specified, the polynucleotide sequences described herein may comprise DNA or RNA.

[0050] Provided herein are methods and compositions for producing synthetic (i.e., de novo synthesized) genes. Libraries comprising synthetic genes can be constructed by a variety of methods further described in detail in other parts of this paper, such as PCA, non-PCA gene assembly methods or hierarchical gene assembly, so that two or more double-stranded polynucleotides are combined ("stitching") to produce larger DNA units (i.e., chassis). The library of a large construct can include polynucleotides having a length of at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb or longer. Large constructs can be constrained by the upper limit of about 5000, 10000, 20000 or 50000 base pairs selected independently. Synthesis of any number of nucleotide sequences encoding polypeptide segments, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins such as antibodies, polypeptide segments from other protein families, including non-coding DNA or RNA, such as regulatory sequences, e.g., promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA) (which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification); DNA molecules synthesized or produced by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The cDNA encoding the gene or gene fragment mentioned herein may include at least one region encoding an exon sequence without the intervening intron sequence found in the corresponding genomic sequence. Alternatively, the corresponding genomic sequence of the cDNA may initially lack an intron sequence.

[0051] De novo polynucleotide synthesis

[0052] Provided herein are methods for synthesizing polynucleotides, the methods generally comprising a series of repeated steps of: applying a protected monomer to a surface of a substrate feature to connect to the surface, a linker, or to a previously deprotected monomer; deprotecting the applied monomer so that it can react with a subsequently applied protected monomer; and applying another protected monomer for connection. One or more intermediate steps include oxidation and / or sulfurization. Optionally, the intermediate step includes a capping step to seal previously deprotected monomers that have not reacted with the protected monomer. Further provided herein are methods comprising one or more washing steps using a solvent or solvent mixture (e.g., a washing preparation), the steps being after at least one or all polynucleotide synthesis steps such as an oxidation step, wherein the solvent or solvent mixture dissolves the main reagent or reagent byproduct of the previous step.

[0053] An exemplary synthesis scheme for synthesizing polynucleotides in the 3' to 5' direction is shown in Figure 1A In step 0, the surface is functionalized with blocked nucleosides, and then deblocked (not shown) to provide unblocked nucleosides attached to the surface. The unblocked nucleosides are then washed 101 with a washing solvent. In step 1, the phosphoramidite nucleosides of 5'DMT protection are coupled with unblocked nucleosides on the surface to form phosphites, thereby producing polynucleotides extending one base. The surface is then washed 102 with a washing solvent, and a capping agent (or multiple agents) is added to block all unreacted 5'OH groups on the surface with acetate groups. The surface is washed after capping 103, and further oxidized in step 3 to generate phosphate. The phosphate is washed 104 with a washing solvent, and deblocked in step 4 by removing the DMT group to generate unblocked polynucleotides. Unblocked polynucleotides are washed 101 with a washing solvent, and steps 1-4 are repeated to synthesize polynucleotides. Alternatively, the polynucleotide is synthesized in the 5' to 3' direction, wherein a 3' blocked phosphoramidite nucleotide is coupled to the 3' OH position of the growing polynucleotide chain.

[0054] Further provided herein is a method comprising a washing step performed before or after another step in the de novo synthesis of polynucleotides, for example, after surface preparation, after phosphoramidite coupling, after oxidation, after capping or after deblocking. The washing step is generally used to remove reagents, solutions, reaction byproducts or solvents remaining in the previous synthesis step or the previous washing. For example, in an oxidation step comprising iodine and an amine base, the subsequent washing step comprises a solvent that dissolves the main component of the reagent (remaining iodine or amine base) or the main byproduct of the reagent (iodide salt of the amine base). The washing step is generally performed before one or more steps, for example, before surface preparation, before phosphoramidite coupling, before oxidation, before capping or before cutting. Multiple washing steps are often used in the polynucleotide synthesis process, for example, multiple washing steps separated by additional step synthesis steps or sequential washing steps. In some cases, the washing step is performed after the deblocking step. In some cases, the washing step is performed after the coupling step. In some cases, the washing step is performed after the oxidation step. In some cases, the washing step is performed after the capping step. In some cases, the washing step is performed after the deblocking step, the coupling step, the oxidation step, and the capping step. In some cases, the washing step comprises washing with a solvent or a solvent mixture. For a given method, in some cases, a single solvent or a plurality of different solvents or solvent mixtures are used for each individual washing step, or for all washing steps in the method. In some cases, the washing step between the synthesis steps is optionally omitted. In some cases, the washing step is performed using a washing solvent comprising one or more solutes.

[0055] Further provided herein is a method in which, after adding nucleoside phosphoramidites, and optionally after capping and one or more washing steps, the polynucleotides bound to the substrate are oxidized with an oxidation solution. The oxidation step includes oxidation of a phosphite triester to a tetracoordinated phosphotriester, a protected precursor of a naturally occurring phosphodiester nucleoside interlink. The oxidation solution typically comprises one or more chemical components. For example, the oxidation solution comprises components such as one or more solvents (such as acetonitrile, acetone, THF or other solvents) and one or more oxidants or catalysts as active components or reagents. The oxidant variously comprises I2, peroxides (such as hydrogen peroxide, mCPBA, TBHP, etc.), dioxirane or other oxidants known in the art that can oxidize phosphotriesters. Oxidation is sometimes carried out under anhydrous conditions using tert-butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some cases, the main component (by mass) of the oxidation solution is a solvent or a solvent mixture. In some cases, the main reagent of the oxidation solution is iodine. In some cases, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base catalyst such as an amine base (e.g., pyridine, lutidine, collidine, N-methylmorpholine or other amine bases) or other weak bases.

[0056] Washing with one or more solvents is generally used to remove the oxidant or oxidation byproducts remaining after the oxidation step. Without being bound by theory, the solvent or solvent mixture increases the solubility of the oxidant in some cases, increases the solubility of the byproducts produced by the oxidant after oxidation, or in some cases, reacts with the solvent to form different oxidants. Alternatively or in combination, in some cases, the solvent or solvent mixture increases the dissolution rate of the oxidant, or increases the dissolution rate of the byproducts produced by the oxidant after oxidation. The oxidation byproducts include salts, salts of weak bases (such as amines), iodide salts or the reaction products of oxidants and solvents. In some cases, solvents that dissolve pyridinium iodide or other amine alkali salts of iodine are used as washing solvents after oxidation. In some cases, the oxidation byproducts include iodine salts of pyridine, lutidine, collidine, N-methylmorpholine or other weak bases. In some cases, the choice of solvent depends on the choice of oxidant; for example, the oxidation solution includes oxidant I2 and a solvent containing acetone.

[0057] The reaction may occur at the O6 oxygen atom of guanosine and the O4 oxygen atom of thymidine / uridine nucleotides. The resulting activated nucleotide derivatives may be further modified by oxidation and nucleophilic aromatic substitution using additional reagents. In some cases, the activated nucleotide derivatives may be further modified with reagents used in capping such as N-methylimidazole or DMAP. In some cases, the activated nucleotide derivatives may be further modified with reagents used in oxidation such as pyridine. In some cases, the activated nucleotide derivatives may be further modified with reagents used in deprotection such as methylamine, ammonia or ethylenediamine. The product obtained after oxidation and capping may also react with amines used for deprotection to further generate unwanted products. The reaction between phosphoramidites and O-containing nucleotides may produce N-substituted guanosine and thymidine / uridine nucleotides, which may form Watson-Crick base pairs with thymidine / uridine and guanosine nucleotides, respectively. The ultimate consequence of undesirable reactivity is G→A and T / U→C mutations.

[0058] Further provided herein is a washing solution, which removes unwanted G→A and T / U→C mutations produced by phosphoramidite coupling to core bases by directly selectively removing unwanted adducts after the coupling step and before the oxidation step. In some cases, the washing solution improves the synthesis uniformity (such as yield, error rate or other performance indicators) on a solid support (such as a chip). In some cases, the washing solution of the present disclosure comprises a solvent, a base and an O-nucleophile. In some cases, the solvent is acetonitrile or THF. In some cases, the base is pyridine, lutidine or collidine. In some cases, the O-nucleophile is water or an organic acid (such as acetic acid, formic acid, propionic acid). In some cases, the O-nucleophile is water. In some cases, the washing solution comprises THF, pyridine, water or a combination thereof. In some cases, the reagent concentration in such washing solutions is measured by the ratio of various components. In some cases, the washing solution includes a certain volume ratio. In some cases, the wash solution comprises a solvent, a base, and an O-nucleophile in a ratio of about 95:3:1, 90:7:3, 80:10:10, 80:20:10, 85:10:5, or 70:20:10. In some cases, the wash solution comprises a solvent, a base, and an O-nucleophile in a ratio of 60-90% solvent, 5-30% base, and 5-30% O-nucleophile. In some cases, the wash solution comprises a solvent, a base, and an O-nucleophile, wherein the ratio of base to O-nucleophile is at least about 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 7:1, 9:1, 10:1, 12:1, or 15:1. In some cases, the washing solution comprises a solvent, a base and an O-nucleophile, wherein the ratio of the base to the O-nucleophile is about 0.5: 1-15: 1, 0.5: 1-3: 1, 1: 1-5: 1, 1: 1-9: 1 or 2: 1-12: 1. In some cases, the concentration of the reagent is measured by molar concentration (M = mol / L). In some cases, the concentration of the O-nucleophile is about 0.01M, 0.02M, 0.05M, 0.08M, 0.1M, 0.2M, 0.5M, 0.8M, 1.0M, 1.2M, 1.2M, 1.5M, 1.8M, 2M, 2.5M or about 3M. In some cases, the O-nucleophile concentration is no more than 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or no more than 3 M. In some cases, the O-nucleophile concentration is at least 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or at least 3 M.In some cases, the O-nucleophile concentration is 0.01-0.1 M, 0.01-0.5 M, 0.01-1.5 M, 0.5-2 M, 0.5-1.0 M, 0.2-1.2 M, 0.8-2.0 M, or 0.5-1 M. In some cases, the base concentration is about 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or about 3 M. In some cases, the base concentration is no more than 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or no more than 3 M. In some cases, the base concentration is at least 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or at least 3 M. In some cases, the base concentration is 0.01-0.1 M, 0.01-0.5 M, 0.01-1.5 M, 0.5-2 M, 0.5-1.0 M, 0.2-1.2 M, 0.8-2.0 M, or 0.5-1 M.

[0059] Provided herein is a chemical polynucleotide synthesis method, wherein the washing solvent after the oxidation step includes a solvent with high solubility for the oxidant I2 or an iodine salt. Table 1 lists exemplary solvents with high solubility for the oxidant I2 or an iodine salt. Provided herein is a chemical polynucleotide synthesis method, wherein the washing solvent after the oxidation reaction includes the solvents listed in Table 1. In some cases, the washing step after the oxidation reaction includes the use of a solvent comprising THF, acetone or other solvents described herein. In some cases, the washing step after the oxidation reaction includes the use of a solvent comprising acetone. In some cases, the washing step after the oxidation reaction includes the use of a solvent comprising a mixture of acetone and THF. In some cases, the same solvent selected as the washing solution after the oxidation reaction is also used as a washing reagent after the additional reaction in the chemical synthesis workflow. In some cases, at least one washing step comprising THF, acetone or another solvent described herein is used after the oxidation reaction, and all other washing steps in the chemical polynucleotide synthesis workflow include the deposition of acetonitrile. Methods are further described herein, wherein the washing solvent is used for its ability to dissolve iodine or iodine salts. Table 1 lists exemplary solubility of iodine at various temperatures in various solvents used for the methods described herein.

[0060] Table 1. Solvents and relative solubility of iodine

[0061]

[0062] *Note that temperatures in degrees Celsius are in parentheses.

[0063] Methods are further described herein, wherein coupling includes the use of protected nucleoside phosphoramidites, such as base-protected nucleoside phosphoramidites. In some cases, the phosphoramidite structural unit causes unwanted reactions at the nucleobase, resulting in branching and mutations. In some cases, methods and reagents block the reaction of guanosine and thymidine / uridine nucleotides with activated phosphoramidites. After activation, phosphoramidites are highly reactive (electrophilic) reagents, and due to the strength of the resulting PO bond, they preferentially react with oxygen nucleophiles. During the reaction with the 5'-OH group of the growing nucleic acid chain, the desired chain extension reaction occurs. However, to a certain extent, this reaction also occurs at the oxygen atoms O6 and O4 of guanosine and thymidine / uridine nucleotides, respectively, in some cases. The activated nucleotide derivatives obtained can be further modified at least in part by oxidation and nucleophilic aromatic substitution with various amines (e.g., methylamine, ammonia or ethylenediamine) used in N-methylimidazole (for capping), DMAP (also used for capping), pyridine (for oxidation) and deprotection. In some cases, the product obtained after oxidation and capping also reacts with various amines used for deprotection. Phosphoramidites and the above-mentioned reaction containing O nucleotides usually result in that the guanosine and thymidine / uridine nucleotides substituted by N- can form Watson-Crick base pairs with thymidine / uridine and guanosine nucleotides respectively. In the context of duplex formation, the results are respectively obvious G->A and T / U->C mutations. In the case of preparing high-quality (e.g., low error rate) polynucleotides, a minimum number of such mutations is required. Nucleic acid synthesis in flow cell is usually carried out using extremely excessive activated phosphoramidites. As a result, only these expensive reagents of negligible amount are actually consumed. In order to improve efficiency and to minimize chemical waste, it is necessary to use these activated phosphoramidite solutions in continuous flow cell reactions in some cases. However, due to the above-mentioned side reactions that activated G and T / U phosphoramidites can occur, the longer the time that these solutions are exposed to the growing polynucleotides, the more branches and mutations of increasing amounts will be introduced in some cases. In some cases, this reactivity prevents the use of multiple coupling / phosphoramidite recovery strategies that save money and waste in high-quality nucleic acid synthesis. In some cases, undesirable side reactions are partially or completely suppressed by attaching a stable protecting group during polynucleotide synthesis, but can be cut at the end, preferably under the same conditions as other protecting groups present in the main synthetic product (protected and fixed polynucleotide). In some cases, protected nucleoside phosphoramidite bases include protected nitrogen atoms. In some cases, protected nucleoside phosphoramidite bases include cytosine, adenine, thymine, uracil or guanine. In some cases, protected nucleoside phosphoramidite bases include thymine or guanine. Exemplary protected dT and dG phosphoramidite structural units AI containing protecting groups at nitrogen atom N3 and N1 are shown below:

[0064]

[0065] Methods are further described herein, wherein after coupling, the phosphoramidite polynucleotide synthesis method includes a capping step. In the capping step, the growing polynucleotide is treated with a capping agent. The capping step is generally used to block unreacted 5'-OH groups bound to the substrate after coupling to prevent further chain extension, thereby preventing the formation of polynucleotides with internal base deletions. In addition, phosphoramidites activated with 1H-tetrazoles generally react with the O6 position of guanosine to a small extent. Without being bound by theory, after oxidation with I2 / water, the byproduct (possibly via O6-N7 migration) undergoes depurination. Apurinic sites can terminate in the final deprotection process of the polynucleotide and be cut, thereby reducing the yield of the full-length product. The O6 modification can be removed by treatment with a capping agent before oxidation with I2 / water. In some cases, including a capping step in the polynucleotide synthesis process reduces the error rate compared to synthesis without capping. As an example, the capping step includes treating the polynucleotide bound to the substrate with a mixture of acetic anhydride and 1-methylimidazole. After the capping step, the substrate is optionally washed. The capping reaction in DNA synthesis prevents the uncoupled DNA fragments from being further extended in the subsequent coupling step, thereby inhibiting the formation of n-1 polymer sequences. The capping reaction is carried out by coupling reactive phosphoramidites ("Unicap") or by reacting carboxylic anhydrides in the presence of a base and an activator. In some cases, the carboxylic anhydride is acetic anhydride. In some cases, the base is lutidine. In some cases, the activator is N-methylimidazole or DMAP. Since the O6 and O4 oxygen atoms of guanosine and thymidine nucleotides are electrophilically activated by acylation, capping by reacting carboxylic anhydrides in the presence of a base and an activator can lead to G→A and T→C mutation reactions. The resulting activated nucleotide derivatives can then be further modified by nucleophilic aromatic substitution with N-methylimidazole or DMAP. The further modified products can be reacted with various amines for deprotection, resulting in the formation of N-substituted guanosine and thymidine nucleotides, which are capable of forming Watson-Crick base pairs with thymidine and guanosine nucleotides (ie, G→A and T / U→C mutations).

[0066] Further provided herein is a capping formulation that inhibits the formation of unwanted G→A and T→C mutations caused by the activation of nucleobases promoted by carboxylic acids. The capping formulation of the present disclosure comprises a solvent, a base, an anhydride and a nucleophile. In some cases, the capping formulation comprises a solvent, a base, an anhydride and an O-nucleophile. In some cases, the capping formulation of the present disclosure comprises a solvent, such as acetonitrile or THF. In some cases, the capping formulation of the present disclosure comprises a base, such as lutidine or collidine. In some cases, the capping formulation of the present disclosure comprises an anhydride, such as acetic anhydride. In some cases, the capping formulation of the present disclosure comprises an activator, such as N-methylimidazole or DMAP. In some cases, the capping formulation of the present disclosure comprises an O-nucleophile, such as acetic acid, methoxyacetic acid or phenoxyacetic acid. The capping solution typically comprises components such as one or more solvents (such as acetonitrile, acetone, THF or other solvents), one or more capping agents and one or more activators (N-methylimidazole or other activators known in the art). Capping agents include acyl halides (e.g., acetyl chloride or other acyl halides), anhydrides (e.g., acetic anhydride or other anhydrides) or other capping agents known in the art that can react with the 5'OH of unprotected nucleobases. In some cases, the main component (by mass) of the capping solution is a solvent or a solvent mixture. In some cases, after the capping step, one or more washings are performed using one or more solvents to remove the remaining capping agent or activator. Without being bound by theory, the solvent or solvent mixture increases the solubility of the capping agent or activator in some cases, increases the solubility of the by-products generated by the capping agent, activator or other reagents, or reacts with the capping agent or activator to form different capping or activating agents in some cases. Alternatively or in combination, in some cases, the solvent or solvent mixture increases the dissolution rate of the capping agent or activator, or increases the dissolution rate of the by-products generated by the capping agent or activator. In some cases, the choice of solvent depends on the choice of the capping agent or activator; for example, the capping solution includes acetic anhydride and a solvent containing tetrahydrofuran. In some cases, any combination of capping agents, activators or other reagents and solvents is used with the methods described herein. In some cases, two or more capping solutions are prepared and then mixed during the polynucleotide synthesis process during the capping step. In some cases, the first capping solution comprises a solvent and an activator. In some cases, the first capping solution comprises acetonitrile / n-methylimidazole in a ratio of 90:10. In some cases, the second capping solution comprises a solvent, a base and a capping agent. In some cases, the second capping solution comprises THF / lutidine / acetic anhydride in a ratio of 80:10:10. In some cases, the first capping solution and the second capping solution are mixed together during the capping step. In some cases, the capping solution comprises a certain volume ratio.In some cases, the capping solution comprises a solvent, a base, and a capping agent in a ratio of about 95:3:1, 90:7:3, 80:10:10, 80:20:10, 85:10:5, or 70:20:10. In some cases, the wash solution comprises a solvent, a base, and a capping agent in a ratio of 60-90% solvent, 5-30% base, and 5-30% capping agent. In some cases, the wash solution comprises a solvent, a base, and a capping agent, wherein the ratio of base to capping agent is at least about 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 7:1, 9:1, 10:1, 12:1, or 15:1. In some cases, the wash solution comprises a solvent, a base, and a capping agent, wherein the ratio of base to capping agent is about 0.5: 1-15: 1, 0.5: 1-3: 1, 1: 1-5: 1, 1: 1-9: 1, or 2: 1-12: 1. In some cases, the concentration of the reagent is measured by molarity (M = mol / L). In some cases, the O-nucleophile concentration is about 0.01M, 0.02M, 0.05M, 0.08M, 0.1M, 0.2M, 0.5M, 0.8M, 1.0M, 1.2M, 1.2M, 1.5M, 1.8M, 2M, 2.5M, or about 3M. In some cases, the O-nucleophile concentration is no more than 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or no more than 3 M. In some cases, the O-nucleophile concentration is at least 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or at least 3 M. In some cases, the O-nucleophile concentration is 0.01-0.1 M, 0.01-0.5 M, 0.01-1.5 M, 0.5-2 M, 0.5-1.0 M, 0.2-1.2 M, 0.8-2.0 M, or 0.5-1 M. In some cases, the base concentration is about 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or about 3 M. In some cases, the base concentration is no more than 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or no more than 3 M.In some cases, the base concentration is at least 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.2 M, 1.2 M, 1.5 M, 1.8 M, 2 M, 2.5 M, or at least 3 M. In some cases, the base concentration is 0.01-0.1 M, 0.01-0.5 M, 0.01-1.5 M, 0.5-2 M, 0.5-1.0 M, 0.2-1.2 M, 0.8-2.0 M, or 0.5-1 M. In some cases, the activator concentration is about 0.001 M, 0.002 M, 0.005 M, 0.008 M, 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.12 M, 0.15 M, 0.18 M, 0.2 M, 0.3 M, or about 0.5 M. In some cases, the activator concentration is at least 0.001 M, 0.002 M, 0.005 M, 0.008 M, 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.12 M, 0.15 M, 0.18 M, 0.2 M, 0.3 M, or at least 0.5 M. In some cases, the activator concentration is no more than 0.001M, 0.002M, 0.005M, 0.008M, 0.01M, 0.02M, 0.05M, 0.08M, 0.1M, 0.12M, 0.15M, 0.18M, ​​0.2M, 0.3M, or no more than 0.5M. In some cases, the activator concentration is 0.001-0.01M, 0.001-0.05M, 0.001-0.15M, 0.05-0.2M, 0.005-0.02M, 0.01-0.1M, 0.08-0.2M, or 0.05-0.1M. In some cases, the capping step is performed after oxidation. In some cases, the capping step is performed before oxidation. In some cases, the capping step is performed before and after oxidation. In some methods, a washing step is performed after oxidation. The second capping step allows the substrate to dry, as residual water from oxidation that may persist can inhibit subsequent coupling. After oxidation, the substrate and growing polynucleotides are optionally washed. In some cases, the oxidation step is replaced with a sulfurization step to obtain polynucleotide thiophosphates, wherein any capping step can be performed after sulfurization. Many reagents are capable of effective sulfur transfer, including but not limited to 3-(dimethylaminomethylene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiolan-3-one 1,1-dioxide (also known as Beaucage reagent) and N,N,N'N'-tetraethylthiuram disulfide (TETD).

[0067] Further provided herein is a method for an extended step including using a coupling solution, wherein the coupling solution comprises one or more chemical components. For example, the coupling solution comprises components such as one or more solvents (such as acetonitrile, acetone, THF or other solvents), one or more monomers and one or more activators (tetrazole or other activators known in the art). Monomers include phosphoramidite nucleosides, chlorophosphites, H-phosphonates, phosphodiesters, phosphotriesters or other activated nucleosides known in the art that can react with unprotected monomers. In some cases, the main component (by mass) of the coupling solution is a solvent or a solvent mixture. In some cases, after the coupling step, one or more solvents are used to wash once or multiple times to remove remaining monomers, activators or by-products. Without being bound by theory, solvents or solvent mixtures can increase the solubility of monomers or activators in some cases, increase the solubility of by-products generated by coupling reagents (monomers, activators, other reagents), or react with solvents to form different coupling agents or activators in some cases. Alternatively or in combination, in some cases, the solvent or solvent mixture increases the dissolution rate of the monomer or activator, or increases the dissolution rate of the by-products generated by the monomer or activator. In some cases, the choice of solvent depends on the choice of monomer or activator; For example, the coupling solution comprises a monomer phosphoramidite and a solvent containing tetrahydrofuran. In some cases, any combination of coupling reagents (monomers, activators or other reagents) and solvents is used together with the systems, methods and compositions described herein. The polynucleotide synthesis method used herein generally includes 1, 2, 3 or more continuous coupling steps.

[0068] Prior to coupling, the nucleosides bound to the substrate are typically deprotected by removing a protecting group, which serves to prevent polymerization. In some cases, failure to fully remove one or more protecting groups may result in errors in the polynucleotide synthetic product. In some cases, the coupling step is repeated twice or more without removing the protecting group. Additional steps include, but are not limited to, capping, oxidation, or cutting.

[0069] Further provided herein is a method for removing the blocking (or deprotection) step including the use of a blocking solution, wherein the blocking solution comprises one or more chemical components. The blocking solution is generally used to remove the protecting group on the 5'OH of a polynucleotide or nucleotide. The 5'OH protecting group is well known in the art, and in some cases includes trityl, DMT (4,4'-dimethoxytrityl) or other protecting groups (including triarylmethyl, triphenylmethyl or other groups), wherein the removal of the protecting group will not otherwise cut or modify the polynucleotide. In some cases, the blocking solution includes components such as one or more solvents (such as acetonitrile, acetone, THF, toluene or other solvents) and one or more blocking agents. The blocking agent includes acid (trifluoroacetic acid or other acid), alkali, light, heat, enzyme or other reagents known in the art that can remove the 5'OH protecting group. In some cases, the main component (by mass) of the blocking solution is a solvent or a solvent mixture. In some cases, after the blocking step, one or more solvents are used to wash once or multiple times to remove the remaining blocking agent. Without being bound by theory, the solvent or solvent mixture in some cases increases the solubility of the deblocking reagent, increases the solubility of the byproduct (cleaved protecting group) generated by the deblocking reagent, or reacts with the solvent to form a different deblocking reagent in some cases. Alternatively or in combination, in some cases, the solvent or solvent mixture increases the dissolution rate of the deblocking reagent, or increases the dissolution rate of the byproduct (cleaved protecting group or other byproduct) generated by the deblocking reagent. In some cases, the choice of solvent depends on the choice of the deblocking reagent or the 5' protecting group; for example, the deblocking solution comprises trifluoroacetic acid and a toluene-containing solvent. Any combination of deblocking reagents, protecting groups, and solvents are used in some cases with the methods described herein.

[0070] In order to make subsequent nucleosides incorporate circulation and occur through coupling, the protected 5 ' end of the polynucleotide of growth combined with substrate must be removed so that the primary hydroxyl can react with the next nucleoside phosphoramidite. In some cases, the protecting group is DMT, and the trichloroacetic acid used in dichloromethane removes blocking. Carrying out the detritylation of extended time or using the acid solution stronger than the acid solution recommended to carry out detritylation can cause the depurination of the polynucleotide combined with solid support to increase, and therefore reduce the productive rate of required full-length product. Method and composition described herein provide controlled de-blocking conditions, thereby limit undesirable depurination reaction. In some cases, the polynucleotide combined with substrate is washed after de-blocking. In some cases, the effective washing after de-blocking helps the synthetic polynucleotide to have the error rate and / or higher productive rate of reduction.

[0071] Exemplary combinations of wash steps and solvents are provided in Table 2, but are not limited thereto, wherein each different wash combination is provided by a different number ("number") reading from left to right in the table.

[0072] Table 2. Exemplary solvent wash steps during de novo polynucleotide synthesis.

[0073]

[0074] The polynucleotides bound to the surface or support can be fixed by their 3' end. It should be understood that the so-called 3' end means a sequence located downstream of the 5' end, for example, 2, 3, 4, 5, 6, 7, 10, 15, 20 or more nucleotides downstream of the 5' end, for example, on the 3' half, one-third or one-quarter of the sequence, for example, less than 2, 3, 4, 5, 6, 7, 10, 15 or 20 nucleotides away from the absolute 3' end; the so-called 5' end means a sequence located upstream of the 3' end, for example, 2, 3, 4, 5, 6, 7, 10, 15, 20 or more nucleotides upstream of the 3' end, for example, on the 5' half, one-third or one-quarter of the sequence, for example, less than 2, 3, 4, 5, 6, 7, 10, 15 or 20 nucleotides away from the absolute 5' end. For example, polynucleotides can be fixed on a support by nucleotide sequences (for example, degenerate binding sequences), connectors or spacers (for example, parts that do not participate in hybridization). In some cases, connectors or spacers comprising nucleosides are homogeneous for single bases. In some cases, connectors or spacers comprising nucleosides are heterogeneous for single bases. In some embodiments, polynucleotides include spacers or connectors to separate polynucleotide sequences from support. Useful spacers or connectors include photo-cleavable connectors or other traditional chemical connectors. In one embodiment, polynucleotides can be attached to a solid support by a cleavable linking portion. For example, solid support can be functionalized to provide a cleavable linker for covalent attachment to polynucleotides. The linker portion can be the length of six or more atoms. Alternatively, the cleavable portion can be in a polynucleotide and can be introduced during in situ synthesis. Suitable cleavable portions can be selected to match the properties of the protecting groups of nucleoside bases, the selection of solid supports and / or the mode of reagent delivery, etc. In an exemplary embodiment, the polynucleotide cut from the solid support contains a free 3'-OH end. Alternatively, after the polynucleotide is cut, the free 3'-OH end can also be obtained by chemical or enzymatic treatment. In multiple embodiments, the present invention relates to methods and compositions for releasing polynucleotides bound to a support or surface into a solution. The cleavable portion can be removed under conditions that will not degrade the polynucleotide. The linker can be cut in two ways: simultaneously under the same conditions as the deprotection step, or after the deprotection step is completed, different conditions or reagents for linker cutting are used and then carried out. Optionally, a capping step is used after any step of the linker / spacer synthesis to prevent additional functionalization of unreacted linkers or spacers. In some cases, two or more capping steps are used during the synthesis of the polynucleotide spacer.

[0075] Provided herein are methods in which a spacer is synthesized by repetition of the following steps: 1) extending a plurality of reactive molecules from a surface by contacting the surface with a base addition solution comprising at least one reactive monomer, wherein the at least one reactive monomer comprises a phosphoramidite nucleoside comprising a 5' protecting group; 2) capping unreacted 5'OH groups of the polynucleotide by contacting the surface with a capping solution; 3) washing the surface at least once with a washing solvent; 4) contacting the surface with an oxidizing solution; 5) washing the surface at least once with a washing solvent; 6) capping unreacted 5'OH groups of the polynucleotide by contacting the surface with a capping solution; 7) removing 5' blocking groups with a deblocking solution; and 8) washing the surface at least once with a washing solvent. Steps 1-8 are repeated in some cases until a plurality of polynucleotide spacers are synthesized. The washing solvent may be a washing solvent described elsewhere herein.

[0076] Provided herein are methods in which one or more capping steps are used to prevent subsequent reactions with unreacted hydroxyls. The capping step is performed in various ways before or after any step of the polynucleotide synthesis described herein. Typically, the capping step is followed by a washing step comprising depositing a washing solvent on the synthesis surface. For example, the capping step is followed by washing with a washing solvent comprising acetone. In some cases, the oxidation step and the capping step are followed by washing with a washing solvent, such as a washing solvent comprising acetone.

[0077] Exemplary combinations of capping steps, wash steps, and solvents are provided in Table 3, but are not limited thereto, wherein each different reaction step and wash combination is provided by a different number ("number") reading from left to right in the table.

[0078] Table 3. Exemplary inclusion of capping steps during de novo polynucleotide synthesis.

[0079]

[0080] Solvents

[0081] Provided herein are methods, systems, compositions and devices for chemical polynucleotide synthesis, including the use of solvents or solvent mixtures. In some cases, solvents or their functional equivalents are used as washing solvents. In some cases, solvents or their functional equivalents are used as reaction solvents. In some cases, based on common intrinsic properties (density, heat capacity, solubility, polarity, miscibility, boiling point, melting point, viscosity, chemical structure or other physical properties) or performance characteristics (ability to dissolve specific reagents or salts, reduction in error rate, chemical reagent degradation resistance, heat mixed with previously washed solvents or reagent solutions, or other performance results) select suitable solvents and solvent mixtures. In the washing step after any step of polynucleotide synthesis, for example, after deblocking, extension, oxidation, capping or any combination thereof, washing solvents are used in different ways.

[0082] Exemplary solvents are further described herein and include hydrocarbons (e.g., hexane, decane, benzene, toluene, xylene, isomers thereof, etc.), ethers (e.g., THF, diethyl ether, methyl tert-butyl ether, etc.), esters (e.g., methyl acetate, ethyl acetate, tert-butyl acetate, etc.), lactones, ketones (e.g., acetone, methyl ethyl ketone, cyclopentanone, etc.), alcohols (e.g., ethanol, butanol, isopropanol, etc.), amides (e.g., DMF, N-methylpyrrolidone or other amides), ureas, carbonates (e.g., diethyl carbonate or other carbonates), carbamates, aldehydes, amines, cyanates, isocyanates, sulfoxides, sulfones, aromatic hydrocarbons, heteroaromatic hydrocarbons, thiols, phosphoramides, nitriles (e.g., acetonitrile), alkynes, olefins, alkanes, halogenated solvents (e.g., tetrachloromethane, dichloromethane, chloroform or other halogenated solvents), silanes, perfluorocarbons (C2-C 18 The washing solvent may be a solvent such as a nitrile, a perfluorinated branched or straight chain alkane, such as perfluorohexane, perfluoroheptane, perfluorodecane, a perfluoroaromatic such as perfluorobenzene, or other perfluorocarbons), a supercritical fluid, an ionic liquid, a compressed gas, etc. In some cases, the washing solvent includes a nitrile, such as acetonitrile. The solvent optionally contains additional components, such as an acid, a base or a salt. In some cases, the solvent used in the non-washing step is the same as the solvent used in the previous or subsequent washing step.

[0083] Further described herein are methods, systems, compositions and devices comprising solvents such as ethers. Exemplary ethers include ether, methyl ethyl ether, dibutyl ether, diisopropyl ether, di(n-propyl) ether, di(tert-butyl) ether, cyclopentyl methyl ether, dimethoxymethane, 1,4-dioxane, ethyl tert-butyl ether, 2-(2-methoxyethoxy) ethanol, morpholine, polyethylene glycol, 2-(2-methoxyethoxy) ethanol, tetrahydrofuran, tetrahydropyran, methyl tert-butyl ether, 2-methyltetrahydrofuran, glycol dimethyl ether, diglycol dimethyl ether and dimethoxyethane. In some cases, the washing solvent includes tetrahydrofuran. In some cases, ether includes C2-C8 ether.

[0084] The solvent typically includes a ketone and is used with the methods, systems and compositions described herein. Exemplary ketones include acetone, acetophenone, butanone, cyclopentanone, cyclohexanone, cyclobutanone, cyclopropanone, ethyl isopropyl ketone, 2-hexanone, isophorone, mesityl oxide, methyl isobutyl ketone, methyl isopropyl ketone, 3-methyl-2-pentanone, 2-pentanone and 3-pentanone. In some cases, the wash solvent includes acetone. In some cases, the ketone includes a C2-C8 ketone.

[0085] Solvents are further described herein, which are "pure" substances in some cases, wherein the solvent contains only trace amounts (<0.1%, w / v) of other chemical components (e.g., salts, solvents, other solutes). In some cases, the solvent comprises water, for example, the solvent comprises (v / v) no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.5%, 1.0%, 2.0% or no more than 5.0% water. In some cases, the purity (w / w) of the solvent is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or at least 99.9% pure. In some cases, the purity (w / w) of the solvent is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or about 99.9% pure. In some cases, the solvent comprises salt or buffer.

[0086] Further described herein are wash solvents comprising acetone having a purity of (<0.1%, w / v) of other chemical components (e.g., salts, solvents, other solutes). In some cases, the wash solvent comprising acetone comprises water, for example, the wash solvent comprising acetone comprises (v / v) no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.5%, 1.0%, 2.0%, or no more than 5.0% water. In some cases, the wash solvent comprising acetone has a purity (w / w) of at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or at least 99.9% pure. In some cases, the wash solvent comprising acetone has a purity (w / w) of about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or about 99.9% pure. In some cases, the wash solvent comprising acetone comprises a salt or buffer.

[0087] The solvent of the mixture comprising two or more solvents is further described herein.For example, solvent includes two solvents mixed with about 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 50:1, 80:10, 99:1 ratio (v / v).In some cases, the mixture of two solvents includes at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or at least 99% a solvent (v / v).In some cases, the mixture of two solvents includes about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or about 99% a solvent (v / v).In some cases, solvent includes a mixture of three or more solvents.

[0088] The solvent of the mixture comprising ketone and additional solvent is further described herein.For example, the solvent comprises ketone and additional solvent mixed with about 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 50:1, 80:10, 99:1 ratio (v / v).In some cases, the mixture of ketone and additional solvent comprises at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or at least 99% ketone (v / v).In some cases, the mixture of ketone and additional solvent comprises about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or about 99% ketone (v / v).In some cases, additional solvent is acetonitrile.

[0089] The solvent of the mixture comprising acetone and additional solvent is further described herein.For example, the solvent comprises acetone and additional solvent mixed with about 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 50:1, 80:10, 99:1 ratio (v / v).In some cases, the mixture of acetone and additional solvent comprises at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or at least 99% acetone (v / v).In some cases, the mixture of acetone and additional solvent comprises about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or about 99% acetone (v / v).In some cases, additional solvent is acetonitrile. In some cases, the additional solvent includes THF. In some cases, the additional solvent is acetonitrile. In some cases, the additional solvent is a solvent mixture of two or more solvents.

[0090] Various solvent temperatures for optimization are further described herein. Solvent temperature can achieve low error rate and increased error rate uniformity. In some cases, solvent temperature can increase the solubility of the main reagent or by-product from the previous step. For example, the temperature of the solvent for washing or reagent solution is at least 0, 1, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 65, 70, 75, 80, 85, 90 or at least 90 degrees Celsius. In some cases, the temperature of the solvent for the wash or reagent solution is no more than 0, 1, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 65, 70, 75, 80, 85 or no more than 90 degrees Celsius. In some cases, the temperature of the solvent for the wash or reagent solution is about 0, 1, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 65, 70, 75, 80, 85 or about 90 degrees Celsius. In some cases, the temperature of the solvent for the wash or reagent solution is about 20 degrees Celsius. In some cases, the temperature of the solvent for washing or reagent solution is about 25 degrees Celsius. In some cases, the temperature of the solvent for washing or reagent solution is about 5 to 90, about 5 to 70, about 5 to 50, about 5 to 30, about 10 to 80, about 10 to 70, about 10 to 40, about 15 to 40, about 15 to 30, about 15 to 25, about 20 to 30, about 20 to 45, about 25 to 50, about 30 to 60, about 40 to 90, about 40 to 70, about 40 to 60 or about 60 to 90 degrees Celsius. In some cases, the temperature of the solvent for washing or reagent solution is about 15 to about 25 degrees Celsius. In some cases, the temperature of the solvent for washing or reagent solution is about 20 to 25 degrees Celsius. In some cases, the temperature of the solvent for washing or reagent solution is about 20 to 35 degrees Celsius. In some cases, the temperature of the solvent used for the wash or reagent solution is about 30 to 45 degrees Celsius. In some cases, the temperature of the solvent varies between two temperatures over time. In some cases, the temperature varies across the surface to form a temperature gradient.

[0091] This article further provides the solvent selected based on polarity index or miscibility with other solvents or reagent solutions.For example, a washing solvent having a polarity index similar to the polarity of the main component of a previous washing solvent or reagent solution is selected.Alternately or in combination, the viscosity of the solvent at a given temperature determines the best washing solvent.Another variable considered in some cases is miscibility.In some cases, if two solvents are "miscible", only one phase will be produced under all ratios for the defined temperature.The polarity index, viscosity, water solubility and miscibility (non-exhaustive) of the various solvents used in the methods described herein are shown in Table 4.In some cases, solvents are combined to further adjust polarity, viscosity or miscibility.

[0092] Table 4. Solvent properties

[0093]

[0094]

[0095]

[0096] Further provided herein are methods, systems, compositions and devices, wherein the viscosity of the solvent for the washing or reagent solution is about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or about 0.9 cP (centipoise). In some cases, the viscosity of the solvent for the washing or reagent solution is at least 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or at least 0.9 cP. In some cases, the viscosity of the solvent for the washing or reagent solution is no more than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or no more than 0.9 cP. In some cases, the viscosity of the solvent for washing or reagent solution is about 0.01 to 0.8, about 0.01 to 0.6, about 0.1 to 0.5, about 0.1 to 0.4, about 0.2 to 0.3, about 0.2 to 0.4, about 0.2 to 0.6, about 0.4 to 0.9, about 0.4 to 0.7, or about 0.5 to 0.9 cP. In some cases, the viscosity of the solvent for washing or reagent solution is about 0.2 to about 0.4 cP. In some cases, the viscosity of the solvent for washing or reagent solution is about 0.1 to about 0.5 cP. In some cases, the viscosity of the solvent for washing or reagent solution is about 0.3 to about 0.4 cP.

[0097] Further provided herein are methods, systems, compositions, and apparatuses wherein the polarity index of the solvent used for the wash or reagent solution is about 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, or about 9. In some cases, the polarity index of the solvent used for the wash or reagent solution is at least 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, or at least 9. In some cases, the polarity index of the solvent used for the wash or reagent solution is no more than 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, or no more than 9. In some cases, the polarity index of the solvent for the wash or reagent solution is about 0 to 9, about 0 to 7, about 1 to 6, about 1 to 5, about 1 to 4, about 1 to 3, about 2 to 6, about 2 to 5, about 2 to 4, about 2 to 3, about 3 to 6, about 3 to 5, about 3 to 4, about 4 to 10, about 4 to 9, about 4 to 8, about 4 to 7, about 5 to 10, about 5 to 9, about 5 to 8, about 5 to 7, or about 5 to 6. In some cases, the polarity index of the solvent for the wash or reagent solution is about 4 to about 6. In some cases, the polarity index of the solvent for the wash or reagent solution is about 5 to about 6. In some cases, the polarity index of the solvent for the wash or reagent solution is about 5 to about 7.

[0098] Error rates in large polynucleotide libraries

[0099] Several methods for evaluating the quality of polynucleotide libraries described herein are described herein, including Sanger sequencing, next generation sequencing or other methods for measuring the quality of polynucleotide libraries. The quality of polynucleotide libraries can be measured by error rate, type representation or loss frequency. The various indices of error rate include deletion, insertion, displacement, mispairing, loss or other differences between predetermined sequences and the polynucleotide sequences measured. In some cases, capping is used to cut off the remaining unreacted molecules after the coupling step, which prevents the polynucleotide from further growing into full-length polynucleotides. In some cases, error rate is described by the number or percentage of full-length polynucleotides. This percentage is usually determined by the efficiency of each coupling step or the average coupling efficiency of a plurality of steps.

[0100] Further provided herein are methods of synthesizing a polynucleotide library wherein the average coupling efficiency is about 80%, 90%, 95%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 99.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% or about 99.99%. In some cases, the average coupling efficiency is at least 80%, 90%, 95%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 99.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, or at least 99.99%. In some cases, such libraries comprise polynucleotides, wherein each polynucleotide is at least 50, 100, 150, 200, 250, or at least 300 bases in length.

[0101] Further provided herein are polynucleotide libraries wherein each polynucleotide comprises at least 50 bases, wherein the percentage of the full-length sequence is about 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or about 90%. In some cases for polynucleotide libraries wherein each polynucleotide comprises at least 50 bases, the percentage of the full-length sequence is at least 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or at least 90%. Further provided herein are polynucleotide libraries wherein each polynucleotide comprises at least 100 bases, wherein the percentage of the full-length sequence is about 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or about 90%. In some cases for polynucleotide libraries wherein each polynucleotide comprises at least 100 bases, the percentage of the full-length sequence is at least 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or at least 90%. Further provided herein are polynucleotide libraries wherein each polynucleotide comprises at least 150 bases, wherein the percentage of the full-length sequence is about 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or about 90%. In some cases for polynucleotide libraries wherein each polynucleotide comprises at least 150 bases, the percentage of the full-length sequence is at least 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or at least 90%. Further provided herein is an error rate for a polynucleotide library wherein each polynucleotide comprises at least 200 bases, wherein the percentage of the full-length sequence is about 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or about 90%.In some cases for polynucleotide libraries wherein each polynucleotide comprises at least 200 bases, the percentage of the full-length sequence is at least 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or at least 90%. Further provided herein is the error rate of a polynucleotide library wherein each polynucleotide comprises at least 300 bases, wherein the percentage of the full-length sequence is about 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88% or about 90%. In some cases for a polynucleotide library in which each polynucleotide comprises at least 300 bases, the percentage of full-length sequence is at least 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, or at least 90%.

[0102] This article further provides the error rate of polynucleotide libraries.In some cases, the total error rate of three groups of different oligonucleotide libraries can be less than 1 error in 900, including 1 error in 1000 bases, or less than 1 error in 1000 bases-wherein the error rate is a comparison of the synthetic sequence and the instructions for synthesizing polynucleotides.In some cases, one or more quality control steps are used in the workflow.For example, the control polynucleotides of various sizes present on the surface are evaluated.In some cases, the length of the control polynucleotide is 10, 20, 30, 50, 70, 90, 100, 150, 200, 250, 300 or more than 300 bases.Control polynucleotides of different sizes are often used.In some cases, the length of the control polynucleotide is 20 bases.In some cases, the length of the control polynucleotide is 90 bases.In some cases, the length of the control polynucleotide is 150 bases.In some cases, the length of the control polynucleotide is 250 bases.

[0103] Further described herein is the method for measuring the error rate in polynucleotide library, such as next generation sequencing.For example, error type (or classification) such as deletion, insertion or mispairing is classified and measured.Usually, error rate is represented for the error type of any number.In some cases, mispairing is classified based on transition type, such as base 1 becomes base 2 (for example, A to T, T to G, C to G, etc.).In some cases, insertion is classified based on the base pair adjacent to insertion, such as base 1+base 2 (for example, A+A corresponds to inserting A after base A).In some cases, deletion is classified based on the base type of deletion, such as base-(for example, A-corresponds to the deletion of A).In some cases, deletion rate is measured as single base deletion or block deletion.Alternatively or in combination, error type is evaluated as the function of base position (relative to synthesis order) on polynucleotide.

[0104] The average error rate of polynucleotides synthesized in a library using the provided system and method can often be less than 1 / 800 base, less than 1 / 900, less than 1 / 1250, less than 1 / 1500, less than 1 / 2000, less than 1 / 3000 or lower. In some cases, the average error rate of polynucleotides synthesized in a library using the provided system and method is less than 1 / 1000. In some cases, the average error rate of polynucleotides synthesized in a library using the provided system and method is less than 1 / 900. In some cases, the average error rate of polynucleotides synthesized in a library using the provided system and method is less than 1 / 800. In some cases, the average error rate of polynucleotides synthesized in a library using the provided system and method is less than 1 / 1100. In some cases, the total error rate of the polynucleotides synthesized in the library using the provided system and method is less than 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 1100, 1 / 1200, 1 / 1250, 1 / 1300, 1 / 1400, 1 / 1500, 1 / 1600, 1 / 1700, 1 / 1800, 1 / 1900, 1 / 2000, 1 / 3000 or lower compared to the predetermined sequence. In some cases, the total error rate of the polynucleotides synthesized in the library using the provided system and method is less than 1 / 1000. In some cases, the total error rate of the polynucleotides synthesized in the library using the provided system and method is less than 1 / 900. In some cases, the total error rate of the polynucleotides synthesized in the library using the provided system and method is less than 1 / 1100.

[0105] In some cases, error correction enzyme can be used for the polynucleotide synthesized in the library using the provided system and method. In some cases, compared with the predetermined sequence, the total error rate of the polynucleotide through error correction can be less than 1 / 1000, 1 / 1500, 1 / 2000, 1 / 3000, 1 / 4000, 1 / 5000, 1 / 6000, 1 / 7000, 1 / 8000, 1 / 9000, 1 / 10000 or lower. In some cases, the total error rate of the polynucleotide synthesized in the library using the provided system and method after error correction can be less than / 1000, 1 / 1500, 1 / 2000, 1 / 3000, 1 / 4000, 1 / 5000, 1 / 6000, 1 / 7000, 1 / 8000, 1 / 9000, 1 / 10000.

[0106] Thus, libraries can be synthesized in which less than 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 1250, 1 / 1500, 1 / 2000, 1 / 2500, 1 / 3000, 1 / 4000, 1 / 5000, 1 / 6000, 1 / 7000, 1 / 8000, 1 / 900, 1 / 1000, 1 / 1250, 1 / 1500, 1 / 2000, 1 / 2500, 1 / 3000, 1 / 4000, 1 / 5000, 1 / 6000, 1 / 7000, 1 / 8000, 1 / 900 000, 1 / 10000, 1 / 12000, 1 / 15000, 1 / 20000, 1 / 25000, 1 / 300000, 1 / 400000, 1 / 500000, 1 / 600000, 1 / 700000, 1 / 800000, 1 / 900000, 1 / 1000000, 1 / 125000, 1 / 150000, 1 / 200000, 1 / 300000, 1 / 400000, 1 / 500000, 1 / 600000, 1 / 700000, 1 / 800000, 1 / 900000, 1 / 1000000 or less base insertion, deletion, substitution or total error rate. The methods and compositions of the present disclosure also relate to large synthetic polynucleotide and gene libraries having low error rates associated with at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%, 99.99% or more of the polynucleotides or genes in at least a subset of the library, thereby relating to error-free sequences compared to a predetermined / preselected sequence. In some cases, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%, 99.99% or more of the polynucleotides or genes in the isolated volume within the library have the same sequence. In some cases, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more in any polynucleotide or gene relevant with more than 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%, 99.99% or more have identical sequence.In some cases, optimize the error rate relevant with the specified locus on polynucleotide or gene.Thus, a given locus or a plurality of selected loci of one or more polynucleotides or genes as part of a large library may each have less than 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 1250, 1 / 1500, 1 / 2000, 1 / 2500, 1 / 3000, 1 / 4000, 1 / 5000, 1 / 6000, 1 / 7000, 1 / 8000, 1 / 9000, 1 / 10000, 1 / 12500, 1 / 1500, 1 / 2000, 1 / 2500 The error rate may be as high as 1 / 1000, 1 / 20000, 1 / 25000, 1 / 30000, 1 / 40000, 1 / 50000, 1 / 60000, 1 / 70000, 1 / 80000, 1 / 90000, 1 / 100000, 1 / 125000, 1 / 150000, 1 / 200000, 1 / 300000, 1 / 400000, 1 / 500000, 1 / 600000, 1 / 700000, 1 / 800000, 1 / 900000, 1 / 1000000 or lower. In various cases, such error-optimized loci can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 5000, 5000, 5000 00, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 30000, 50000, 75000, 100000, 500000, 1000000, 2000000, 3000000 or more loci. 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 40000, 50000, 60000, 70000, 80000, 9000, 10000, 30000, 75000, 100000, 500000, 1000000, 2000000, 3000000 or more polynucleotides or genes.

[0107] The error rates can be achieved with or without error correction. The error rates can be achieved in the entire library, or in more than 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%, 99.99% or more of the library.

[0108] Polynucleotide libraries as described herein can be measured in terms of uniformity, the measure of polynucleotide species representation. Uniformity can be measured on the basis of clusters and devices. In some cases, 99% of polynucleotides have an abundance within or about 2x of the average abundance of about 0.05, 0.1, 0.2, 0.5, 0.7, 1, 1.2, 1.5, 1.7x. In some cases, 97% of polynucleotides have an abundance within or about 2x of the average abundance of about 0.05, 0.1, 0.2, 0.5, 0.7, 1, 1.2, 1.5, 1.7x. In some cases, 60%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the sequences have an abundance within 2x of the mean. In some cases, 60%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the sequences have an abundance within 1.5x of the mean. In some cases, 60%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the sequences have an abundance within 1x of the mean.

[0109] The error rate of the whole surface can be reduced using the washing solvent provided herein.For example, in some cases, the flow effect caused by the solvent moving on the surface can cause residual reagent to stay on the surface, or solubility effect, which causes the difference of local reagent concentration on the surface.In some cases, the mobile reagent solution of the first part of the contact surface has different properties (reagent concentration, temperature, viscosity or other properties) from the same reagent solution of the second part of the contact surface at a later time point; This can cause the error rate of the first part of the surface and the second part to be different (error gradient).This can cause the reaction to be incomplete, or in some cases can cause undesirable side reactions in an uneven manner on the whole surface, thereby causing the error of the polynucleotide synthesized.In some cases, the washing solvent provided herein improves the solubility of reagent or reagent by-product, which reduces or eliminates the reagent concentration gradient on the whole surface.Solid surface can be horizontal or vertical, wherein orientation is relative to the flow of fluid through the solid surface.In other cases, the washing solvent provided herein promotes the favorable interaction with the residual reagent solution of the contact surface. In some cases, the difference in error rate across the surface is less than 1 / 100, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 1100, 1 / 1250, 1 / 1500, 1 / 2000, 1 / 5000, or less than 1 / 10000. In some cases, the difference in error rate across the surface is at least 1 / 100, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 1100, 1 / 1250, 1 / 1500, 1 / 2000, 1 / 5000, or less than 1 / 10000. In some cases, the difference in error rate across the surface is about 1 / 100, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 1100, 1 / 1250, 1 / 1500, 1 / 2000, 1 / 5000, or about 1 / 10000.

[0110] In some cases, methods, compositions, systems and devices described herein include surfaces, such as flow cells, wherein clusters are distributed between inlets and outlets, which allow for exchange of reagent solutions and washings with the seat for polynucleotide synthesis. In some cases, multiple inlets and outlets are used. In some cases, the flow cell is horizontal, wherein solvent and reagent solution flow vertically relative to a vector defined by gravity. In some cases, one or more washings performed with solvents as described herein result in a uniform error rate between clusters near the inlet and clusters away from the inlet. In some cases, the difference in error rate between the inlet and outlet is about 0.0001%, 0.001%, 0.002%, 0.005%, 0.008%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, 0.8% or about 1.0% per micron. In some cases, the difference in error rate between the inlet and the outlet is no more than 0.0001%, 0.001%, 0.002%, 0.005%, 0.008%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, 0.8%, or no more than 1.0% per micron. In some cases, the difference in error rate between the inlet and the outlet is at least 0.0001%, 0.001%, 0.002%, 0.005%, 0.008%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, 0.8%, or at least 1.0% per micron.

[0111] When fluid flows from inlet to outlet, various flow rates are used herein for reagent solution or washing solvent. In some cases, flow rate is about 5,10,15,20,50,75,100,125,150,200,250,300,350,400 or about 500uL / s. In some cases, flow rate is at least 5,10,15,20,50,75,100,125,150,200,250,300,350,400 or at least 500uL / s. In some cases, flow rate is no more than 5,10,15,20,50,75,100,125,150,200,250,300,350,400 or no more than 500uL / s. In some cases, the flow rate is about 5 to 500uL / s, about 10 to 400uL / s, about 20 to 300uL / s, about 50 to 500uL / s, about 50 to 400uL / s, about 50 to 300uL / s, about 75 to 300uL / s, about 100 to 400uL / s, about 200 to 500uL / s, or about 40 to 350uL / s. In some cases, the flow rate is about 40 to 350uL / s. In some cases, the flow rate is about 75 to 250uL / s. In some cases, the flow rate is about 50 to 400uL / s.

[0112] Apparatus for polynucleotide synthesis

[0113] Provided herein are structures having surfaces having a plurality of features (locuses) for polynucleotide synthesis or extension. The structures may include, but are not limited to, plates, membranes, strips, bars, or beads. With respect to structures in array form, each feature in a portion of the structure may include a substantially planar feature, hole, or channel. In some cases, polynucleotides are synthesized at clusters of loci for polynucleotide extension, released, and subsequently subjected to an amplification reaction, such as PCR. An exemplary workflow for synthesizing polynucleotides from clusters is described in Figure 2 . A silicon plate 201 includes a plurality of clusters 203. Within each cluster are a plurality of loci 221. Polynucleotides are synthesized 207 de novo from clusters 203 on plate 201. The polynucleotides are cleaved 211 and removed 213 from the plate to form a population of released polynucleotides 215. The population of released polynucleotides 215 is then amplified 217 to form a library 219 of amplified polynucleotides.

[0114] In some cases, the ratio of the width of the hole described herein to the depth (or height) is 20 to 0.01, wherein the width is the measured value of the width at the narrowest section of the hole. In some cases, the ratio of the width of the hole described herein to the depth (or height) is 20 to 0.05, wherein the width is the measured value of the width at the narrowest section of the hole. In some cases, the ratio of the width of the hole described herein to the depth (or height) is 1 to 0.01, wherein the width is the measured value of the width at the narrowest section of the hole. In some cases, the ratio of the width of the hole described herein to the depth (or height) is 0.5 to 0.01, wherein the width is the measured value of the width at the narrowest section of the hole. In some cases, the width of the hole described herein to the depth (or height) ratio is about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, 1, 2, 5, 10 or 20.

[0115] In some cases, the diameter of the hole described herein is 20 to 0.01 with a depth (or height) ratio, wherein the diameter is the measured value of the diameter at the narrowest section of the hole. In some cases, the diameter of the hole described herein is 20 to 0.05 with a depth (or height) ratio, wherein the diameter is the measured value of the diameter at the narrowest section of the hole. In some cases, the diameter of the hole described herein is 1 to 0.01 with a depth (or height) ratio, wherein the diameter is the measured value of the diameter at the narrowest section of the hole. In some cases, the diameter of the hole described herein is 0.5 to 0.01 with a depth (or height) ratio, wherein the diameter is the measured value of the diameter at the narrowest section of the hole. In some cases, the diameter of the hole described herein is about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, 1, 2, 5, 10 or 20 with a depth (or height) ratio.

[0116] In some cases, the structures described herein comprise a plurality of pores, wherein the height or depth of the pores is from about 10 nm to about 10 μm, from about 10 nm to about 1 μm, from about 10 nm to about 500 nm, from about 10 nm to about 100 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, or from about 50 nm to about 100 nm. In some cases, the height of the pores is no more than 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In some cases, the height of the pores is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

[0117] In some cases, the structures described herein comprise a plurality of pores, wherein the pores have a width of about 10 nm to about 10 μm, about 10 nm to about 1 μm, about 10 nm to about 500 nm, about 10 nm to about 100 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm. In some cases, the width of the pores is no more than 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In some cases, the width of the pores is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

[0118] In some cases, the structures described herein comprise a plurality of pores, wherein the diameter of the pores is from about 10 nm to about 10 μm, from about 10 nm to about 1 μm, from about 10 nm to about 500 nm, from about 10 nm to about 100 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, or from about 50 nm to about 100 nm. In some cases, the diameter of the pores is no more than 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In some cases, the diameter of the pores is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

[0119] In some cases, the spots or substantially planar features described herein have a diameter of about 50 nm to about 1000 nm, about 50 nm to about 900 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm.

[0120] In some cases, the width of channel as described herein is 20 to 0.01 with respect to depth (or height), wherein channel is the measured value of width at the narrowest section of channel. In some cases, the width of channel as described herein is 20 to 0.05 with respect to depth (or height), wherein width is the measured value of width at the narrowest section of channel. In some cases, the width of channel as described herein is 1 to 0.01 with respect to depth (or height), wherein width is the measured value of width at the narrowest section of channel. In some cases, the width of channel as described herein is 0.5 to 0.01 with respect to depth (or height), wherein width is the measured value of width at the narrowest section of hole. In some cases, the width of channel as described herein is about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, 1, 2, 5, 10 or 20 with respect to depth (or height).

[0121] In some cases, the diameter of the channel described herein is 20 to 0.01 with a depth (or height) ratio, wherein the diameter is the measurement of the diameter at the narrowest section of the channel. In some cases, the diameter of the channel described herein is 20 to 0.05 with a depth (or height) ratio, wherein the diameter is the measurement of the diameter at the narrowest section of the channel. In some cases, the diameter of the channel described herein is 1 to 0.01 with a depth (or height) ratio, wherein the diameter is the measurement of the diameter at the narrowest section of the channel. In some cases, the diameter of the channel described herein is 0.5 to 0.01 with a depth (or height) ratio, wherein the diameter is the measurement of the diameter at the narrowest section of the channel. In some cases, the diameter of the channel described herein is about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, 1, 2, 5, 10 or 20 with a depth (or height) ratio.

[0122] In some cases, the structures described herein comprise a plurality of channels, wherein the height or depth of the channels is from about 10 nm to about 10 μm, from about 10 nm to about 1 μm, from about 10 nm to about 500 nm, from about 10 nm to about 100 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, or from about 50 nm to about 100 nm. In some cases, the height of the channels is no more than 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In some cases, the height of the channel is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

[0123] In some cases, the structures described herein comprise a plurality of channels, wherein the channels have a width of about 10 nm to about 10 μm, about 10 nm to about 1 μm, about 10 nm to about 500 nm, about 10 nm to about 100 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm. In some cases, the width of the channels is no more than 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In some cases, the width of the channel is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

[0124] In some cases, the structures described herein comprise a plurality of channels, wherein the channels have a diameter of about 10 nm to about 10 μm, about 10 nm to about 1 μm, about 10 nm to about 500 nm, about 10 nm to about 100 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm. In some cases, the diameter of the channels is no more than 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In some cases, the diameter of the pores is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

[0125] In some cases, the width of a feature (e.g., a substantially planar feature, hole, channel, or other feature that supports polynucleotide synthesis) is about 10 nm to about 10 μm, about 100 nm to about 10 μm, about 200 nm to about 1 μm, about 50 nm to about 500 nm, about 50 nm to about 200 μm, or about 10 nm to about 100 nm, e.g., about 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or 10 nm. In some cases, the width of a feature is no more than about 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, or 10 nm. In some cases, the distance between the centers of two adjacent features is about 10 nm to about 10 μm, 20 nm to about 5 μm, about 50 nm to about 2 nm, about 100 nm to about 1 μm, about 200 nm to about 500 nm, about 200 nm to about 1 μm, about 200 nm to about 750 nm, or about 300 nm to about 600 nm, for example, about 500 nm. In some cases, the total width of the feature is about 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the total width of the feature is about 10 nm to 1 μm, 20 nm to 500 nm, or 50 nm to 100 nm.

[0126] Surfaces for polynucleotide synthesis

[0127] Provided herein are rigid or flexible structures for polynucleotide synthesis. In the case of a rigid structure, provided herein are devices having structures (eg, plates) for generating polynucleotide libraries. Figure 1D An exemplary structure 109 is shown in , where the structure 109 has approximately the same size dimensions as a standard 96-well plate: 140 mm x 90 mm. The structure 109 contains clusters grouped in 24 regions or sub-domains 115, each sub-domain 115 containing an array of 256 clusters 110. Figure 1E An expanded view of an exemplary subdomain 115 is shown in FIG. In the expanded view of the four clusters ( Figure 1E ), the Y-axis cluster spacing (center-to-center distance of adjacent clusters) of a single cluster 110 is 1079.210 μm or 1142.694 μm, and the X-axis cluster spacing is 1125 μm. Figure 1F An illustrative cluster 110 is depicted in , where the Y-axis seat spacing (center-to-center distance of adjacent seats) is 63.483 μm, and the X-axis seat spacing is 75 μm. The seat width at the longest portion (eg, the diameter of a circular seat) is 50 μm, and the distance between seats is 24 μm. Figure 1FThe number of seats 111 in an exemplary cluster is 121. The seats can be flat, holes or channels.

[0128] In the case of a flexible structure, devices are provided herein where the flexible structure comprises a continuous loop 105 wrapped around one or more fixed structures (e.g., a pair of rollers 106) or a discontinuous flexible structure 107 wrapped around a separate fixed structure (e.g., a pair of rolls 108). Figure 1B-1C In some cases, the structure comprises multiple regions for polynucleotide synthesis.

[0129] In some cases, each feature supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another feature. Provided herein are surfaces comprising at least 10, 100, 256, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 30,000, 40,000, 50,000 or more clusters. Provided herein are surfaces comprising more than 2,000, 5,000, 10,000, 20,000, 30,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 5,000,000 or 10,000,000 or more different features. In some cases, each cluster comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more features. In some cases, each cluster comprises 50 to 500, 50 to 200, 50 to 150 or 100 to 150 features. In some cases, each cluster comprises 100 to 150 features. In an exemplary arrangement, each cluster comprises 109, 121, 130 or 137 features. In some cases, each structure in a feature supports synthesis of a polynucleotide population having different sequences from a polynucleotide population that grows on another structure in the same feature. Features are provided herein, and in some cases they each comprise at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000 or more than 200,000 different structures. In some cases, each feature comprises approximately 10 to approximately 500, approximately 50 to approximately 250, approximately 10 to approximately 1000 or approximately 1 to approximately 50 structures.

[0130] In some cases, the distance between the centers of two adjacent loci within a cluster is about 10 μm to about 500 μm, about 10 μm to about 200 μm, or about 10 μm to about 100 μm. In some cases, the distance between the centers of two adjacent loci is greater than about 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the distance between the centers of two adjacent loci is less than about 200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In some cases, each locus has a width of about 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, each locus has a width of about 0.5 μm to 100 μm, about 0.5 μm to 50 μm, about 10 μm to 75 μm, or about 0.5 μm to 50 μm.

[0131] In some cases, the density of clusters within the device is at least or about 1 cluster / 100 mm 2 , 1 cluster / 10mm 2 , 1 cluster / 5mm 2 , 1 cluster / 4mm 2 , 1 cluster / 3mm 2 , 1 cluster / 2mm 2 , 1 cluster / 1mm 2 , 2 clusters / 1mm 2 , 3 clusters / 1mm 2 , 4 clusters / 1mm 2 , 5 clusters / 1mm 2 , 10 clusters / 1mm 2 , 50 clusters / 1mm 2 or higher. In some cases, the device comprises about 1 cluster / 10 mm 2 To about 10 clusters / 1mm 2In some cases, the distance between the centers of two adjacent clusters is less than about 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or 2000 μm, or 5000 μm. In some cases, the distance between the centers of two adjacent clusters is about 50 μm to about 100 μm, about 50 μm to about 200 μm, about 50 μm to about 300 μm, about 50 μm to about 500 μm, and about 100 μm to about 2000 μm. In some cases, the distance between the centers of two adjacent clusters is from about 0.05 mm to about 50 mm, from about 0.05 mm to about 10 mm, from about 0.05 mm to about 5 mm, from about 0.05 mm to about 4 mm, from about 0.05 mm to about 3 mm, from about 0.05 mm to about 2 mm, from about 0.1 mm to 10 mm, from about 0.2 mm to 10 mm, from about 0.3 mm to about 10 mm, from about 0.4 mm to about 10 mm, from about 0.5 mm to 10 mm, from about 0.5 mm to about 5 mm, or from about 0.5 mm to about 2 mm. In some cases, each cluster has a diameter or width of about 0.5 to 2 mm, about 0.5 to 1 mm, or about 1 to 2 mm along one dimension. In some cases, each cluster has a diameter or width along one dimension of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mm. In some cases, each cluster has an inner diameter or width along one dimension of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mm.

[0132] Provided herein are features having a width of 10 nm to 1 μm at the longest segment. In some cases, the width of the feature at the longest segment is about 10, 20, 30, 35, 40, 45, 50, 55, or 60 nm. In some cases, the feature is a channel with multiple segments, wherein the center-to-center distance of each segment is 5 to 50 nm. In some cases, the center-to-center distance of each segment is 5, 10, 15, 20, or 25 nm, respectively.

[0133] The number of different polynucleotides synthesized on the surface of the structures described herein generally depends on the number of different features available in the substrate. In some cases, the density of features within a cluster of the substrate is at least or about 1 feature / mm 2 , 10 features / mm 2 , 25 features / mm 2 , 50 features / mm 2 , 65 features / mm 2 , 75 features / mm 2 , 100 features / mm 2 , 130 features / mm2 , 150 features / mm 2 , 175 features / mm 2 , 200 features / mm 2 , 300 features / mm 2 , 400 features / mm 2 , 500 features / mm 2 , 1,000 features / mm 2 , 2,000 features / mm 2 , 5,000 features / mm 2 , 10,000 features / mm 2 , 100,000 features / mm 2 , 1,000,000 features / mm 2 or more than 1,000,000 features / mm 2 In some cases, the substrate comprises about 10 features / mm 2 Up to approximately 500 features / mm 2 , about 25 features / mm 2 Up to approximately 400 features / mm 2 , about 50 features / mm 2 Up to approximately 500 features / mm 2 , about 100 features / mm 2 Up to approximately 500 features / mm 2 , about 150 features / mm 2 Up to approximately 500 features / mm 2 , about 10 features / mm 2 Up to approximately 250 features / mm 2 , about 50 features / mm 2 Up to approximately 250 features / mm 2 , about 10 features / mm 2 Up to about 200 features / mm 2 or about 50 features / mm 2 Up to about 200 features / mm 2 .

[0134] In some cases, the density of features within a cluster of the substrate is at least or about 1 feature / μm 2 , 10 features / μm 2 , 25 features / μm 2 , 50 features / μm 2 , 65 features / μm 2 , 75 features / μm 2 , 100 features / μm 2 , 130 features / μm 2, 150 features / μm 2 , 175 features / μm 2 , 200 features / μm 2 , 300 features / μm 2 , 400 features / μm 2 , 500 features / μm 2 , 1,000 features / μm 2 , 2,000 features / μm 2 , 5,000 features / μm 2 , 10,000 features / μm 2 , 100,000 features / μm 2 , 1,000,000 features / μm 2 or more than 1,000,000 features / μm 2 In some cases, the substrate contains about 10 features / μm 2 Up to about 500 features / μm 2 , about 25 features / μm 2 Up to about 400 features / μm 2 , about 50 features / μm 2 Up to about 500 features / μm 2 , about 100 features / μm 2 Up to about 500 features / μm 2 , about 150 features / μm 2 Up to about 500 features / μm 2 , about 10 features / μm 2 To about 250 features / μm 2 , about 50 features / μm 2 To about 250 features / μm 2 , about 10 features / μm 2 Up to about 200 features / μm 2 or about 50 features / μm 2 Up to about 200 features / μm 2. In some cases, the distance between the centers of two adjacent features within a cluster is about 10 μm to about 500 μm, about 10 μm to about 200 μm, or about 10 μm to about 100 μm. In some cases, the distance between the centers of two adjacent features is greater than about 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the distance between the centers of two adjacent features is less than about 200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In some cases, the distance between the centers of two adjacent features within a cluster is about 10 nm to about 1000 nm, about 10 nm to about 500 nm, 10 nm to about 200 nm, or about 10 nm to about 100 nm. In some cases, the distance between two centers of adjacent features is greater than about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some cases, the distance between the centers of two adjacent features is less than about 500 nm, 200 nm, 150 nm, 100 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In some cases, the structures described herein allow at least about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or at least about 10 12 features, wherein each feature supports one oligonucleotide. In some cases, the structures described herein allow at least about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or at least about 10 12 Each feature supports a plurality of different oligonucleotides. In some cases, in less than about 6, 5, 4, 3, 2, or 1 m 2 The structures described herein support 109 polynucleotides.

[0135] In some cases, the structures described herein are synthesized to produce more than 2,000, 5,000, 10,000, 20,000, 30,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 0,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000, 100,000,000 or more non-identical polynucleotides. In some cases, the structure is synthesized to contain more than 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1 , 400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000, 100,000,000 or more polynucleotides encoding different sequences provide support. In some cases, at least a portion of the polynucleotides have the same sequence or are configured to be synthesized with the same sequence. In some cases, the structure provides a surface environment for the growth of a polynucleotide having at least about 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 1,000, 2,000 bases, or more. In some cases, the structure provides a surface environment for the growth of polynucleotides, each of which has at least about 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 1,000, 2,000 bases, or more.In some cases, the structure provides a surface environment for the growth of a polynucleotide having 50 to 2,000 bases, 50 to 1,000, 50 to 500, 50 to 250, or 100 to 1,000, 100 to 500, or 100 to 300 bases.

[0136] In some cases, polynucleotide is synthesized on the different features of structure, wherein each feature supports synthesis of polynucleotide colony.In some cases, each feature supports synthesis of polynucleotide colony with different sequence from the polynucleotide colony grown on another seat.In some cases, the feature of structure is located in multiple clusters.In some cases, structure comprises at least 10,500,1,000,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,11,000,12,000,13,000,14,000,15,000,20,000,30,000,40,000,50,000 or more clusters. In some cases, the structure comprises more than 2,000, 5,000, 10,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,100,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 1,600,000, 1,700,000, 1,800,000, 1,900,000, 2 , 000,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000 or 10,000,000 or more different features. In some cases, each cluster comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150 or more features (seats). In some cases, each cluster comprises 50 to 500, 100 to 150 or 100 to 200 features. In some cases, each cluster comprises 109, 121, 130 or 137 features. In some cases, each cluster comprises 5, 6, 7, 8, 9, 10, 11 or 12 features. In some cases, polynucleotides from different features within a cluster have sequences of continuous longer polynucleotides encoding a predetermined sequence when assembled.

[0137] In some cases, the structures described herein are about the size of a standard 96-well plate, for example, about 100 to 200 mm by about 50 to 150 mm. In some cases, the structures described herein have a diameter less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 100 mm or 50 mm. In some cases, the diameter of the substrate is about 25 mm to 1000 mm, about 25 mm to about 800 mm, about 25 mm to about 600 mm, about 25 mm to about 500 mm, about 25 mm to about 400 mm, about 25 mm to about 300 mm or about 25 mm to about 200. Non-limiting examples of substrate size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm. In some cases, the substrate has at least about 100 mm. 2 , 200mm 2 , 500mm 2 、1,000mm 2 、2,000mm 2 、5,000mm 2 、10,000mm 2 、12,000mm 2 、15,000mm 2 、20,000mm 2 、30,000mm 2 、40,000mm 2 、50,000mm 2 The substrate may be a substrate having a thickness of about 50 mm to about 2000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 200 mm to about 1000 mm, or about 250 mm to about 1000 mm. Non-limiting examples of thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm, and 925 mm. In some cases, the thickness of the substrate varies with diameter and depends on the composition of the substrate. For example, a structure comprising a material other than silicon may have a thickness different from a silicon structure of the same diameter. The structural thickness may depend on the mechanical strength of the material used, and the structure must be thick enough to support its own weight during operation without breaking. In some cases, the structure exceeds about 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any one dimension.

[0138] Surface Architecture

[0139] This article provides a device comprising protrusions and / or recessed features. A benefit with this type of feature is that the surface area used to support the synthesis of polynucleotides increases. In some cases, the device with protrusions and / or recessed features is referred to as a three-dimensional substrate. In some cases, the three-dimensional device comprises one or more channels. In some cases, one or more seats comprise a channel. In some cases, the channel can be deposited with reagents by a deposition device such as a polynucleotide synthesizer. In some cases, reagents and / or fluids are collected in a larger hole that is communicated with one or more channel fluids. For example, the device comprises a plurality of channels corresponding to a plurality of seats with a cluster, and the plurality of channels are communicated with a hole fluid of the cluster. In some methods, the polynucleotide library is synthesized in a plurality of seats of the cluster.

[0140] In some cases, the structure is configured to allow controlled flow and mass transfer path for polynucleotide synthesis on the surface. In some cases, the structure of the device allows controlled and uniform distribution of mass transfer path, chemical exposure number of times and / or washing efficacy in polynucleotide synthesis process. In some cases, the structure of the device allows to increase scanning efficiency, such as by providing a volume that is enough for increasing polynucleotides, so that the volume excluded by the polynucleotides of increase accounts for the initial available volume that can be used for or is suitable for increasing polynucleotides and is no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. In some cases, the three-dimensional structure allows the controlled flow of fluid, thereby allowing the rapid exchange of chemical exposure.

[0141] Provided herein are methods for synthesizing nucleic acids, such as RNA or DNA, in amounts of 1 fM, 5 fM, 10 fM, 25 fM, 50 fM, 75 fM, 100 fM, 200 fM, 300 fM, 400 fM, 500 fM, 600 fM, 700 fM, 800 fM, 900 fM, 1 pM, 5 pM, 10 pM, 25 pM, 50 pM, 75 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM or more. In some cases, the polynucleotide library can span about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a gene. A gene can vary by up to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%.

[0142] Different polynucleotides can encode at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of the sequence of a gene together. In some cases, polynucleotides can encode 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the sequence of a gene. In some cases, polynucleotides can encode 80%, 85%, 90%, 95% or more of the sequence of a gene.

[0143] In some cases, isolation is achieved by physical structure. In some cases, isolation is achieved by differential functionalization of the surface to generate activation and passivation areas for polynucleotide synthesis. Differential functionalization can also be achieved by alternating hydrophobicity on the entire device surface, thereby causing a water contact angle effect that can cause reagent beads or wetting that can cause deposition. The use of larger structures can reduce cross-contamination of reagents from splashes and adjacent spots to different polynucleotide synthesis positions. In some cases, reagents are deposited to different polynucleotide synthesis positions using devices such as polynucleotide synthesizers. Substrates with three-dimensional features are configured in a manner that allows the synthesis of a large number of polynucleotides (e.g., more than about 10,000) with a low error rate (e.g., less than about 1:500, 1:1000, 1:1500, 1:2,000; 1:3,000; 1:5,000; or 1:10,000). In some cases, the device comprises a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, or 500 features / mm 2 characteristics.

[0144] The hole of device can have the width, height and / or volume identical or different with another hole of substrate.The channel of device can have the width, height and / or volume identical or different with another channel of substrate.In some cases, the width of cluster is about 0.05mm to about 50mm, about 0.05mm to about 10mm, about 0.05mm to about 5mm, about 0.05mm to about 4mm, about 0.05mm to about 3mm, about 0.05mm to about 2mm, about 0.05mm to about 1mm, about 0.05mm to about 0.5mm, about 0.05mm to about 0.1mm, about 0.1mm to 10mm, about 0.2mm to 10mm, about 0.3mm to about 10mm, about 0.4mm to about 10mm, about 0.5mm to 10mm, about 0.5mm to about 5mm or about 0.5mm to about 2mm. In some cases, the width of the hole comprising the cluster is from about 0.05mm to about 50mm, from about 0.05mm to about 10mm, from about 0.05mm to about 5mm, from about 0.05mm to about 4mm, from about 0.05mm to about 3mm, from about 0.05mm to about 2mm, from about 0.05mm to about 1mm, from about 0.05mm to about 0.5mm, from about 0.05mm to about 0.1mm, from about 0.1mm to 10mm, from about 0.2mm to 10mm, from about 0.3mm to about 10mm, from about 0.4mm to about 10mm, from about 0.5mm to 10mm, from about 0.5mm to about 5mm or from about 0.5mm to about 2mm. In some cases, the width of the cluster is less than or about 5mm, 4mm, 3mm, 2mm, 1mm, 0.5mm, 0.1mm, 0.09mm, 0.08mm, 0.07mm, 0.06mm or 0.05mm. In some cases, the width of the cluster is about 1.0 to 1.3mm. In some cases, the width of the cluster is about 1.150mm. In some cases, the width of the hole is less than or about 5mm, 4mm, 3mm, 2mm, 1mm, 0.5mm, 0.1mm, 0.09mm, 0.08mm, 0.07mm, 0.06mm or 0.05mm. In some cases, the width of the hole is about 1.0 to 1.3mm. In some cases, the width of the hole is about 1.150mm. In some cases, the width of the cluster is about 0.08mm. In some cases, the width of the hole is about 0.08mm. The width of the cluster can refer to the cluster in two-dimensional or three-dimensional substrate.

[0145] In some cases, the height of the pores is about 20 μm to about 1000 μm, about 50 μm to about 1000 μm, about 100 μm to about 1000 μm, about 200 μm to about 1000 μm, about 300 μm to about 1000 μm, about 400 μm to about 1000 μm, or about 500 μm to about 1000 μm. In some cases, the height of the pores is less than about 1000 μm, less than about 900 μm, less than about 800 μm, less than about 700 μm, or less than about 600 μm.

[0146] In some cases, the device comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of the channels is about 5 μm to about 500 μm, about 5 μm to about 400 μm, about 5 μm to about 300 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 10 μm to about 50 μm. In some cases, the height of the channels is less than 100 μm, less than 80 μm, less than 60 μm, less than 40 μm, or less than 20 μm.

[0147] In some cases, the diameter of the channel, the seat (e.g., in a substantially planar substrate), or both the channel and the seat (e.g., in a three-dimensional device in which the seat corresponds to the channel) is about 1 μm to about 1000 μm, about 1 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to about 100 μm, or about 10 μm to about 100 μm, such as about 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In some cases, the diameter of the channel, the seat, or both the channel and the seat is less than about 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In some cases, the distance between the centers of two adjacent channels, seats, or both channels and seats is about 1 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 5 μm to about 30 μm, for example, about 20 μm.

[0148] Surface material

[0149] Provided herein is a device comprising a surface, wherein the surface is modified to support polynucleotide synthesis at a predetermined position, and has low error rate, low omission rate, high yield and high oligonucleotide presentation. In some cases, the surface of the device for polynucleotide synthesis provided herein is made of a variety of materials that can be modified to support de novo polynucleotide synthesis reactions. In some cases, the device has enough conductivity, for example, it is possible to form a uniform electric field across the entire device or a portion thereof. Device as described herein may include flexible materials. Exemplary flexible materials include but are not limited to modified nylon, unmodified nylon, nitrocellulose and polypropylene. Device as described herein may include rigid materials. Exemplary rigid materials include but are not limited to glass, fused quartz, silicon, silicon dioxide, silicon nitride, metal nitride, metal silicide, metal carbide, metal oxide, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof) and metals (e.g., gold, platinum). In some cases, the metal oxide comprises TiO2, Ta2O5, Nb2O5, Al2O3, BaO, Y2O3, HfO2, SrO, or other metal oxides known in the art. In some cases, the metal carbide comprises TiC, WC, ThC2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbides known in the art. In some cases, the metal nitride comprises GaN, InN, BN, Be3N2, Cr2N, MoN, Si3N4, TaN, Th2N2, VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitrides known in the art. The devices disclosed herein are in some cases made of materials comprising silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, the devices disclosed herein are made using a combination of the materials listed herein or any other suitable materials known in the art.

[0150] A list of the tensile strengths of exemplary materials described herein is provided below: nylon (70 MPa), nitrocellulose (1.5 MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa), agarose (1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane (PDMS) (3.9-10.8 MPa). The tensile strength of the solid supports described herein can be 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 MPa. The tensile strength of the solid supports described herein can be about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 270 MPa or more. In some cases, the devices described herein comprise a solid support for polynucleotide synthesis in the form of a flexible material such as a tape or flexible sheet that can be stored in a continuous loop or scroll.

[0151] Young's modulus is a measure of a material's resistance to elastic (recoverable) load deformation. A list of Young's moduli for the stiffness of exemplary materials described herein is provided below: nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10 GPa), polydimethylsiloxane (PDMS) (1-10 GPa). The Young's modulus of the solid supports described herein can be 1 to 500, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 GPa. The Young's modulus of the solid supports described herein can be about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa or more. Since the relationship between flexibility and stiffness is inverse to each other, flexible materials have a low Young's modulus and their shape changes significantly under load.

[0152] In some cases, the device disclosed herein comprises a silicon dioxide base and a silicon oxide surface layer. Alternatively, the device can have a silicon oxide matrix. The surface of the device provided herein can be textured, resulting in an increase in the total surface area for polynucleotide synthesis. The device described herein can comprise at least 5%, 10%, 25%, 50%, 80%, 90%, 95% or 99% silicon. The device disclosed herein can be made of silicon on insulator (SOI) wafers.

[0153] Surface modification

[0154] In various cases, surface modification is used to chemically and / or physically alter a surface by either additive or subtractive processes to change one or more chemical and / or physical properties of a device surface or selected sites or regions of a device surface. For example, surface modification includes, but is not limited to: (1) changing the wetting properties of a surface; (2) functionalizing a surface, i.e., providing, modifying, or replacing surface functional groups; (3) defunctionalizing a surface, i.e., removing surface functional groups; (4) otherwise changing the chemical composition of a surface, such as by etching; (5) increasing or decreasing surface roughness; (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties different from those of the surface; and / or (7) depositing particles on a surface.

[0155] In some cases, adding a chemical layer (referred to as an adhesion promoter) on top of the surface facilitates the structural patterning of seats on the substrate surface. Exemplary surfaces for applying adhesion promoters include, but are not limited to, glass, silicon, silicon dioxide, and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy. In some cases, a second chemical layer is deposited on the surface of the substrate. In some cases, the second chemical layer has a low surface energy. In some cases, the surface energy of the chemical layer coated on the surface supports the positioning of small droplets on the surface. Depending on the selected patterning arrangement, the proximity of the seat and / or the fluid contact area at the seat is variable.

[0156] In some cases, the device surface or resolved loci to which the nucleic acid or other part is deposited (e.g., for polynucleotide synthesis) is smooth or substantially planar (e.g., two-dimensional), or has irregularities, such as raised or recessed features (e.g., three-dimensional features). In some cases, the device surface is modified with one or more different compound layers. Such modified layers of interest include, but are not limited to, inorganic layers and organic layers, such as metals, metal oxides, polymers, small organic molecules, etc. Non-limiting polymer layers include peptides, proteins, nucleic acids or their mimetics (e.g., peptide nucleic acids, etc.), polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyvinylamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and any other suitable compounds described herein or known in the art. In some cases, the polymer is a heteropolymer. In some cases, the polymer is a homopolymer. In some cases, the polymer comprises a functional moiety or is conjugated.

[0157] In some cases, the resolved loci of the device are functionalized using one or more parts that increase and / or reduce the surface energy. In some cases, the parts are chemically inert. In some cases, the parts are configured to support the desired chemical reactions, such as one or more processes in a polynucleotide synthesis reaction. The surface energy or hydrophobicity of the surface is a factor that determines the affinity of the nucleotides attached to the surface. In some cases, the device functionalization method may include: (a) providing a device having a surface comprising silicon dioxide; and (b) silanizing the surface using a suitable silanizing agent (e.g., an organofunctional alkoxysilane molecule) as described herein or known in the art.

[0158] In some cases, the organofunctional alkoxysilane molecules include dimethylchloro-octadecyl-silane, methyldichloro-octadecyl-silane, trichloro-octadecyl-silane, trimethyl-octadecyl-silane, triethyl-octadecyl-silane, or any combination thereof. In some cases, the device surface comprises functionalization with polyethylene / polypropylene (functionalized by gamma irradiation or chromic acid oxidation and reduction to a hydroxyalkyl surface), highly cross-linked polystyrene-divinylbenzene (derivatized by chloromethylation and aminated to a benzylamine functional surface), nylon (terminal aminohexyl groups are directly reactive), or etching with reduced polytetrafluoroethylene. Other methods and functionalizing agents are described in U.S. Pat. No. 5,474,796, which is incorporated herein by reference in its entirety.

[0159] In some cases, the device surface is functionalized by contacting the device surface with a derivatizing composition containing a mixture of silanes under reaction conditions effective to couple the silanes to the device surface, typically via reactive hydrophilic moieties present on the device surface. Silanization is generally performed by self-assembly using organofunctional alkoxysilane molecules to cover the surface.

[0160] A variety of siloxane functionalizing agents currently known in the art may also be used, for example to reduce or increase surface energy. Organofunctional alkoxysilanes can be classified according to their organic functionality.

[0161] Provided herein are patterned devices that may include reagents that can be coupled to nucleosides. In some cases, the device may be coated with an active agent. In some cases, the device may be coated with a passivating agent. Exemplary active agents included in the coating materials described herein include, but are not limited to, N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxy undecyl triethoxysilane, n-decyl triethoxysilane, (3-aminopropyl) trimethoxysilane, (3-aminopropyl) triethoxysilane, 3-glycidyloxypropyl trimethoxysilane (GOPS), 3-iodo-propyl trimethoxysilane, butyl-aldehyde-trimethoxysilane, dimeric secondary aminoalkylsiloxane, (3-aminopropyl) trimethoxysilane, 1-Aminopropyl)-trimethoxysilane, (3-glycidyloxypropyl)-dimethyl-ethoxysilane, glycidyloxy-trimethoxysilane, (3-mercaptopropyl)-trimethoxysilane, 3-4-epoxycyclohexyl-ethyltrimethoxysilane and (3-mercaptopropyl)-methyl-dimethoxysilane, allyltrichlorosilane, 7-oct-1-enyltrichlorosilane or bis(3-trimethoxysilylpropyl)amine.

[0162] Exemplary passivating agents included in the coating materials described herein include, but are not limited to, perfluorooctyltrichlorosilane; tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane; 1H,1H,2H,2H-fluorooctyltriethoxysilane (FOS); trichloro(1H,1H,2H,2H-perfluorooctyl)silane; tert-butyl-dimethyl-silane; CYTOP TM ;Fluorinert TM ; perfluorooctyltrichlorosilane (PFOTCS); perfluorooctyldimethylchlorosilane (PFODCS); perfluorodecyltriethoxysilane (PFDTES); pentafluorophenyl-dimethylpropylchloro-silane (PFPTES); perfluorooctyltriethoxysilane; perfluorooctyltrimethoxysilane; octylchlorosilane; dimethylchloro-octadecyl-silane; methyldichloro-octadecyl-silane; trichloro-octadecyl-silane; trimethyl-octadecyl-silane; triethyl-octadecyl-silane; or octadecyltrichlorosilane.

[0163] In some cases, the functionalizing agent includes a hydrocarbon silane, such as octadecyltrichlorosilane. In some cases, the functionalizing agent includes 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl / trimethoxysilane, and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.

[0164] Provided herein is a device for polynucleotide synthesis, which comprises a structure made of any one or more materials in a variety of materials. In some cases, the material for making substrate / solid support shows low-level polynucleotide binding. In some cases, a material transparent to visible light and / or ultraviolet light can be adopted. Materials (conductors) with enough electrical conductivity can be used, for example, materials that can form a uniform electric field across the entire substrate / solid support described herein or a part thereof. In some cases, such materials can be electrically grounded. In some cases, substrate or solid support can be conductive or insulating. The material can be chemical resistant and heat resistant, to support chemical reaction or biochemical reaction, such as a series of polynucleotide building-up reactions.

[0165] In some cases, the conductive or semiconductive material (semiconductor) includes but is not limited to one or more of silicon titanium nitride, titanium nitride, tungsten nitride, tantalum nitride, silicon tantalum nitride, titanium, platinum silicide or other conductive materials. In some cases, the material includes but is not limited to one or more of aluminum carbide, carbide, nitride, oxide, silicide, silicon nitride, phosphide or other non-metal or metalloid used as conductive material components. In some cases, exemplary materials include (but are not limited to) tungsten, cobalt, iridium, molybdenum, nickel, platinum, rhenium, ruthenium, tantalum, titanium or other metal elements used as conductive material components. In some cases, the material includes a mixture of metal, non-metal or metalloid. In some cases, dopants are added to semiconductive materials. Dopants include but are not limited to phosphorus, antimony, arsenic, boron, aluminum, indium or other elements that meet the specifications. Conductors, semiconductors or insulators can be made using a combination of materials listed herein or any other suitable materials known in the art.

[0166] For rigid materials, specific materials of interest include: glass; fused quartz; silicon, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, etc.); metals (e.g., gold, platinum, etc.). The structure can be made of a material selected from silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), and glass. The substrate / solid support, microstructure, reactor, or other polynucleotide synthesis structure therein can be made using a combination of the materials listed herein or any other suitable materials known in the art.

[0167] Exemplary flexible materials for the structures described herein include, but are not limited to, nylon (unmodified nylon, modified nylon, transparent nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resins, transparent PVC foil, transparent foil for printers, poly(methyl methacrylate) (PMMA), methacrylate copolymers, styrene polymers, high refractive index polymers, fluoropolymers, polyethersulfones, polyimides containing alicyclic structures, rubber, fabrics, metal foils, and any combination thereof. Various plasticizers and modifiers can be used with the polymer substrate material to achieve selected flexibility properties.

[0168] Flexible structures as described herein may include plastic materials. In some cases, the structure includes thermoplastic materials. Non-limiting examples of thermoplastic materials include acrylic acid, acrylonitrile butadiene styrene, nylon, polylactic acid, polybenzimidazole, polycarbonate, polyether sulfone, polyether ether ketone, polyether imide, polyethylene, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polyvinyl chloride and polytetrafluoroethylene. In some cases, the substrate includes thermoplastic materials in polyaryletherketone (PEAK) family. Non-limiting examples of PEAK thermoplastics include polyetherketone (PEK), polyetherketoneketone (PEKK), poly(etherketoneketone) (PEEKK), polyetheretherketone (PEEK) and polyetherketoneetherketoneketone (PEKEKK). In some cases, the structure includes thermoplastic materials compatible with toluene. In some cases, the flexibility of plastic materials is increased by adding plasticizers. The example of plasticizer is a plasticizer based on ester, such as phthalates. Phthalate plasticizers include di(2-ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), di-n-butyl phthalate (DnBP, DBP), butyl benzyl phthalate (BBzP), diisodecyl phthalate (DIDP), dioctyl phthalate (DOP, DnOP), diisooctyl phthalate (DIOP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP) and di-n-hexyl phthalate. In some cases, modifying thermoplastic polymers by copolymerization or by adding non-reactive side chains to the monomers prior to polymerization also increases flexibility.

[0169] Provided herein is a flexible structure that may further include a fluoroelastomer. A material with about 80% fluoroelastomer is referred to as FKM. Fluoroelastomers include perfluoroelastomers (FFKM) and tetrafluoroethylene / propylene rubber (FEPM). Fluoroelastomers have five known types. Type 1 FKM is composed of vinylidene fluoride (VDF) and hexafluoropropylene (HFP), and their fluorine content is generally about 66% by weight. Type 2 FKM is composed of VDF, HFP and tetrafluoroethylene (TFE), and generally has about 68% to 69% fluorine. Type 3 FKM is composed of VDF, TFE and perfluoromethyl vinyl ether (PMVE), and generally has about 62% to 68% fluorine. Type 4 FKM is composed of propylene, TFE and VDF, and generally has about 67% fluorine. Type 5 FKM is composed of VDF, HFP, TFE, PMVE and ethylene.

[0170] In some cases, the substrate disclosed herein comprises computer readable material. Computer readable material includes, but is not limited to, magnetic media, reel-to-reel tape, cassette tape, cassette tape, floppy disk, paper media, film, microfilm, continuous tape (e.g., tape), and any medium suitable for storing electronic instructions. In some cases, the substrate comprises a magnetic reel-to-reel tape or a magnetic tape. In some cases, the substrate comprises a flexible printed circuit board.

[0171] The structures described herein can be transparent to visible light and / or ultraviolet light. In some cases, the structures described herein have sufficient conductivity to form a uniform electric field across the entire structure or a portion thereof. In some cases, the structures described herein are thermally conductive or heat-insulating. In some cases, the structure is chemically resistant and heat-resistant to support chemical reactions, such as polynucleotide synthesis reactions. In some cases, the substrate is magnetic. In some cases, the structure comprises a metal or a metal alloy.

[0172] The structures used for polynucleotide synthesis can be more than 1, 2, 5, 10, 30, 50 feet long or longer in any dimension. In the case of flexible structures, the flexible structures are optionally stored in a wound state, such as a roll. In the case of large structures (e.g., greater than 1 foot in length), the structures can be stored vertically or horizontally.

[0173] Material Deposition Systems

[0174] Provided herein are systems and devices for depositing and storing biomolecules on structures described herein. In some cases, the biomolecule is a polynucleotide storing coded information in its sequence. In some cases, the system includes a structural surface supporting biomolecule attachment and / or a device for applying biomolecules to a substrate surface. In one example, the device for applying biomolecules is a polynucleotide synthesizer. In some cases, the system includes a device (e.g., a flow cell) for using a fluid to process a substrate. In some cases, the system includes a device for moving a substrate between an application device and a treatment device. For a situation where the substrate is rolled to a reel, the system may include two or more reels, which allow different parts of the substrate to be accessed to an application device and an optional treatment device at different times.

[0175] Figure 3 A first example of a polynucleotide material deposition system for polynucleotide synthesis is shown in . The system includes a material deposition device that moves in the XY direction to align with the position of the substrate. The material deposition device can also move in the Z direction to seal with the substrate to form a resolved reactor. The resolved reactor is configured to allow transfer of fluids (including polynucleotides and / or reagents) from the substrate to the capping element, and / or vice versa. Figure 3 As shown, the fluid can pass through either or both of the substrate and the capping element, and includes but is not limited to coupling reagents, capping reagents, oxidants, deblocking agents, acetonitrile and nitrogen. Examples of devices capable of high-resolution droplet deposition include print heads of inkjet printers and laser printers. Devices useful in the systems and methods described herein achieve a resolution of about 100 dots per foot (DPI) to about 50,000 DPI, about 100 DPI to about 20,000 DPI, about 100 DPI to about 10,000 DPI, about 100 DPI to about 5,000 DPI, about 1,000 DPI to about 20,000 DPI, or about 1,000 DPI to about 10,000 DPI. In some cases, the device has a resolution of at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 12,000 DPI, or 20,000 DPI.High resolution deposition by the device is related to the number and density of individual nozzles corresponding to features of the substrate.

[0176] Figure 4An exemplary process workflow for synthesizing polynucleotides from scratch on a substrate using a polynucleotide synthesizer is shown in FIG. Droplets containing polynucleotide synthesis reagents are released from a material deposition device to a substrate in a stepwise manner, wherein the material deposition device has a piezoelectric ceramic material and an electrode to convert an electrical signal into a mechanical signal for releasing the droplets. The droplets release one nucleobase at a time at a specific position on the substrate surface to generate a plurality of synthetic polynucleotides having a predetermined sequence of coded data. In some cases, the synthesized polynucleotides are stored on the substrate. The polynucleotide reagents can be deposited on the substrate surface in a non-continuous or drop-on-demand method. Examples of such methods include electromechanical transfer methods, electrothermal transfer methods, and electrostatic attraction methods. In the electromechanical transfer method, a piezoelectric element deformed by an electric pulse causes the droplets to be ejected. In the electrothermal transfer method, bubbles are generated in the chamber of the device, and the expansion force of the bubbles causes the droplets to be ejected. In the electrostatic attraction method, the droplets are ejected onto the substrate using electrostatic attraction. In some cases, the dripping frequency is about 5KHz to about 500KHz; about 5KHz to about 100KHz; about 10KHz to about 500KHz; about 10KHz to about 100KHz; or about 50KHz to about 500KHz. In some cases, the frequency is less than about 500KHz, 200KHz, 100KHz or 50KHz.

[0177] The size of the small droplets distributed is related to the resolution of the device. In some cases, the device deposits small droplets of reagents with a size of about 0.01 pl to about 20 pl, about 0.01 pl to about 10 pl, about 0.01 pl to about 1 pl, about 0.01 pl to about 0.5 pl, about 0.01 pl to about 0.01 pl, or about 0.05 pl to about 1 pl. In some cases, the size of the small droplets is less than about 1 pl, 0.5 pl, 0.2 pl, 0.1 pl or 0.05 pl. The size of the small droplets distributed by the device is related to the diameter of the deposition nozzle, wherein each nozzle is capable of depositing the reagent on the feature of the substrate. In some cases, the deposition device of the polynucleotide synthesizer comprises about 100 to about 10,000 nozzles; about 100 to about 5,000 nozzles; about 100 to about 3,000 nozzles; about 500 to about 10,000 nozzles; or about 100 to about 5,000 nozzles. In some cases, the deposition device comprises more than 1,000, 2,000, 3,000, 4,000, 5,000 or 10,000 nozzles. In some cases, each material deposition device comprises multiple nozzles, wherein each nozzle is optionally configured to correspond to a feature on a substrate. Each nozzle can deposit a reagent component different from another nozzle. In some cases, each nozzle deposits a droplet of one or more features covering a substrate. In some cases, one or more nozzles are angled. In some cases, multiple deposition devices are stacked side by side to achieve a multiple increase in flux. In some embodiments, the gain is 2x, 4x, 8x or more. The example of a deposition device is Samba Printhead (Fujifilm). Samba Printhead can be used with Samba Web Administration Tool (SWAT).

[0178] The number of deposition sites can be increased by using the same deposition device and rotating it at a sword angle to a certain extent. By rotating the deposition device, each nozzle sprays with a certain amount of delay time corresponding to the sword angle. This asynchronous spraying causes crosstalk between the nozzles. Therefore, when the droplets are sprayed at a sword angle different from 0 degrees, the volume of the droplets from the nozzles can be different.

[0179] In some arrangements, the configuration of the polynucleotide synthesis system allows continuous polynucleotide synthesis process, which utilizes the flexibility of substrate to advance with a roll-to-roll process. This synthesis process is operated in a continuous production line mode, wherein the position of the substrate is rotated using one or more reels to advance the substrate through each stage of polynucleotide synthesis. In an exemplary embodiment, the polynucleotide synthesis reaction comprises a rolling substrate: by a solvent bath below the deposition device for phosphoramidite deposition, by an oxidant bath, by an acetonitrile wash bath, and by removing a blocking bath. Alternatively, other wash baths comprising alternative washing solvents are used, such as acetone, toluene, THF or other washing solvents. Optionally, the band is also passed through a capping bath. The roll-to-roll process allows the final product of the substrate comprising the synthesized polynucleotide to be easily gathered on a take-up reel, at which the final product can be transported for further processing or storage.

[0180] In some arrangements, polynucleotide synthesis is performed in a continuous process as the continuous flexible belt is conveyed along a conveyor belt system. Similar to a reel-to-reel process, polynucleotide synthesis on a continuous belt is operated in a production line manner, where the substrate travels through the various stages of polynucleotide synthesis during conveyance. However, in a conveyor belt process, the continuous belt returns to the polynucleotide synthesis step without the need to roll and unroll the belt, as in a reel-to-reel process. In some arrangements, the polynucleotide synthesis step is divided into sections, and the continuous belt is conveyed through each section once or multiple times in a cycle. For example, a polynucleotide synthesis reaction may include (1) conveying the substrate through a solvent bath below a deposition device for phosphoramidite deposition, through an oxidant bath, through an acetonitrile wash bath, and through a closed bath in a cycle; then (2) repeating the cycle to obtain a synthesized polynucleotide of a predetermined length. Alternatively, other wash baths containing alternative wash solvents are used, such as acetone, THF, or other wash solvents. After polynucleotide synthesis, the flexible substrate is removed from the conveyor belt system and optionally rolled up for storage. It can be rolled around a roll for storage.

[0181] In an exemplary arrangement, a flexible substrate comprising a thermoplastic material is coated with a nucleoside coupling agent. The coating is patterned into features such that each feature has a diameter of about 10 μm and a center-to-center distance between two adjacent features of about 21 μm. In this case, the feature size is sufficient to accommodate a 0.2 pl sessile drop volume during the polynucleotide synthesis deposition step. In some cases, the feature density is about 2.2 billion features / m 2 (1 feature / 441x 10 -12 m 2 ). In some cases, 4.5m 2 The substrate contains about 10 billion features, each 10 μm in diameter.

[0182] In another exemplary arrangement, a substrate comprising nanostructures is coated with a nucleoside coupling reagent. The coating is patterned into features such that each feature has a diameter of about 10 nm to about 200 nm and a center-to-center distance between two adjacent features of about 10 nm to about 200 nm. In this case, during the polynucleotide synthesis deposition step, multiple features accommodate a 0.2 pl sessile drop volume. In some cases, a feature diameter of about 50 nm and a center-to-center distance between two adjacent features of about 100 nm result in a feature density of about 10 billion features / m 2 (1 feature / 100x 10 -12 m 2 ).

[0183] The material deposition apparatus described herein may include about 2,048 nozzles, each nozzle depositing about 100,000 droplets per second at 1 nucleobase per droplet. For each deposition apparatus, at least about 1.75 x 10 13 In some cases, 100 to 500 nucleobase polynucleotides are synthesized. In some cases, 200 nucleobase polynucleotides are synthesized. Optionally, about 1.75 x 10 13 The rate of base synthesis is at least about 262.5 x 10 9 polynucleotides.

[0184] In some arrangements, the device for applying one or more reagents to the substrate during the synthesis reaction is configured to deposit reagents and / or nucleotide monomers for the synthesis based on nucleoside phosphoramidites. The reagents for polynucleotide synthesis include reagents and wash buffer for polynucleotide extension. As a non-limiting example, the device deposits cleaning reagents, coupling reagents, capping reagents, oxidants, deblocking agents, acetonitrile, washing solvents, gases such as nitrogen and any combination thereof. In addition, the device optionally deposits reagents for preparing and / or maintaining substrate integrity. In some cases, the polynucleotide synthesizer deposits droplets with a diameter less than about 200 μm, 100 μm or 50 μm with a volume less than about 1000, 500, 100, 50 or 20 pl. In some cases, the polynucleotide synthesizer deposits about 1 to 10,000, 1 to 5,000, 100 to 5,000 or 1,000 to 5,000 droplets per second.

[0185] In some arrangements, during the polynucleotide synthesis process, the substrate is placed in a flow cell and / or sealed in a flow cell. The flow cell can provide a continuous or discontinuous flow of a liquid, such as those liquids contained in the substrate for the reagents (e.g., oxidants and / or solvents) necessary for the reaction. The flow cell can provide a continuous or discontinuous flow of a gas (e.g., nitrogen) to dry the substrate generally by enhancing the evaporation of a volatile substrate. A variety of auxiliary devices can be used to improve drying and reduce residual moisture on the substrate surface. Examples of such auxiliary drying devices include, but are not limited to, vacuum sources, pressure reducing pumps, and vacuum tanks. In some cases, the polynucleotide synthesis system comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20) flow cells and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20) substrates. In some cases, the flow cell is configured to keep reagents during one or more steps of a building-up reaction and provide reagents to the substrate. In some cases, the flow cell includes a lid that slides on the top of the substrate and can be clamped in place to form a pressure-tight seal around the edge of the substrate. In some cases, the flow cell is used to generate the polynucleotide product.Sufficient sealing includes but is not limited to allowing about 1,2,3,4,5,6,7,8,9 or 10 atmospheric pressures of the seal.In some cases, the lid of the flow cell is opened to allow access to an application device such as a polynucleotide synthesizer.In some cases, one or more steps of the polynucleotide synthesis method are carried out on the substrate in the flow cell, without the need to carry out the transportation of the substrate.The flow cell is optionally used / operated in any direction, including vertical or horizontal directions.In some cases, one or more washings are used in a horizontally oriented flow cell to cause the error rate of the polynucleotide product to decrease.

[0186] In some arrangements, the device for treating substrate with fluid comprises a spray bar. Nucleotide monomers are applied to the substrate surface with an application device, and the spray bar subsequently uses the nozzle of the spray bar to spray the substrate surface with one or more treatment reagents. In some arrangements, the nozzle is arranged in sequence to be associated with the different treatment steps in the polynucleotide synthesis process. The chemical substance used for different process steps can be changed in the spray bar to easily adapt to the variation between the steps of the synthesis method or the synthesis method. In some cases, when the substrate moves through the spray bar, the spray bar continuously sprays a given chemical substance on the substrate surface. In some cases, the spray bar is deposited on the substrate of large area, very much like the spray bar used in the lawn sprinkler. In some cases, the spray bar nozzle is positioned to provide a uniform treatment material coating to a given area of ​​the substrate.

[0187] In some cases, the polynucleotide synthesis system comprises one or more elements that can be used for downstream processing of synthetic polynucleotides. As an example, the system comprises a temperature control element, such as a thermal cycler. In some cases, the temperature control element is used together with a plurality of analytical reactors to carry out polynucleotide assembly such as PCA and / or polynucleotide amplification such as PCR.

[0188] In some cases, a polynucleotide with a light-labile protective group is synthesized, wherein the hydroxyl groups generated on the surface are closed by the light-labile protective group. When the surface is exposed to ultraviolet light, for example, by a photolithography mask, a pattern of free hydroxyl groups can be generated on the surface. These hydroxyl groups can react with the photoprotected nucleoside phosphoramidites according to the phosphoramidite chemistry. A second photolithography mask can be used, and the surface can be exposed to ultraviolet light to generate a second pattern of hydroxyl groups, followed by coupling with the 5'-photoprotected nucleoside phosphoramidites. Similarly, a pattern can be generated and the oligomer chain can be extended. Without being bound by theory, the instability of the photocleavable group depends on the wavelength and the polarity of the solvent used, and the rate of photocleavage can be affected by the duration of exposure and the intensity of light. The method can utilize many factors, for example, the accuracy of mask alignment, the efficiency of photoprotective group removal, and the yield of the phosphoramidite coupling step. In addition, the leakage of undesirable light to adjacent sites can be minimized. The density of each spot synthetic oligomer can be monitored by adjusting the load of the lead nucleoside on the synthetic surface.

[0189] The surface of the substrate providing support for polynucleotide synthesis can be chemically modified to allow the polynucleotide chain of synthesis to be cut from the surface. In some cases, the polynucleotide chain is cut while the polynucleotide is deprotected. In some cases, the polynucleotide chain is cut after the polynucleotide chain is deprotected. In an exemplary scheme, trialkoxysilylamine such as (CH3CH2O)3Si-(CH2)2-NH2 reacts with the surface SiOH group of substrate, then reacts with succinic anhydride and amine to produce amide bonds and free OH supporting the growth of polynucleotide chains. Cutting includes gas cutting with alkali such as ammonia or methylamine. In some cases, once released from the surface, polynucleotide is assembled into larger polynucleotide, which is sequenced and decoded to extract the information stored.

[0190] Provided herein are systems and methods for synthesizing high density polynucleotides on a substrate in a short period of time. In some cases, the substrate is a flexible substrate. In some cases, at least about 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 In some cases, at least about 10 x 10 8 , 10x 10 9 , 10x 10 10 , 10x 10 11 or 10x 10 12In some cases, each synthesized polynucleotide comprises at least about 20, 50, 100, 200, 300, 400 or 500 nuclear bases. In some cases, the total average error rate of synthesizing these bases is less than about 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 1000, 1 / 2000, 1 / 5000, 1 / 10000, 1 / 15000, 1 / 20000 base. In some cases, these error rates are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or more of the synthesized polynucleotide. In some cases, these at least 90%, 95%, 98%, 99%, 99.5% or more of the synthesized polynucleotides are indistinguishable from the predetermined sequence encoded therefrom. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 200. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 1,000. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 2,000. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 3,000. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 5,000. Various types of error rates include mismatches, deletions, insertions and / or substitutions of the polynucleotides synthesized on the substrate. The term "error rate" refers to the comparison of the total amount of the synthesized polynucleotides with the sum of the predetermined polynucleotide sequences. In some cases, the synthesized polynucleotides disclosed herein include tethers of 12 to 25 bases. In some cases, the tether comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases.

[0191] The disclosed suitable method for synthesizing polynucleotide on substrate is phosphoramidite method, it is included in the coupling step (for example, extension step) that forms phosphite triester bond between phosphoramidite structural unit and the nucleoside that is attached to substrate, phosphoramidite structural unit (i.e. nucleoside phosphoramidite) is controlled to be added in the polynucleotide chain of growth.In some cases, nucleoside phosphoramidite is provided to the substrate of activation.In some cases, nucleoside phosphoramidite is provided to the substrate with activator.In some cases, nucleoside phosphoramidite is provided to substrate with 1.5,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,50,60,70,80,90,100 times or more times of excessive relative to the nucleoside bonded with substrate.In some cases, the interpolation of nucleoside phosphoramidite is carried out in anhydrous environment (for example, in anhydrous acetonitrile). After adding and linking the nucleoside phosphoramidite in the coupling step, the substrate is optionally washed. In some cases, the coupling step is repeated one or more additional times, optionally with washing steps between adding the nucleoside phosphoramidite to the substrate.

[0192] Polynucleotide assembly

[0193] Polynucleotides can be designed to span a large region of a predetermined sequence of coding information together. In some cases, the synthesized polynucleotides are connected by ligation to generate larger polynucleotides. An example of ligation is polymerase chain assembly (PCA). In some cases, at least a portion of the polynucleotides are designed to include an additional region of a substrate bound as a universal primer. For PCA reactions, pre-synthesized polynucleotides include overlaps with each other (e.g., 4, 20, 40 or more bases with overlapping sequences). During the polymerase cycle, the polynucleotides anneal with the complementary fragments and are then filled in by the polymerase. Therefore, depending on which polynucleotides are found to each other, each cycle randomly increases the length of each fragment. The complementarity between the fragments allows the formation of a complete large-span double-stranded DNA. In some cases, after the PCA reaction is completed, a mismatch repair detection enzyme is used to perform an error correction step to remove the mismatch in the sequence. Once a larger target sequence fragment is generated, the fragment can be amplified. For example, in some cases, a target sequence comprising 5' and 3' end adapter sequences is amplified in a polymerase chain reaction (PCR), and the PCR includes a modified primer hybridized with the adapter sequence. In some cases, the modified primer comprises one or more uracil bases. The use of modified primers allows the removal of primers by an enzymatic reaction focused on the bases targeted for modification and / or by the gaps left by the enzymes of the modified base pairs from the fragment cutting. What remains is a double-stranded amplification product lacking the adapter sequence residue. In this way, multiple amplification products can be generated in parallel with the same set of primers to generate different double-stranded DNA fragments.

[0194] Error correction can be performed to the polynucleotide synthesized and / or the product of assembly. The exemplary strategy for error correction relates to site-directed mutagenesis to correct errors by overlap extension PCR, which is optionally combined with two or more rounds of cloning and sequencing. In some cases, double-stranded polynucleotides with mispairing, protrusion and small ring, chemically altered bases and / or other heteroduplexes are selectively removed from the correctly synthesized polynucleotide population. In some cases, error correction is performed using recognition and in conjunction with or next to the protein / enzyme of the mispairing or unpaired base in the double-stranded polynucleotide, to produce single-strand or double-strand breaks or to start chain transfer transposition events. The non-limiting examples of the protein / enzyme for error correction include endonucleases (T7 endonuclease I, Escherichia coli endonuclease V, T4 endonuclease VII, mung bean nuclease, cell Escherichia coli endonuclease IV, UVDE), restriction enzymes, glycosylases, ribonucleases, mispairing repair enzymes, resolvase, helicase, ligase, mispairing specific antibodies and variants thereof. The example of specific error correction enzyme includes T4 endonuclease 7, T7 endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL, cleavage enzyme (cleavase), CELI and HINF1. In some cases, DNA mismatch binding protein MutS (Thermus aquaticus (Thermus aquaticus)) is used to remove the failed product from the synthetic product population. In some cases, error correction is carried out using a correction enzyme (Correctase). In some cases, error correction is carried out using SURVEYOR endonuclease (Transgenomic), which is a mismatch-specific DNA endonuclease that scans the known and unknown mutations and polymorphisms of heteroduplex DNA.

[0195] Computer Systems

[0196] In various aspects, any system described herein is operably connected to a computer and optionally automated locally or remotely by a computer. In various cases, the methods and systems of the present invention further include a software program on a computer system and its use. Therefore, computerized control of synchronization of the distribution / vacuuming / refilling functions (such as choreographing and synchronizing material deposition device movement, distribution action and vacuum actuation) is within the scope of the present invention. In some cases, the computer system is programmed to engage between the base sequence specified by the user and the position of the material deposition device to deliver the correct reagent to the specified area of ​​the substrate.

[0197] Figure 5 The computer system 500 shown in FIG. 5 can be understood as a logical device capable of reading instructions from a medium 511 and / or a network port 505, which can optionally be connected to a server 509. Figure 5The system shown may include a CPU 501, a disk drive 503, optional input devices such as a keyboard 515 and / or a mouse 516, and an optional monitor 507. Data communication with a server at a local or remote location may be achieved via the communication medium shown. The communication medium may include any means of transmitting and / or receiving data. For example, the communication medium may be a network connection, a wireless connection, or an Internet connection. Such a connection may provide communication via the World Wide Web. It is contemplated that data related to the present disclosure may be transmitted via such a network or connection, thereby being received and / or viewed by a party 522.

[0198] Figure 6 1 is a block diagram illustrating a first example architecture of a computer system 1000 that may be used in conjunction with example embodiments of the present invention. Figure 6 As shown, the example computer system may include a processor 602 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ (F) -S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 AppleA4™ processor, Marvell PXA 930™ processor or functionally equivalent processors. Multiple execution threads can be used for parallel processing. In some cases, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across a network comprising multiple computers, cellular phones and / or personal data assistant devices.

[0199] like Figure 6 As shown, cache memory 604 may be connected to or incorporated into processor 602 to provide high-speed storage of instructions or data that are recently or frequently used by processor 602. Processor 602 is connected to north bridge 606 via processor bus 608. North bridge 606 is connected to random access memory (RAM) 610 via memory bus 612 and manages access of processor 602 to RAM 610. North bridge 606 is also connected to south bridge 614 via chipset bus 616. South bridge 614 is in turn connected to peripheral bus 618. The peripheral bus may be, for example, PCI, PCI-X, PCI Express or other peripheral bus. North bridge and south bridge are generally referred to as processor chipsets and manage data transfer between processor, RAM and peripheral components on peripheral bus 618. In some alternative architectures, the functionality of the north bridge may be incorporated into the processor rather than using a separate north bridge chip.

[0200] In some cases, the system 600 may include an accelerator card 622 attached to the peripheral bus 618. The accelerator may include a field programmable gate array (FPGA) or other hardware for accelerating a process. For example, the accelerator may be used for adaptive data reconstruction or for evaluating algebraic expressions used in extended set processing.

[0201] Software and data are stored in external memory 624 and may be loaded into RAM 610 and / or cache 604 for use by the processor. System 600 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally equivalent operating systems, and application software running on top of the operating system for managing data storage and optimization according to the exemplary embodiments of the present invention.

[0202] In this example, system 600 also includes network interface cards (NICs) 620 and 621 connected to the peripheral bus to provide a network interface to external storage such as network attached storage (NAS) and other computer systems that can be used for distributed parallel processing.

[0203] Figure 7 700 is a diagram showing a network 700 having multiple computer systems 702a and 702b, multiple cell phones and personal data assistants 702c, and network attached storage (NAS) 704a and 704b. In an example embodiment, the systems 702a, 702b, and 702c can manage data storage and optimize data access to data stored in the network attached storage (NAS) 704a and 704b. Mathematical models can be used for the data and evaluated using distributed parallel processing across the computer systems 702a and 702b and the cell phones and personal data assistant system 702c. The computer systems 702a and 702b and the cell phones and personal data assistant system 702c can also provide parallel processing for adaptive data reconstruction of data stored in the network attached storage (NAS) 704a and 704b. Figure 7 Only one example is shown, and a wide variety of other computer architectures and systems can be used with multiple examples of the present invention. For example, blade servers can be used to provide parallel processing. Processor blades can be connected via a backplane to provide parallel processing. Storage can also be connected to the backplane via a separate network interface or as a network attached storage (NAS).

[0204] In some example embodiments, a processor may maintain a separate memory space and transmit data through a network interface, backplane or other connector for parallel processing by other processors. In other cases, some or all processors may use a shared virtual address memory space.

[0205] Figure 8 8 is a block diagram of a multi-processor computer system 800 using a shared virtual address storage space according to an example embodiment. The system includes multiple processors 802a-f that can access a shared memory subsystem 804. In the system, multiple programmable hardware storage algorithm processors (MAPs) 806a-f are incorporated into the memory subsystem 804. Each MAP 806a-f may include a memory 808a-f and one or more field programmable gate arrays (FPGAs) 810a-f. MAP provides a configurable functional unit and can provide a specific algorithm or part of an algorithm to the FPGA 810a-f for processing in close coordination with the corresponding processor. For example, in the example instance, the MAP can be used to evaluate algebraic expressions related to the data model and to perform adaptive data reconstruction. In this example, each MAP can be globally accessed by all processors for these purposes. In one configuration, each MAP can use direct memory access (DMA) to access the associated memory 808a-f, allowing it to perform tasks independently and asynchronously from the respective microprocessors 802a-f. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

[0206] The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems may be used in conjunction with the example examples, including systems using any combination of general purpose processors, coprocessors, FPGAs and other programmable logic devices, systems on a chip (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some cases, all or part of the computer system may be implemented in software or hardware. Any kind of data storage media may be used in conjunction with the example examples, including random access memory, hard drives, flash memory, tape drives, disk arrays, network attached storage (NAS), and other local or distributed data storage devices and systems.

[0207] Implementation

[0208] Provided herein are methods for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; d) depositing a washing solvent on the surface, wherein the washing solvent comprises a ketone, an ester, an ether, a hydrocarbon, or a functional equivalent thereof; and e) repeating steps bd to synthesize a plurality of polynucleotides. Further provided herein are methods, wherein the method further comprises depositing a capping solution on the surface, wherein capping prevents coupling of unblocked nucleosides. Further provided herein are methods, wherein the method further comprises depositing a deblocking solution on the surface, wherein deblocking allows coupling of the polynucleotide to the nucleoside. Further provided herein are methods, wherein the surface is washed with the washing solvent after one or more steps. Further provided herein are methods, wherein the surface is washed with acetonitrile after one or more steps. Further provided herein are methods, wherein the surface is washed with the washing solvent after each step after the coupling step. Further provided herein are methods, wherein the surface is washed with the washing solvent after each step after the capping step. Further provided herein are methods, wherein the surface is washed with the washing solvent after each step after the deblocking step. Further provided herein are methods, wherein the at least one nucleoside comprises a phosphoramidite. Further provided herein are methods, wherein the at least one nucleoside comprises a 5' blocking group. Further provided herein are methods, wherein the at least one nucleoside comprises a 3' blocking group. Further provided herein are methods, wherein the wash solvent is functionally equivalent to the major component by volume of the previously contacted reagent solution. Further provided herein are methods, wherein the wash solvent comprises acetone, tetrahydrofuran, ethyl acetate, toluene, benzene, ethanol, or a combination thereof. Further provided herein are methods, wherein the wash solvent comprises a ketone, an ether, or a functional equivalent thereof. Further provided herein are methods, wherein the structure is a plate, a tape, a belt, or a bead. Further provided herein are methods, wherein the oxidizing solution comprises iodine. Further provided herein are methods, wherein the polynucleotide or nucleoside comprises DNA or RNA.

[0209] Provided herein are methods for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface, wherein each of the at least one nucleoside comprises a blocking group; c) depositing an oxidizing solution on the surface; d) depositing a washing solvent on the surface, wherein the washing solvent comprises acetone or a functional equivalent thereof, or wherein the washing solvent comprises tetrahydrofuran or a functional equivalent thereof; e) depositing a deblocking solution on the surface to remove the blocking group, wherein the removal of the blocking group allows the nucleoside to couple to the polynucleotide; and f) repeating steps be to synthesize a plurality of polynucleotides. Further provided herein are methods, wherein the method further comprises contacting the surface with a capping solution, wherein capping prevents extension of unblocked nucleosides. Further provided herein are methods, wherein the washing solvent is functionally equivalent to the major component by volume of the previously contacted reagent solution. Further provided herein are methods, wherein the surface is washed with the washing solvent after one or more steps. Further provided herein are methods, wherein the surface is washed with acetonitrile after one or more steps. Further provided herein are methods, wherein the surface is washed with the wash solvent after each step after the coupling, capping or deblocking step. Further provided herein are methods, wherein the at least one nucleoside comprises a phosphoramidite. Further provided herein are methods, wherein the at least one nucleoside comprises a 5' blocking group. Further provided herein are methods, wherein the at least one nucleoside comprises a 3' blocking group. Further provided herein are methods, wherein the structure is a plate, a tape, a belt or a bead. Further provided herein are methods, wherein the oxidizing solution comprises iodine. Further provided herein are methods, wherein the polynucleotide or nucleoside comprises DNA or RNA.

[0210] Provided herein are methods for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; d) depositing a wash solvent on the surface, wherein I2 or an iodine salt has a greater solubility or increased dissolution rate in the wash solvent compared to acetonitrile; and e) repeating steps bd to synthesize a plurality of polynucleotides. Further provided herein are methods, wherein the method further comprises depositing a deblocking solution on the surface, wherein deblocking allows the polynucleotide to be coupled to the nucleoside. Further provided herein are methods, wherein the surface is washed with the wash solvent after one or more steps. Further provided herein are methods, wherein the surface is washed with acetonitrile after one or more steps. Further provided herein are methods, wherein the surface is washed with the wash solvent after each step after the coupling or deblocking step. Further provided herein are methods, wherein the wash solvent is functionally equivalent to the major component by volume of the reagent solution previously contacted. Further provided herein are methods, wherein the at least one nucleoside comprises a phosphoramidite. Further provided herein are methods, wherein the at least one nucleoside comprises a 5' blocking group. Further provided herein are methods, wherein the at least one nucleoside comprises a 3' blocking group. Further provided herein are methods, wherein the structure is a plate, tape, belt, or bead. Further provided herein are methods, wherein the oxidizing solution comprises iodine. Further provided herein are methods, wherein the oxidizing solution further comprises an amine base. Further provided herein are methods, wherein the amine base is selected from pyridine, lutidine, collidine, N-methylmorpholine, or a functional equivalent thereof.

[0211] Further provided herein are methods wherein the polynucleotide or nucleoside comprises DNA or RNA.

[0212] Provided herein is a method for synthesizing polynucleotides, comprising: a) providing a predetermined sequence for a plurality of preselected polynucleotides; b) providing a substrate comprising a surface; c) synthesizing the plurality of preselected polynucleotides extending from the surface, wherein the synthesis comprises one or more washing steps with a washing solvent after depositing an oxidizing solution on the surface, wherein the washing solvent dissolves active components or byproducts of the oxidizing solution, wherein the washing solvent comprises a ketone, an ester, an ether, a hydrocarbon, or a functional equivalent thereof; and d) assembling the plurality of preselected polynucleotides. Further provided herein is a method, wherein the method further comprises washing before or after depositing the oxidizing solution on the surface, wherein washing comprises depositing the washing solvent on the surface. Further provided herein is a method, wherein the washing solvent comprises a ketone, an ether, or a functional equivalent thereof. Further provided herein is a method, wherein the washing solvent comprises acetone, tetrahydrofuran, or a functional equivalent thereof. Further provided herein is a method, wherein the oxidizing solution comprises iodine.

[0213] Further provided herein are methods, wherein the oxidizing solution further comprises an amine base. Further provided herein are methods, wherein the amine base is selected from pyridine, lutidine, collidine, N-methylmorpholine or a functional equivalent thereof. Further provided herein are methods, wherein the polynucleotide or nucleoside comprises DNA or RNA.

[0214] Provided herein is a method for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; d) depositing a capping solution on the surface before or after depositing the oxidizing solution on the surface; and e) repeating steps bd to synthesize a plurality of polynucleotides. Further provided is a method, further comprising depositing the capping solution on the surface before and after depositing the oxidizing solution on the surface. Further provided is a method, further comprising depositing a washing solvent after the following steps: (i) coupling at least one nucleoside to the polynucleotide attached to the surface; (ii) depositing the capping solution; and (iii) depositing the oxidizing solution. Further provided is a method, wherein the washing solvent deposited after depositing the oxidizing solution comprises a ketone, an ester, an ether, a hydrocarbon, or a functional equivalent thereof. Further provided is a method, wherein the washing solvent deposited after depositing the oxidizing solution comprises a ketone, an ether, or a functional equivalent thereof. Further provided is a method, wherein the washing solvent deposited after depositing the oxidizing solution comprises a ketone, an ether, or a functional equivalent thereof. Further provided is a method, wherein the washing solvent deposited after depositing the oxidizing solution comprises acetone, THF, or a functional equivalent thereof. Further provided is a method, wherein the wash solvent deposited after the coupling step or the capping step comprises acetonitrile. Further provided is a method, wherein the wash solvent deposited after the coupling step or the capping step comprises acetone, tetrahydrofuran, or a functional equivalent thereof. Further provided is a method, wherein the wash solvent comprises a ketone, an ester, an ether, a hydrocarbon, or a functional equivalent thereof. Further provided is a method, wherein the wash solvent comprises a ketone, an ether, or a functional equivalent thereof. Further provided is a method, wherein the wash solvent comprises acetone, tetrahydrofuran, or a functional equivalent thereof. Further provided is a method, wherein the capping solution comprises an acyl halide or anhydride. Further provided is a method, wherein the capping solution comprises acetyl chloride or acetic anhydride. Further provided is a method, wherein the capping solution comprises an amine base. Further provided is a method, wherein the polynucleotide or nucleoside comprises DNA or RNA.

[0215] The following examples are described to more clearly illustrate the principles and practices of the examples disclosed herein to those skilled in the art, and should not be interpreted as limiting the scope of any claimed examples. Unless otherwise specified, all parts and percentages are by weight.

[0216] Example

[0217] Example 1: Polynucleotide Synthesis with Acetone Wash Step

[0218] The substrate surface (e.g., silicon plate) is functionalized with 3-glycidyloxypropyltrimethoxysilane (GOPS), a chemical group that can react with phosphoramidites to support the attachment and synthesis of polynucleotide libraries. The synthesis of polynucleotides is accomplished by repetition of the following steps: 1) extending a plurality of polynucleotides from the surface by contacting the surface with a base addition solution comprising at least one reactive monomer, wherein the at least one reactive monomer is a 3' phosphoramidite nucleobase (A, T, G, or C) comprising a 5' blocking group; 2) washing the surface at least once; 3) contacting the surface with an oxidizing solution containing iodine; 4) washing the surface at least once; and 5) removing the 5' blocking group with a deblocking solution; and 6) washing the surface at least once. Steps 1-6 are repeated until a plurality of polynucleotides are synthesized.

[0219] The above-mentioned polynucleotide synthesis method uses two different sets of conditions. In condition A, acetone is used as a washing agent to complete the washing step. In condition B, acetonitrile is used as a washing agent to complete the washing step. The error rate of the polynucleotide library under both conditions is analyzed using next generation sequencing, and the results by error type are shown in Table 5. The error rate of condition A is reduced, and the percentage reduction observed for single base deletion is the largest.

[0220] Table 5. Comparison of error rates for conditions A and B.

[0221] Error Type Condition A Condition B Insertion rate 0.000227 0.000315 Mismatch rate 0.000798 0.000905 Single base deletion rate 0.000539 0.001117 Total error rate 0.001564 0.002337

[0222] The error rates for single-base deletions and block deletions were further examined as a function of columns across the surface, where each column contains a series of clusters. Fig. 9A ) provides a better comparison than condition B ( Fig. 9B ) lower deletion rate and higher deletion uniformity in cluster columns for condition A. Inspection of all clusters on the surface (plate) also demonstrates similarity to condition B ( Fig.9D ) compared to condition A( Fig. 9C ) has a higher uniformity of single-base deletion rates among clusters on the surface.

[0223] Example 2: Polynucleotide synthesis using acetone wash after oxidation

[0224] A two-dimensional polynucleotide synthesis device was assembled into a flow cell, which was connected to a flow cell (Applied Biosystems ("ABI394 DNA Synthesizer"). The surface of the polynucleotide synthesis device was functionalized with 3-glycidyloxypropyltrimethoxysilane (GOPS) and used to synthesize a polynucleotide library using the synthesis methods described herein. The universal sequence of nucleic acids synthesized on the surface is 5'-##-3', where # represents thymidine-succinylhexanamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker that can release the polynucleotide from the surface during deprotection. The 3' end of the polynucleotide is attached to the surface, and the TTTTTTTTTT sequence between the linker and the surface represents the polynucleotide spacer. The synthesis of the spacer is performed by repetition of the following steps: 1) extending a plurality of reactive molecules (initially GOPS and then in a subsequent step a nucleic acid or polynucleotide) from the surface by contacting the surface with a base addition solution comprising at least one reactive monomer, wherein the at least one reactive monomer is a 3' phosphoramidite thymine comprising a 5' blocking gene; 2) capping the unreacted 5'OH group of thymine by contacting the surface with a capping solution; 3) washing the surface at least once with acetone; 4) contacting the surface with an iodine-containing oxidizing solution; 5) washing the surface at least once with acetone; 6) capping the unreacted 5'OH group of thymine by contacting the surface with a capping solution; 7) removing the 5' blocking group with a deblocking solution; and 8) washing the surface at least once with acetone. Steps 1-8 are repeated until a plurality of polynucleotide spacers are synthesized. A cleavable linker group (##) is then coupled to the 5' end of the polynucleotide spacer, and the surface is then washed at least once with acetone.

[0225] Extension beyond the linker / spacer to synthesize a polynucleotide is performed by repetition of the following steps: 1) contacting the surface with a base addition solution comprising at least one reactive monomer, wherein the at least one reactive monomer is a 3' phosphoramidite nucleobase (A, T, G, or C) comprising a 5' blocking group; 2) contacting the surface with an iodine-containing oxidizing solution; 3) washing the surface at least once with acetone; and 4) removing the 5' blocking group with a deblocking solution; and 5) washing the surface at least once with acetone. Steps 1-5 are repeated until a plurality of polynucleotides are synthesized. After polynucleotide synthesis, the polynucleotide is cleaved from the surface by deprotection overnight in gaseous ammonia for further analysis.

[0226] The polynucleotide libraries were synthesized with high uniformity. Several methods were used to evaluate the quality of the polynucleotide libraries, including Sanger sequencing and next generation sequencing. The total error rate of the three different oligonucleotide libraries was about 1 / 1000 or less ( Fig.10The quality of the polynucleotide library was evaluated using next generation sequencing. The error rates of the error classes deletion, insertion, and mismatch were measured ( Fig.11A ). For example, the substitution rate was further evaluated as a function of the reference base and the observed base. Substitutions occurred at a uniform rate, with a slight increase in the rate of G to A substitutions and the rate of G to T substitutions. ( Fig. 11B The deletion rate for each base type was measured; the deletion rate for all bases was comparable, approximately 0.002 ( Fig. 11C ). G+G insertion errors increased relative to A+A, C+C, and T+T insertion errors ( Fig.11D Insertion, mismatch, single deletion, and block deletion rates were also evaluated as a function of base position on the polynucleotide. The observed error rates were uniform across all positions of the synthesized polynucleotide, with the x-axis associated with the base distance from the surface ( Fig.11E ). To evaluate loss, clusters from each region of the board ( Fig. 12A ) were evaluated for average read counts, with a total of 8.7 million reads (Table 6). The average read count for all clusters was approximately 400 and was highly uniform in the x (1-24) and y (AP) directions of the plate. Fig. 12B In the distribution curve of , the loss rate is less than 0.0% and the uniformity is 1.7 times the mean. In another depiction of the error rate, the read count is in the cluster ( Fig. 12C , above) and multiple devices ( Fig. 12C , lower figure) are highly uniform.

[0227] Table 6. Polynucleotide library measurements

[0228]

[0229] Example 3: Washing solution to remove unwanted G→A and T / U→C mutations.

[0230] Washing solution was prepared as described herein, comprising THF, pyridine and water in a ratio of 80:20:10. According to the general procedure of Examples 1 and 2, polynucleotide samples were synthesized using phosphoramidite chemistry. Sample A was washed after coupling, and the washing solution comprised THF / pyridine / water, followed by acetone, and the second sample B was washed with acetone after coupling. Deprotection of the first sample with methylamine and washing steps with a washing solution comprising THF, pyridine and O-nucleophile (water) resulted in no T→C and G→A mutation rates above the noise level (ratio less than 1 / 5000) being detected by NGS. Deprotection of the second sample with methylamine and washing steps with acetonitrile or acetone resulted in T→C and G→A mutation rates greater than 1 / 1500. The uniformity of the synthesis performed using washing solution was greatly improved. The results showed that within the detection limit, the phosphoramidite / tetrazolyl material deposited in the previous coupling reaction by printing was completely removed. For sample A, the uniformity of the obtained polynucleotides was improved. For sample A, approximately >90% of all records had between 220 and 670 reads. For sample B, approximately >90% of all records had between 30 and 940 reads.

[0231] Table 7. Washing solution results

[0232] sample Lost (%) Uniformity Total reads (millions) Mean ± SD A 0.1 6.9 12 518±229 B 0.1 1.9 10.1 436±88

[0233] Example 4: Capping to remove unwanted G→A and T / U→C mutations.

[0234] Capping solution 1 is prepared as described herein, which comprises THF, lutidine, acetic anhydride in a ratio of 80:10:10, and acetic acid (10 g / L) is added. Capping solution 2 comprising acetonitrile and n-methylimidazole in a ratio of 90:10 is also prepared. According to the general procedure of Examples 1 and 2, polynucleotide samples are synthesized using phosphoramidite chemistry. Sample A is washed with a mixture of solution 1 and 2, while the second sample B is washed with capping solution in the absence of acetic acid. Deprotection of the first sample with methylamine and washing steps with a capping solution comprising an O-nucleophile (acetic acid) result in no T→C and G→A mutation rates above the noise level (ratio of 1 / 10101) being detected by NGS. Deprotection of the second sample with methylamine and washing steps with a capping solution without O-nucleosides result in T→C and G→A mutation rates of 1 / 3058. The addition of alternative O-nucleophiles, such as sigma acceptor-substituted acetic acid derivatives, such as methoxyacetic acid and phenoxyacetic acid, also improved the efficiency of the capping reaction.

[0235] Example 5: Capping sequence

[0236] Following the general procedure of Examples 1 and 2, Sample A was washed with the capping solution after oxidation, while Sample B was washed with the capping solution before oxidation. For Sample A, the procedure was to wash -

[0237] Acetone-Ox-Acetone-CapMix (from Example 4)-Acetone-Deblocking-

[0238] Acetone. The single base deletion rate of sample B is 1 / 1196, while the single base deletion rate of sample A is 1 / 5450.

[0239] Example 6: Base-protected phosphoramidites

[0240] Using the general procedures of Examples 1 and 2, protected dT and dG phosphoramidite building blocks AI containing protecting groups at nitrogen atoms N3 and N1 were incorporated into polynucleotides. After deprotection of the resulting polynucleotide product with MeNH2, T->C and G->A mutations above the noise level (ratios less than 1 / 5000) could not be detected according to the limitations of NGS, while control experiments using standard unprotected nucleoside phosphoramidites resulted in mutation rates greater than 1 / 1500. The use of base-protected nucleoside phosphoramidites resulted in lower error rates.

[0241] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions may be employed in the process of implementing the present invention. It is intended that the scope of the present invention be limited to the appended claims, thereby covering methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for synthesizing a polynucleotide, comprising: a) providing a structure comprising a surface; b) coupling at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution on the surface; d) depositing a wash solvent on said surface, wherein the wash solvent comprises a ketone, an ester, an ether, a hydrocarbon or a functional equivalent thereof; and e) repeating steps bd to synthesize multiple polynucleotides.

2. The method of claim 1, wherein the washing solvent comprises acetone, tetrahydrofuran, ethyl acetate, toluene, benzene, ethanol or a combination thereof.

3. The method of claim 1, wherein the wash solvent comprises a ketone, an ether, or a functional equivalent thereof.

4. The method of claim 3, wherein the washing solvent comprises acetone or a functional equivalent thereof.

5. The method of claim 1, wherein the wash solvent is functionally equivalent to a major component by volume of the previously contacted reagent solution.

6. The method of claim 1, wherein the surface is washed with the washing solvent after one or more of steps b) to d).

7. The method of claim 1, wherein the surface is washed with acetonitrile after one or more of steps b) to d).

8. The method of claim 1, wherein the surface is washed with the washing solvent after each step after the coupling step.

9. The method of claim 1, wherein the surface is washed with the washing solvent after each step after the deblocking step.

10. The method of claim 1, wherein the method further comprises depositing a capping solution on the surface, wherein capping prevents coupling of unblocked nucleosides.

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