Compositions, systems, and methods for making and using encapsulated freeze-dried microspheres

Abstract

The present disclosure relates to a composition comprising a shell surrounding a core, the core comprising one or more lyophilized microspheres. Also described herein is a method comprising preparing one or more lyophilized microspheres and coating one or more lyophilized microspheres with a shell under conditions effective to encapsulate the one or more lyophilized microspheres. The present disclosure further relates to a system comprising one or more compositions described herein and one or more lyophilized cakes, the one or more compositions and the one or more lyophilized cakes being combined under conditions effective to form a rehydration system. Also described herein is a method of controlling the release of one or more encapsulated microspheres, comprising preparing a composition described herein and mixing the composition with a rehydration solution under first conditions effective to control the release of one or more lyophilized microspheres from the composition.

Description

[Technical field]

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 174,325, filed April 13, 2021, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates generally to compositions, systems, and methods for making and using encapsulated lyophilized microspheres. [Background technology]

[0003] Many current sequencing platforms use "sequencing by synthesis" ("SBS") technology and fluorescence-based methods for detection. As a complement to SBS, alternative sequencing methods and improved sample preparation processes that allow for more cost-effective, rapid, and convenient sequencing and nucleic acid detection are desirable.

[0004] Current protocols for SBS techniques routinely use sample preparation processes that convert DNA or RNA into libraries of fragmented, sequenceable templates. Sample preparation methods often require multiple steps, material transfers, and expensive equipment to effect fragmentation, and are therefore often difficult, tedious, expensive, and inefficient.

[0005] A library containing polynucleotides is generally prepared by any suitable method that attaches oligonucleotide adaptors to target polynucleotides. Sequencing can result in the determination of the sequence of the entire or part of the target polynucleotide. The number of steps required to convert nucleic acids into adaptor-modified templates in solution ready for clustering and sequencing can be reduced, and in some instances minimized, by the use of transposase-mediated fragmentation and tagging. This process, called "tagmentation", involves modification of nucleic acids with a transposome complex that includes a transposase enzyme complexed with an adaptor that includes a transposon end sequence, as described, for example, in WO 2016 / 130704. Methods for immobilization and amplification prior to sequencing are described, for example, in U.S. Pat. No. 8,053,192, WO 2016 / 130704, U.S. Pat. No. 8,895,249, and U.S. Pat. No. 9,309,502. The library of fragments can be used to prepare clustered arrays of nucleic acid colonies by solid-phase amplification, more specifically solid-phase isothermal amplification, as described in U.S. Patent Application Publication No. 2005 / 0100900, U.S. Patent No. 7,115,400, WO 00 / 18957 and WO 98 / 44151.

[0006] Sequencing can be performed using any suitable sequencing technique, and methods for determining the sequence of immobilized and amplified adaptor-target-adaptor molecules, including strand resynthesis, are known in the art and are described, for example, in U.S. Pat. No. 8,053,192, WO 2016 / 130704, U.S. Pat. No. 8,895,249, and U.S. Pat. No. 9,309,502. SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand by the repetitive addition of nucleotides to a template strand. In conventional methods of SBS, a single nucleotide monomer can be provided to a target nucleotide in the presence of a polymerase at each delivery. Exemplary SBS systems and methods are described in U.S. Patent Application Publication Nos. 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2012 / 0270305, and 2013 / 0260372, U.S. Patent No. 7,057,026, WO 05 / 065814, U.S. Patent Application Publication Nos. 2005 / 0100900, WO 06 / 064199, and WO 07 / 010,251, and U.S. Patent Application Publication No. 2013 / 0079232.

[0007] The stability of reagents involved in sample preparation, including PCR, for example, varies depending on a variety of factors. Historically, reagents have been wet, which often involves freezing for shipping and storage. Moving to dry reagents may allow for ambient shipping and storage, but dry reagents may be more sensitive to environmental conditions than wet reagents. If reagents are exposed to undesirable environmental conditions in manufacturing, shipping, storage, or during library preparation, the quality and efficiency of the resulting library may be affected. Similarly, the pH of reagents such as SBS buffers fluctuates during sequencing, and stabilization of these buffers needs to be improved to enhance SBS performance. Reagents involved in sample preparation can be very sensitive to changes in humidity, light, and moisture, and as a result are notoriously difficult to keep stable.

[0008] Furthermore, freeze-dried microspheres that may be used for sample preparation often degrade when exposed to mechanical stress during transport and storage, and their outer shell may be adversely shed. The resulting powders may have problems with clogging of the membranes used for sample preparation, leading to variations in the desired final concentration after rehydration has been achieved. Also, static charges are a risk for dispensing and dry formulation of microspheres.

[0009] Triboelectric charging is achieved by (frictional) contact through particle-particle and / or particle-wall interactions. Charge transfer occurs during contact, resulting in two oppositely charged objects after separation. Static electricity is achieved by the ability of a material (particle or wall) to dissipate static charge, which is related to the electrical conductivity of the material.

[0010] Lyophilized microspheres are typically manufactured from non-conductive materials (e.g., trehalose). The need to handle and store lyophilized microspheres in a dry environment arises from the limited resistance of the microspheres to ambient humidity. Thus, in a dry environment, static behavior and triboelectric charging are expected in lyophilized microspheres, which are manifested by the adhesion of the microspheres to the walls of the container. The risk associated with static electricity is that the microspheres are difficult to handle for dry loading into cartridges. Upon dry storage, the microspheres tend to adhere to the walls of the cartridge, which leads to cross-contamination between cartridge wells and inaccuracies during rehydration.

[0011] Therefore, there is a need for improved sample preparation compositions and processes.In particular, there is a need for sequencing reagents with improved stability, and related methods that show improved workflow and efficiency of tagmented library generation, and thus increased lead enrichment of resulting libraries.There is also a need for compositions and methods that improve lead enrichment of resulting libraries and simplify workflow.

[0012] The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0013] The first embodiment relates to a composition comprising a shell surrounding a core, the core comprising one or more lyophilized microspheres.

[0014] In one embodiment, the shell comprises one or more of carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0015] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises a static relief material, a moisture barrier material, or a combination thereof. In one embodiment, the shell additive is a static relief material present in an amount of 40% w / w or less of the shell. In one embodiment, the shell additive is a moisture barrier material present in an amount of 90% w / w or less of the shell. In one embodiment, the shell additive is present in an amount of at least 10% w / w of the shell. In one embodiment, the shell additive is in an amount of about 10% w / w to about 90% w / w of the shell. In one embodiment, the shell additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity promoter, a stearic acid derivative, an oleic acid derivative, a lauric acid derivative, a polyether compound, an amino acid, tocopherol acetate, piperidyl sebacate, a sodium salt, a buffer, a chelating agent, an imidazolium salt, a polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearic acid derivative or the oleic acid derivative is selected from magnesium stearate, triglycerol stearate, Span® 60, Tween® 60, glycerol trioleate, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof.In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, or polyquaternium, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole), or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0016] In one embodiment, the core comprises one or more reagents selected from one or more enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In one embodiment, the reagent is a polymerase. In one embodiment, the volume of the reagent in the core is about 0.1 μL to about 50 μL.

[0017] In one embodiment, the shell comprises a reagent.

[0018] In one embodiment, the core further comprises one or more additional agents, the additional agents being selected from one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, and any combination thereof. In one embodiment, when the additional agent comprises one or more sugars, the sugars are selected from trehalose, mannitol, cyclodextrin, dextran, sucrose, or any combination thereof. In another embodiment, the additional agent comprises one or more amino acids having hydrophobic side chains. In yet another embodiment, when the additional agent comprises one or more polymers, the polymers are selected from polyvinylpyrrolidone, polyvinyl alcohol, or any combination thereof.

[0019] In one embodiment, the core comprises a core additive. In one embodiment, the core additive comprises a static relief material. In one embodiment, the core additive is a static relief material present in an amount of 25% w / w concentration or less of the core. In one embodiment, the core additive is present in an amount of at least 0.5% w / w concentration of the core. In one embodiment, the core additive is in an amount of about 2% w / w to about 10% w / w of the core. In one embodiment, the core additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), a conductivity enhancer, an ammonium salt, an imidazolium salt, a polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, or polyquaternium, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole), or a combination thereof. In one embodiment, the composition is used to perform multiple simultaneous assay reactions. In one embodiment, the shell comprises more than one lyophilized microsphere, and the reagents in the more than one lyophilized microsphere are different.

[0020] A second aspect relates to a method, comprising providing one or more lyophilized microspheres, and coating the one or more lyophilized microspheres with a shell under conditions effective to encapsulate the one or more lyophilized microspheres.

[0021] In one embodiment, the method further comprises coating the shell with an outer layer under conditions effective to surround the encapsulated microspheres with the outer layer, hi one embodiment, the coating is carried out for a period of time sufficient to provide the outer layer with a specified thickness.

[0022] In one embodiment, the shell, the outer layer, or both the shell and the outer layer comprise one or more of carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0023] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises a static relief material, a moisture barrier material, or a combination thereof. In one embodiment, the shell additive is a static relief material present in an amount of 40% w / w or less of the shell. In one embodiment, the shell additive is a moisture barrier material present in an amount of 90% w / w or less of the shell. In one embodiment, the shell additive is present in an amount of at least 10% w / w of the shell. In one embodiment, the shell additive is in an amount of about 10% w / w to about 90% w / w of the shell. In one embodiment, the shell additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity promoter, a stearic acid derivative, an oleic acid derivative, a lauric acid derivative, a polyether compound, an amino acid, tocopherol acetate, piperidyl sebacate, a sodium salt, a buffer, a chelating agent, an imidazolium salt, a polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearic acid derivative or the oleic acid derivative is selected from magnesium stearate, triglycerol stearate, Span® 60, Tween® 60, glycerol trioleate, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof.In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, or polyquaternium, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole), or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0024] In one embodiment, the shell surrounds a core, and the core comprises one or more reagents selected from one or more enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In one embodiment, the reagent is a polymerase. In one embodiment, the volume of the reagent in the core is about 0.1 μL to about 50 μL.

[0025] In one embodiment, the shell comprises a reagent.

[0026] In one embodiment, the encapsulated microspheres have a diameter of about 100 μm to 1000 μm.

[0027] In one embodiment, the core further comprises one or more additional agents, the additional agents comprising one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, or any combination thereof. In one embodiment, when the additional agent comprises one or more sugars, the sugars are selected from trehalose, mannitol, cyclodextrin, dextran, sucrose, or any combination thereof. In another embodiment, the additional agent comprises one or more amino acids having hydrophobic side chains. In yet another embodiment, when the additional agent comprises a polymer, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, or any combination thereof.

[0028] In one embodiment, the core comprises a core additive. In one embodiment, the core additive comprises a static relief material. In one embodiment, the core additive is a static relief material present in an amount of 25% w / w concentration or less of the core. In one embodiment, the core additive is present in an amount of at least 0.5% w / w concentration of the core. In one embodiment, the core additive is in an amount of about 2% w / w to about 10% w / w of the core. In one embodiment, the core additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), a conductivity enhancer, an ammonium salt, an imidazolium salt, a polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polyquaternium or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole) or a combination thereof.

[0029] In one embodiment, the method further comprises contacting a reagent with the sample to perform multiple simultaneous assay reactions, hi one embodiment, the shell comprises more than one lyophilized microsphere and the reagents in the more than one lyophilized microsphere are different.

[0030] A third aspect relates to a system comprising one or more compositions described herein and one or more lyophilized cakes, wherein the one or more compositions and the one or more lyophilized cakes are combined under conditions effective to form a rehydrated system.

[0031] In one embodiment, the system further comprises one or more shell layers disposed between the one or more encapsulated microspheres and the one or more lyophilized cakes. In another embodiment, the shell layer comprises a material selected from carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0032] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises a static relief material, a moisture barrier material, or a combination thereof. In one embodiment, the shell additive is a static relief material present in an amount of 40% w / w or less of the shell. In one embodiment, the shell additive is a moisture barrier material present in an amount of 90% w / w or less of the shell. In one embodiment, the shell additive is present in an amount of at least 10% w / w of the shell. In one embodiment, the shell additive is in an amount of about 10% w / w to about 90% w / w of the shell. In one embodiment, the shell additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity promoter, a stearic acid derivative, an oleic acid derivative, a lauric acid derivative, a polyether compound, an amino acid, tocopherol acetate, piperidyl sebacate, a sodium salt, a buffer, a chelating agent, an imidazolium salt, a polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearic acid derivative or the oleic acid derivative is selected from magnesium stearate, triglycerol stearate, Span® 60, Tween® 60, glycerol trioleate, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof.In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, or polyquaternium, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole), or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0033] A fourth aspect relates to a method for controlling the release of one or more encapsulated microspheres, the method comprising the steps of providing a composition as described herein and mixing the composition with a rehydration solution under first conditions effective to control the release of one or more lyophilized microspheres from the composition.

[0034] In one embodiment, the method further comprises changing the first conditions to a second condition. In one embodiment, the change in the first conditions comprises one or more of changing the temperature, changing the exposure time, changing the pH of the rehydration solution, or changing the position of the encapsulated microspheres in the rehydration solution. In another embodiment, the temperature in the first condition and / or the second condition is from about 10° C. to about 90° C. In yet another embodiment, the pH in the rehydration solution is from about 6.0 to about 10.0.

[0035] In one embodiment, the first conditions are effective to release a first lyophilized microsphere. In another embodiment, the second conditions are effective to release a second lyophilized microsphere, the content of the second lyophilized microsphere being different from the content of the first lyophilized microsphere. In another embodiment, the alteration of the first conditions allows for the sequential release of one or more lyophilized microspheres.

[0036] In one embodiment, the shell comprises carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, an oxygen scavenger, alginate, chitosan, a starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0037] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises a static relief material. In one embodiment, the shell additive is a static relief material present in an amount of 40% w / w concentration or less of the shell. In one embodiment, the shell additive is present in an amount of at least 10% w / w concentration of the shell. In one embodiment, the shell additive is in an amount of about 10% w / w to about 90% w / w of the shell. In one embodiment, the shell additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity enhancer, a stearic acid derivative, an oleic acid derivative, a lauric acid derivative, a polyether compound, an amino acid, a tocopherol acetate, a piperidyl sebacate, a sodium salt, a buffer, a chelating agent, an imidazolium salt, a polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearic acid derivative or oleic acid derivative is selected from magnesium stearate, triglycerol stearate, Span® 60, Tween® 60, glycerol trioleate, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof.In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salts or polyquaterniums or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole) or combinations thereof. In one embodiment, the shell additive comprises an ammonium salt, copolymer, polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0038] In one embodiment, the core comprises one or more reagents selected from one or more enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In one embodiment, the reagent is a polymerase.

[0039] In one embodiment, the first change in conditions is effective to release two or more lyophilized microspheres, the two or more lyophilized microspheres comprising different reagents. In one embodiment, the volume of the reagent in the core is about 0.1 μL to about 50 μL.

[0040] In one embodiment, the shell further comprises a reagent.

[0041] In one embodiment, the core and / or the rehydration solution further comprises one or more additional agents, the additional agents being selected from one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, or any combination thereof. In one embodiment, when the additional agent comprises a sugar, the sugar is selected from trehalose, mannitol, cyclodextrin, dextran, sucrose, or any combination thereof. In another embodiment, the additional agent comprises one or more amino acids having hydrophobic side chains. In yet another embodiment, when the additional agent comprises a polymer, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, or any combination thereof.

[0042] In one embodiment, the core comprises a core additive. In one embodiment, the core additive comprises a static relief material. In one embodiment, the core additive is a static relief material present in an amount of 25% w / w concentration or less of the core. In one embodiment, the core additive is present in an amount of at least 0.5% w / w concentration of the core. In one embodiment, the core additive is in an amount of about 2% w / w to about 10% w / w of the core. In one embodiment, the core additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), a conductivity enhancer, an ammonium salt, an imidazolium salt, a polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polyquaternium or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole) or a combination thereof.

[0043] In one embodiment, the method further comprises providing an additional composition described herein and mixing the additional composition under third conditions effective to control the release of the one or more lyophilized microspheres from the additional composition.

[0044] In one embodiment, the method further comprises contacting a reagent with the sample to perform multiple simultaneous assay reactions. In one embodiment, the shell comprises more than one lyophilized microsphere, and the reagents in the more than one lyophilized microsphere are different. In one embodiment, the method further comprises providing one or more lyophilized cakes, and rehydrating the one or more lyophilized cakes.

[0045] In accordance with the present disclosure, the compositions, systems, and methods described herein have many advantages including, for example, increased stability of the microspheres, macroencapsulation allowing for multi-run cartridges, and microencapsulation allowing for simplified workflow and reduced number of reagent wells.

[0046] There is great interest and an unmet need for encapsulating freeze-dried microspheres to increase the stability of sequencing reagents and simplify workflow. The present disclosure describes compositions, systems, and methods for encapsulating freeze-dried reagents that allow for sequential release of freeze-dried reagents. One method for allowing sequential release of freeze-dried reagents is by temperature-triggered release, for example, by immersing microsphere-filled gelatin capsules in paraffin wax. Such an approach allows for release of microspheres at different temperatures, for example, from about 30° C. to about 50° C. for natural gelatin capsules and from about 50° C. to about 90° C. for coated capsules. Similarly, such an approach allows for time-triggered release by the addition of additives to the rehydration solution, for example, amino acids that can slow the rate of rehydration of the cake.

[0047] The compositions, systems, and methods described herein have many advantages. For example, encapsulated freeze-dried microspheres provide antistatic protection by neutralizing charge and reducing triboelectric affinity, thereby reducing the complexity of metering and handling in manufacturing (e.g., mesoporous silica, ionic liquids, quaternary amines). Static charge has been identified as the highest risk in dispensing and dry compounding of microspheres, as it significantly impacts the metering and mixing of dry microsphere powders during manufacturing. The encapsulation of microspheres described in the compositions, systems, and methods described herein neutralizes the charge of the microspheres, for example, by coating the particles with a neutral material that has low triboelectric affinity, thereby greatly improving the stability of sequencing.

[0048] Similarly, the compositions, systems, and methods described herein provide oxygen protection through low oxygen permeable polymer coatings (e.g., polyvinyl alcohol and / or oxygen scavengers in the coating). Similarly, the compositions, systems, and methods described herein provide moisture protection through the application of amphiphilic coatings (e.g., amino acids and / or PVP copolymers). The compositions, systems, and methods described herein further provide protection from mechanical stresses, for example, by preventing or reducing fragmentation during manufacturing (e.g., by providing a shell with 40% solute content). Such protective coatings increase the mechanical robustness of the microspheres and their contents during manufacturing and shipping, and eliminate shedding of powder from the microspheres that could otherwise result in powder clogging the membrane.

[0049] The compositions, systems, and methods described herein may further provide protection from light exposure, since the reagents are protected from light exposure, thereby reducing light constraints on manufacturing. Encapsulation of lyophilized microspheres can improve sequencing quality, enable one-pot library preparation, and simplify manufacturing. For example, microspheres with coatings or shells may be opaque or contain dyes or other additives that otherwise prevent or reduce the amount of light entering the core of the microsphere.

[0050] The pH of SBS buffers is known to change over a sequencing run. The compositions, systems, and methods described herein can use encapsulation of particles that are otherwise responsive to pH changes to stabilize these particles (e.g., buffers) and enhance SBS performance. The compositions, systems, and methods described herein can further improve control of the pH of solutions (e.g., uptake mixes ("ICMs")) that may change over time while on the instrument. Different solutions may be used throughout the length of an SBS cycle, which can take hours, and thus reagents present in the solution are prone to decomposition upon environmental exposure. This is accomplished by developing pH-sensitive microspheres that release when the buffer drops below a certain pH, releasing ions and returning the buffer to the desired pH. Similarly, the compositions, systems, and methods described herein can improve control of the external charge of the microspheres to facilitate dispensing, prevent or reduce stratification in the mixed bulk, and even allow separation of the reagent components SBS Cleave Mix to prevent or reduce and / or control undesired interactions in a single pot or well. For example, a cleave mix may benefit from separation of reagents to reduce the temperature sensitivity of the mixed reagents achieved in the compositions, systems, and methods described herein. Similarly, the compositions, systems, and methods described herein protect the polymerase during fully functionalized nucleotide ("ffN") polishing, when polishing is involved, and protect light-sensitive ffNs from photodegradation, especially when the environmental conditions involved in polishing degrade the enzyme.

[0051] The problem of using two incompatible competing polymerases (polishing polymerase and sequencing polymerase) to modify deblocked lyophilized ffNs in one well (incorporation mix reagent well) can be addressed by spatially and temporally separating the polymerases using the compositions, systems, and methods described herein. In particular, the problem can be solved by encapsulating one polymerase (the sequencing polymerase, since this polymerase is used after the polishing polymerase) in a water-soluble, slowly dissolving film (e.g., polyvinyl alcohol). The problem of timing the release of the sequencing polymerase from the capsule to coincide with the completion of the polishing process can be addressed by adjusting the components and their relative amounts in the water-soluble film. To achieve a finer level of control, additives that are temperature or light responsive can also be used.

[0052] Lyophilization of ffN achieves increased stability compared to their liquid forms, but results in elevated 3'OH levels, increasing prephasing and reducing run quality. Laboratory use of the polishing workflow can be complicated. The polishing mix (ffN, polishing polymerase, polishing oligo, Mg) is prepared, combined separately, incubated at an elevated temperature of 50°C (to facilitate the polishing reaction) for up to 1 hour, and then added to the remainder of the incorporation mix where the sequencing polymerase is found. This level of complexity for the user and the sequencer means that this workflow is undesirable in its current form, and even less desirable at large scale. A solution with minimal and / or no user touchpoints, as complex as or less complex than the current sequencer workflow, as described in the compositions, systems, and methods of the present disclosure, is a viable improvement over existing workflows.

[0053] The only capture reagent well is dispensed with free "polishing microspheres" (which may contain ffN, polishing polymerase, polishing oligo, and magnesium enzyme cofactor). Also present in this well are sequencing polymerase microspheres; however, these are encapsulated in a water-soluble timed dissolving film. This configuration allows for multiple advantages, including, for example, a reduction in well number. Following the current separate preparation of the polishing mix followed by mixing with the Grand ICM mix, individual wells may be required for the polishing reagents. Utilizing the encapsulation compositions, systems, and methods described herein, it is easy to perform multiple sequential reactions in one well, thereby minimizing the number of wells. This also impacts the cartridge footprint, with knock-on benefits regarding environmental impacts including, for example, plastic use and incinerator waste. The compositions, systems, and methods described herein can be easily scaled while also resulting in reduced fluidics and valving, thereby reducing the complexity and associated costs of the sequencer. When a rehydration buffer, such as water, is dispensed into the wells, the released polishing microspheres rapidly dissolve and the polishing reaction begins to modify the unblocked ffNs. This rehydration buffer also begins to dissolve the water-soluble film that encapsulates the sequencing polymerase.

[0054] The compositions, systems, and methods described herein allow for benefits in addition to those mentioned above. For example, using lyophilized and separated lyophilized materials means that additional cofactors for the enzymes, such as magnesium, can be added to the microspheres themselves, rather than having a separate additional rehydration buffer. This can allow reagents of different concentrations and / or types of enzymes, all of which require or benefit from different amounts of cofactors, salts, pH, etc., to be rehydrated with water alone or by atmospheric water capture. This facilitates a reduction in the amount of plastic used in the sequencing process, as well as the associated carbon footprint, given the reduction in the weight of the reagents when in concentrated and / or lyophilized form.

[0055] The encapsulation method described herein can be applied to allow an easy way to adjust the reagent concentration. For example, a smaller capsule may contain a smaller amount of lyophilized reagent compared to a larger capsule, and multiples of this capsule may be placed in a well according to the user's requirements. This facilitates improved user flexibility in terms of throughput without potential errors that occur in dilution / concentration calculations. A unit-based approach, where X capsules = Y runs, allows this flexibility in a more controlled manner. Such an approach can also give the user flexibility in terms of sequencing depth. Applications involving deep sequencing, e.g., cancer screening, may use many capsules, while surface screening, e.g., MRSA, may use fewer capsules.

[0056] The compositions, systems, and methods described herein achieve an improved level of control over reagent release (e.g., rehydration of a first reagent followed by delayed rehydration of one or more subsequent reagents after a period of time), as well as mechanical protection, buffer stabilization, charge control, combination of two or more different reagents in a single microsphere, single well, or single pot, and photoprotection. In particular, the controlled timed release of reagents by the use of encapsulated lyophilized microspheres described herein enables one-pot for library preparation. The inhibition of tagmentation by reagents involved in PCR is addressed by encapsulating the PCR reagents and releasing them at a predetermined time.

[0057] To address the problems of static electricity and triboelectric charging, it has been proposed to incorporate additives either directly into the microspheres (as a lyophilized matrix) or as a coating that encapsulates the microspheres. The rationale for the additives is, first, to prevent or reduce charge accumulation (increased conductivity) and, second, to reduce the surface charge by diffusion and / or dissipation.

[0058] The prevention or reduction of charge accumulation can be achieved by increasing the ion concentration, crystallinity or salinity, which proceeds competitively under the shielding by water molecules, and as a result, the tendency to accept charge is reduced, or the charge dissipation is increased. Water shielding also induces a reduction in the angle of internal friction, and as a result, a reduction in electrostatic charging.

[0059] Such additives are exemplified as sodium salts (i.e. sodium chloride, sodium bisulfite, sodium citrate), Trizma (Tris.HCl), MOPS, HEPES, ammonium salts (tetraalkylammonium chlorides, Efka® IO 6783 or tris(hydroxyethyl)alkylammonium chlorides), imidazolium salts (i.e. Efka® IO 6786 or 1-ethyl-3-methylimidazolium salts, polyquaterniums or copolymers of vinylpyrrolidone and vinylimidazolium exemplified as Luviquat® FC550, FC370), polyaniline, amino acids (isoleucine, leucine, phenylalanine). On the other hand, reducing the surface charge can also be achieved by a lubricating effect on the surface of the microspheres by lowering the contact angle with the container wall (i.e. stainless steel) or by reducing interparticle friction.

[0060] Further such additives are exemplified as stearic acid derivatives (i.e., magnesium stearate, triglycerol stearate, Span® 60, Tween® 60), oleic acid derivatives (i.e., glycerol trioleate, Tween® 80), lauric acid derivatives (i.e., lauric acid diethanolamide, Tween® 20, sodium laureth sulfate), amino acids, tocopherol acetate, piperidyl sebacate, and Makon® 17R4 (polyethylene glycol / polypropylene glycol block copolymer). [Brief description of the drawings]

[0061] [Figure 1]FIG. 1 illustrates types of encapsulation. [Figure 2A] 2A-2E are schematic diagrams of core reagent formulations used in embodiments of the compositions, systems, and methods described herein: FIG. 2A is a schematic diagram of sample extraction, FIG. 2B is a schematic diagram of library preparation, FIG. 2C is a schematic diagram of enrichment, FIG. 2D is a schematic diagram of clustering, and FIG. 2E is a schematic diagram of sequencing. [Figure 2B] 2A-2E are schematic diagrams of core reagent formulations used in embodiments of the compositions, systems, and methods described herein: FIG. 2A is a schematic diagram of sample extraction, FIG. 2B is a schematic diagram of library preparation, FIG. 2C is a schematic diagram of enrichment, FIG. 2D is a schematic diagram of clustering, and FIG. 2E is a schematic diagram of sequencing. [Figure 2C] 2A-2E are schematic diagrams of core reagent formulations used in embodiments of the compositions, systems, and methods described herein: FIG. 2A is a schematic diagram of sample extraction, FIG. 2B is a schematic diagram of library preparation, FIG. 2C is a schematic diagram of enrichment, FIG. 2D is a schematic diagram of clustering, and FIG. 2E is a schematic diagram of sequencing. [Figure 2D] 2A-2E are schematic diagrams of core reagent formulations used in embodiments of the compositions, systems, and methods described herein: FIG. 2A is a schematic diagram of sample extraction, FIG. 2B is a schematic diagram of library preparation, FIG. 2C is a schematic diagram of enrichment, FIG. 2D is a schematic diagram of clustering, and FIG. 2E is a schematic diagram of sequencing. [Figure 2E] 2A-2E are schematic diagrams of core reagent formulations used in embodiments of the compositions, systems, and methods described herein: FIG. 2A is a schematic diagram of sample extraction, FIG. 2B is a schematic diagram of library preparation, FIG. 2C is a schematic diagram of enrichment, FIG. 2D is a schematic diagram of clustering, and FIG. 2E is a schematic diagram of sequencing. [Diagram 3] FIG. 1 illustrates the various stresses that microspheres are subjected to during manufacturing, shipping, storage, and upon opening. [Figure 4A]FIG. 4A shows an embodiment of the present disclosure in which gelatin capsules are filled with microspheres and the gelatin capsules (filled with microspheres) can be coated with a jacket (e.g., paraffin wax). FIG. 4A shows an OTS gelatin capsule filled with microspheres (102) and an OTS gelatin capsule filled with microspheres and quickly dipped into hot wax (104). FIG. 4B shows an OTS gelatin capsule filled with microspheres that melt at 37° C. (106) and an OTS gelatin capsule filled with microspheres that melt at 58° C. and quickly dipped into hot wax (108). [Figure 4B] FIG. 4A shows an embodiment of the present disclosure in which gelatin capsules are filled with microspheres and the gelatin capsules (filled with microspheres) can be coated with a jacket (e.g., paraffin wax). FIG. 4A shows an OTS gelatin capsule filled with microspheres (102) and an OTS gelatin capsule filled with microspheres and quickly dipped into hot wax (104). FIG. 4B shows an OTS gelatin capsule filled with microspheres that melt at 37° C. (106) and an OTS gelatin capsule filled with microspheres that melt at 58° C. and quickly dipped into hot wax (108). [Diagram 5] FIG. 1 shows the results of paraffin wax coating of gelatin capsules allowing the release of encapsulated lyophilized microspheres at different temperatures. [Figure 6] FIG. 1 shows the compatibility test results of gelatin in Nextera Flex tagmentation. [Figure 7A] Figure 7A shows the time controlled release for a migration-free response. Figure 7B shows the rehydration of an exemplary composition described herein. [Figure 7B]Figure 7A shows the time controlled release for a migration-free response. Figure 7B shows the rehydration of an exemplary composition described herein. [Figure 8A] FIG. 8 shows that the encapsulated lyophilized microspheres described in the compositions, systems, and methods of the present disclosure enable a one-pot ligation protocol. FIG. 8A shows the steps of the one-pot ligation protocol. FIG. 8B shows the time-dependent release of microspheres. FIG. 8C shows data for Reagent A, Reagent B, and an additional reagent, Reagent B'. [Figure 8B] FIG. 8 shows that the encapsulated lyophilized microspheres described in the compositions, systems, and methods of the present disclosure enable a one-pot ligation protocol. FIG. 8A shows the steps of the one-pot ligation protocol. FIG. 8B shows the time-dependent release of microspheres. FIG. 8C shows data for Reagent A, Reagent B, and an additional reagent, Reagent B'. [Figure 8C] FIG. 8 shows that the encapsulated lyophilized microspheres described in the compositions, systems, and methods of the present disclosure enable a one-pot ligation protocol. FIG. 8A shows the steps of the one-pot ligation protocol. FIG. 8B shows the time-dependent release of microspheres. FIG. 8C shows data for Reagent A, Reagent B, and an additional reagent, Reagent B'. [Figure 9A]FIG. 9A illustrates one embodiment of the encapsulated freeze-dried microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase. FIG. 9A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 9B illustrates a sequencing polymerase microsphere (404) containing a sequencing polymerase. FIG. 9C illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) in a single well (408). FIG. 9D illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) rehydrated with water in a single well (408) at elevated temperature. [Figure 9B] FIG. 9A illustrates one embodiment of the encapsulated freeze-dried microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase. FIG. 9A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 9B illustrates a sequencing polymerase microsphere (404) containing a sequencing polymerase. FIG. 9C illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) in a single well (408). FIG. 9D illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) rehydrated with water in a single well (408) at elevated temperature. [Figure 9C]FIG. 9A illustrates one embodiment of the encapsulated freeze-dried microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase. FIG. 9A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 9B illustrates a sequencing polymerase microsphere (404) containing a sequencing polymerase. FIG. 9C illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) in a single well (408). FIG. 9D illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) rehydrated with water in a single well (408) at elevated temperature. [Figure 9D] FIG. 9A illustrates one embodiment of the encapsulated freeze-dried microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase. FIG. 9A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 9B illustrates a sequencing polymerase microsphere (404) containing a sequencing polymerase. FIG. 9C illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) in a single well (408). FIG. 9D illustrates an encapsulated polymerase microsphere (406) and an all-in-one polishing microsphere (402) rehydrated with water in a single well (408) at elevated temperature. [Figure 10A]FIG. 10A illustrates an embodiment of the encapsulated lyophilized microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase, and the same unit dose may be repeated across different reagents to achieve a larger dose. FIG. 10A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 10B illustrates a sequencing polymerase microsphere (404) containing sequencing polymerase. FIG. 10C illustrates an encapsulated polymerase microsphere (406) (×3 units) and an all-in-one polishing microsphere (402) (×3 units) in a single well (408). FIG. 10D illustrates the same unit dose repeated across different reagents to achieve an overall ×3 (triple or 3 times) dose. [Figure 10B] FIG. 10A illustrates an embodiment of the encapsulated lyophilized microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase, and the same unit dose may be repeated across different reagents to achieve a larger dose. FIG. 10A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 10B illustrates a sequencing polymerase microsphere (404) containing sequencing polymerase. FIG. 10C illustrates an encapsulated polymerase microsphere (406) (×3 units) and an all-in-one polishing microsphere (402) (×3 units) in a single well (408). FIG. 10D illustrates the same unit dose repeated across different reagents to achieve an overall ×3 (triple or 3 times) dose. [Figure 10C]FIG. 10A illustrates an embodiment of the encapsulated lyophilized microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase, and the same unit dose may be repeated across different reagents to achieve a larger dose. FIG. 10A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 10B illustrates a sequencing polymerase microsphere (404) containing sequencing polymerase. FIG. 10C illustrates an encapsulated polymerase microsphere (406) (×3 units) and an all-in-one polishing microsphere (402) (×3 units) in a single well (408). FIG. 10D illustrates the same unit dose repeated across different reagents to achieve an overall ×3 (triple or 3 times) dose. [Figure 10D] FIG. 10A illustrates an embodiment of the encapsulated lyophilized microspheres described in the disclosed compositions, systems, and methods, where the all-in-one polishing microsphere may contain ffN, polishing polymerase, and polishing oligos, while the sequencing polymerase microsphere may contain sequencing polymerase, and the same unit dose may be repeated across different reagents to achieve a larger dose. FIG. 10A illustrates an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. FIG. 10B illustrates a sequencing polymerase microsphere (404) containing sequencing polymerase. FIG. 10C illustrates an encapsulated polymerase microsphere (406) (×3 units) and an all-in-one polishing microsphere (402) (×3 units) in a single well (408). FIG. 10D illustrates the same unit dose repeated across different reagents to achieve an overall ×3 (triple or 3 times) dose. [Figure 11A]FIG. 11A illustrates an embodiment of the encapsulated freeze-dried microsphere described in the disclosed compositions, systems, and methods. FIG. 11A illustrates an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos. FIG. 11B illustrates a sequencing polymerase microsphere (404) that may contain a sequencing polymerase and may be encapsulated (406) and in a single well with the all-in-one polishing microsphere (402). FIG. 11C illustrates that the encapsulated freeze-dried microsphere (406) may be rehydrated with water at 50° C. FIG. 11D illustrates that after 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins. FIG. 11E illustrates that the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. In FIG. 11F, all the microspheres have completely dissolved and the ICM is then ready for use. [Figure 11B] FIG. 11A illustrates an embodiment of the encapsulated freeze-dried microsphere described in the disclosed compositions, systems, and methods. FIG. 11A illustrates an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos. FIG. 11B illustrates a sequencing polymerase microsphere (404) that may contain a sequencing polymerase and may be encapsulated (406) and in a single well with the all-in-one polishing microsphere (402). FIG. 11C illustrates that the encapsulated freeze-dried microsphere (406) may be rehydrated with water at 50° C. FIG. 11D illustrates that after 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins. FIG. 11E illustrates that the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. In FIG. 11F, all the microspheres have completely dissolved and the ICM is then ready for use. [Figure 11C] FIG. 11A illustrates an embodiment of the encapsulated freeze-dried microsphere described in the disclosed compositions, systems, and methods. FIG. 11A illustrates an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos. FIG. 11B illustrates a sequencing polymerase microsphere (404) that may contain a sequencing polymerase and may be encapsulated (406) and in a single well with the all-in-one polishing microsphere (402). FIG. 11C illustrates that the encapsulated freeze-dried microsphere (406) may be rehydrated with water at 50° C. FIG. 11D illustrates that after 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins. FIG. 11E illustrates that the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. In FIG. 11F, all the microspheres have completely dissolved and the ICM is then ready for use. [Figure 11D]FIG. 11A illustrates an embodiment of the encapsulated freeze-dried microsphere described in the disclosed compositions, systems, and methods. FIG. 11A illustrates an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos. FIG. 11B illustrates a sequencing polymerase microsphere (404) that may contain a sequencing polymerase and may be encapsulated (406) and in a single well with the all-in-one polishing microsphere (402). FIG. 11C illustrates that the encapsulated freeze-dried microsphere (406) may be rehydrated with water at 50° C. FIG. 11D illustrates that after 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins. FIG. 11E illustrates that the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. In FIG. 11F, all the microspheres have completely dissolved and the ICM is then ready for use. [Figure 11E] FIG. 11A illustrates an embodiment of the encapsulated freeze-dried microsphere described in the disclosed compositions, systems, and methods. FIG. 11A illustrates an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos. FIG. 11B illustrates a sequencing polymerase microsphere (404) that may contain a sequencing polymerase and may be encapsulated (406) and in a single well with the all-in-one polishing microsphere (402). FIG. 11C illustrates that the encapsulated freeze-dried microsphere (406) may be rehydrated with water at 50° C. FIG. 11D illustrates that after 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins. FIG. 11E illustrates that the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. In FIG. 11F, all the microspheres have completely dissolved and the ICM is then ready for use. [Figure 11F] FIG. 11A illustrates an embodiment of the encapsulated freeze-dried microsphere described in the disclosed compositions, systems, and methods. FIG. 11A illustrates an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos. FIG. 11B illustrates a sequencing polymerase microsphere (404) that may contain a sequencing polymerase and may be encapsulated (406) and in a single well with the all-in-one polishing microsphere (402). FIG. 11C illustrates that the encapsulated freeze-dried microsphere (406) may be rehydrated with water at 50° C. FIG. 11D illustrates that after 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins. FIG. 11E illustrates that the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. In FIG. 11F, all the microspheres have completely dissolved and the ICM is then ready for use. [Figure 12] FIG. 1 shows details of the manufacturing process for the encapsulated lyophilized microspheres described in the compositions, systems, and methods of the present disclosure. [Figure 13] FIG. 1 describes the production and point of use of the encapsulated lyophilized microspheres described in the compositions, systems, and methods of the present disclosure. [Figure 14A] 14A illustrates the manufacture and point of use of encapsulated freeze-dried microspheres and cakes described in the compositions, systems, and methods of the present disclosure. FIG. 14A illustrates the manufacture and point of use of encapsulated freeze-dried microspheres. FIG. 14B illustrates an embodiment of a first cake, wax, and second cake in a tube. [Figure 14B]14A illustrates the manufacture and point of use of encapsulated freeze-dried microspheres and cakes described in the compositions, systems, and methods of the present disclosure. FIG. 14A illustrates the manufacture and point of use of encapsulated freeze-dried microspheres. FIG. 14B illustrates an embodiment of a first cake, wax, and second cake in a tube. [Figure 15A] Figures describing the application of SBS for the compositions, systems, and methods of the present disclosure. Figure 15A shows an AOM SBS cleave mix with Pd (550) in the core and THP (552) in the shell. Figure 15B shows an ffN / Pol bead with Pol (554) in the core and ffN (556) in the shell. Figure 15C shows photoprotection of ffN with ffN (560) in the core and a light-blocking shell (558). [Figure 15B] Figures describing the application of SBS for the compositions, systems, and methods of the present disclosure. Figure 15A shows an AOM SBS cleave mix with Pd (550) in the core and THP (552) in the shell. Figure 15B shows an ffN / Pol bead with Pol (554) in the core and ffN (556) in the shell. Figure 15C shows photoprotection of ffN with ffN (560) in the core and a light-blocking shell (558). [Figure 15C] Figures describing the application of SBS for the compositions, systems, and methods of the present disclosure. Figure 15A shows an AOM SBS cleave mix with Pd (550) in the core and THP (552) in the shell. Figure 15B shows an ffN / Pol bead with Pol (554) in the core and ffN (556) in the shell. Figure 15C shows photoprotection of ffN with ffN (560) in the core and a light-blocking shell (558). [Figure 16A]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16B]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16C]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16D]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16E]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16F]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16G]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16H]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 16I]High throughput sequencing screening of additives. A particular additive such as Efka® IO 6783 is titrated to find the concentration limit. Figure 16A shows phasing and prephasing metrics for various additives. Figure 16B shows error rates and Q30 for various additives. Figure 16C shows intensity of all lanes and all channels for additives in Figures 16A and 16B. Figure 16D shows phasing and prephasing metrics for various additives. Figure 16E shows error rates and Q30 for various additives. Figure 16F shows intensity of all lanes and all channels for additives in Figures 16D and 16E. Figure 16G shows sequencing by titration of Efka® IO 6783, specifically phasing and prephasing metrics. Figure 16H shows error rates and Q30 for Efka® IO 6783. Figure 16I shows intensity of all lanes and all channels for Figures 16G and 16H. [Figure 17A] Figure 17 shows the stability of ffC spiked with 1% additive in liquid format compared to control after 1-2 days of incubation at 60°C. Figure 17 shows HPLC analysis of ffC incubated with additives described herein and heat staged at 60°C for 1 and 2 days. The decrease in ffc peak area and increase in 3'OH and DiP indicate degradation of ffN. The effect of additives is compared to control. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​a second ffN. Figure 17E shows the 3'OH of a second ffN. Figure 17F shows the diphosphate of a second ffN. [Figure 17B]Figure 17 shows the stability of ffC spiked with 1% additive in liquid format compared to control after 1-2 days of incubation at 60°C. Figure 17 shows HPLC analysis of ffC incubated with additives described herein and heat staged at 60°C for 1 and 2 days. The decrease in ffc peak area and increase in 3'OH and DiP indicate degradation of ffN. The effect of additives is compared to control. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​a second ffN. Figure 17E shows the 3'OH of a second ffN. Figure 17F shows the diphosphate of a second ffN. [Figure 17C] Figure 17 shows the stability of ffC spiked with 1% additive in liquid format compared to control after 1-2 days of incubation at 60°C. Figure 17 shows HPLC analysis of ffC incubated with additives described herein and heat staged at 60°C for 1 and 2 days. The decrease in ffc peak area and increase in 3'OH and DiP indicate degradation of ffN. The effect of additives is compared to control. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​a second ffN. Figure 17E shows the 3'OH of a second ffN. Figure 17F shows the diphosphate of a second ffN. [Figure 17D] Figure 17 shows the stability of ffC spiked with 1% additive in liquid format compared to control after 1-2 days of incubation at 60°C. Figure 17 shows HPLC analysis of ffC incubated with additives described herein and heat staged at 60°C for 1 and 2 days. The decrease in ffc peak area and increase in 3'OH and DiP indicate degradation of ffN. The effect of additives is compared to control. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​a second ffN. Figure 17E shows the 3'OH of a second ffN. Figure 17F shows the diphosphate of a second ffN. [Figure 17E] Figure 17 shows the stability of ffC spiked with 1% additive in liquid format compared to control after 1-2 days of incubation at 60°C. Figure 17 shows HPLC analysis of ffC incubated with additives described herein and heat staged at 60°C for 1 and 2 days. The decrease in ffc peak area and increase in 3'OH and DiP indicate degradation of ffN. The effect of additives is compared to control. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​a second ffN. Figure 17E shows the 3'OH of a second ffN. Figure 17F shows the diphosphate of a second ffN. [Figure 17F] Figure 17 shows the stability of ffC spiked with 1% additive in liquid format compared to control after 1-2 days of incubation at 60°C. Figure 17 shows HPLC analysis of ffC incubated with additives described herein and heat staged at 60°C for 1 and 2 days. The decrease in ffc peak area and increase in 3'OH and DiP indicate degradation of ffN. The effect of additives is compared to control. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​a second ffN. Figure 17E shows the 3'OH of a second ffN. Figure 17F shows the diphosphate of a second ffN. [Figure 18] FIG. 1 shows DNA recombinase activity of exclusion amplification (ExAmp) solutions incubated with additives described herein. [Figure 19A] 19A and 19B show the clustering performance of ExAmp spiked with coating materials by cBOT first base assay, specifically, the ExAmp average intensity (FIG. 19A) and clustering function (FIG. 19B) of ExAmp solutions incubated with additives described herein by cBOT first base uptake kinetics. [Figure 19B]19A and 19B show the clustering performance of ExAmp spiked with coating materials by cBOT first base assay, specifically, the ExAmp average intensity (FIG. 19A) and clustering function (FIG. 19B) of ExAmp solutions incubated with additives described herein by cBOT first base uptake kinetics. [Figure 20] FIG. 1 shows charging potential (Keyence) measurements (at 3% RH) of powdered lyophilized cakes of ExAmp containing different concentrations of additive (matrix). [Figure 21] FIG. 1 shows charging potential (Keyence) measurements (at 40% RH) of powdered lyophilized cakes of ExAmp containing different concentrations of additive (matrix). [Figure 22] FIG. 1 shows charging potential (Keyence) measurements (at 3% RH) of powdered lyophilized cakes of ExAmp containing different concentrations of additive (matrix). [Figure 23] FIG. 1 shows charging potential (Keyence) measurements (at 40% RH) of powdered lyophilized cakes of ExAmp containing different concentrations of additive (matrix). [Figure 24] FIG. 1 shows charging potential (Keyence) measurements (at 3% and 40% RH) of powdered lyophilized cakes of ExAmp containing different concentrations of salt / buffer (matrix). [Figure 25A]Figure 25A shows Atto and FSCN (fluorescein) microspheres containing additives in a matrix format. The antistatic properties of the additives are evaluated by adhesion of the microspheres to the container and their charge density is measured by GranuCharge. A low Δq value indicates low triboelectric charging. The matrix format of 1% Efka® IO 6783 minimizes triboelectric charging. Figure 25A shows the results of the first set of additives tested (Atto 20%, +1% Efka® IO 6783, +1.5% Tris.HCl, and +1% Tween 20) in terms of both visual results (top) and percent loss (bottom). Figure 25B shows the results of the second set of additives tested (FSCN 20% Ctrl, +1% Efka® IO 6783, +2% Efka® IO 6783, and +1% Efka® IO 6786), both in terms of visual results (top) and percent loss (bottom). Figure 25C shows that Atto and FSCN (both 20% trehalose) were dry formulated utilizing the antistatic agent Efka® IO 6783 in a matrix format. [Figure 25B]Figure 25A shows Atto and FSCN (fluorescein) microspheres containing additives in a matrix format. The antistatic properties of the additives are evaluated by adhesion of the microspheres to the container and their charge density is measured by GranuCharge. A low Δq value indicates low triboelectric charging. The matrix format of 1% Efka® IO 6783 minimizes triboelectric charging. Figure 25A shows the results of the first set of additives tested (Atto 20%, +1% Efka® IO 6783, +1.5% Tris.HCl, and +1% Tween 20) in terms of both visual results (top) and percent loss (bottom). Figure 25B shows the results of the second set of additives tested (FSCN 20% Ctrl, +1% Efka® IO 6783, +2% Efka® IO 6783, and +1% Efka® IO 6786), both in terms of visual results (top) and percent loss (bottom). Figure 25C shows that Atto and FSCN (both 20% trehalose) were dry formulated utilizing the antistatic agent Efka® IO 6783 in a matrix format. [Figure 25C]Figure 25A shows Atto and FSCN (fluorescein) microspheres containing additives in a matrix format. The antistatic properties of the additives are evaluated by adhesion of the microspheres to the container and their charge density is measured by GranuCharge. A low Δq value indicates low triboelectric charging. The matrix format of 1% Efka® IO 6783 minimizes triboelectric charging. Figure 25A shows the results of the first set of additives tested (Atto 20%, +1% Efka® IO 6783, +1.5% Tris.HCl, and +1% Tween 20) in terms of both visual results (top) and percent loss (bottom). Figure 25B shows the results of the second set of additives tested (FSCN 20% Ctrl, +1% Efka® IO 6783, +2% Efka® IO 6783, and +1% Efka® IO 6786), both in terms of visual results (top) and percent loss (bottom). Figure 25C shows that Atto and FSCN (both 20% trehalose) were dry formulated utilizing the antistatic agent Efka® IO 6783 in a matrix format. [Figure 26A] ffN microspheres containing additives in matrix form. The antistatic properties of the additives are evaluated by adhesion to the container and measured by GranuCharge. Figure 26A shows the visual results of the first set of additives (ffN+25%T Ctrl, +1% Efka® IO 6783, +1% Efka® IO 6786, +1.5% Tris.HCl, +2% Isoleucine). Figure 26B shows the visual results of the second set of additives (2nd ffN+20%T Ctrl, +0.5% LDA, +1% Makon® 17R4, +1.5% Kollidon® VA64, +2% Kollicoat® Protect). Figure 26C shows the charge density of the various additives tested in Figures 26A and 26B. [Figure 26B]ffN microspheres containing additives in matrix form. The antistatic properties of the additives are evaluated by adhesion to the container and measured by GranuCharge. Figure 26A shows the visual results of the first set of additives (ffN+25%T Ctrl, +1% Efka® IO 6783, +1% Efka® IO 6786, +1.5% Tris.HCl, +2% Isoleucine). Figure 26B shows the visual results of the second set of additives (2nd ffN+20%T Ctrl, +0.5% LDA, +1% Makon® 17R4, +1.5% Kollidon® VA64, +2% Kollicoat® Protect). Figure 26C shows the charge density of the various additives tested in Figures 26A and 26B. [Figure 26C] ffN microspheres containing additives in matrix form. The antistatic properties of the additives are evaluated by adhesion to the container and measured by GranuCharge. Figure 26A shows the visual results of the first set of additives (ffN+25%T Ctrl, +1% Efka® IO 6783, +1% Efka® IO 6786, +1.5% Tris.HCl, +2% Isoleucine). Figure 26B shows the visual results of the second set of additives (2nd ffN+20%T Ctrl, +0.5% LDA, +1% Makon® 17R4, +1.5% Kollidon® VA64, +2% Kollicoat® Protect). Figure 26C shows the charge density of the various additives tested in Figures 26A and 26B. [Figure 27A]FIG. 27 shows FSCN (fluorescein) and MB (methylene blue) microspheres coated with Eudragit® L100 and magnesium stearate. The electrostatic and triboelectric charging behavior of the microspheres is mitigated by the presence of the coating as shown by GranuCharge measurements and microsphere dry blend experiments. FIG. 27A shows an exemplary shell and core according to the present disclosure. FIG. 27B shows 5-7% weight gain from coating. FIG. 27C shows static reduction in coated vs. uncoated compositions. FIG. 27D shows increased moisture barrier in coated vs. uncoated compositions. FIG. 27E shows triggered release in coated compositions. [Figure 27B] FIG. 27 shows FSCN (fluorescein) and MB (methylene blue) microspheres coated with Eudragit® L100 and magnesium stearate. The electrostatic and triboelectric charging behavior of the microspheres is mitigated by the presence of the coating as shown by GranuCharge measurements and microsphere dry blend experiments. FIG. 27A shows an exemplary shell and core according to the present disclosure. FIG. 27B shows 5-7% weight gain from coating. FIG. 27C shows static reduction in coated vs. uncoated compositions. FIG. 27D shows increased moisture barrier in coated vs. uncoated compositions. FIG. 27E shows triggered release in coated compositions. [Figure 27C]FIG. 27 shows FSCN (fluorescein) and MB (methylene blue) microspheres coated with Eudragit® L100 and magnesium stearate. The electrostatic and triboelectric charging behavior of the microspheres is mitigated by the presence of the coating as shown by GranuCharge measurements and microsphere dry blend experiments. FIG. 27A shows an exemplary shell and core according to the present disclosure. FIG. 27B shows 5-7% weight gain from coating. FIG. 27C shows static reduction in coated vs. uncoated compositions. FIG. 27D shows increased moisture barrier in coated vs. uncoated compositions. FIG. 27E shows triggered release in coated compositions. [Figure 27D] FIG. 27 shows FSCN (fluorescein) and MB (methylene blue) microspheres coated with Eudragit® L100 and magnesium stearate. The electrostatic and triboelectric charging behavior of the microspheres is mitigated by the presence of the coating as shown by GranuCharge measurements and microsphere dry blend experiments. FIG. 27A shows an exemplary shell and core according to the present disclosure. FIG. 27B shows 5-7% weight gain from coating. FIG. 27C shows static reduction in coated vs. uncoated compositions. FIG. 27D shows increased moisture barrier in coated vs. uncoated compositions. FIG. 27E shows triggered release in coated compositions. [Figure 27E]FIG. 27 shows FSCN (fluorescein) and MB (methylene blue) microspheres coated with Eudragit® L100 and magnesium stearate. The electrostatic and triboelectric charging behavior of the microspheres is mitigated by the presence of the coating as shown by GranuCharge measurements and microsphere dry blend experiments. FIG. 27A shows an exemplary shell and core according to the present disclosure. FIG. 27B shows 5-7% weight gain from coating. FIG. 27C shows static reduction in coated vs. uncoated compositions. FIG. 27D shows increased moisture barrier in coated vs. uncoated compositions. FIG. 27E shows triggered release in coated compositions. [Figure 28A] Figures 28A and 28B show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. Figures 28A and 28B show images of 800 μm FSCN Wurster-Spray multiple (Figure 28A) and single (Figure 28B) microspheres coated with 20% Kollidon VA64 and Efka® IO 6783 and PEG (#6). Figures 28C and 28D show images of Cryo-Ion-Mill SEM multiple (Figure 28C) and single (Figure 28D) microspheres coated with Rec / BSA at 15% with Kollidon VA64 and Efka® IO 6783 and PEG (#8). Figures 28E and 28F show Cryo-Ion-Mill SEM images of multiple (Figure 28E) and single (Figure 28F) microspheres of ffN coated with Kollidon VA64 and Efka® IO 6783 and PEG (#11) 10%. [Figure 28B]Figures 28A and 28B show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. Figures 28A and 28B show images of 800 μm FSCN Wurster-Spray multiple (Figure 28A) and single (Figure 28B) microspheres coated with 20% Kollidon VA64 and Efka® IO 6783 and PEG (#6). Figures 28C and 28D show images of Cryo-Ion-Mill SEM multiple (Figure 28C) and single (Figure 28D) microspheres coated with Rec / BSA at 15% with Kollidon VA64 and Efka® IO 6783 and PEG (#8). Figures 28E and 28F show Cryo-Ion-Mill SEM images of multiple (Figure 28E) and single (Figure 28F) microspheres of ffN coated with Kollidon VA64 and Efka® IO 6783 and PEG (#11) 10%. [Figure 28C]Figures 28A and 28B show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. Figures 28A and 28B show images of 800 μm FSCN Wurster-Spray multiple (Figure 28A) and single (Figure 28B) microspheres coated with 20% Kollidon VA64 and Efka® IO 6783 and PEG (#6). Figures 28C and 28D show images of Cryo-Ion-Mill SEM multiple (Figure 28C) and single (Figure 28D) microspheres coated with Rec / BSA at 15% with Kollidon VA64 and Efka® IO 6783 and PEG (#8). Figures 28E and 28F show Cryo-Ion-Mill SEM images of multiple (Figure 28E) and single (Figure 28F) microspheres of ffN coated with Kollidon VA64 and Efka® IO 6783 and PEG (#11) 10%. [Figure 28D]Figures 28A and 28B show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. Figures 28A and 28B show images of 800 μm FSCN Wurster-Spray multiple (Figure 28A) and single (Figure 28B) microspheres coated with Kollidon VA64, Efka® IO 6783, and PEG (#6) at 20%. Figures 28C and 28D show Cryo-Ion-Mill SEM images of multiple (Figure 28C) and single (Figure 28D) microspheres coated with Rec / BSA at 15% with Kollidon VA64, Efka® IO 6783, and PEG (#8). Figures 28E and 28F show Cryo-Ion-Mill SEM images of multiple (Figure 28E) and single (Figure 28F) microspheres of ffN coated with Kollidon VA64 and Efka® IO 6783 and PEG (#11) 10%. [Figure 28E]Figures 28A and 28B show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. Figures 28A and 28B show images of 800 μm FSCN Wurster-Spray multiple (Figure 28A) and single (Figure 28B) microspheres coated with 20% Kollidon VA64 and Efka® IO 6783 and PEG (#6). Figures 28C and 28D show images of Cryo-Ion-Mill SEM multiple (Figure 28C) and single (Figure 28D) microspheres coated with Rec / BSA at 15% with Kollidon VA64 and Efka® IO 6783 and PEG (#8). Figures 28E and 28F show Cryo-Ion-Mill SEM images of multiple (Figure 28E) and single (Figure 28F) microspheres of ffN coated with Kollidon VA64 and Efka® IO 6783 and PEG (#11) 10%. [Figure 28F]Figures 28A and 28B show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. Figures 28A and 28B show images of 800 μm FSCN Wurster-Spray multiple (Figure 28A) and single (Figure 28B) microspheres coated with 20% Kollidon VA64 and Efka® IO 6783 and PEG (#6). Figures 28C and 28D show images of Cryo-Ion-Mill SEM multiple (Figure 28C) and single (Figure 28D) microspheres coated with Rec / BSA at 15% with Kollidon VA64 and Efka® IO 6783 and PEG (#8). Figures 28E and 28F show Cryo-Ion-Mill SEM images of multiple (Figure 28E) and single (Figure 28F) microspheres of ffN coated with Kollidon VA64 and Efka® IO 6783 and PEG (#11) 10%. [Figure 29A] 29A shows ffN microspheres containing additives in coating form. The antistatic properties of the additives are evaluated by adhesion to the container and measured by GranuCharge. FIG. 29A shows a visual representation of various ffN microspheres containing additives in coating form. FIG. 29B shows the charge density of the various ffN microspheres in FIG. 29A. [Figure 29B] 29A shows ffN microspheres containing additives in coating form. The antistatic properties of the additives are evaluated by adhesion to the container and measured by GranuCharge. FIG. 29A shows a visual representation of various ffN microspheres containing additives in coating form. FIG. 29B shows the charge density of the various ffN microspheres in FIG. 29A. [Figure 30A]FIG. 30A shows a visual representation of various DNA recombinase / BSA microspheres containing additives as a coating format. The antistatic properties of the additives are evaluated by adhesion to a container and measured by GranuCharge. FIG. 30B shows the charge density of the various DNA recombinase / BSA microspheres of FIG. 30A. [Figure 30B] FIG. 30A shows a visual representation of various DNA recombinase / BSA microspheres containing additives as a coating format. The antistatic properties of the additives are evaluated by adhesion to a container and measured by GranuCharge. FIG. 30B shows the charge density of the various DNA recombinase / BSA microspheres of FIG. 30A. [Figure 31A] 31A shows the stability of the compositions described herein. FIG. 31A shows ffN microspheres. FIG. 31B shows 5% Kollidon® VA64 in a dry matrix. FIG. 31C shows 5% Makon® 17R4 in a dry matrix. FIG. 31D shows 5% Efka® 6783 in a dry matrix. FIG. 31E shows 10% Kollicoat® Protect in a dry matrix. FIG. 31F shows 7.5% isoleucine (bottom right) in a dry matrix under various moisture and time conditions. [Figure 31B]31A shows the stability of the compositions described herein. FIG. 31A shows ffN microspheres. FIG. 31B shows 5% Kollidon® VA64 in a dry matrix. FIG. 31C shows 5% Makon® 17R4 in a dry matrix. FIG. 31D shows 5% Efka® 6783 in a dry matrix. FIG. 31E shows 10% Kollicoat® Protect in a dry matrix. FIG. 31F shows 7.5% isoleucine (bottom right) in a dry matrix under various moisture and time conditions. [Figure 31C] 31A shows the stability of the compositions described herein. FIG. 31A shows ffN microspheres. FIG. 31B shows 5% Kollidon® VA64 in a dry matrix. FIG. 31C shows 5% Makon® 17R4 in a dry matrix. FIG. 31D shows 5% Efka® 6783 in a dry matrix. FIG. 31E shows 10% Kollicoat® Protect in a dry matrix. FIG. 31F shows 7.5% isoleucine (bottom right) in a dry matrix under various moisture and time conditions. [Figure 31D] 31A shows the stability of the compositions described herein. FIG. 31A shows ffN microspheres. FIG. 31B shows 5% Kollidon® VA64 in a dry matrix. FIG. 31C shows 5% Makon® 17R4 in a dry matrix. FIG. 31D shows 5% Efka® 6783 in a dry matrix. FIG. 31E shows 10% Kollicoat® Protect in a dry matrix. FIG. 31F shows 7.5% isoleucine (bottom right) in a dry matrix under various moisture and time conditions. [Figure 31E]31A shows the stability of the compositions described herein. FIG. 31A shows ffN microspheres. FIG. 31B shows 5% Kollidon® VA64 in a dry matrix. FIG. 31C shows 5% Makon® 17R4 in a dry matrix. FIG. 31D shows 5% Efka® 6783 in a dry matrix. FIG. 31E shows 10% Kollicoat® Protect in a dry matrix. FIG. 31F shows 7.5% isoleucine (bottom right) in a dry matrix under various moisture and time conditions. [Fig. 31F] 31A shows the stability of the compositions described herein. FIG. 31A shows ffN microspheres. FIG. 31B shows 5% Kollidon® VA64 in a dry matrix. FIG. 31C shows 5% Makon® 17R4 in a dry matrix. FIG. 31D shows 5% Efka® 6783 in a dry matrix. FIG. 31E shows 10% Kollicoat® Protect in a dry matrix. FIG. 31F shows 7.5% isoleucine (bottom right) in a dry matrix under various moisture and time conditions. [Figure 32A] 32A and 32B show the results of measuring the resistance of microspheres to relative humidity by dynamic vapor sorption. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. FIG. 32A shows the results for ffN Ctrl (18% T, 2% HCD). FIG. 32B shows the results for 2% Kollicoat® Protect matrix (10% dry). FIG. 32C shows the results for +1% Efka® IO 6783 matrix (5% dry). FIG. 32D shows the results for 1% Kollidon® VA64 matrix (5% dry). FIG. 32E shows the results for +1.5% Trizma matrix (7.5% dry). FIG. 32F shows the results for +1.5% isoleucine matrix (7.5% dry). [Figure 32B]32A and 32B show the results of measuring the resistance of microspheres to relative humidity by dynamic vapor sorption. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. FIG. 32A shows the results for ffN Ctrl (18% T, 2% HCD). FIG. 32B shows the results for 2% Kollicoat® Protect matrix (10% dry). FIG. 32C shows the results for +1% Efka® IO 6783 matrix (5% dry). FIG. 32D shows the results for 1% Kollidon® VA64 matrix (5% dry). FIG. 32E shows the results for +1.5% Trizma matrix (7.5% dry). FIG. 32F shows the results for +1.5% isoleucine matrix (7.5% dry). [Figure 32C] 32A and 32B show the results of measuring the resistance of microspheres to relative humidity by dynamic vapor sorption. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. FIG. 32A shows the results for ffN Ctrl (18% T, 2% HCD). FIG. 32B shows the results for 2% Kollicoat® Protect matrix (10% dry). FIG. 32C shows the results for +1% Efka® IO 6783 matrix (5% dry). FIG. 32D shows the results for 1% Kollidon® VA64 matrix (5% dry). FIG. 32E shows the results for +1.5% Trizma matrix (7.5% dry). FIG. 32F shows the results for +1.5% isoleucine matrix (7.5% dry). [Fig. 32D]32A and 32B show the results of measuring the resistance of microspheres to relative humidity by dynamic vapor sorption. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. FIG. 32A shows the results for ffN Ctrl (18% T, 2% HCD). FIG. 32B shows the results for 2% Kollicoat® Protect matrix (10% dry). FIG. 32C shows the results for +1% Efka® IO 6783 matrix (5% dry). FIG. 32D shows the results for 1% Kollidon® VA64 matrix (5% dry). FIG. 32E shows the results for +1.5% Trizma matrix (7.5% dry). FIG. 32F shows the results for +1.5% isoleucine matrix (7.5% dry). [Figure 32E] 32A and 32B show the results of measuring the resistance of microspheres to relative humidity by dynamic vapor sorption. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. FIG. 32A shows the results for ffN Ctrl (18% T, 2% HCD). FIG. 32B shows the results for 2% Kollicoat® Protect matrix (10% dry). FIG. 32C shows the results for +1% Efka® IO 6783 matrix (5% dry). FIG. 32D shows the results for 1% Kollidon® VA64 matrix (5% dry). FIG. 32E shows the results for +1.5% Trizma matrix (7.5% dry). FIG. 32F shows the results for +1.5% isoleucine matrix (7.5% dry). [Fig. 32F]32A and 32B show the results of measuring the resistance of microspheres to relative humidity by dynamic vapor sorption. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. FIG. 32A shows the results for ffN Ctrl (18% T, 2% HCD). FIG. 32B shows the results for 2% Kollicoat® Protect matrix (10% dry). FIG. 32C shows the results for +1% Efka® IO 6783 matrix (5% dry). FIG. 32D shows the results for 1% Kollidon® VA64 matrix (5% dry). FIG. 32E shows the results for +1.5% Trizma matrix (7.5% dry). FIG. 32F shows the results for +1.5% isoleucine matrix (7.5% dry). [Figure 33A] Figure 33 shows the results of Kollicoat® Protect and VA64 coatings which resulted in improved moisture protection of Rec / BSA MS. Figure 33A shows the results of uncoated Rec / BSA (AP1) control at 20% RH limit. Figure 33B shows Rec / BSA coated with 15% protect and Efka® IO 6783 at 30% RH limit. Figure 33C shows Rec / BSA coated with 15% Kollidon® VA64 and Efka® IO 6783 at 20-30% RH limit. Figure 33D shows images of the coated compositions of Figures 33A-33C after exposure to humidity. [Figure 33B]Figure 33 shows the results of Kollicoat® Protect and VA64 coatings which resulted in improved moisture protection of Rec / BSA MS. Figure 33A shows the results of uncoated Rec / BSA (AP1) control at 20% RH limit. Figure 33B shows Rec / BSA coated with 15% protect and Efka® IO 6783 at 30% RH limit. Figure 33C shows Rec / BSA coated with 15% Kollidon® VA64 and Efka® IO 6783 at 20-30% RH limit. Figure 33D shows images of the coated compositions of Figures 33A-33C after exposure to humidity. [Figure 33C] Figure 33 shows the results of Kollicoat® Protect and VA64 coatings which resulted in improved moisture protection of Rec / BSA MS. Figure 33A shows the results of uncoated Rec / BSA (AP1) control at 20% RH limit. Figure 33B shows Rec / BSA coated with 15% protect and Efka® IO 6783 at 30% RH limit. Figure 33C shows Rec / BSA coated with 15% Kollidon® VA64 and Efka® IO 6783 at 20-30% RH limit. Figure 33D shows images of the coated compositions of Figures 33A-33C after exposure to humidity. [Figure 33D]Figure 33 shows the results of Kollicoat® Protect and VA64 coatings which resulted in improved moisture protection of Rec / BSA MS. Figure 33A shows the results of uncoated Rec / BSA (AP1) control at 20% RH limit. Figure 33B shows Rec / BSA coated with 15% protect and Efka® IO 6783 at 30% RH limit. Figure 33C shows Rec / BSA coated with 15% Kollidon® VA64 and Efka® IO 6783 at 20-30% RH limit. Figure 33D shows images of the coated compositions of Figures 33A-33C after exposure to humidity. [Figure 34A] Figure 34 shows that the coating minimizes moisture absorption of Rec / BSA microspheres. Figure 34A shows images of various microspheres under different humidity conditions. Figure 34B shows the effect of % RH on uncoated vs. coated Rec-BSA microspheres at 30%, 55%, and 80% RH for 24 hours at 35°C in 30 mg samples graded on aluminum pans. Notably, the Kollicoat® Protect coating performed better than Kollidon® VA64 in terms of moisture barrier. [Figure 34B] Figure 34 shows that the coating minimizes moisture absorption of Rec / BSA microspheres. Figure 34A shows images of various microspheres under different humidity conditions. Figure 34B shows the effect of % RH on uncoated vs. coated Rec-BSA microspheres at 30%, 55%, and 80% RH for 24 hours at 35°C in 30 mg samples graded on aluminum pans. Notably, the Kollicoat® Protect coating performed better than Kollidon® VA64 in terms of moisture barrier. [Diagram 35] FIG. 1 shows the concept of solubility screening. [Diagram 36] FIG. 1 shows results for Kollidon® VA64, Efka®, Eudragit®, which are soluble in spray coating solution (15% water / solvent) and buffer. [Figure 37A] Figure 37A shows the diphosphate levels of specific ffCs in the presence of polymer (Figure 37A), the 3'OH and diphosphate levels of specific ffCs in the presence of polymer (Figure 37B), and the diphosphate levels of specific ffCs in the presence of polymer (Figure 37C). [Figure 37B] Figure 37A shows the diphosphate levels of specific ffCs in the presence of polymer (Figure 37A), the 3'OH and diphosphate levels of specific ffCs in the presence of polymer (Figure 37B), and the diphosphate levels of specific ffCs in the presence of polymer (Figure 37C). [Figure 37C] Figure 37A shows the diphosphate levels of specific ffCs in the presence of polymer (Figure 37A), the 3'OH and diphosphate levels of specific ffCs in the presence of polymer (Figure 37B), and the diphosphate levels of specific ffCs in the presence of polymer (Figure 37C). [Figure 38] FIG. 1 shows the fluorescence intensity of specific ffCs in the presence of polymers. [Figure 39] FIG. 1 shows the fluorescence intensity of specific ffCs in the presence of polymers. [Figure 40A] Figure 40 shows the results of coating material screening based on sequencing suitability. Figure 40A shows the phase and prephasing metrics of the various coating materials tested. Figure 40B shows the error rate and Q30 of the various coatings tested. Figure 40C shows the intensity of all lanes and all channels for the coating materials described in Figures 40A and 40B. [Figure 40B]Figure 40 shows the results of coating material screening based on sequencing suitability. Figure 40A shows the phase and prephasing metrics of the various coating materials tested. Figure 40B shows the error rate and Q30 of the various coatings tested. Figure 40C shows the intensity of all lanes and all channels for the coating materials described in Figures 40A and 40B. [Figure 40C] Figure 40 shows the results of coating material screening based on sequencing suitability. Figure 40A shows the phase and prephasing metrics of the various coating materials tested. Figure 40B shows the error rate and Q30 of the various coatings tested. Figure 40C shows the intensity of all lanes and all channels for the coating materials described in Figures 40A and 40B. [Figure 41A] Figures 41A-B show the DNA recombinase activity and clustering performance (cBOT) of ExAmp in the presence of polymers described herein, and the results of screening the polymers for DNA recombinase activity. Figure 41A shows the DNA recombinase activity of ExAmp (TCS1 v1.0) solutions spiked with polymers. Figure 41B shows the DNA recombinase activity after incubation at 40°C for 24 hours. [Figure 41B] Figures 41A-B show the DNA recombinase activity and clustering performance (cBOT) of ExAmp in the presence of polymers described herein, and the results of screening the polymers for DNA recombinase activity. Figure 41A shows the DNA recombinase activity of ExAmp (TCS1 v1.0) solutions spiked with polymers. Figure 41B shows the DNA recombinase activity after incubation at 40°C for 24 hours. [Figure 42A]Figure 42A shows the results of DNA binding protein activity of ExAmp in the presence of polymers described herein after staging, and the results of DNA binding protein activity of ExAmp in the presence of polymers described herein after staging. Figure 42A shows the DNA binding protein 3 activity of polymer spiked ExAmp (TCX1 v1.0) solutions. Figure 42B shows the DNA binding protein activity after 24 hours incubation at 40°C. [Figure 42B] Figure 42A shows the results of DNA binding protein activity of ExAmp in the presence of polymers described herein after staging, and the results of DNA binding protein activity of ExAmp in the presence of polymers described herein after staging. Figure 42A shows the DNA binding protein 3 activity of polymer spiked ExAmp (TCX1 v1.0) solutions. Figure 42B shows the DNA binding protein activity after 24 hours incubation at 40°C. [Figure 43A] Figure 43 shows that microencapsulation improves the stability of SBS reagents. Figure 43A shows improved solubility. Figure 43B shows improved enzyme compatibility. Figure 34C shows improved ffN compatibility. Figure 34D shows improved sequencing. [Figure 43B] Figure 43 shows that microencapsulation improves the stability of SBS reagents. Figure 43A shows improved solubility. Figure 43B shows improved enzyme compatibility. Figure 34C shows improved ffN compatibility. Figure 34D shows improved sequencing. [Figure 43C] Figure 43 shows that microencapsulation improves the stability of SBS reagents. Figure 43A shows improved solubility. Figure 43B shows improved enzyme compatibility. Figure 34C shows improved ffN compatibility. Figure 34D shows improved sequencing. [Fig. 43D]Figure 43 shows that microencapsulation improves the stability of SBS reagents. Figure 43A shows improved solubility. Figure 43B shows improved enzyme compatibility. Figure 34C shows improved ffN compatibility. Figure 34D shows improved sequencing. [Diagram 44] FIG. 1 shows the physical characterization of encapsulated microspheres. [Figure 45A] FIG. 45 shows that the quality of the tested ffNs may not be affected by the spray-coating process: peak areas (FIG. 45A) and 3′OH levels (FIG. 45B) were comparable to the control and no diphosphate was detected. [Figure 45B] FIG. 45 shows that the quality of the tested ffNs may not be affected by the spray-coating process: peak areas (FIG. 45A) and 3′OH levels (FIG. 45B) were comparable to the control and no diphosphate was detected. [Figure 46A] Figure 46 shows that shell encapsulation improves moisture barrier and mitigates static electricity. Figure 46A shows a natural microsphere after exposure to moisture conditions. Figure 46B shows a matrix microsphere after exposure to moisture conditions. Figure 46C shows a core-shell microsphere coated with a composition described herein after exposure to moisture conditions. [Figure 46B] Figure 46 shows that shell encapsulation improves moisture barrier and mitigates static electricity. Figure 46A shows a natural microsphere after exposure to moisture conditions. Figure 46B shows a matrix microsphere after exposure to moisture conditions. Figure 46C shows a core-shell microsphere coated with a composition described herein after exposure to moisture conditions. [Figure 46C]Figure 46 shows that shell encapsulation improves moisture barrier and mitigates static electricity. Figure 46A shows a natural microsphere after exposure to moisture conditions. Figure 46B shows a matrix microsphere after exposure to moisture conditions. Figure 46C shows a core-shell microsphere coated with a composition described herein after exposure to moisture conditions. [Figure 47] FIG. 1 illustrates the method described herein. [Figure 48] FIG. 1 illustrates the method described herein. [Figure 49A] Figure 49A shows triple coated microspheres with sequential pH triggers for release. Figure 49A shows the results of run 1, run 2, and run 3 of a composition with coated microspheres with three shells: shell 1: Eudragit S100 (fluorescein), shell 2: Eudragit L100 (light blue), and shell 3: Eudragit L100-55 (rhodamine). Figure 49B shows the migration of coated microspheres from Figure 49A from pH 5 to pH 6, pH 7, and pH 8. Figure 49C shows the release of various coating compositions under different pH conditions. [Figure 49B] Figure 49A shows triple coated microspheres with sequential pH triggers for release. Figure 49A shows the results of run 1, run 2, and run 3 of a composition with coated microspheres with three shells: shell 1: Eudragit S100 (fluorescein), shell 2: Eudragit L100 (light blue), and shell 3: Eudragit L100-55 (rhodamine). Figure 49B shows the migration of coated microspheres from Figure 49A from pH 5 to pH 6, pH 7, and pH 8. Figure 49C shows the release of various coating compositions under different pH conditions. [Figure 49C]Figure 49A shows triple coated microspheres with sequential pH triggers for release. Figure 49A shows the results of run 1, run 2, and run 3 of a composition with coated microspheres with three shells: shell 1: Eudragit S100 (fluorescein), shell 2: Eudragit L100 (light blue), and shell 3: Eudragit L100-55 (rhodamine). Figure 49B shows the migration of coated microspheres from Figure 49A from pH 5 to pH 6, pH 7, and pH 8. Figure 49C shows the release of various coating compositions under different pH conditions.

[0062] It is to be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The first embodiment relates to a composition comprising a shell surrounding a core, the core comprising one or more lyophilized microspheres.

[0064] It should be understood that certain aspects, modes, embodiments, variations, and features of the present disclosure are described below with various levels of detail to provide a substantial understanding of the present technology. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art. The use of the term "including" and other forms is not limiting. The use of the term "having" and other forms is not limiting. When used in this disclosure, whether in transitional phrases or in the body of the claims, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, these terms should be interpreted as synonymous with the phrases "having at least" or "including at least".

[0065] The terms "substantially," "approximately," "about," "relatively," or other such similar terms, which may be used throughout this disclosure, including the claims, are used to describe and take into account small variations from a reference or parameter, such as due to variations in processing. Such small variations include zero point variations from a reference or parameter. For example, the variation can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0066] It will be further understood that certain features described herein, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0067] The terms "connected," "contacted," and / or "coupled" include a variety of arrangements and assemblies, including, but not limited to, (1) directly joining one component to another without any intervening components between them (i.e., the components are in direct physical contact) and (2) joining one component to another with one or more components between them, provided that a component that is "connected" or "in contact" or "coupled" to another component is in some operative communication (e.g., electrical, fluid, physical, optical, etc.) with the other component (optionally with the presence of one or more additional components between them). Components that are in direct physical contact with each other may or may not be in electrical and / or fluid contact with each other. Furthermore, two components that are electrically connected, electrically coupled, optically connected, optically coupled, fluidly connected, or fluidly coupled may or may not be in direct physical contact, and one or more other components may be located between the two connected components.

[0068] As described herein, the term "array" may include a collection of conductive channels or molecules that may be attached to one or more solid substrates such that the conductive channels or molecules may be differentiated from one another based on their location. The arrays described herein may include different molecules, each located in a different distinguishable location (e.g., different conductive channels) on the solid substrate. Alternatively, the array may include separate solid substrates, each with a different molecule, and the different probe molecules may be differentiated according to the location of the solid substrate on the surface to which the solid substrate is attached, or based on the location of the solid substrate in a liquid, such as a fluid stream. Examples of arrays in which separate substrates are located on a surface include wells with beads, as described in U.S. Pat. No. 6,355,431, U.S. Patent Application Publication No. 2002 / 0102578, and WO 00 / 63437, all of which are incorporated herein by reference in their entireties. The molecules of the array may be nucleic acid primers, nucleic acid probes, nucleic acid templates, or nucleic acid enzymes, such as polymerases and exonucleases.

[0069] As described herein, the term "attached" can include when two things are joined, fixed, attached, connected, or bonded to each other. A reaction component, such as a polymerase, can be attached to a solid phase component, such as a conductive channel, by covalent or non-covalent bonds. As described herein, the phrase "covalently attached" or "covalently bonded" refers to the formation of one or more chemical bonds characterized by the sharing of electron pairs between atoms. Non-covalent bonds do not involve the sharing of electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0070] As described herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogs of either DNA or RNA made from nucleotide analogs. As used herein, these terms also encompass complementary cDNA or copy DNA generated from an RNA template, for example, by the action of reverse transcriptase. In one embodiment, the nucleic acid to be analyzed, for example, by sequencing using the described system, is immobilized on a substrate (e.g., a substrate in a flow cell or one or more beads on a substrate such as a flow cell). The term immobilization as used herein is intended to encompass direct or indirect, covalent or non-covalent attachment, unless otherwise indicated explicitly or by context. The analyte (e.g., nucleic acid) may remain immobilized or attached to the support under conditions for which the support is intended to be used, such as in nucleic acid sequencing applications. In one embodiment, the template polynucleotide is one of multiple template polynucleotides attached to the substrate. In one embodiment, the plurality of template polynucleotides attached to the substrate comprises a cluster of copies of a library polynucleotide described herein.

[0071] Nucleic acids include naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize to nucleic acids in a sequence-specific manner or can be used as templates to replicate specific nucleotide sequences. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone linkages, including any of a variety known in the art, such as peptide nucleic acid (PNA) or locked nucleic acid (LNA). Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

[0072] In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of deoxyribose may be attached to the N-1 of a pyrimidine or the N-9 of a purine.

[0073] Nucleic acids can contain any of the various analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural bases. Natural deoxyribonucleic acids can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine, and ribonucleic acids can have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Useful non-natural bases that can be included in nucleic acids are known in the art.

[0074] The term nucleotide as used herein may include natural nucleotides, their analogs, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and other molecules known as nucleotides. As used herein, a nucleotide may include a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. A nucleotide may be, for example, a monomeric unit of a nucleic acid sequence to identify subunits present in a DNA or RNA chain. A nucleotide may also include molecules that are not necessarily present in a polymer, such as molecules that can be incorporated into a polynucleotide in a template-dependent manner by a polymerase. A nucleotide may include, for example, a nucleoside unit having zero, one, two, three or more phosphates on the 5' carbon. Tetraphosphate, pentaphosphate, and hexaphosphate nucleotides may be useful, as may nucleotides having more than six phosphates on the 5' carbon, such as seven, eight, nine, ten, or more. Examples of naturally occurring nucleotides include, but are not limited to, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP.

[0075] Non-natural nucleotides include nucleotide analogs such as those that are not present in a natural biological system or that are not substantially incorporated into a polynucleotide by a polymerase in its natural environment, e.g., a non-recombinant cell expressing the polymerase. Non-natural nucleotides include those that are incorporated into a polynucleotide chain by a polymerase at a rate that is substantially faster or slower than the rate at which another nucleotide, such as a natural nucleotide that base pairs with the same Watson-Crick complementary base, is incorporated into the chain by the polymerase. For example, a non-natural nucleotide may be incorporated at a rate that is at least 2-fold different, 5-fold different, 10-fold different, 25-fold different, 50-fold different, 100-fold different, 1000-fold different, 10000-fold different, or more different than the rate at which a natural nucleotide is incorporated. A non-natural nucleotide may be capable of being further extended after being incorporated into a polynucleotide. Examples include nucleotide analogs with a 3' hydroxyl or nucleotide analogs with a reversible terminator moiety at the 3' position that can be removed to allow a polynucleotide incorporating the nucleotide analog to be further extended. Examples of reversible terminator moieties are described, for example, in U.S. Patent Nos. 7,427,673, 7,414,116, and 7,057,026, as well as WO 91 / 06678 and WO 07 / 123744, each of which is incorporated herein by reference in its entirety. It should be understood that in some instances, nucleotide analogs having a 3' terminator moiety or lacking a 3' hydroxyl (such as dideoxynucleotide analogs) can be used under conditions in which the polynucleotide incorporating the nucleotide analog is not further extended. In some instances, the nucleotide may not include a reversible terminator moiety, or the nucleotide does not include an irreversible terminator moiety, or the nucleotide does not include any terminator moiety at all.

[0076] The present disclosure encompasses nucleotides that contain a fluorescent label (or any other detection tag) that can be used in any of the methods disclosed herein, either by themselves or incorporated into or associated with a larger molecular structure or conjugate.

[0077] The fluorescent label may comprise a compound selected from any known fluorescent species, for example, rhodamine or cyanine. The fluorescent label disclosed herein may be attached at any position on the nucleotide base and may optionally comprise a linker. The function of the linker generally aids in the chemical attachment of the fluorescent label to the nucleotide. In certain embodiments, Watson-Crick base pairing can still be performed on the resulting analog. The linker group may be used to covalently attach the dye to the nucleoside or nucleotide. The linker moiety may be of sufficient length to connect the nucleotide to the compound, such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by nucleic acid replicating enzymes. Thus, the linker may also comprise a spacer unit. The spacer distances the nucleotide base from, for example, the cleavage site or the label.

[0078] As used herein, a "nucleoside" is structurally similar to a nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is one in which a label is linked to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" is used herein in its ordinary sense as understood by those of skill in the art. Examples include, but are not limited to, ribonucleosides that contain a ribose moiety, and deoxyribonucleosides that contain a deoxyribose moiety. Modified pentose moieties are those in which an oxygen atom is replaced with a carbon and / or a carbon is replaced with a sulfur or oxygen atom. A "nucleoside" is a monomer that may have a substituted base and / or sugar moiety.

[0079] The term "purine base" is used herein in its ordinary sense as understood by those of skill in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those of skill in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0080] The term substrate (or solid support), as used herein, may include any inert substrate or matrix to which a nucleic acid may be attached, such as, for example, a glass surface, a plastic surface, latex, dextran, a polystyrene surface, a polypropylene surface, a polyacrylamide gel, a gold surface, and a silicon wafer. For example, the substrate may be a glass surface (e.g., the planar surface of a flow cell channel). In one embodiment, the substrate may include an inert substrate or matrix that has been "functionalized," such as by applying a layer or coating of an intermediate material that includes reactive groups that allow for covalent attachment to a molecule, such as a polynucleotide. The support may include a polyacrylamide hydrogel supported on an inert substrate, such as glass. The molecule (e.g., a polynucleotide) may be covalently attached directly to the intermediate material (e.g., the hydrogel). The support may include a plurality of particles or beads, each having a different attached analyte.

[0081] As used herein, "derivative" or "analog" refers to a synthetic nucleotide or nucleoside derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Bucher, NUCLEOTIDE ANALOGS (John Wiley & Son, 1980) and Uhlmann et al., "Antisense Oligonucleotides: A New Therapeutic Principle," Chemical Reviews 90:543-584 (1990), both of which are incorporated herein by reference in their entirety. Nucleotide analogs may also include modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, and phosphoramidate linkages. As used herein, "derivative," "analog," and "modified" may be used interchangeably and are encompassed by the terms "nucleotide" and "nucleoside" as described herein.

[0082] The compositions, systems, and methods described herein include a shell surrounding a core, and the core may include one or more lyophilized microspheres (i.e., the compositions may include encapsulated lyophilized microspheres).

[0083] As described herein, "encapsulate," "encapsulated," and "encapsulation" include the inclusion of one or more microspheres as described herein. Microencapsulation as described herein refers to embedding at least one component, e.g., an active agent, in at least one other material, e.g., a shell material. Encapsulation according to the present disclosure includes, but is not limited to, bulk encapsulation, macroencapsulation, microencapsulation, nanoencapsulation, single molecule, and ion encapsulation. According to the present disclosure, the compositions, systems, and methods described herein have many advantages including, for example, increased stability of the microspheres, use of macroencapsulation to allow for multi-run cartridges, and use of microencapsulation to allow for simplified workflow and reduced number of reagent wells. The compositions, systems, and methods described herein use encapsulation of particles that are otherwise responsive to pH changes to stabilize these buffers and improve SBS performance. The compositions, systems, and methods described herein also use encapsulation to reduce the risk of electrostatic charges that would otherwise cause difficulties in dispensing and dry compounding of microspheres during manufacturing.

[0084] As used herein, "microsphere" includes spherical particles, including a shell and a core, having a diameter of 0.1 μm to 1,000 μm. For example, the microspheres can have a diameter of about 0.1 μm, 0.5 μm, 1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, or any diameter between about 0.1 μm and about 1,000 μm. In one embodiment, the encapsulated microspheres have a diameter between about 100 μm and 1000 μm.

[0085] Microspheres are generally composed of a polymer shell, such as a biodegradable polymer. Microspheres according to the present disclosure include those prepared by conventional techniques known to those skilled in the art. For example, microspheres can be prepared by freezing a liquid into frozen pellets, and then placing the frozen microspheres in a dryer, such as a rotary dryer.

[0086] As described herein, a "macrosphere" may include a plurality of microspheres. Macrospheres are generally larger in diameter than microspheres, e.g., from 0.1 mm to 1,000 mm. The macrospheres described herein may have a diameter of, for example, about 0.1 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 50 mm, 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, 600 mm, 700 mm, 800 mm, 900 mm, 1,000 mm, or any diameter from about 0.1 mm to about 1,000 mm. In one embodiment, the macrospheres (e.g., macrocapsules) have dimensions ranging from about 5 mm x 5 mm x 9 mm to about 7 cm x 7 cm x 2 cm.

[0087] Macrospheres according to the present disclosure include those prepared by conventional techniques known to those of skill in the art. The compositions, systems, and methods described herein may include a single lyophilized microsphere, or may include multiple lyophilized microspheres, thereby forming a macrosphere. For example, the compositions described herein may include anywhere between 1 and more than 1,000,000 lyophilized microspheres. In one embodiment, the composition includes one lyophilized microsphere, or less than 100 lyophilized microspheres, or less than 500 lyophilized microspheres, or any number between about 1 and about 1,000,000 microspheres. In one embodiment, when the shell surrounds more than one lyophilized microsphere, the reagents in the core of the lyophilized microspheres are different.

[0088] As described herein, a "shell" includes a composition that surrounds a core. In one embodiment, the shell includes the outer layer of a microsphere, or alternatively, the outer layer of a macrosphere. In one embodiment, the shell includes a shell material selected from, for example, carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof. In one example, the shell may include, but is not limited to, starch, cellulose, hydrocolloids, alginate, collagen, and any combination thereof. The amount of shell material includes, for example, any amount suitable to produce a desired shell result. In one embodiment, the shell material is present in an amount of about 1 wt% to about 100 wt% of the shell. For example, the shell material can be present at about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 100 wt%, or any amount therebetween of the shell. In one embodiment, the shell material is present in an amount of about 10 wt% to about 90 wt%, or about 10 wt% to about 80 wt%, or about 10 wt% to about 70 wt%, or about 10 wt% to about 60 wt%, or about 10 wt% to about 50 wt% of the shell.

[0089] The shell may comprise one layer or multiple layers of various compositions, as described herein. For example, the shell may comprise one layer, two layers, three layers, four layers, five layers, six layers, seven layers, eight layers, nine layers, ten layers, or more than ten layers. Each layer may comprise the same or different materials as the other layers present in the shell.

[0090] The shells described herein may, in one embodiment, include a shell additive. The shell additive may be present in an amount of about 0.01% w / w of the shell to about 99% w / w of the shell. In one embodiment, the shell additive is present in an amount of about 10% w / w of the shell to about 90% w / w of the shell. In one embodiment, the shell additive is present in an amount of about 10% w / w of the shell to about 40% w / w of the shell. In one embodiment, the shell additive is a static relief material present in an amount of 40% w / w of the shell or less. In one embodiment, the shell additive is a moisture barrier material present in an amount of 90% w / w of the shell or less. In one embodiment, the shell additive is present in an amount of at least 10% w / w of the shell. For example, the shell additive may, in one embodiment, be present in an amount of 0.1% w / w of the shell to about 15.0% w / w of the shell. For example, the shell additive may be present in amounts of about .01% w / w, 0.05% w / w, 0.1% w / w, 0.5% w / w, 1.0% w / w, 1.5% w / w, 2.0% w / w, 2.5% w / w, 3.0% w / w, 3.5% w / w, 4.0% w / w, 4.5% w / w, 5.0% w / w, 5.5% w / w, 6.0% w / w, 6.5% w / w, 7.0% w / w, 7.5% The amount of shell additive may be present in an amount of 0.1 to 1.0% w / w, 8.0% w / w, 8.5% w / w, 9.0% w / w, 9.5% w / w, 10.0% w / w, 10.5% w / w, 11.0% w / w, 11.5% w / w, 12.0% w / w, 12.5% ​​w / w, 13.0% w / w, 13.5% w / w, 14.0% w / w, 14.5% w / w, 15% w / w, or any amount therebetween. The amount of shell additive may be any suitable amount to reduce triboelectric charging of the compositions described herein and / or provide a suitable moisture barrier. The amount of shell additive may be adjusted to accommodate a particular reagent or combination of reagents or to accommodate a particular microsphere composition.

[0091] In one embodiment, the shell additive comprises a static relief material, a moisture barrier material, or a combination thereof. In one embodiment, the shell additive is a static relief material present in an amount of 40% w / w concentration of the shell or less. In one embodiment, the shell additive is a moisture barrier material present in an amount of 90% w / w concentration of the shell or less. In one embodiment, the shell additive is present in an amount of at least 10% w / w concentration of the shell. In one embodiment, the shell additive is in an amount of about 10% w / w to about 90% w / w of the shell. In one embodiment, the shell additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity enhancer, a stearic acid derivative, an oleic acid derivative, a lauric acid derivative, a polyether compound, an amino acid, a tocopherol acetate, a piperidyl sebacate, a sodium salt, a buffer, a chelating agent, an imidazolium salt, a polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearic acid derivative or oleic acid derivative is selected from magnesium stearate, triglycerol stearate, Span® 60, Tween® 60, glycerol trioleate, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof.In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salts or polyquaterniums or Luviquat® (copolymer of vinylpyrrolidone and quaternized vinylimidazole) or combinations thereof. In one embodiment, the shell additive comprises an ammonium salt, copolymer, polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof. The shell may be comprised of a polymer, and the maximum concentration of the polymer in the shell may be about 90% (in dry form). One or more static relief additives may be added to the polymer coating, and the shell additive may be present in the shell in the dry form in the range of about 10% to about 40%.

[0092] In various embodiments, the shell additive may include a beneficial combination of compounds for improved and unexpected compatibility with SBS reagents. For example, the shell additive may include a polyether compound and a polymer and / or copolymer, or alternatively, a polyether compound, PVA, and / or a polymer and / or copolymer. In one embodiment, the shell additive includes polyethylene glycol, Kollidon® VA64, and Efka® IO 6783, or chemical equivalents thereof. In another embodiment, the shell additive includes polyethylene glycol and Kollidon® VA64, or chemical equivalents thereof. In another embodiment, the shell additive includes polyethylene glycol, Kollicoat® Protect, and Efka® IO 6783, or chemical equivalents thereof. In yet another embodiment, the shell additive includes polyethylene glycol and Kollicoat® Protect, or chemical equivalents thereof. In one embodiment, the ammonium salt acts as a conductivity promoter. In one embodiment, the imidazolium salt acts as a conductivity promoter.

[0093] As used herein, a "core" or "core region" includes any material within the surrounding shell. A core according to the present disclosure comprises one or more lyophilized microspheres.

[0094] As used herein, the term "compatible" means capable of existing or occurring together without conflict, i.e., without substantially decreasing the performance or activity of one or more substances that exist or occur together. Similarly, as used herein, the term "incompatible" means not capable of existing or occurring together without conflict, i.e., without substantially decreasing the performance or activity of one or more substances that exist or occur together.

[0095] Lyophilization according to the present disclosure includes conventional methods known to those of skill in the art. Lyophilization is also referred to herein as freeze-drying. In this disclosure, the terms "lyophilize" or "lyophilizate" are used as equivalent terms to "lyophilised", "lyophilisate", or "freeze-dried", for example, with respect to the compositions, systems, or methods described herein.

[0096] Lyophilizable formulations can be reconstituted into a solution, suspension, emulsion, or any other form suitable for administration or use.Lyophilizable formulations are typically first prepared as liquid, then frozen and lyophilized.The total liquid volume before lyophilization can be less than, equal to, or greater than the final reconstituted volume of the lyophilized formulation.Preferably, the final reconstituted volume of the lyophilized formulation is less than the total liquid volume before lyophilization.The lyophilization process is known to those skilled in the art and typically involves the sublimation of water from the frozen formulation under controlled conditions.

[0097] The lyophilized formulations can be stored at a wide range of temperatures. The lyophilized formulations can be refrigerated below 25°C, e.g., at 2-8°C, or stored at room temperature (e.g., about 25°C). Preferably, the lyophilized formulations can be stored below about 25°C, more preferably at about 4-20°C, below about 4°C, below about -20°C, about -40°C, about -70°C, or about -80°C. The stability of the lyophilized formulations can be determined in several ways known in the art, e.g., by visual appearance of the microspheres and / or cake and / or by moisture content. The compositions of the present disclosure can also withstand temperature fluctuations that may occur during shipping, e.g., up to 70°C for short periods of time.

[0098] Lyophilized formulations are typically rehydrated for use (interchangeably referred to herein as "reconstituted") by the addition of an aqueous solution to dissolve the lyophilized formulation. A wide variety of aqueous solutions, including water, saline, or another electrolyte or non-electrolyte diluent, can be used to reconstitute the lyophilized formulation. Preferably, the lyophilized microspheres described herein are reconstituted using water. Lyophilized formulations can be rehydrated with a solution containing water (e.g., USP WFI, or water for injection) or bacteriostatic water (e.g., USP WFI with 0.9% benzyl alcohol). However, solutions containing additives, buffers, excipients, and / or carriers can also be used and are described herein.

[0099] Freeze-dried or lyophilized formulations are typically prepared from liquids, i.e., from solutions, suspensions, emulsions, etc. Thus, the liquid undergoing freeze-drying or lyophilization preferably contains all the components desired in the final reconstituted liquid formulation. As a result, upon rehydration or reconstitution, the freeze-dried or lyophilized formulation becomes the desired liquid formulation upon reconstitution. Core additives and / or shell additives, if present, may be incorporated into the reagent upon rehydration of the compositions described herein.

[0100] In one embodiment, the core comprises one or more reagents, such as, but not limited to, one or more enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In a preferred embodiment, the core is not an aqueous medium.

[0101] As used herein, the term "reagent" describes a single agent or a mixture of two or more agents useful for reacting with, interacting with, diluting, or adding to a sample, and may include agents used in nucleic acid reactions, including, for example, buffers, chemicals, enzymes, polymerases, primers, including those having a size of less than 50 base pairs, template nucleic acids, nucleotides, labels, dyes, or nucleases. Reagents described herein may, in certain embodiments, include enzymes such as polymerases, ligases, recombinases, or transposases, antibodies, epitopes, binding partners such as streptavidin, avidin, biotin, lectins, or carbohydrates, or other biochemically active molecules. Other exemplary reagents include reagents for biochemical protocols, such as nucleic acid amplification protocols, affinity-based assay protocols, enzymatic assay protocols, sequencing protocols, and / or protocols for the analysis of biological fluids. According to some of the embodiments disclosed herein, the reagents may include one or more types of beads, particularly magnetic beads, depending on the particular workflow and / or downstream application.

[0102] In one embodiment, the reagent according to the present disclosure is a polymerase. The polymerase according to the present disclosure may include any polymerase that can tolerate the incorporation of phosphate-labeled nucleotides. Examples of polymerases that may be useful according to the present disclosure include, but are not limited to, phi29 polymerase, Klenow fragment, DNA polymerase I, DNA polymerase III, GA-1, PZA, phi15, Nf, G1, PZE, PRD1, B103, GA-1, 9oN polymerase, Bst, Bsu, T4, T5, T7, Taq, Vent, RT, pol beta, and pol gamma. Polymerases engineered to have specific properties may also be used. In one example, the core region may include, but is not limited to, polishing microspheres, sequencing microspheres, and any combination thereof. The polishing microspheres described herein may include, but are not limited to, ffN, a polymerase useful for polishing ("polishing polymerase"), an oligo useful for polishing ("polishing oligo"), a magnesium enzyme cofactor, and any combination thereof. In another example, a sequencing microsphere may include, but is not limited to, a polymerase useful for sequencing ("sequencing polymerase").

[0103] The primers disclosed herein comprise a nucleic acid molecule that can hybridize to a target sequence of interest. In some embodiments, the primer may function as a substrate onto which nucleotides can be polymerized by a polymerase. However, in some instances, the primer can be incorporated into a synthesized nucleic acid strand to provide a site to which another primer can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid molecule. The primer can comprise any combination of nucleotides or analogs thereof. In some instances, the primer is a single-stranded oligonucleotide or polynucleotide.

[0104] Non-limiting examples of nucleic acid molecules that can be encapsulated within the microsphere include those described above, e.g., DNA, such as genomic or cDNA, RNA, such as mRNA, sRNA, or rRNA, or hybrids of DNA and RNA. The core may further comprise labeled nucleotides.

[0105] The term "salts" may include salts prepared from toxic or non-toxic acids or bases, including inorganic acids and bases, and organic acids and bases. Salts can be prepared, for example, from pharma- ceutically acceptable non-toxic acids, including inorganic and organic acids.

[0106] Any surfactant known to those skilled in the art may be used as a reagent in the core. The surfactant may be non-ionic or ionic (specifically cationic or anionic), or zwitterionic. Examples of suitable surfactants include, but are not limited to, polyacrylate surfactants, silicone surfactants, and / or other commercially available surfactants or detergents. Examples of cationic surfactants are cetyldimethylammonium acetamide, octadecyldimethylammonium acetamide, tetradecyldimethylammonium acetamide, dodecyldimethylammonium acetamide, cetyltrimethylammonium, octadecyltrimethylammonium, tetradecyltrimethylammonium, dodecyltrimethylammonium, dimethyldioctadecylammonium, dioctadecyldimethylammonium, and mixtures thereof. Suitable sources of these cations for cationic surfactants include, but are not limited to, alkyl trimethyl ammonium salts such as cetyl trimethyl ammonium bromide (CTAB) or cetyl trimethyl ammonium chloride (CTAC), cetyl pyridinium chloride (CPC), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cetyl dimethyl ammonium acetamide bromide, or other cationic surfactants, including lipids. Alternatively, the surfactant may be benzyl hexadecyl dimethyl ammonium chloride (BHDC). The core may include an anionic surfactant that includes an anionic functional group at one end, such as sulfate, sulfonate, phosphate, and carboxylate functional groups. An example of an anionic surfactant is sodium dodecyl sulfate. The core may include a neutral surfactant, such as polyethylene glycol lauryl ether.

[0107] The core may also or alternatively include enzyme inhibitors, molecular probes, crowding agents, organic osmolytes, cyclodextrins, adenosine triphosphate (ATP), ethylenediaminetetraacetic acid (EDTA), creatine kinase, creatine phosphate, palladium, lipoic acid, hexaethylene glycol, trihydroxypropanephosphine, sodium ascorbate, or any combination thereof. Enzyme inhibitors as described herein include any molecule that binds to an enzyme and reduces its activity. Molecular probes as described herein include, for example, digoxigenin, 8-anilinonaphthalene-1-sulfonic acid ("ANS"), porphyrins, BODIPY, cyanines, or any combination thereof. Crowding agents as described herein include any crowding agent known to those of skill in the art. Examples include, but are not limited to, polyethylene glycol, ficoll, dextran, and serum albumin.

[0108] Those skilled in the art of sequencing will understand that there are additional reagents that may be useful in the compositions, systems, and methods of the present disclosure that are not explicitly described herein.

[0109] The cores described herein may further comprise, in one embodiment, one or more additional agents. The one or more additional agents in the core improve the ability to control the release of one or more lyophilized microspheres. In one embodiment, the additional agent is selected from one or more sugars, amino acids, polymers, mesoporous silica, quaternary amines, or any combination thereof. In one embodiment, when the additional agent comprises a sugar, the sugar is selected from trehalose, mannitol, cyclodextrin, dextran, sucrose, or any combination thereof. In another embodiment, when the additional agent comprises an amino acid, the amino acid has a hydrophobic side chain. In another embodiment, when the additional agent comprises a polymer, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, or any combination thereof. In some embodiments, the additional agent may be, for example, one or more copolymers, ionic liquids, or any combination thereof. The additional agent may be added in any amount suitable to produce the desired effect, for example, from about 0.1 wt% to about 50 wt% of the core. In one embodiment, the concentration of the additional agent in the core is about 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or any amount therebetween.

[0110] In one embodiment, the core further comprises a core additive. The core additive may be present in an amount from about 0.01% w / w of the core to about 100% w / w of the core. For example, the core additive may be present in an amount from 0.1% w / w of the core to about 20.0% w / w of the core, in one embodiment. In one embodiment, the core additive may be from about 2% w / w to about 10% w / w of the core. For example, core additives are approximately .01%w / w, 0.05%w / w, 0.1%w / w, 0.5%w / w, 1.0%w / w, 1.5%w / w, 2.0%w / w, 2.5%w / w, 3.0%w / w, 3.5%w / w, 4.0%w / w, 4.5%w / w, 5.0%w / w, 5.5%w / w, 6.0%w / w, 6.5%w / w, 7.0%w / w, 7.5%w / w, 8.0%w / w, 8.5%w / w, 9.0%w / w, 9.5%w / w, 10.0%w / w, 1 It may be present in an amount of 0.5% w / w, 11.0% w / w, 11.5% w / w, 12.0% w / w, 12.5% ​​w / w, 13.0% w / w, 13.5% w / w, 14.0% w / w, 14.5% w / w, 15.0% w / w, 15.5% w / w, 16.0% w / w, 16.5% w / w, 17.0% w / w, 17.5% w / w, 18.0% w / w, 18.5% w / w, 19.0% w / w, 19.5% w / w, 20.0% w / w, or any amount therebetween. The amount of the core additive may be any suitable amount for reducing triboelectric charging of the compositions described herein. The amount of the core additive may be adjusted to accommodate a particular reagent or combination of reagents, or to accommodate a particular microsphere composition.

[0111] In one embodiment, the core additive comprises a static relief material. In one embodiment, the core additive is a static relief material present in an amount of 25% w / w concentration of the core or less. In one embodiment, the core additive is present in an amount of at least 0.5% w / w concentration of the core. In one embodiment, the core additive is in an amount of about 2% w / w to about 10% w / w of the core. In one embodiment, the core additive is a water insoluble additive, a water soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), a conductivity enhancer, an ammonium salt, an imidazolium salt, a polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polyquaternium or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole) or a combination thereof. In one embodiment, the compositions described herein can be made from about 20% lyophilized formulations (i.e., the formulations contain 20% lyophilized excipients such as trehalose and other additives). Thus, additives (static relief or moisture protection) can be incorporated and / or spiked into the lyophilized formulations and then dried to obtain the appropriate concentration in the dry format. In one embodiment, the ammonium salt acts as a conductivity promoter. In one embodiment, the imidazolium salt acts as a conductivity promoter.

[0112] The core additives described herein may, in one embodiment, comprise water-insoluble additives, water-soluble additives, enteric additives, or any combination thereof. In one embodiment, the core additives may comprise one or more of sodium salts, buffers, chelating agents, ammonium salts, imidazolium salts, polyanilines, or any combination thereof. In one embodiment, one or more water-soluble core additives are added to the core. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In another embodiment, the buffer is Trizma (Tris.HCl). In one embodiment, the ammonium salt is selected from tetraalkylammonium chlorides, tris(hydroxyethyl)alkylammonium chlorides, or any combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salts or polyquaterniums or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole) or any combination thereof. In another embodiment, one or more water-soluble additives such as Efka® IO 6783, Efka® IO 6786, Tween® 80, Makon® 17R4, lauric acid diethanolamide, or any combination of one or more of these additives may be included in the core additive composition. In another embodiment, one or more water-insoluble additives such as glycerol trioleate, polyaniline, piperidyl sebacate, amino acids, vitamin E (tocopherol acetate), Span® 60, or any combination of one or more of these additives may be included in the core additive composition. In one embodiment, Efka® IO 6783 is used as a core additive in an amount suitable to reduce the triboelectric charging behavior of the composition (e.g., an amount of about 5% w / w of the core).

[0113] The composition (i.e., the encapsulated lyophilized microspheres) can be of any suitable size or volume suitable for encapsulating one or more reagents and suitable for use in library preparation for sequencing. In one embodiment, the composition has a volume of reagent in the core region of about 0.1 μL to about 50 μL. For example, the composition (i.e., the encapsulated lyophilized microspheres) can have an active reagent volume of about 0.1 μL, 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, or any volume between about 0.1 μL and about 50 μL. In one embodiment, the active reagent volume is between about 10 μL and about 40 μL. The composition (i.e., the encapsulated lyophilized microspheres) can have, for example, a diameter of about 2 μm to about 120 μm, e.g., a diameter of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, or 120 μm, or a diameter within a range defined by any two of the foregoing values.

[0114] The compositions described herein may include additional reagents in the shell of the microsphere. In one embodiment, the encapsulated microsphere includes a reagent or additive in the shell of the microsphere. The reagent in the shell may include, for example, any of the reagents or additives described above. In one embodiment, the shell does not include a nucleic acid molecule, for example, the shell does not include DNA. In one embodiment, the shell includes more than one reagent, or alternatively, includes more than one additive.

[0115] Similarly, the compositions described herein (e.g., encapsulated lyophilized microspheres) can include a low oxygen permeable polymer coating, such as polyvinyl alcohol and / or an oxygen scavenger. Similarly, the compositions described herein (e.g., encapsulated lyophilized microspheres) can include an amphiphilic coating, such as an amino acid and / or a PVP copolymer. The encapsulated lyophilized microspheres described herein can further provide protection from mechanical stress, for example, by preventing or reducing fragmentation in manufacturing, for example, with a shell with a solute content of 40%. The compositions described herein (e.g., encapsulated lyophilized microspheres) can further provide protection from light exposure, since they can protect the reagents from light exposure, thereby reducing light constraints in manufacturing.

[0116] The compositions described herein can be used for multiple sequential simultaneous assays including lysis, DNA analysis, RNA analysis, protein analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, SBS technology, transposase-accessible chromatin region analysis by sequencing (ATAC-seq), processively conserved transposition (CPT-seq), single-cell combinatorial indexed sequencing (SCI-seq), or single-cell genome amplification, or any combination thereof performed sequentially. In one embodiment, the compositions are used to perform multiple simultaneous assay reactions. The compositions, systems, and methods described herein (e.g., encapsulation in lyophilized microspheres) can, in one embodiment, improve sequencing quality, enable one-pot library preparation, and simplify manufacturing. As used herein, the term "one-pot reaction" can also be referred to as "transfer-free reaction."

[0117] The compositions, systems, and methods described herein may be prepared for various stages of sequencing, including but not limited to sample extraction, library preparation, enrichment, clustering, and sequencing. In a sample extraction composition, the core may include enzymes, salts, detergents, buffers, and any combination thereof. Sample extraction may be performed at a pH of about 7.5 in a reaction volume of about 1 mL to about 5 mL. In a library preparation composition, the core may include enzyme inhibitors, salts, primers, enzymes, nucleotides, organic osmolytes, magnetic beads, and any combination thereof. Library preparation may be performed at a pH of about 7 in a reaction volume of about 0.05 mL. In an enrichment composition, the core may include nucleotides, molecular probes, enzymes, magnetic beads, crowding agents, and any combination thereof. Enrichment may be performed at a pH of about 8.5 in a reaction volume of about 0.1 mL to about 0.2 mL. In a clustering composition, the core may include salts, enzymes, one or more nucleotides, small molecules, detergents, primers, and any combination thereof. Clustering may be performed at a pH of about 8.6 in a reaction volume of about 1 mL to about 5 mL. In the sequencing composition, the core may include labeled nucleotides, fluorophores, detergents, salts, enzymes, small molecules, and any combination thereof. Sequencing may be performed at a pH of about 7 to about 10 in a reaction volume of about 30 mL to about 100 mL.

[0118] In one embodiment, the shell may rehydrate under a pH of 1-14. In one embodiment, the shell may include one or more shell layers, each layer may rehydrate under the same or different conditions. For example, the shell may include multiple layers that rehydrate under different conditions. In one embodiment, the shell may include two or more layers (e.g., three or more layers) that release at different pH levels, for example, one layer may release at pH 5, one layer may release at pH 5.5, one layer may release at pH 6, one layer may release at pH 6.5, one layer may release at pH 7, one layer may release at pH 7.5, and / or one layer may release at pH 8.

[0119] The core may include any number of reagents different from those described herein, or any reagent that may be useful to facilitate the utility of a sequencing system, for example, SBS technology.

[0120] In one embodiment, a biological sample is contacted with the composition. The biological sample may include, for example, whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal discharge, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudate, exudate, cyst fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, fluids containing single or multiple cells, fluids containing cellular organelles, fluidized tissue, fluidized organisms, fluids containing multicellular organisms, biological swabs, and biological washes. Biological samples can include nucleic acids, such as DNA, genomic DNA, RNA, mRNA, or analogs thereof, nucleotides, e.g., deoxyribonucleotides, ribonucleotides, or analogs thereof, e.g., analogs having terminator moieties, such as those described in Bentley et al., "Accurate whole human genome sequencing using reversible terminator chemistry," Nature 456:53-59 (2008), WO 2013 / 131962, U.S. Patent No. 7,057,026, WO 2008 / 042067, WO 2013 / 117595, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, U.S. Patent No. 7,405,281, and U.S. Patent Application Publication No. 20080108082, which are incorporated by reference in their entireties.

[0121] A second aspect relates to a method, comprising providing one or more lyophilized microspheres, and coating the one or more lyophilized microspheres with a shell under conditions effective to encapsulate the one or more lyophilized microspheres.

[0122] This embodiment is carried out according to the previous embodiment, particularly with regard to one or more lyophilized microspheres and their coating, shell, core and encapsulation characteristics.

[0123] Any suitable method can be used to form the microspheres. Standard microsphere manufacturing techniques are known to those skilled in the art and include preparing frozen pellets and placing the pellets in a dryer as described herein. Various microspheres are contemplated by the compositions, systems, and methods of the present disclosure, including, for example, time-sustained release, immediate pulse, timed pulse release, organic acid Diffucaps® Bead, and alkaline buffer Diffucaps® Bead microspheres. There are also various types of encapsulation that are encompassed by the compositions, systems, and methods described herein, including, but not limited to, bulk encapsulation, microencapsulation, nanoencapsulation, single molecule encapsulation, and ionic encapsulation.

[0124] To produce the compositions described herein, modifications to standard microsphere production are used. For example, one or more additional supply buffer tanks and one or more suitable nozzles and / or nozzle plates may be added to standard microsphere production equipment. Other modifications, particularly to the solidification system, can be made to produce various types of shells, including compounds such as hydrocolloids, alginates, and pectins, among others, as described herein.

[0125] In one embodiment, two liquid solutions are prepared: one for the core and one for the shell. A double nozzle system (i.e., a single or multiple nozzle system with an annular gap nozzle) can be installed to allow the initiation of production. Additional factors are important and can be adjusted based on the size and type of composition to be prepared. For example, the interfacial tension, the viscosity of the core and shell, the nozzle diameter ratio of the inner and outer nozzles, and the pressure ratio of the core and shell can all be considered and adjusted.

[0126] In one embodiment, an airbrush is used to generate the encapsulated freeze-dried microspheres. In one embodiment, a filtration membrane may be added to reduce the amount of freeze-dried microspheres that leave the chamber during airbrushing. In another embodiment, an aerosol generator is used.

[0127] According to the compositions, systems, and methods described herein, a liquid may be formed followed by storage at ambient conditions for 1-2 days, and then the microspheres may be spray frozen and stored at -80° C. The lyophilized microspheres may be placed in a tray or tumble dryer, followed by dry dispensing of the microspheres into consumables and / or capsules, and finally heat sealing with foil on the plastic consumables.

[0128] The composition (e.g., encapsulated lyophilized microspheres) may be coated with one or more additional compositions to provide enhanced control of microsphere release. In one embodiment, the method further comprises coating the shell with an outer layer under conditions effective to surround the encapsulated microspheres with the outer layer.

[0129] The composition may be dipped into the wax coating at a specific thickness for a specific time to avoid the composition melting and to eliminate the risk of the coating becoming too thick. In one embodiment, the coating is carried out for a period of time sufficient to provide a specified thickness to the outer layer. In one embodiment, the specified thickness is, for example, about 50 μm. In other embodiments, the specified thickness is less than 50 μm or greater than 50 μm. In one example, a capsule is filled with one or more lyophilized microspheres and the capsule is dipped into hot wax to provide the outer layer. This process allows for the release of the microspheres at two different temperatures, the capsule may melt at about 30° C. to about 50° C., while the wax coating may melt at about 50° C. to about 80° C.

[0130] In one embodiment, the outer layer comprises one or more of carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0131] This aspect can be applied to allow reagent concentration adjustment. For example, a smaller capsule may contain a smaller amount of lyophilized reagent compared to a larger capsule, and multiples of this capsule may be placed in a well according to user requirements. In one embodiment, a unit-based approach is applied, where X capsules = Y runs.

[0132] A third aspect relates to a system comprising one or more compositions described herein and one or more lyophilized cakes, wherein the one or more compositions and the one or more lyophilized cakes are combined under conditions effective to form a rehydrated system.

[0133] This embodiment is carried out according to the previous embodiment, particularly with regard to one or more lyophilized microspheres and their coating, shell, core and encapsulation characteristics.

[0134] In one embodiment, the system further comprises one or more shell layers disposed between the one or more encapsulated microspheres and the one or more lyophilized cakes. In one embodiment, the shell layer comprises a material selected from carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), furalin, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0135] A fourth aspect relates to a method for controlling the release of one or more encapsulated microspheres, the method comprising the steps of providing a composition as described herein and mixing the composition with a rehydration solution under first conditions effective to control the release of one or more lyophilized microspheres from the composition.

[0136] This embodiment is carried out according to the previous embodiment, particularly with regard to one or more lyophilized microspheres and their coating, shell, core and encapsulation characteristics.

[0137] In one embodiment, the method further comprises changing the first condition to a second condition, hi one embodiment, the change in the first condition comprises one or more of changing the temperature, changing the exposure time, changing the pH of the rehydration solution, or changing the position of the encapsulated microspheres in the rehydration solution.

[0138] As described herein, in one embodiment, a capsule is filled with one or more lyophilized microspheres and dipped into a coating (e.g., hot wax) to provide an outer layer. This process allows for controlled release of the microspheres at different temperatures in a migration-free reaction. The capsule allows for release of the microspheres at a temperature of about 30°C to about 50°C, preferably about 30°C to about 40°C, while the coating (e.g., hot wax) allows for release of the microspheres at a temperature of about 50°C to about 90°C, preferably about 55°C to about 85°C. The coating may be of hot wax, preferably paraffin wax. In one embodiment, the temperature in the first condition and / or the second condition is about 10°C to about 90°C, preferably about 30°C to about 50°C, or about 50°C to about 90°C, depending on the composition of the shell and / or the outer coating.

[0139] Similarly, the modification of the first condition can be accomplished by adding one or more additives to the rehydration solution or the composition described herein using the additives described herein. In one embodiment, the core and / or alternatively the rehydration solution further comprises one or more additional agents. The additional agent may be any single additional agent described above or any combination of two or more additional agents described above. For example, an amino acid alone or in combination with another amino acid can be used to modify the first condition to promote controlled release of the microspheres. The content of the one or more additives varies and depends on the composition and reaction conditions (e.g., time, temperature, and pH) used. In one embodiment, the one or more additives are about 0.1 wt% to 40 wt% of the composition and / or rehydration solution. For example, the concentration of one or more additives may be 0.1 wt%, 0.5 wt%, 1.0 wt%, 5.0 wt%, 10.0 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, or any amount therebetween.

[0140] The rehydration (or reconstitution) solution used herein may include water, deionized water, saline, acidic solution, basic solution, detergent solution and / or buffer, and may follow the rehydration solutions described above. In a preferred embodiment, the rehydration solution is water. In one embodiment, the reagents described herein with various concentrations, types of enzymes, and different amounts of cofactors, salts, pH, etc., can be rehydrated with water alone or with atmospheric water capture. Additional additives described herein can be provided in the rehydration solution to further improve the control of the release of the microspheres.

[0141] In one embodiment, the pH of the rehydration solution is about 6.0 to about 10.0. The pH of the rehydration solution can be, for example, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any amount therebetween. The rehydration time varies depending on the content of the composition and the reaction conditions (e.g., reagents, temperature, pH). In one embodiment, the rehydration time can be 0.1 seconds to 10 hours. For example, the rehydration time can be about 0.1 seconds, 1 second, 10 seconds, 30 seconds, 45 seconds, 60 seconds, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 5 hours, 8 hours, 10 hours, or any time therebetween.

[0142] In one embodiment, the first conditions are effective to release the first lyophilized microspheres, which in this embodiment are in accordance with the lyophilized microspheres and compositions described herein.

[0143] In one embodiment, the second conditions are effective to release a second lyophilized microsphere, the content of which is different from the content of the first lyophilized microsphere, the second lyophilized microsphere in this embodiment being in accordance with the lyophilized microspheres and compositions described herein.

[0144] In one embodiment, the first change in conditions is effective to release two or more lyophilized microspheres, the two or more lyophilized microspheres comprising different reagents. In one embodiment, the contents of a first lyophilized microsphere comprises a reagent that is different from the reagent of a second lyophilized microsphere, thereby reducing the temperature sensitivity of the mixed reagent.

[0145] In one embodiment, the method further comprises providing an additional composition according to the compositions described herein, and mixing the additional composition under third conditions effective to control the release of one or more lyophilized microspheres from the additional composition. The lyophilized microspheres in this embodiment are according to the lyophilized microspheres and compositions described herein. In one embodiment, the reagent components in the SBS Cleave Mix are separated into at least two different lyophilized microspheres, thereby preventing or reducing and / or controlling undesirable interactions.

[0146] The "alteration" of the first, second, or third conditions described herein includes any change in one or more conditions in the encapsulated microspheres, or alternatively in the rehydration solution. In one embodiment, the alteration of conditions allows for the sequential release of one or more lyophilized microspheres. One way to allow for the sequential release of lyophilized reagents is by temperature-triggered release, for example, by immersing gelatin capsules filled with microspheres in paraffin wax, as described herein. Such an approach allows for the release of microspheres at different temperatures, for example, from about 30°C to about 50°C for natural gelatin capsules and from about 50°C to about 90°C for coated capsules. Similarly, such an approach allows for time-triggered release by adding additives to the rehydration solution, for example, amino acids. Other reaction characteristics may be altered in addition to or instead of time and / or temperature. For example, pH and humidity may be altered to further control the release of one or more encapsulated microspheres and reagents contained therein.

[0147] In another embodiment, the compositions, systems, and methods described herein protect the polymerase in the first composition (e.g., encapsulated lyophilized microspheres). Protection of the polymerase in the first composition during fully functionalized nucleotide ("ffN") polishing can, in one embodiment, protect light-sensitive ffN from photodegradation.

[0148] The problem of timing the release of a reagent, e.g., a sequencing polymerase, from encapsulation to coincide with the completion of a desired reaction, e.g., a polishing process, can be addressed by adjusting the various components and the relative amounts of those components in the compositions and rehydration solutions described herein. To achieve a finer level of control, additives that are temperature or light responsive can also be used. For example, the problem of using two incompatible competing polymerases (polishing and sequencing) to amend deblocked lyophilized ffN in one well (the uptake mix reagent well) can be addressed by spatially and temporally separating the polymerases using the compositions, systems, and methods of the present disclosure.

[0149] In one example, two capsules may be in a single tube, and the capsules may melt with different triggers. In another example, two or more capsules may be stacked along the y-axis or x-axis in a capillary, and a second capsule is dissolved as the first capsule is released upon contact with liquid, and then a third capsule is dissolved as the second capsule is released upon contact with liquid, and this is repeated for as many capsules as there are in a particular stack. These embodiments may be triggered by temperature changes, such as heat, as well. In yet another example, a tube may contain a cake formed by freeze-drying, wax may be pipetted into the tube, and capsules may be dripped in. A user may first add a liquid that dissolves the capsules, and then as the temperature increases, the wax melts and the cake rehydrates. In another example, a tube may contain a first cake formed by freeze-drying, and then wax is pipetted into the tube. A second liquid is then deposited, freeze-drying is repeated, and then new wax (e.g., with a different melting temperature) is deposited. As the wax melts in turn, the user adds liquid to rehydrate the different cakes.

[0150] In one embodiment, the method further comprises providing one or more lyophilized cakes and rehydrating the one or more lyophilized cakes.

[0151] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail herein (unless such concepts are mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein.

[0152] In this disclosure, reference is made to the accompanying drawings which form a part hereof and which show, by way of illustration, specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the disclosure, and it is to be understood that other embodiments may be utilized and that structural, logical, and electrical changes may be made without departing from the scope of the disclosure. Thus, the following description of exemplary embodiments should not be construed in a limiting sense.

[0153] The present disclosure may be further illustrated by reference to the following examples. EXAMPLES

[0154] The following examples are intended to illustrate, but not to limit the scope of the disclosure, which is set forth in the appended claims.

[0155] Example 1 - Production of encapsulated lyophilized microspheres. Here, encapsulated freeze-dried microspheres are produced.

[0156] Microspheres can be produced using standard techniques. Frozen pellets are first prepared. Liquid flows into a vessel with a nozzle diameter at a liquid flow rate and vibration frequency. Deflection power (electrostatic ring) is used at a given tower temperature and height to produce frozen pellets. Spray towers can be used to prepare microspheres. Trays are placed in a tray dryer and microspheres in bulk form are placed in a dryer, such as a rotary dryer. Microsphere production for the encapsulated freeze-dried microspheres described herein may be built on standard microsphere production techniques or, alternatively, may be modified from standard microsphere production techniques.

[0157] As shown in FIG. 1, there are various types of encapsulation that are encompassed by the compositions, systems, and methods described herein, including, but not limited to, bulk encapsulation, microencapsulation, nanoencapsulation, single molecule encapsulation, and ionic encapsulation.

[0158] One embodiment of the method for encapsulation used according to the present disclosure includes wax coating of the microspheres. An airbrush is used to apply the coating to the microspheres contained in the chamber. The airbrush can generate air currents in the chamber, resulting in some microspheres exiting the chamber. Mitigation includes adding a filter membrane to prevent the microspheres from exiting the chamber or to reduce the likelihood of the microspheres exiting the chamber. An aerosol generator may be used instead of or in addition to the airbrush to reduce air current generation. An aerosol generator may make it more difficult to coat viscous solutions.

[0159] Example 2 - Encapsulated lyophilized microsphere composition. Here, exemplary formulations according to the present compositions, systems, and methods of the present disclosure are shown. The compositions described herein can be prepared for various stages of sequencing, including but not limited to sample extraction, library preparation, enrichment, clustering, and sequencing, as shown in Figure 2.

[0160] In the sample extraction composition, the core may comprise an enzyme, a salt, a detergent, a buffer, and any combination thereof. The sample extraction may be performed at a pH of about 7.5. The reaction volume may be about 1 mL to about 5 mL. In the library preparation composition, the core may comprise an enzyme inhibitor, a salt, a primer, an enzyme, a nucleotide, an organic osmolyte, a magnetic bead, and any combination thereof. The library preparation may be performed at a pH of about 7. The reaction volume may be about 0.05 mL. In the enrichment composition, the core may comprise one or more nucleotides, a molecular probe, an enzyme, a magnetic bead, a crowding agent, and any combination thereof. The enrichment may be performed at a pH of about 8.5. The reaction volume may be about 0.1 mL to about 0.2 mL. In the clustering composition, the core may comprise a salt, an enzyme, a nucleotide, a small molecule, a detergent, a primer, and any combination thereof. The clustering may be performed at a pH of about 8.6. The reaction volume may be about 1 mL. In the sequencing composition, the core may include labeled nucleotides, fluorophores, detergents, salts, enzymes, small molecules, and any combination thereof. Sequencing may be performed at a pH of about 7 to about 10. The reaction volume may be about 30 mL to about 100 mL.

[0161] In embodiments of the compositions, systems, and methods described herein, various blends of lyophilization excipients may be added to the core, including, for example, sugars, amino acids, and polymers. Sugars may include trehalose (10-25%), mannitol (1-10%), cyclodextrin (1-10%), dextran (1-10%), sucrose (1-10%), or any combination thereof. Polymers may include polyvinylpyrrolidone (1-10%), polyvinyl alcohol (1-10%), or combinations thereof. Mesoporous silica and / or quaternary amines may be added to reduce affinity for triboelectric charging.

[0162] In embodiments of the compositions, systems, and methods described herein, various combinations of shell components may be added to the shell. Various examples of shell components include carrageenan, shellac, trehalose (20-40%), paraffin wax, gelatin, HPMC, furalin, oxygen scavengers, alginate, chitosan, gelatin, starch film, benzoxaborole-PVA, pectin, polyvinylpyrrolidone (1-5%), polyvinyl alcohol (1-10%), or any combination thereof. In certain embodiments, the materials used are DNA-free so as not to contaminate the reaction and to avoid the use of valuable sequencing resources.

[0163] As shown in FIG. 3, there are various stresses that microspheres are subjected to. During manufacturing, for a period of time (e.g., 2-3 days), the microspheres are exposed to moisture before packaging and further to triboelectric charging during filling and finishing. During shipping and storage, the microspheres are exposed to temperature fluctuations over a period that can reach several months (e.g., 3-6 months). Once the consumable is opened (even if opened for less than 10 minutes), there are various new stresses on the microspheres, including, for example, ambient humidity, which can affect the microspheres within seconds of exposure. Furthermore, manufacturing, shipping and storage all expose the microspheres to additional mechanical stresses, such as shock and vibration. The compositions, systems, and methods described herein (e.g., encapsulation of lyophilized microspheres) mitigate the stresses otherwise experienced by non-lyophilized and non-encapsulated microspheres.

[0164] Additionally, static charge can be a significant risk in dispensing and drying formulations of microspheres. The encapsulation of the microspheres described in the compositions, systems, and methods described herein significantly reduces that risk and greatly improves sequencing stability.

[0165] Similarly, the compositions, systems, and methods described herein provide encapsulated lyophilized microspheres with improved oxygen protection by application of a low oxygen permeable polymer coating (e.g., polyvinyl alcohol, oxygen scavengers in the coating). Similarly, the compositions, systems, and methods described herein provide encapsulated lyophilized microspheres with improved moisture protection by application of an amphiphilic coating (e.g., amino acid and PVP copolymer). The encapsulated lyophilized microspheres described herein may further provide protection from mechanical stress, for example, by preventing or reducing fragmentation during manufacture (e.g., shells with 40% solute content). Such protective coatings increase the mechanical robustness of the microspheres and their contents during manufacture and shipping, and reduce or eliminate shedding of powder from the microspheres.

[0166] The encapsulated lyophilized microspheres described herein can further provide protection from light exposure, since they can protect reagents from light exposure, thereby reducing manufacturing light constraints. Encapsulation can improve sequencing quality, enable one-pot library preparation, and simplify manufacturing. Benefits and applications of encapsulation of lyophilized microspheres by the compositions, systems, and methods described herein. SBS applications of the encapsulated lyophilized microspheres described herein are described in Figures 15A-15C.

[0167] An AOM SBS cleave mix with Pd (550) in the core and THP (552) in the shell is shown in FIG. 15A, where the Pd cleave mix may require separation of Pd and THP to reduce temperature sensitivity of the mixed reagents. ffN / Pol beads with Pol (554) in the core and ffN (556) in the shell are shown in FIG. 15B, where the polymerase may be protected during ffN polishing, if polishing is required. Photoprotection of ffN by ffN (560) in the core and a light-tight shell (558) is shown in FIG. 15C, where the light-sensitive ffN (found in the core (560)) is protected from photodegradation by the light-tight shell (558).

[0168] Example 3 - Sequential release of lyophilized reagents. There is great interest in encapsulating lyophilized microspheres to increase the stability of sequencing reagents and simplify workflow. The present disclosure describes compositions, systems, and methods for encapsulating lyophilized reagents in microspheres, which may enable sequential release of the lyophilized reagents, for example, as shown in Figures 47 and 48. In a first method shown in Figure 47, one or more lyophilized microspheres are provided (602), and the one or more lyophilized microspheres are coated with a shell (604) under conditions effective to encapsulate the one or more lyophilized microspheres. In another method shown in Figure 48, a composition as described herein is provided (702), and the composition is mixed with a rehydration solution under first release conditions effective to control the release of the one or more lyophilized microspheres from the composition (704).

[0169] One method that allows for sequential release of lyophilized reagents is by temperature triggered release, for example by immersing microsphere-filled gelatin capsules in paraffin wax. Such an approach allows for release of microspheres at different temperatures, for example, from about 30°C to about 50°C (e.g., 37°C) for natural gelatin capsules and from about 50°C to about 90°C (e.g., 58°C) for coated capsules. Similarly, such an approach allows for time triggered release by addition of additives to the rehydration solution, for example, amino acids that can slow the rate of rehydration of the cake.

[0170] Here, encapsulated freeze-dried microspheres are used to allow for sequential release of freeze-dried reagents. As shown in Figures 4A-4B, in one embodiment of the present disclosure, gelatin capsules can be loaded with microspheres and the microsphere-loaded gelatin capsules can be immersed in hot paraffin wax for a period of time (or coated with any suitable outer layer). Figure 5 shows temperature controlled release of a migration-free reaction. As shown in Figures 4A-4B and 5, the paraffin wax coating of the gelatin capsule allows for the release of microspheres at different temperatures. Figure 4A shows an OTS gelatin capsule (102) loaded with microspheres and an OTS gelatin capsule (104) loaded with microspheres and quickly dipped into hot wax. Figure 4B shows an OTS gelatin capsule (106) loaded with microspheres melted at 37°C and an OTS gelatin capsule (108) loaded with microspheres melted at 58°C and quickly dipped into hot wax. The gelatin capsules (102) and / or (106) can release the microspheres at about 30° C. to about 50° C. (e.g., 37° C. and / or 50° C.), while the paraffin wax coatings (104) and / or (108) can release the microspheres at about 50° C. to about 90° C. (e.g., 58° C. and / or 85° C.). In this embodiment, the capsule is filled with the microspheres and the capsule is quickly dipped into the hot wax, which should be fast enough so that the capsule does not begin to melt and to limit the thickness of the wax shell.

[0171] Figure 6 shows compatibility testing of gelatin in Nextera Flex tagmentation. Approximately 80 mM gelatin can be tolerated in tagmentation reactions without reducing library yield. Gelatin (used in OTC capsules) was titrated in Nextera Flex reactions (n=3). Flex was performed according to the standard protocol (100 ng input, 5 cycles of amplification). Yields were measured after two-sided SPRI on Qubit. Yields were normalized to a no gelatin control. Results are compared to a no DNA / no gelatin control.

[0172] Figures 7A-7B show time controlled release for a migration-free reaction. Figure 7A shows the percentage of excipients and their respective rehydration times. Figure 7B shows the rehydration of an exemplary composition described herein during drying (202), after 1 minute (204), after 2 minutes (206), after 5 minutes (208), after 10 minutes (210) and after 12 minutes (212). The addition of amino acid excipients allows for delayed release of the lyophilized material. Different amounts of excipients were added at different rehydration times. In one example, as shown in Figures 7A-7B, the addition of 1% leucine and 4% trehalose resulted in a rehydration time of 12 minutes (as shown in (202), (204), (206), (208), (210), and (212)). A variety of other amino acid excipients were tested, including trehalose alone, phenylalanine in combination with trehalose, and isoleucine in combination with trehalose, at various concentrations resulting in different rehydration times. Thus, there are a variety of materials that can be added to the encapsulated lyophilized microspheres prior to rehydration and / or alternatively to the rehydration solution during rehydration to allow for improved control and stability, as well as delayed release of the contents of the microspheres, including amino acids, PVP copolymers, mesoporous silica, ionic liquids, quaternary amines, polyvinyl alcohol, oxygen scavengers, phenylalanine, leucine, isoleucine, polyvinyl alcohol, alginates, chitosan, carrageenan, gelatin, HPMC, paraffin wax, starch film, benzoxaborole-PVA, and pectin. Further examples of additives and tools that can be used to control the release and triggering mechanisms of the encapsulated lyophilized microspheres of the present disclosure include polyvinyl alcohol, alginate, chitosan, gelatin, carrageenan, POD, hydrogels, starch films, benzoxaborole-PVA, capsules, waxes, pectin, metal organic frameworks, CNTs, modified RBCs, polymer matrices, and logic gated photocages. The various additives and tools described herein promote the stability of the encapsulated lyophilized microspheres, allow macroencapsulation to enable multi-run cartridges, and allow microencapsulation to enable simplified workflows, for example with fewer reagent wells.

[0173] Example 4 - Encapsulated lyophilized microspheres for one-pot ligation protocol and controlled release of encapsulated lyophilized microspheres. The encapsulated lyophilized microspheres described herein enable a one-pot ligation protocol as shown in Figures 8A-8C. Steps of the one-pot ligation protocol are illustrated in Figure 8A. Figure 8B shows the time-dependent release of microspheres in a single pot (306) with two different microspheres: (302) corresponding to reagent A in Figure 8A and (304) corresponding to reagent B in Figure 8A. Examples of active reagent volumes, number of microspheres, active reagent composition, encapsulation shell composition, and encapsulation release triggers for reagent A, reagent B, and additional reagents, reagent B' are shown in Figure 8C.

[0174] Mechanical protection, buffer stabilization, charge control, reagent combination (e.g., combination of two or more different reagents in a single microsphere, single well, single pot), and light protection, along with delayed release of encapsulated lyophilized microspheres (e.g., rehydration of a first reagent in a microsphere followed by rehydration of one or more subsequent reagents in the same rehydration solution after a period of time in the same or different microspheres), are achieved through the compositions, systems, and methods described herein. In particular, the controlled timed release of reagents through the use of encapsulated lyophilized microspheres described herein allows for one-pot library preparation. The inhibition of tagmentation by reagents involved in PCR is resolved by encapsulating the PCR reagents and releasing them at a predetermined time. Figures 8A-8C show the presence of encapsulated lyophilized microspheres with different core reagents in a single rehydration solution. Reagent A may be rehydrated in less than one minute, for example, while Reagent B rehydrates after five minutes in this example, thus avoiding undesired reactions between Reagent A and Reagent B, allowing Reagent A and Reagent B to be in a single pot or well, allowing transfer-free reactions. This concept may be used for more than two types of reagents in a single pot or well. For example, Reagent A, Reagent B, and Reagent B' as depicted in Figures 8A-8C, each containing one or more different encapsulated lyophilized microspheres with one or more different reagents, may be combined in a single well or pot and rehydrated in a time-dependent manner. Varying amounts of active reagents, numbers of microspheres, as well as compositions of the reagents themselves, along with varying compositions of the shell, may be used for various periods of time, all in a single pot or well. This discovery allows the various steps in PCR for sequencing applications to all be performed in the same pot or well, uniquely allowing transfer-free reactions.

[0175] The compositions, systems, and methods described herein allow for benefits in addition to those mentioned above. For example, using lyophilized materials and separated lyophilized materials means that additional cofactors for the enzymes, such as magnesium, can be added to the microspheres themselves, rather than having a separate additional rehydration buffer. This can allow all the reagents, of different concentrations, types of enzymes, all of which require or benefit from different amounts of cofactors, salts, pH, etc., to be rehydrated with water alone or with atmospheric water capture. This facilitates a reduction in the amount of plastic used in the sequencing process, as well as the associated carbon footprint, given the reduction in the weight of the reagents when in concentrated and / or lyophilized form.

[0176] Encapsulation methods can be applied to allow an easy way to adjust reagent concentrations. For example, smaller capsules may contain less lyophilized reagent compared to larger capsules, and multiples of these capsules may be placed in the wells according to the user's needs. This facilitates increased user flexibility in terms of throughput, without potential errors that arise in dilution / concentration calculations. A unit-based approach, where X capsules = Y runs, allows this flexibility in a more controlled manner. Another option includes deeper sequencing or longer sequencing (2 x 500) runs, where three times the number of capsules ("3X") can be used, while rapid surface screening tests may use several times fewer capsules ("X").

[0177] Example 5 - Amendment of deblocked lyophilized ffN in one well. As shown in Figures 9A-9D, 10A-10D and 11A-11F, the problem of using two incompatible competing polymerases (polishing polymerase and sequencing polymerase) to modify deblocked lyophilized ffNs in one well (e.g., the incorporation mix reagent well) can be addressed by separating the polymerases spatially and temporally. The problem of separating the two polymerases spatially and temporally can be solved by encapsulating one polymerase (the sequencing polymerase, since this polymerase is used after the polishing polymerase) in a water-soluble, slowly dissolving film (e.g., polyvinyl alcohol). The problem of timing the release of the sequencing polymerase from the capsule to coincide with the completion of the polishing process can be addressed by adjusting the components and their relative amounts in the water-soluble film. To achieve a finer level of control, additives that are temperature or light responsive can also be used.

[0178] Lyophilization of ffNs achieves increased stability compared to their liquid forms, but results in elevated 3'OH levels, increasing prephasing and thereby reducing run quality. Laboratory use of the polishing workflow can be complicated. The polishing mix (containing ffNs, polishing polymerase, polishing oligos, and Mg) is prepared, combined separately, incubated at an elevated temperature of 50°C (to facilitate the polishing reaction) for up to 1 hour, and then added to the remainder of the incorporation mix where the sequencing polymerase is found. This level of complexity for the user and the sequencer means that this workflow is undesirable in its current form, and even less desirable at large scale. A solution with minimal and / or no user touchpoints, as complicated as or less complicated than the current sequencer workflow, is a viable option.

[0179] The compositions, systems, and methods of the present disclosure propose a viable alternative. The only capture reagent well may be dispensed with free "polishing microspheres" (which may include ffN, polishing polymerase, polishing oligo, and magnesium enzyme cofactor). This well may also have sequencing polymerase microspheres present. However, these may be encapsulated in a water-soluble timed dissolving film, as shown in Figures 19A-9D and 10A-10D. This configuration allows for multiple advantages, including, for example, a reduction in well number. Following the current separate preparation of the polishing mix, followed by mixing with the Grand ICM mix, individual wells may be required for the polishing reagents. Utilizing the encapsulation compositions, systems, and methods described herein, it becomes easy to perform multiple sequential reactions in one well, thereby minimizing the number of wells. This also impacts the cartridge footprint, with knock-on benefits regarding environmental impacts, including, for example, plastic use and incinerator waste. The compositions, systems, and methods described herein can be easily scaled while also resulting in reduced fluidics and valving, thereby reducing the complexity and associated costs of the sequencer. When a rehydration buffer, such as water, is dispensed into the wells, the released polishing microspheres rapidly dissolve and the polishing reaction begins to modify the unblocked ffNs. This rehydration buffer also begins to dissolve the water-soluble film that encapsulates the sequencing polymerase, see, e.g., Figures 10A-10D and 11A-11F.

[0180] An all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos is shown, for example, in FIG. 9A. A sequencing polymerase microsphere (404) containing a sequencing polymerase is shown, for example, in FIG. 9B. An encapsulated polymerase microsphere (406) and all-in-one polishing microsphere (402) in a single well (408) is shown in FIG. 9C. The encapsulated polymerase microsphere (406) and all-in-one polishing microsphere (402) can be placed in the single well (408) at elevated temperature and rehydrated with water, as shown in FIG. 9D.

[0181] Further shown in Figure 10A is an all-in-one polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligos. A sequencing polymerase microsphere (404) containing a sequencing polymerase is shown, for example, in Figure 10B. An encapsulated polymerase microsphere (406) (x3 units) and an all-in-one polishing microsphere (402) (x3 units) in a single well (408) are shown in Figure 10C. The same unit dose can be repeated across different reagents to achieve an overall x3 dose, as shown in Figure 10D.

[0182] Further shown in FIG. 11A is an all-in-one polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligos, for example. A sequencing polymerase microsphere (404) may be encapsulated (406) and may contain a sequencing polymerase and may be in a single well alongside the all-in-one polishing microsphere (402), as shown in FIG. 11C. The encapsulated lyophilized microsphere (406) may be rehydrated with water at 50° C., as shown in FIG. 11C. After 1 hour, the all-in-one microsphere (402) begins to dissolve and polishing begins, as shown in FIG. 11D. As shown in FIG. 11E, the all-in-one microsphere (402) dissolves, polishing is completed, and the encapsulated polymerase microsphere (406) dissolves after a delay. The microspheres dissolve completely and the ICM is then ready to use, as shown in FIG. 11F.

[0183] Example 6 - Controlled release considerations and advantages of one or more encapsulated lyophilized microspheres. The pH of SBS buffers is known to change over the course of a sequencing run, and the compositions, systems, and methods described herein use encapsulation of particles that are otherwise responsive to pH changes to stabilize these buffers and improve SBS performance.

[0184] The compositions, systems, and methods described herein have many advantages. For example, encapsulated freeze-dried microspheres provide antistatic protection by neutralizing charge and reducing triboelectric affinity, thereby reducing the handling complexity of metering and manufacturing (e.g., by using mesoporous silica, ionic liquids, quaternary amines). Static charge can be a significant risk for microspheres, as it significantly affects the metering and mixing of dry microsphere powders during manufacturing. Encapsulation can be used to neutralize the charge of microspheres by coating the particles with a neutral material that has low triboelectric affinity.

[0185] The compositions, systems, and methods described herein further improve control of the pH of a solution that may change over time while on an instrument (e.g., an ICM) by developing pH-sensitive microspheres that release when the buffer drops below a certain pH, releasing ions and returning the buffer to the desired pH. Similarly, the compositions, systems, and methods described herein improve control of the external charge of the microspheres to facilitate dispensing, reduce if not prevent stratification in the mixed bulk, and further allow separation of the reagent components SBS Cleave Mix to reduce temperature sensitivity of the mixed reagents and reduce or prevent and / or control undesirable interactions. Similarly, the compositions, systems, and methods described herein provide encapsulated lyophilized microspheres that protect the polymerase during ffN polishing, if polishing is used, and protect the photosensitive ffN from photodegradation, especially if the environmental conditions involved in polishing degrade the enzyme.

[0186] Various applications of the compositions, systems, and methods (e.g., encapsulated freeze-dried microspheres) described herein. In one example, two or more capsules may be adjacent to each other in a single tube, and these capsules may be dissolved by different triggers. In another example, two or more capsules may be stacked along the y-axis or x-axis in a capillary tube, and a second capsule is dissolved when the first capsule is released upon contact with a liquid, and then a third capsule is dissolved when the second capsule is released upon contact with a liquid, and this is repeated for as many capsules as there are in a particular stack. These embodiments may be triggered by temperature changes, such as heat. In yet another example, a tube may contain a cake formed by freeze-drying, wax may be pipetted into the tube, and capsules may be dripped. A user may first add a liquid that dissolves the capsules, and then as the temperature increases, the wax melts and the cake rehydrates. In another example, a tube may contain a first cake formed by freeze-drying, and then wax may be pipetted into the tube. A second liquid is deposited, the freeze-drying is repeated, and then new wax (e.g., with a different melting temperature) is deposited. As the waxes melt in sequence, the user adds liquid to rehydrate the different cakes.

[0187] The manufacturing process for one embodiment of the encapsulated freeze-dried microspheres described herein is detailed in FIG. 12. The liquid formulation is made in bulk, followed by storage at ambient conditions for a period of time, e.g., 1-2 days, and then the microspheres are spray frozen and stored at -80°C. The freeze-dried microspheres are then placed in a tray or rotary dryer, followed by dry dispensing of the microspheres into consumables and / or capsules, and finally heat sealing with foil on the plastic consumables. The capsules may be placed next to each other in a single tube and melted with different triggers, or may be stacked in a capillary tube and melted when the previous capsule is released and comes into contact with liquid, as described herein (this embodiment may be triggered by heat as well).

[0188] The manufacturing and point of use for one embodiment of the encapsulated lyophilized microspheres described herein is shown in Figure 13. In one embodiment, during manufacturing, one or more reagents are liquid dispensed into the consumable, the consumable is freeze-dried, hot wax is dispensed, the wax is cooled to solidify the barrier, and capsules are added to the consumable. A lyophilized cake may be formed by lyophilization, and then the wax may be pipetted into a tube and the capsules may be dropped. In one embodiment, at the point of use, the user adds liquid, which dissolves the capsule and rehydrates the lyophilized microspheres from the capsule, and the temperature is increased to melt the wax and rehydrate the lyophilized cake.

[0189] The manufacturing and point of use for one embodiment of the encapsulated freeze-dried microspheres and cakes described herein are shown in Figures 14A-14B. In one embodiment, during manufacturing, one or more reagents are liquid dispensed into the consumable, the consumable is freeze-dried, hot wax is dispensed, and the wax is cooled to solidify the barrier. This process is repeated for additional layers. A freeze-dried cake may be formed by freeze-drying, then wax may be pipetted into a tube and the capsules may be dropped. A second liquid is deposited and freeze-drying is repeated, followed by a new wax deposit with a different melting temperature. In one embodiment, at the point of use, a user adds liquid, a first temperature melts the first wax and rehydrates the first cake, and a second temperature melts the second wax and rehydrates the second cake. This process is repeated for additional layers.

[0190] Example 7 - Discussion of significant findings of the compositions described herein. Important aspects of this disclosure include four major research and development activities: (1) additive compatibility screening and sequencing for ffN, (2) additive compatibility screening for ExAmp and clustering, (3) incorporation of additives into the lyophilization matrix to mitigate static electricity, and (4) incorporation of additives into the coating of microspheres to mitigate static electricity.

[0191] In this example and in Examples 8-13, we focus on water-soluble additives as examples, but water-insoluble additives can be used as well. These include metal nanoparticles, graphene fillers, carbon nanotubes, and mesoporous silica, which can be incorporated into the freeze-dried or dry reagents described herein. These additives are typically used in solid plastics or polymers for electronic applications.

[0192] In pharmaceutical or biotechnological applications, the use of such additives is constrained by their impact on compatibility with the active ingredient. Dry reagents in the form of lyophilized microspheres contain enzymes, oligonucleotides and ffNs (nucleotides). It is therefore essential that the choice of additives in the reagent microspheres is determined by the compatibility of the additives and their ability to mitigate the static behavior of the dry microspheres. In particular, the lyophilized microspheres described herein benefit from being handled in a dry environment that favors the accumulation of electrostatic charges. Incorporation of such additives in the form of a matrix or coating on the microspheres helps to mitigate static and triboelectric charging during dry filling and blending. Upon rehydration, the additives in the matrix or coating of the lyophilized microspheres are expected to be soluble in aqueous solutions as well. Nevertheless, when an in situ method of separation of the additives exists, i.e., a filtration membrane, the water-insoluble additives mentioned above can be technically applied as well as coating the microspheres.

[0193] In Table 1, the additives were screened based on their solubility in aqueous solutions as well as in organic solvents (ie, isopropanol).

[0194] [Table 1]

[0195] Organic solvent solubility can be beneficial when the additive is applied to lyophilized microspheres as a coating. In addition to solubility in aqueous solutions (i.e., MOPS and ICM buffers), it is important to find that the additive does not change the pH of the buffer used to store or stabilize the active ingredient.

[0196] In this example, water-soluble additives such as Efka® IO 6783, 6786, Tween® 80, Makon® 17R4, lauric acid diethanolamide, etc., may be beneficial as they can be incorporated directly into the matrix of the lyophilized microspheres. Water-insoluble additives such as glycerol trioleate, polyaniline, piperidyl sebacate, vitamin E (tocopherol acetate), Span® 60 can be incorporated into the microspheres using DMSO.

[0197] Example 8 - Screening additive suitability for ffN and sequencing. Additives were spiked into the incorporation mixture (IMX) containing ffN and polymerase to screen their suitability for sequencing performance. Their detrimental effects were monitored by increased phasing, prephasing metrics and error rates, as well as reduced Q30. As shown in Figures 16A-16I, lauric acid diethanolamide, Makon® 17R4 and Efka® IO 6783 may be suitable, as the sequencing results were comparable to the control (non-spiked IMX).

[0198] Promising materials such as Efka® IO 6783 were screened at different concentrations spiked into IMX to gauge the limit of suitability (i.e., 2% w / v).

[0199] Additives such as ethoxylated PEI and Luviquat® FC550, which are reported to be used as antistatic agents, may not be compatible with sequencing. The results of sequencing the additives are summarized in Table 2 below, which can help down-select antistatic additives for SBS microspheres (ffN and polymerase).

[0200] [Table 2]

[0201] In addition to spiking IMX, we also screened the compatibility of additives with ffN (Figures 17A-17F). Two blue ffCs from different sequencing platforms were selected for their temperature sensitivity to deblocking (3'OH generation) and triphosphate hydrolysis (DiP generation). Additives were incubated with ffN aqueous formulations (or rehydrated lyophilized formulations) at 60°C for 1 and 2 days. A significant increase in 3'OH and DiP as well as a decrease in peak area were observed compared to the control. Except for Tris.HCl, promising additives such as Efka® IO 6783 may be compatible with ffN.

[0202] Example 9 - Screening additive suitability for ExAmp and clustering. Similarly, the same additives were incubated with an aqueous ExAmp formulation (TCX V1.0). The activity of DNA recombinase (the sensitive enzyme component of ExAmp) was evaluated in the presence of 0.1% and 1% w / v additives (FIG. 18). Most of the additives produced activity comparable to the control, except for Efka® IO 6786, Luviquat®, and Eudragit®.

[0203] The clustering function of ExAmp was evaluated using the first base incorporation kinetics of cBOT (Figures 19A-19B). The results confirmed the DNA recombinase activity assay. Incompatible additives such as Luviquat® and Eudragit® show low clustering strength. On the other hand, Ekfa IO 6783 was slightly detrimental to DNA recombinase activity and clustering function, especially when ExAmp is staged at higher temperatures (i.e., 40°C for 1 day). Therefore, the concentration of Efka® IO 6783 can benefit from being kept below about 1% w / v in the ExAmp solution (or rehydrated microspheres).

[0204] These additives were also incorporated into the ExAmp as lyophilized cakes. To measure the antistatic properties of the additives, the cakes were powdered and their charge potentials were measured using a Keyence (as a matrix format). In a dry environment (3% relative humidity), the static was more pronounced (Figure 20). It is unexpected that Efka® IO 6783 is effective even at 0.1% w / v in a 20% lyophilized formulation.

[0205] Additionally, Kollidon® VA64, Efka® IO 6786, and Luviquat® are effective at 0.1% and 1% w / v, however, Luviquat® and Efka® IO 6786 were not well compatible with ExAmp.

[0206] At higher relative humidity (40% RH, FIG. 21), the charging potential of the powdered lyophilized cake decreased, indicating little electrostatic and triboelectric charging behavior.

[0207] Due to interest in a larger library of antistatic additives, water insoluble additives were investigated in this ExAmp compatibility assay. To aid in the water solubility of these hydrophobic additives, DMSO was also added at the same concentration as the additives. The lyophilized formulation of TCX V1.0 may contain 7.5% HPBCD, which also aids in the water solubility of the additives.

[0208] Figures 22 and 23 show the charging potential measurements of powdered lyophilized cakes of ExAmp at 3% and 40% relative humidity, respectively. Lauric acid diethanolamide, coumarin 6, tocopherol acetate, bis(4-piperidyl sebacate) and even DMSO were shown to reduce the charging potential of the ExAmp powder.

[0209] The effect of lyophilized formulation excipients on the charge potential of ExAmp powder cake was investigated (Figure 24). Many of these excipients are salts and buffers that are used in lyophilized formulations anyway. HEPES, MOPS and tetraalkylammonium chlorides were found to be effective at both concentrations (0.1% and 1% w / v spiked into a 20% lyophilized formulation) to reduce the charge potential of ExAmp powder. This unexpected finding is useful for the design of lyophilized formulations, especially those using salts / buffers, which are also expected to mitigate the electrostatic and triboelectric charging of the lyophilized microsphere format. Notably, they were also shown to be compatible with DNA recombinase activity in ExAmp.

[0210] Example 10 - Incorporation of additives into lyophilized matrices to mitigate static electricity. To test the antistatic effect of additives in microspheres, these additives were freeze-dried together as a matrix format. Figures 25A-25C show the incorporation of additives into Atto microspheres as well as fluorescein (FSCN) microspheres (both containing 20% ​​trehalose) as a matrix format. The antistatic properties of the additives are evaluated by the adhesion of the microspheres to the container and their charge density is measured by GranuCharge. A low Δq value indicates low triboelectric charging. The matrix format of 1% Efka® IO 6783 minimized triboelectric charging. Figure 25A shows the results of the first set of additives tested (Atto 20%, +1% Efka® IO 6783, +1.5% Tris.HCl, and +1% Tween 20) in terms of both visual results (top) and percent loss (bottom). Figure 25B shows the results of the second set of additives tested (FSCN 20% Ctrl, +1% Efka® IO 6783, +2% Efka® IO 6783, and +1% Efka® IO 6786) in terms of both visual results (top) and percent loss (bottom). Figure 25C shows that Atto and FSCN (both at 20% trehalose) are dry formulated utilizing the antistatic agent Efka® IO 6783 in a matrix format.

[0211] The adhesion of microspheres to the glass and plastic (polypropylene) walls of the container indicates their static and triboelectric behavior. The lyophilized formulation matrix spiked with 1% Efka® IO 6783 appears to exhibit low static and triboelectric charging. Charge density measurements using GranuCharge confirmed the antistatic and triboelectric behavior (low q0 and Δq values) of Efka® IO 6783. Furthermore, both Atto and FSCN microspheres can be blended more uniformly when spiked with Efka® IO 6783.

[0212] The electrostatic and triboelectric charging behavior of ffN microspheres was screened in the presence of additives (as matrix form). Efka® IO 6783, lauric acid diethanolamide and isoleucine appeared to be effective in glass containers. Nevertheless, Efka® IO 6783, Efka® IO 6786 and Makon® 17R4 appeared to be effective in plastic containers. GranuCharge measurements gave the charge density of microspheres containing additives in matrix form (Figures 26A-26C). ffN microspheres containing additives as matrix form are shown in Figures 26A-26C. The antistatic properties of the additives are evaluated by adhesion to the container and measured by GranuCharge. Figure 26A shows the visual results of the first set of additives (ffN+25%T Ctrl, +1% Efka® IO 6783, +1% Efka® IO 6786, +1.5% Tris.HCl, +2% Isoleucine). Figure 26B shows the visual results of the second set of additives (2nd ffN+20%T Ctrl, +0.5% LDA, +1% Makon® 17R4, +1.5% Kollidon® VA64, +2% Kollicoat® Protect). Figure 26C shows the charge density of the various additives tested in Figures 26A and 26B.

[0213] The low charge density from Efka® IO 6783 and isoleucine-containing microspheres confirmed the behavior in glass containers.

[0214] Example 11 - Incorporation of additives into the coating of microspheres to mitigate static electricity. Typically, organic solvents are used in the spray coating process of microspheres, so water-insoluble additives such as magnesium stearate can be applied as a coating on the surface of the microspheres. Figures 27A-27E show that Eudragit® L100 and magnesium stearate coatings reduced the charge density of the microspheres measured by GranuCharge. As shown by the matrix format, adhesion of the coated microspheres onto glass and plastic containers is greatly reduced by the presence of the additive in the coating. A dry blend of the microspheres (core: trehalose and pigment) can be achieved by coating. Nevertheless, magnesium stearate can be a challenge due to its hydrophobicity upon rehydration. If such additives can be separated in situ by centrifugation or filtration membrane, magnesium stearate can be an attractive antistatic additive for microspheres.

[0215] Based on ffN, sequencing, and ExAmp compatibility screening, Efka® IO 6783, Kollidon® VA64, Kollicoat® IR, and PEG were selected as alternative water-soluble coating materials. Figures 28A-28F show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with Kollidon® VA64, Efka® IO 6783, and PEG at different coating levels. The presence of the coating encapsulating the lyophilized microspheres can be observed. Images of multiple (Figure 28A) and single (Figure 28B) microspheres of 800 μm FSCN Wurster-Spray coated with 20% Kollidon VA64 and Efka® IO 6783 and PEG (#6) are shown in Figures 28A and 28B. CryoIon-Mill SEM images of multiple (FIG. 28C) and single (FIG. 28D) microspheres of Rec / BSA coated with 15% Kollidon VA64 and Efka® IO 6783 and PEG (#8) are shown in Figures 28C and 28D. CryoIon-Mill SEM images of multiple (FIG. 28E) and single (FIG. 28F) microspheres of ffN coated with 10% Kollidon VA64 and Efka® IO 6783 and PEG (#11) are shown in Figures 28E and 28F.

[0216] The electrostatic and triboelectric behavior of the coated microspheres was evaluated in comparison with uncoated control microspheres (FIGS. 29A-29B). The uncoated control microspheres showed high triboelectric charging in the container adhesion experiments and GranuCharge measurements, but the presence of the coating appeared to reduce the charge density of the microspheres. In particular, the presence of Efka® IO 6783 reduced the adhesion of the coated microspheres to the glass container. Nevertheless, the coating containing Kollicoat® Protect appeared to show lower triboelectric charging as measured by GranuCharge.

[0217] The same coating formulation (A: Kollidon® VA64 vs. C: Kollicoat® Protect) was applied onto DNA recombinase / BSA microspheres. Different coating levels were investigated in the coating process of these microspheres (Figures 30A-30B). A coating level of 7.5% (weight gain due to coating) did not significantly improve the adhesion of the microspheres onto glass and plastic containers. Nevertheless, at a higher coating level of 15%, the adhesion of the coated microspheres onto plastic containers was greatly reduced. This was evidenced by the lower charge density Δq values ​​from GranuCharge measurements, which indicated a reduction in triboelectric charging. Notably, the Kollicoat® Protect coating appeared to be more effective on glass containers, but both coating materials (Kollidon® VA64 and Kollicoat® Protect) reduced the adhesion of the coated microspheres onto plastic containers when they contained Efka® IO 6783.

[0218] The stability of compositions described herein containing ffN microspheres, 5% Kollidon® VA64 in dry matrix, 5% Makon® 17R4 in dry matrix, 5% Efka® 6783 in dry matrix, 10% Kollicoat® Protect in dry matrix, and 7.5% isoleucine in dry matrix under various moisture and time conditions is shown in Figures 31A-31F.

[0219] The results of measuring the resistance of the microspheres to relative humidity by dynamic vapor sorption are shown in Figures 32A-32F. Isoleucine in the matrix increases the humidity resistance of ffN microspheres. The results of ffN Ctrl (18% T, 2% HCD) are shown in Figure 32A, 2% Kollicoat® Protect matrix (10% dry) are shown in Figure 32B, +1% Efka® IO 6783 matrix (5% dry) are shown in Figure 32C, 1% Kollidon® VA64 matrix (5% dry) are shown in Figure 32D, +1.5% Trizma matrix (7.5% dry) are shown in Figure 32E, and +1.5% isoleucine matrix (7.5% dry) are shown in Figure 32F.

[0220] The results of Kollicoat® Protect and Kollidon® VA64 coatings, which resulted in improved moisture protection of Rec / BSA MS, are shown in Figures 33A-33D. The coatings minimize moisture absorption of Rec / BSA microspheres (Figures 34A-34B). Rec / BSA microspheres were tested using two different coating materials: Kollidon® VA64 (#8) and Kollicoat® Protect (#9). The coating thickness was adjusted by the coating level or how much coating weight was applied on the surface of the microspheres. In this case, coating levels of 7.5% and 15% were demonstrated, which was controlled by the amount of coating material sprayed on the microspheres. Notably, the Kollicoat® Protect coating performed better than Kollidon® VA64 in terms of moisture barrier. These are unexpected findings. The microspheres with a coating level of 15% show an improvement compared to the uncoated control. In particular, they show 10% higher resistance to humidity (by dynamic vapor sorption, DVS), slower moisture absorption at different humidity levels, and less shrinkage and melting of the microspheres with the coating. Microspheres with 7.5% and 15% coating levels containing Kollicoat® Protect perform better than Kollidon® VA64.

[0221] The discovery of the present disclosure has led to a number of unexpected results. For example, additives compatible with ExAmp activity have been identified. Second, additives compatible with ffN stability have been identified. Third, additives compatible with sequencing have been identified. Fourth, additives that exhibit antistatic properties (minimizing triboelectric charging) in dry format as lyophilized microspheres (matrix format) have been identified. Fifth, additives that exhibit antistatic properties (minimizing triboelectric charging) as coated microspheres (blend of excipients as coating formulation) have been identified.

[0222] Example 12 - Shell and Core Additive Considerations. Various shell additives have been found to improve the stability of both the shell and the core of the compositions described herein.

[0223] Water-soluble polymers such as hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone and polyvinyl alcohol are used in film coating of pharmaceutical tablets. These materials are used according to the present disclosure due to their commercial availability. Time-delayed release may be exemplified by HPMC or Methocel coating.

[0224] Cationic and anionic charged polymers are used as film coatings in pharmaceutical coatings for their enteric or enteric protection. Cationic charged polymers are exemplified as aminodimethyl or diethyl methacrylate copolymers, which are commercially known as Eudragit® E and Kollicoat® Smartseal 30D. Anionic charged polymers are exemplified as methacrylic acid copolymers or Eudragit® S / L, where L100-55, L100 and S100 are used for triggered release at pH 5.5, 7.0 and 8.0, respectively. Other anionic polymers are cellulose derivatives, such as carboxymethylcellulose (Akucel), cellulose acetate phthalate (Aquacoat CPD), cellulose acetate butyrate (Eastman CAB). These charged polymers are used according to the present disclosure due to their commercial availability.

[0225] Water-insoluble polymers are typically designed to provide sustained or extended release of drugs. As coating materials, these polymers can be blended with water-soluble polymers such as PEG or PVP, whereby the latter form pores (porogens) to allow the drug to diffuse slowly from the core. These water-insoluble polymers are exemplified as ethyl cellulose (Ethocel), cellulose acetate (Opadry CA), ammonio methacrylate copolymer (Eudragit® RS100 / RL100), and polyvinyl acetate (Kollicoat® SR). These polymers are used according to the present disclosure due to their commercial availability.

[0226] In summary, materials used for controlled release may include materials for immediate release, such as, for example, Opadry AMB(II), Opadry II(TF), AquaPolish, Starch 1500, Methocel E3, E5, Kollidon® VA64, Kollicoat® Protect / IR, and Soluplus. Other materials for controlled release may include sustained release materials, such as Eudragit® RL PO, Eudragit® RS PO, Methocel E15, K4M, Kollidon® SR(30D), Surelease, Ethocel / Klucel, Methocel E15, K4M, Aquacoat, Opadry CA, etc. Other materials for controlled release may include delayed release materials such as Kollicoat® MAE-100, Eudragit® L100, Eudragit® S100, Sureteric, HPMC acetate succinate, or CMC.

[0227] Typically, polymers used in moisture barrier film coatings exhibit low water permeability, which is often exhibited by water-insoluble polymers. Nevertheless, water-soluble polymers such as Kollidon® VA64, Kollicoat® Protect, Eudragit® L100 / S100 have been investigated for their moisture absorption as well. In many cases, high coating thickness / levels (≧20%) or the addition of hydrophobic / water-insoluble additives such as stearic acid help to reduce moisture absorption.

[0228] Further materials that may be used include those listed in Table 3 below. These materials are reviewed based on their chemical and physical properties. Their potential functions for moisture barrier and static mitigation are estimated based on the specifications and chemical properties of commercial products.

[0229] [Table 3]

[0230] Example 13 - Core-shell format: spray solvent and An excipient that is soluble in the rehydration buffer.

[0231] The concept of solubility screening is shown in FIG.

[0232] The results for Kollidon® VA64, Efka®, and Eudragit®, which are soluble in the spray coating solution (15% water / solvent) and buffer, are shown in FIG.

[0233] The results of the polymer solubility and pH screening are listed in Table 4. Polymers such as Kollicoat® IR, Protect, Kollidon® VA64, Methocel and Metolose are preferred due to their solubility in aqueous solutions.

[0234] [Table 4]

[0235] For coating applications, polymers that are not soluble in organic solvents (e.g., IPA) can be formulated as solid dispersions in water / alcohol mixtures. For example, Kollicoat® IR and Methocel are not soluble in solvents and use little water, the parameters of which are listed in Table 5 below.

[0236] Adding IPA to presolubilized Kollicoat® maintains 2.5% polymer in suspension (25% water / IPA). Reducing the water content results in precipitation. Adding IPA to presolubilized HPMC maintains 1.4% HPMC in solution (14% water). Replacing IPA with ethanol (and removing acetone) was found to improve the formulation to maintain the polymer in a stable solid dispersion that is deemed sprayable for coating processes.

[0237] [Table 5]

[0238] Many of the polymers tested did not significantly lower the pH of IMX at 1%, except Eudragit®. Kollidon® V64 and K30 are soluble in both aqueous solutions and IPA at 10% w / v, which is suitable for the associated spray coating process. The other polymers tested are not soluble in IPA. Water / solvent mixtures may be beneficial, whereby more than 15% v / v of water may be used. Most polymers lower the pH of IMX, whereby Eudragit® L100 is more effective and HPMC is less effective (which is preferred) (see Table 6 below).

[0239] [Table 6]

[0240] In the presence of the polymers described herein, there were equivalent 3'OH and diphosphate levels of ffC (as shown in Figures 38, 39, 40A-40C, 41A-41B, 42A-42B, and 43A-43D).

[0241] The results of screening coating materials based on sequencing compatibility are shown in Table 7.

[0242] [Table 7]

[0243] The results of screening the polymers for DNA recombinase activity and clustering performance (cBOT) described herein are shown in Figures 45A-45B and 46A-46C. The quality of the tested ffNs was not substantially affected by the spray coating process: peak areas (Figure 45A) and 3'OH levels (Figure 45B) were comparable to the control, and no diphosphate was detected. Figures 46A-46C show that shell encapsulation was shown to improve moisture barrier and mitigate static electricity (Figures 46A-46C).

[0244] The comparable DNA recombinase and DNA binding protein activities of ExAmp in the presence of polymers described herein after staging are shown in Figures 41A-41B and 42A-42B.

[0245] A summary of polymer coating materials based on ExAmp and clustering capabilities is shown in Table 8.

[0246] [Table 8]

[0247] Example 14 - Improving the stability of SBS reagents by microencapsulation. Microencapsulation to improve the stability of SBS reagents is shown in Figures 43A-43D. Formulations to reduce static and increase moisture barrier are selected down through a screening pipeline.

[0248] Table 9 shows a material summary of the compositions described herein.

[0249] [Table 9]

[0250] Both ffN and DNA recombinase / BSA microspheres were used (approximately 20% w / v) and the target coating level resulted in a weight gain of 10-15%. Coating formulations tested for dry content: Formulation A: PEG (plasticizer), Kollidon R Formulation B: PEG, Kollidot® VA64, Efka® IO 6783, Formulation C: PEG, Kollicoat® Protect, Efka® IO 6783, and Formulation D: PEG, Kollicoat® Protect. Physical characterization, staging, and activity testing of nearly fully encapsulated microspheres are shown in FIG.

[0251] The quality of the ffN was not substantially affected by the spray-coating process: peak areas and 3'OH levels were comparable to the control, and no diphosphate was detected (see Figures 45A-45B).

[0252] Shell encapsulation improves moisture barrier and mitigates static electricity (see Figures 46A-46C). As shown and described in Figures 46A-46C, matrix encapsulation may be limited to static mitigation, whereas core-shell may address both.

[0253] As shown in Figures 49A-49C, for sequential release based on a pH trigger (e.g., pH 5, pH 6, pH 7, pH 8), triple-coated (e.g., with Eudragit® S100 (fluorescein) in shell 1, Eudragit® L100 (light blue) in shell 2, and Eudragit® L100-55 (rhodamine) in shell 3) spheres provided improved results.

[0254] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, and the like, can be made therein without departing from the spirit of the invention, and therefore are deemed to be within the scope of the invention as defined in the following claims.

Claims

1. 1. A composition comprising: A shell surrounding a core, the shell comprises a shell additive, the shell additive comprising a static mitigation material, a moisture barrier material, or a combination thereof; A composition, wherein the core comprises one or more lyophilized microspheres.

2. 10. The composition of claim 1, wherein the shell additive is a static relief material present in an amount up to 40% w / w concentration of the shell.

3. 10. The composition of claim 1, wherein the shell additive is a moisture barrier material present in an amount up to 90% w / w concentration of the shell.

4. 10. The composition of claim 1, wherein the shell additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity promoter, a stearic acid derivative, an oleic acid derivative, a lauric acid derivative, a polyether compound, an amino acid, tocopherol acetate, piperidyl sebacate, a sodium salt, a buffer, a chelating agent, an imidazolium salt, a polyaniline, or any combination thereof.

5. 5. The composition of claim 4, wherein the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof.

6. 5. The composition of claim 4, wherein the amino acids are selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof.

7. 5. The composition of claim 4, wherein the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof.

8. 5. The composition of claim 4, wherein the buffer is Trizma, Tris. HCl, or a combination thereof.

9. 5. The composition of claim 4, wherein the ammonium salt is selected from a tetraalkylammonium chloride, a tris(hydroxyethyl)alkylammonium chloride, or a combination thereof.

10. 5. The composition of claim 4, wherein the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salts, or polyquaterniums, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole), or combinations thereof.

11. 10. The composition of claim 1, wherein the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

12. 2. The composition of claim 1, wherein the core comprises one or more reagents selected from one or more enzymes, salts, surfactants, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof.

13. The composition of claim 12 , wherein the reagent is a polymerase.

14. 2. The composition of claim 1, wherein the core further comprises one or more additional agents, the additional agents being selected from one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, and any combination thereof.

15. 15. The composition of claim 14, wherein when the additional agent comprises one or more sugars, the sugars are selected from trehalose, mannitol, cyclodextrin, dextran, sucrose, or any combination thereof.

16. The composition of claim 14 , wherein the additional agent comprises one or more amino acids having a hydrophobic side chain.

17. A composition comprising: A shell surrounding a core, the core comprises a core additive; The composition, wherein the core additive comprises a static relief material.

18. 20. The composition of claim 17, wherein the core additive is a static relief material present in an amount up to 25% w / w concentration of the core.

19. 18. The composition of claim 17, wherein the core additive is in an amount of about 2% w / w to about 10% w / w of the core.

20. 20. The composition of claim 17, wherein the core additive comprises one or more of a polymer, a copolymer, a block copolymer, a second polyvinyl alcohol (PVA), a conductivity promoter, an ammonium salt, an imidazolium salt, a polyether compound, or any combination thereof.

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