Reagent compositions, methods, cartridges and systems

Abstract

The present disclosure relates to a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition. Further disclosed is a composition comprising a dissolvable first shell and a dissolvable second shell, the dissolvable second shell comprising one or more reagents. Further disclosed is a method of controlling the release of one or more reagents using the compositions described herein. The present disclosure further relates to a cartridge comprising a reagent reservoir comprising the compositions described herein. Further disclosed is a system for controlling the release of one or more reagents comprising the compositions described herein.

Description

[Technical field]

[0001] The present disclosure relates generally to compositions, methods, cartridges, and systems for controlling the release of reagents. [Background technology]

[0002] 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 and library preparation processes that allow for more cost-effective, rapid, and convenient sequencing and nucleic acid detection are desirable.

[0003] Current protocols for SBS techniques routinely use sample preparation processes that convert DNA or RNA into libraries of fragmented templates suitable for sequencing. Sample preparation methods often require multiple steps, material transfers, and expensive equipment to effect fragmentation, and are therefore often difficult, tedious, expensive, and inefficient.

[0004] 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, or in some cases 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 adaptors that include transposon end sequences, as described, for example, in WO 2016 / 130704. Methods of immobilization and amplification prior to sequencing are described, for example, in U.S. Pat. No. 8,053,192. The library of templates 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. Pat. Pub. No. 2005 / 0100900.

[0005] Sequencing can be performed using any suitable sequencing technique, and methods for sequencing 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. SBS techniques generally involve 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 may 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. Pat. Publication No. 2007 / 0166705.

[0006] There are various problems that hinder the efficiency of sample preparation compositions and processes for sequencing. For example, it is difficult to time the dissolution of multiple reagents in a common well. There are problems in distinguishing different reagents in terms of dissolution time. There are also problems with purifying atmospherically captured water for sample and library preparation compositions and processes for sequencing.

[0007] Thus, there is a need for improved sample preparation compositions and processes, in particular sequencing, sample and library preparation reagents with improved stability, and related compositions that demonstrate improved workflow and efficiency of tagmented library generation, thus increasing read enrichment of the resulting libraries and simplifying the workflow.

[0008] The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0009] A first aspect relates to a composition comprising a shell surrounding an inner compartment, the inner compartment comprising one or more reagents, the shell releasing the inner compartment when the shell is exposed to a first release condition, and the inner compartment releasing the one or more reagents when the inner compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0010] In one embodiment, the internal compartment prevents release of one or more reagents when the shell is exposed to a first release condition. In one embodiment, the first release condition occurs before the second release condition. In another embodiment, the second release condition occurs after the first release condition.

[0011] In one embodiment, the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In another embodiment, the second release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In one embodiment, one or both of the first and second release conditions comprises a change in temperature. In another embodiment, the change in temperature is to a temperature above about 25° C. In another embodiment, the change in temperature is to a temperature below about 25° C.

[0012] In one embodiment, the shell releases the internal compartment when the shell is exposed to at least one additional shell release condition, one or more of the at least one additional shell release conditions being different from the first release condition. In one embodiment, the internal compartment prevents release of the one or more reagents when the shell is exposed to the at least one additional shell release condition. In another embodiment, the internal compartment releases one or more reagents when the internal compartment is exposed to at least one additional internal compartment release condition, one or more of the at least one additional internal compartment release condition being different from the second release condition.

[0013] In one embodiment, the shell has a shell width and the interior compartment has an interior compartment width, and the shell width is different from the interior compartment width. In one embodiment, the shell width is from about 1 micrometer to about 1,000 micrometers. In another embodiment, the interior compartment width is from about 1 micrometer to about 1,000 micrometers.

[0014] In one embodiment, the shell comprises a water soluble compound, hi one embodiment, the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0015] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof. In one embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof.

[0016] In one embodiment, the composition further comprises a water purification compound, which in one embodiment comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0017] In one embodiment, the inner compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In one embodiment, the one or more reagents are lyophilized. In one embodiment, the inner compartment comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the inner compartment comprises a plurality of microspheres comprising a single reagent.

[0018] A second aspect relates to a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents.

[0019] In one embodiment, the first shell is an outer shell. In one embodiment, the second shell is an inner shell. In one embodiment, the first shell dissolves when the composition is exposed to a first release condition. In one embodiment, the second shell prevents the release of one or more reagents when the composition is exposed to a first release condition. In another embodiment, the second shell dissolves when exposed to a second release condition.

[0020] In one embodiment, the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In another embodiment, the second release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In one embodiment, one or both of the first and second release conditions comprises a change in temperature. In another embodiment, the change in temperature is to a temperature above about 25° C. In yet another embodiment, the change in temperature is to a temperature below about 25° C.

[0021] In one embodiment, the first shell dissolves when the first shell is exposed to at least one additional first shell release condition, one or more of the at least one additional first shell release conditions being different from the first release condition. In another embodiment, the second shell prevents the release of one or more reagents when the second shell is exposed to at least one additional first shell release condition. In one embodiment, the second shell releases one or more reagents when the second shell is exposed to at least one additional second shell release condition, one or more of the at least one additional second shell release condition being different from the second release condition.

[0022] In one embodiment, the first shell has a first shell width and the second shell has a second shell width, the first shell width being different from the second shell width. In another embodiment, the first shell width is from about 1 micrometer to about 1,000 micrometers. In yet another embodiment, the second shell width is from about 1 micrometer to about 1,000 micrometers.

[0023] In one embodiment, the first shell comprises a water soluble compound, hi another embodiment, the first shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0024] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof. In another embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof.

[0025] In one embodiment, the composition further comprises a water purification compound, hi one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0026] In one embodiment, the second shell comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In another embodiment, the one or more reagents are lyophilized. In one embodiment, the second shell comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the second shell comprises a plurality of microspheres comprising a single reagent.

[0027] A third aspect relates to a composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound.

[0028] In one embodiment, the water purification compound is located between the dissolvable first shell and the dissolvable second shell.

[0029] In one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof. In one embodiment, the first shell is an outer shell. In another embodiment, the second shell is an inner shell.

[0030] A fourth aspect relates to a method of controlling the release of one or more reagents, the method comprising providing a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, exposing the composition to a first release condition to release the internal compartment, and exposing the internal compartment to a second release condition to release the one or more reagents, the first release condition being different from the second release condition.

[0031] In one embodiment, the internal compartment prevents release of one or more reagents when the shell is exposed to a first release condition. In one embodiment, the first release condition occurs before the second release condition. In another embodiment, the second release condition occurs after the first release condition.

[0032] In one embodiment, the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In another embodiment, the second release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In one embodiment, one or both of the first and second release conditions comprises a change in temperature. In another embodiment, the change in temperature is to a temperature above about 25° C. In yet another embodiment, the change in temperature is to a temperature below about 25° C.

[0033] In one embodiment, the first release conditions comprise a pH of about 1.0 to about 10.0. In another embodiment, the second release conditions comprise a pH of about 1.0 to about 10.0. In one embodiment, the second release conditions are effective to release a plurality of reagents, the content of at least one reagent being different from the content of at least one other reagent. In one embodiment, exposing the shell to the first release conditions and exposing the interior compartment to the second release conditions occur sequentially.

[0034] In one embodiment, the shell releases the internal compartment when the shell is exposed to at least one additional shell release condition, where one or more of the at least one additional shell release condition is different from the first release condition, hi another embodiment, the internal compartment prevents release of one or more reagents when the shell is exposed to the at least one additional shell release condition.

[0035] In one embodiment, the inner compartment releases one or more reagents when the inner compartment is exposed to at least one additional inner compartment release condition, and one or more of the at least one additional inner compartment release conditions is different from the second release condition. In another embodiment, the shell has a shell width and the inner compartment has an inner compartment width, and the shell width is different from the inner compartment width. In another embodiment, the shell width is from about 1 micrometer to about 1,000 micrometers. In yet another embodiment, the inner compartment width is from about 1 micrometer to about 1,000 micrometers.

[0036] In one embodiment, the shell comprises a water soluble compound, hi one embodiment, the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0037] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof. In one embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof.

[0038] In one embodiment, the method further comprises providing a water purification compound, hi one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0039] In one embodiment, the inner compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In one embodiment, the one or more reagents are lyophilized. In one embodiment, the inner compartment comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the inner compartment comprises a plurality of microspheres comprising a single reagent.

[0040] A fifth aspect relates to a method of controlling the release of one or more reagents, the method comprising providing a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents, exposing the composition to first release conditions to dissolve the first shell, and exposing the composition to second release conditions to release the second shell, the first release conditions being different from the second release conditions.

[0041] In one embodiment, the second shell prevents the release of one or more reagents when the composition is exposed to a first release condition. In one embodiment, the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In one embodiment, the second release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. In one embodiment, one or both of the first and second release conditions comprises a change in temperature. In another embodiment, the change in temperature is to a temperature greater than about 25° C. In yet another embodiment, the change in temperature is to a temperature of about 25° C. or less.

[0042] In one embodiment, the first shell dissolves when the first shell is exposed to at least one additional first shell release condition, one or more of the at least one additional first shell release condition being different from the first release condition. In another embodiment, the second shell prevents the release of one or more reagents when the second shell is exposed to at least one additional first shell release condition. In another embodiment, the second shell releases one or more reagents when the second shell is exposed to at least one additional second shell release condition, one or more of the at least one additional second shell release condition being different from the second release condition. In one embodiment, the first shell has a first shell width and the second shell has a second shell width, the first shell width being different from the second shell width. In another embodiment, the first shell width is from about 1 micrometer to about 1,000 micrometers. In yet another embodiment, the second shell width is from about 1 micrometer to about 1,000 micrometers.

[0043] In one embodiment, the first shell comprises a water soluble compound, hi one embodiment, the first shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0044] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof. In one embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof.

[0045] In one embodiment, the method further comprises providing a water purification compound. In one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated charcoal, or any combination thereof. In one embodiment, the inner shell comprises one or more dry reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In one embodiment, the one or more reagents are lyophilized. In one embodiment, the inner shell comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the inner shell comprises a plurality of microspheres comprising a reagent. In one embodiment, the first shell is the outer shell. In another embodiment, the second shell is the inner shell.

[0046] A sixth aspect relates to a method of controlling the release of one or more reagents, the method comprising providing a composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound, exposing the composition to a first release condition to dissolve the water purification compound, exposing the composition to a second condition to dissolve the first shell, and exposing the composition to a third release condition to dissolve the second shell, the first release condition being different from the second release condition.

[0047] In one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof. In one embodiment, the first shell is an outer shell. In another embodiment, the second shell is an inner shell.

[0048] A seventh aspect relates to a method comprising providing a capsule in a well at a first temperature, providing a liquid having a temperature in the well, increasing the temperature of the liquid to a second temperature, decreasing the temperature of the liquid from the second temperature to a third temperature, and releasing one or more reagents from the capsule.

[0049] In one embodiment, the capsule comprises a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition. In one embodiment, the capsule comprises a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents. In one embodiment, the capsule comprises a composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound. In one embodiment, the second temperature is greater than about 25° C. In one embodiment, the third temperature is less than or equal to about 25° C.

[0050] In one embodiment, the method further comprises providing a water purification compound, hi one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0051] In one embodiment, the capsule comprises a water soluble compound, hi one embodiment, the capsule comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0052] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof. In one embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof.

[0053] In one embodiment, the inner compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In one embodiment, the one or more reagents are lyophilized. In one embodiment, the inner compartment comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the inner compartment comprises a plurality of microspheres comprising a single reagent. In one embodiment, the first temperature is different from the third temperature. In another embodiment, the first temperature is the same as the third temperature.

[0054] An eighth aspect relates to a method comprising dissolving an outer shell of a capsule in a well at a first temperature, where the well contains a liquid and the capsule contains an outer shell, a water purification compound, an inner shell and one or more reagents, and where dissolving the outer shell of the capsule releases the water purification compound, and raising the temperature of the well to a second temperature, dissolving the inner shell, thereby releasing the one or more reagents.

[0055] In one embodiment, dissolving the outer shell of the capsule in the well comprises flowing a liquid into the well. In another embodiment, dissolving the inner shell comprises increasing the pH of the liquid to above 7.0. In another embodiment, dissolving the inner shell comprises lowering the pH of the liquid to below 7.0. In yet another embodiment, the inner shell is dissolved by a second temperature. In another embodiment, the inner shell dissolves after a minimum time. In one embodiment, the minimum time is 5 minutes.

[0056] In one embodiment, the second temperature is greater than about 25° C. In another embodiment, the method further comprises reducing the second temperature to a third temperature. In one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0057] In one embodiment, the shell comprises a water soluble compound, hi one embodiment, the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0058] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof. In one embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof.

[0059] In one embodiment, the inner compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In one embodiment, the one or more reagents are lyophilized. In one embodiment, the inner compartment comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the inner compartment comprises a plurality of microspheres comprising a single reagent.

[0060] A ninth embodiment relates to a cartridge comprising a reagent reservoir, the reagent reservoir comprising a composition, the composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0061] In one embodiment, the cartridge comprises a water purification compound, hi one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0062] In one embodiment, the first release condition is exposure to a liquid.

[0063] In one embodiment, the second release condition is exposure to a temperature greater than about 25°C.

[0064] A tenth embodiment relates to a cartridge comprising a reagent reservoir, the reagent reservoir comprising a composition, the composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents.

[0065] In one embodiment, the cartridge comprises a water purification compound, hi one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0066] In one embodiment, the first release condition is exposure to a liquid. In one embodiment, the second release condition is exposure to a temperature greater than about 25° C. In one embodiment, the first shell is an outer shell. In one embodiment, the second shell is an inner shell.

[0067] An eleventh embodiment relates to a system for controlling the release of one or more reagents, the system comprising a well, a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition, and a liquid.

[0068] In one embodiment, the liquid is present in the well. In another embodiment, the composition is present in the well. In another embodiment, the system further comprises a temperature controller over the well.

[0069] In one embodiment, the system further comprises a water purification compound, hi one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0070] A twelfth aspect relates to a system for controlling the release of one or more reagents. The system comprises a well, a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents, and a liquid.

[0071] In one embodiment, a liquid is present in the well. In one embodiment, the composition is present in the well. In one embodiment, the system further comprises a temperature controller over the well.

[0072] In one embodiment, the system further comprises a water purification compound. In one embodiment, the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof. In one embodiment, the first shell is an outer shell. In one embodiment, the second shell is an inner shell.

[0073] A thirteenth embodiment relates to a method, comprising: flowing a liquid having a temperature into a well, the well comprising a capsule, the capsule comprising a first shell surrounding a water purification compound and a second shell surrounding one or more reagents, the first shell releasing the water purification compound when exposed to a first release condition, the second shell releasing the one or more reagents when exposed to a second release condition, the first release condition being different from the second release condition, the water purification compound substantially or completely decomposing when exposed to a decomposition condition, exposing the first shell to the first release condition, thereby releasing the water purification compound, exposing the water purification compound to the decomposition condition, thereby substantially or completely decomposing the water purification compound, and exposing the second shell to the second release condition, thereby releasing the one or more reagents.

[0074] In one embodiment, the first release condition is exposure to a liquid. In another embodiment, the decomposition condition is an elevated temperature of the liquid. In one embodiment, the elevated temperature is about 25° C. or higher. In one embodiment, the decomposition condition is the same as the second release condition. In one embodiment, flowing a liquid, exposing the first shell to a first release condition, and exposing the water purification compound to a decomposition condition are performed sequentially. In another embodiment, flowing a liquid, exposing the first shell to a first release condition, exposing the water purification compound to a decomposition condition, and exposing the second shell to a second release condition are performed sequentially.

[0075] In accordance with the present disclosure, the compositions, methods, cartridges and systems described herein have numerous advantages.

[0076] The challenge of time-staggering the dissolution of multiple reagent capsules in a common well can be overcome using a sequential release system as described herein. The challenge of distinguishing different reagents in terms of dissolution time can be addressed by using a combination of water-soluble films of different thicknesses, different compositions, and different release triggers to provide the reagent releases described herein. The challenge of purifying atmospherically captured water can be overcome using water purification compounds such as sodium dichloroisocyanurate, which can be integrated into the reagent capsule compositions, methods, cartridges, and systems described herein. [Brief description of the drawings]

[0077] [Figure 1] 1 illustrates water purification compounds in a continuous workflow. Water purification compounds may be placed in the atmospheric capture water tank as ground water purification tablets or as mini-tablets compounded in a reagent capsule, the size of which is proportional to the amount of liquid the capsule will rehydrate. [Diagram 2] FIG. 1 illustrates the manufacture of a reagent capsule with water purification compounds formulated within the reagent capsule. [Diagram 3]1 shows how the size of the water purification compounds corresponds to the rehydration volume or volume of the final reagent mix. The full reagent capsule is shown on the left and the reagent component capsules are shown on the right. [Figure 4] FIG. 1 shows the workflow of encapsulated reagent microspheres with water purifying compounds. [Diagram 5] 1 shows a composition design with a common well configuration. Water purification compounds may be placed in the tank or along with the composition, tablets or capsules. [Figure 6] FIG. 1 shows an embodiment of a composition described herein having a shell surrounding an internal compartment. [Figure 7] FIG. 1 illustrates one embodiment of a composition described herein that comprises three separate compositions: a first shell surrounding an inner compartment, a second shell surrounding a second inner compartment, and a third shell surrounding a third inner compartment. [Figure 8] FIG. 1 depicts a composition described herein under one or more release conditions described herein. [Figure 9] FIG. 1 shows one embodiment of the compositions described herein, particularly one or more reagents in lyophilized microspheres. [Figure 10] FIG. 1 shows one embodiment of the compositions described herein, particularly one or more reagents in lyophilized microspheres. [Figure 11] 1 is a flow chart illustrating one embodiment described herein of a method for controlling the release of one or more reagents. [Figure 12] 1 is a flow chart illustrating one embodiment described herein of a method for controlling the release of one or more reagents. [Figure 13] 1 is a flow chart illustrating one embodiment described herein of a method for controlling the release of one or more reagents. [Figure 14] 1 is a flow chart illustrating one aspect of a method described herein. [Figure 15]1 is a flow chart illustrating one aspect of a method described herein. [Figure 16] 1 is a flow chart illustrating one aspect of a method described herein.

[0078] 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

[0079] A first aspect relates to a composition comprising a shell surrounding an inner compartment, the inner compartment comprising one or more reagents, the shell releasing the inner compartment when the shell is exposed to a first release condition, and the inner compartment releasing the one or more reagents when the inner compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0080] 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".

[0081] 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.

[0082] 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.

[0083] The terms "connected," "contacted," and / or "coupled" encompass 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 (possibly 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.

[0084] 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.

[0085] 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. These terms as used herein 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 applications requiring nucleic acid sequencing. 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 a substrate comprises a cluster of copies of a library polynucleotide described herein.

[0086] 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)).

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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 No. 7,427,673, which is incorporated by reference in its entirety. It should be understood that in some embodiments, nucleotide analogs having a 3' terminator moiety or lacking a 3' hydroxyl (such as dideoxynucleotide analogs) can be used under conditions where the polynucleotide incorporating the nucleotide analog is not further extended. In some embodiments, 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. In one embodiment, the 3'-hydroxy blocking group is a reversible blocking group.

[0091] The term "cluster" refers to a discrete site on a solid support that is composed of a plurality of identical immobilized nucleic acid strands and a plurality of identical immobilized complementary nucleic acid strands. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of the clusters.

[0092] As used herein, the term "different" refers to nucleic acid that has different nucleotide sequences.Two or more nucleic acids can have different nucleotide sequences along their entire length.Alternatively, two or more nucleic acids can have different nucleotide sequences along a substantial portion of their length.For example, two or more nucleic acids can have different target nucleotide sequence portions, but also have the same universal sequence regions.

[0093] As used herein, a "library" is a population of polynucleotides derived from a given source or sample. A library contains a plurality of target polynucleotides.

[0094] Modified nucleotides described herein include those having a purine or pyrimidine base and a sugar moiety having a 3'-hydroxy blocking group. In one embodiment, the modified nucleotide is linked to a detectable label. In one embodiment, the detectable label comprises a fluorophore. 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 itself or incorporated into or associated with a larger molecular structure or conjugate. Additional examples of detectable labels are described in U.S. Patent No. 7,541,444, which is incorporated herein by reference in its entirety.

[0095] The fluorescent label may comprise a compound selected from any known fluorescent chemical species, such as rhodamine or cyanine. The fluorescent label disclosed herein may be attached to any position on the nucleotide base and may optionally comprise a linker. In one embodiment, the modified nucleotide is linked to the detectable label via a cleavable linker. The function of the linker is generally to aid 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.

[0096] The linker may be cleavable, and the cleavage site may be located at a position on the linker that results in a portion of the linker remaining attached to the nucleotide base after cleavage, or the entire linker being removed from the nucleotide base. Exemplary linkers include azide and allyl-containing cleavable moieties, disulfide linkers, acid labile moieties (including dialkoxybenzyl moieties, Sieber linkers, indole moieties, t-butyl Sieber moieties), electrophilically cleavable moieties, nucleophilically cleavable moieties, photocleavable moieties, cleavage under reducing conditions, cleavage under oxidative conditions, cleavage by the use of a safety catch moiety, and cleavage by an elimination mechanism. Examples of such moieties are described in WO 03 / 048387, which is incorporated herein by reference in its entirety.

[0097] The composition may comprise different modified nucleotides linked to different detectable labels. In some embodiments, four different modified nucleotides may be linked to four different detectable labels. Alternatively, four different modified nucleotides may be labeled with two different detectable labels (e.g., for two-channel sequencing by synthesis) or with a single detectable label (e.g., for one-channel sequencing by synthesis).

[0098] 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 has been replaced by a carbon and / or a carbon has been replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that may have a substituted base and / or sugar moiety.

[0099] The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled 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).

[0100] The term substrate (or solid support), as used herein, may include any inert substrate or matrix to which nucleic acids may be attached, such as, for example, glass surfaces, plastic surfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. 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 molecules, such as polynucleotides. The support may include a polyacrylamide hydrogel supported on an inert substrate, such as glass. Molecules (e.g., polynucleotides) may be covalently attached directly to the intermediate material (e.g., hydrogel). The support may include a plurality of particles or beads, each with a different binding analyte.

[0101] 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, N. "Nucleotide Analogs. Synthesis and Biological Function" Angewandte Chemie 97:564 (1980), which is incorporated herein by reference in its 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.

[0102] As used herein, the terms "solid phase" or "surface" are used to mean either a flat surface, such as a glass, silica or plastic microscope slide, or similar flow cell device; a planar array of beads, where one or two primers are bound to the beads and the beads are amplified; or an array of beads on a surface after the beads have been amplified.

[0103] As used herein, "substantially free of" a substance (including, e.g., a crowding agent or a nucleic acid) refers to a composition having less than 10% of the substance, less than 5% of the substance, less than 4% of the substance, less than 3% of the substance, less than 2% of the substance, or less than 1% of the substance.

[0104] As described herein, a "shell" includes a composition that surrounds an internal compartment. The internal compartment described herein includes one or more reagents. As described herein, the shell in the composition releases the internal compartment when the shell is exposed to a first release condition. The internal compartment of the composition described herein releases one or more reagents when the internal compartment is exposed to a second release condition. The internal compartment may, for example, have its own internal compartment shell that surrounds one or more reagents. The first release condition may be different from the second release condition.

[0105] In one embodiment, the shell comprises a water-soluble compound. In one embodiment, the shell comprises a material selected from, for example, one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof. In one embodiment, the shell comprises one or more of polymethacrylate-based copolymers, acrylic polymers, water-soluble polymers, poly(N-isopropylacrylamide), pluronics, waxes, photochromic markers such as azobenzene, spirobenzopyran, gold nanoparticles, polyvinyl alcohol (PVA), poly(lactic-co-glycolic acid) (PLGA), alginate, gellan, poly(disulfide), metal-organic frameworks (MOFs), and any combination thereof. Examples of useful copolymers include those derived from esters of acrylic and methacrylic acid, polyvinyl alcohol-polyethylene glycol graft copolymers, and combinations of polyvinyl acetate phthalate (Phthalavin enteric coating polymer) and a plasticizer.

[0106] The amount of shell material includes, for example, any amount suitable to produce the desired shell result. In one embodiment, the shell material is present in an amount of about 1% to about 100% by weight of the shell. For example, the shell material can be present in about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% by weight of the shell, or any amount therebetween. In one embodiment, the shell material is present in an amount of about 10% to about 90% by weight of the shell, or about 10% to about 80% by weight, or about 10% to about 70% by weight, or about 10% to about 60% by weight, or about 10% to about 50% by weight.

[0107] As used herein, "encapsulate" and "encapsulated" and "encapsulation" include enclosing one or more compositions described herein. As used herein, microencapsulation refers to embedding at least one component, such as an active agent, in at least one other material, such as a shell material. Encapsulation according to the present disclosure includes, but is not limited to, bulk encapsulation, matrix encapsulation, macroencapsulation, microencapsulation, nanoencapsulation, monomolecular and ionic encapsulation.

[0108] According to the present disclosure, the compositions, methods, cartridges and systems described herein have numerous advantages and benefits including, for example, increased reagent stability, 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, methods, cartridges and systems described herein use encapsulation of particles that would otherwise respond to pH changes to stabilize these buffers to improve SBS performance. The compositions, methods, cartridges and systems described herein also use encapsulation to reduce the risk of electrostatic charges that would otherwise cause difficulties in dispensing and dry blending reagents during manufacturing. High electrostatic charges make it more difficult to predict the distribution of lyophilized contents in the wells. For example, high electrostatic charges can cause the well contents not to settle to the bottom of the well, which contributes to the difficulty in obtaining the desired rehydration of the lyophilized contents. The shells described herein may include, for example, biodegradable polymers.

[0109] The first release condition described herein may, in one embodiment, include a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. The first release condition may be based on a particular temperature, pH, duration, or position suitable for dissolving the shell or releasing the inner compartment.

[0110] The second release condition described herein may, in one embodiment, include a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof. The second release condition may be based on a specific temperature, pH, duration, or location suitable for dissolving the internal compartment or releasing one or more reagents. The first release condition and the second release condition are separate and independent of each other.

[0111] "Modifying" any of the conditions described herein (e.g., first release condition or second release condition) includes any change in one or more conditions in the composition or any change in the environment surrounding the composition (e.g., rehydration solution or other surrounding solution). In one embodiment, modifying the conditions allows for sequential release of any compound in the composition or release of one or more reagents in the internal compartment. One way to allow for sequential release of reagents is by temperature-triggered release. Other reaction characteristics may be altered in addition to or instead of temperature, and / or time. For example, pH and humidity may be altered to further control the release of one or more compounds, components and reagents therein. The conditions may be altered any number of times to generate any number of different conditions.

[0112] In one embodiment, an additional composition is provided and mixed under a third condition effective to control the release of one or more reagents from the additional composition. In one embodiment, the reagent components are separated, thereby preventing and controlling undesirable interactions. The third condition or other subsequent condition described herein may, in one embodiment, include a temperature-controlled release condition, a pH-controlled release condition, a time-controlled release condition, a position-controlled release condition, or any combination thereof. The third condition or other subsequent release condition may be based on a particular temperature, pH, duration, or location suitable for dissolving the internal compartment or releasing one or more reagents therein. The third release condition is separate and independent of the first release condition and the second release condition.

[0113] In one embodiment, for example, as shown in FIG. 6 and FIG. 7, the composition includes a shell 100 (e.g., 100a, 100b, 100c, etc.) surrounding an inner compartment 102 (e.g., 102a, 102b, 102c, etc.). The shell 100 may be referred to herein as a first shell or a dissolvable first shell or an outer shell. The inner compartment 102 includes at least one reagent. The inner compartment 102 may also, or alternatively, include one or more water purification compounds. The inner compartment 102 may be referred to herein as a second shell or a dissolvable second shell and may include an inner shell. The shell 100 may, for example, release the inner compartment 102 when the shell 100 is exposed to a first release condition. The inner compartment 102 may, for example, release one or more reagents located inside the inner compartment 102. The present composition may include multiple compositions or may be used in conjunction with one or more additional compositions as described above, including a shell 100 (e.g., 100a, 100b, 100c, etc.) and an internal compartment 102 (e.g., 102a, 102b, 102c) as shown in Figure 7, which may include different reagents, the same reagents, or substantially the same reagents. Furthermore, the shell 100 (e.g., 100a, 100b, 100c, etc.) and an internal compartment 102 (e.g., 102a, 102b, 102c) may respond to different release conditions, the same release conditions, or substantially similar release conditions. The release conditions may be in accordance with those described herein.

[0114] In one example, as shown in FIG. 8, the shell 100 surrounds an internal compartment 102, which contains multiple reagents 104. The multiple reagents may be the same type of reagent or different types of reagents, and may be dry or substantially dry (e.g., lyophilized), as shown in FIGS. 8-10. The composition may release the internal compartment 102 when subjected to a first release condition. The composition may release one or more reagents 104 when subjected to a second release condition. In one embodiment, the first release condition may release the first reagent 104a into the surrounding liquid environment. In another embodiment, the second release condition may release the second reagent 104b, optionally after the first reagent 104a is released.

[0115] As shown in FIG. 9, the reagent 104 may be disposed within or formed as a microsphere, for example. The reagent 104 may contain multiple reagents, which may be the same type of reagent or different types of reagents. FIG. 9 shows an embodiment in which the reagent 104 includes three reagents: reagent 104a is located within the outer layer or shell (also referred to herein as the inner shell) of the reagent 104; reagent 104b is located within the middle layer or shell of the reagent 104; and reagent 104c is located in the core of the reagent 104. Any of the reagents 104a, 104b, and / or 104c, along with any additional reagents, may be organized into concentric rings in the microsphere, or may alternatively be adjacent to one another.

[0116] FIG. 10 shows an embodiment of the reagent 104, where the reagent 104 is a microencapsulated lyophilized microsphere. In such an example, the reagent 104 may contain a single type of reagent or multiple reagent types. The encapsulated lyophilized microspheres described herein may contain one, two, three or more types of reagents. The encapsulated lyophilized microspheres described herein may contain an outer layer or shell 104a (also referred to herein as an inner shell) and a core (e.g., 104b or 104c), as shown in FIGS. 9 and 10, each of which may optionally contain one or more reagents, the same or different. In one embodiment, the core of the microsphere contains a reagent (e.g., as shown as 104b in FIG. 10) and may be located inside the microsphere shell, which may contain the same or different reagent as the core (e.g., as shown as 104a in FIG. 10). The lyophilized microspheres may further comprise a third reagent or any number of additional reagents to make the microspheres useful for the applications described herein. In one embodiment, the core of the microsphere contains a reagent (e.g., as shown as 104c in FIG. 9), which may be located inside the middle layer of the microsphere (e.g., as shown as 104b in FIG. 9), which are both located inside the outer layer or shell of the microsphere (e.g., as shown as 104c in FIG. 9). Each reagent may be different. In one embodiment, the first reagent may be different from the second reagent. In one embodiment, the first reagent may be different from the third reagent. In other embodiments, the second reagent may be different from the third reagent. Alternatively, the reagents in the lyophilized microspheres may be the same or substantially similar. For example, the first reagent may be the same as or substantially similar to the third reagent. The first reagent may in turn be the same as or substantially similar to the second reagent. The second reagent may be the same as or substantially similar to the third reagent.In certain embodiments, the diameter of reagent 104b is between 470 μm and 500 μm, for example, about 484 μm.In certain embodiments, the shell has a thickness of between 4 μm and 5 μm, for example, about 4.6 μm.

[0117] Each reagent in the compositions described herein may respond to different release conditions. In certain embodiments, the first, second and / or third reagents may respond to different release conditions. In certain embodiments, the first and third reagents respond to different release conditions. In certain embodiments, the first and second reagents respond to different release conditions. In other embodiments, the second and third reagents respond to different release conditions. Alternatively, the first, second and / or third reagents may respond to the same release condition or substantially similar release conditions. In certain embodiments, the first and third reagents respond to the same release condition or substantially similar release conditions. In certain embodiments, the first and second reagents respond to the same release condition or substantially similar release conditions. In certain embodiments, the second and third reagents respond to the same release condition or substantially similar release conditions.

[0118] In one embodiment, the shell releases the internal compartment when the shell is exposed to at least one additional shell release condition, one or more of the at least one additional shell release conditions being different from the first release condition. In one embodiment, the internal compartment prevents the release of one or more reagents when the shell is exposed to at least one additional shell release condition. In another embodiment, the internal compartment releases one or more reagents when the internal compartment is exposed to at least one additional internal compartment release condition, one or more of the at least one additional internal compartment release condition being different from the second release condition. The additional shell release condition and internal compartment release condition may be in addition to or instead of the first and second release conditions.

[0119] The compositions, methods, cartridges and systems described herein provide for timed release such that various components and reagents can be released at different times, e.g., continuously or otherwise in a controlled manner. The release rate can be adjustable to allow for controlled release of the composition components and reagents. The release rate can be any suitable period of time. For example, the shell or inner compartment can be released or dissolved over a short period of time, such as within 1 minute (e.g., less than 1 second, 1 second, 10 seconds, 20 seconds, 30 seconds, 45 seconds, 60 seconds, or any period of time in between). Alternatively, the shell or inner compartment can be released or dissolved over an intermediate period of time, such as 1 minute to 30 minutes (e.g., 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or any period of time in between). Alternatively, the shell or inner compartment may be released or dissolved over an extended period of time, such as greater than 30 minutes (e.g., 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, more than 120 minutes, or any period in between). For example, a shell or inner compartment described herein may be released quickly (e.g., in less than 1 minute) at low pH (e.g., between about 2-6), while the same shell may be released slowly (e.g., in about 30 minutes or more) at high pH (e.g., between about 10-14). Similarly, a shell or inner compartment described herein may be released slowly (e.g., in about 30 minutes or more) at low pH (e.g., between about 2-6), while the same shell may be released quickly (e.g., in less than 1 minute) at high pH (e.g., between about 10-14). Similarly, a shell or inner compartment described herein may release quickly (e.g., in less than one minute) at an elevated temperature (e.g., above about 25° C.), while the same shell or inner compartment may release slowly (e.g., in about 30 minutes or more) at a lower temperature (e.g., below about 25° C.). In another example, a shell or inner compartment described herein may release slowly (e.g., in about 30 minutes or more) at an elevated temperature (e.g., above about 25° C.), while the same shell or inner compartment may release quickly (e.g., in less than one minute) at a lower temperature (e.g., below about 25° C.).In one embodiment, one or both of the first and second conditions include a change in temperature. The temperature may be increased, for example, to a temperature above about 25° C. Alternatively, the temperature may be decreased, for example, to a temperature below about 25° C.

[0120] In one embodiment, the inner compartment prevents the release of one or more reagents when the shell is exposed to a first release condition. As described herein, the second release condition (i.e., the inner compartment releases one or more reagents) may be the same as the first release condition (i.e., the shell releases the inner compartment). The shell and the inner compartment, in one embodiment, may release under the same conditions. In such a case, the shell and the inner compartment may release or dissolve at different times. Alternatively, the shell and the inner compartment, in one embodiment, may release under different conditions. In one embodiment, the first release condition occurs before the second release condition. In another embodiment, the second release condition occurs after the first release condition.

[0121] As described herein, preventing the release of one or more reagents when the shell is exposed to a first condition includes preventing release for at least an order of magnitude longer than under a second release condition. As described herein, preventing the release of one or more reagents includes both completely preventing release of the reagents and substantially slowing the release of the reagents (i.e., preventing the release of one or more reagents includes substantially preventing release).

[0122] In one embodiment, the shell has a shell width and the interior compartment has an interior compartment width, and the shell width is different from the interior compartment width. In one embodiment, the shell width is from about 1 micrometer to about 1,000 micrometers. In another embodiment, the interior compartment width is from about 1 micrometer to about 1,000 micrometers. The shell width can be, for example, about 1 micrometer, about 10 micrometer, about 25 micrometer, about 50 micrometer, about 75 micrometer, about 100 micrometer, about 125 micrometer, about 150 micrometer, about 175 micrometer, about 200 micrometer, about 225 micrometer, about 250 micrometer, about 275 micrometer, about 300 micrometer, about 325 micrometer, about 350 micrometer, about 375 micrometer, about 400 micrometer, about 450 micrometer, about 500 micrometer, about 550 micrometer, about 600 micrometer, about 650 micrometer, about 700 micrometer, about 750 micrometer, about 800 micrometer, about 850 micrometer, about 900 micrometer, about 950 micrometer, about 1,000 micrometer, or any amount therebetween. In one embodiment, the shell width is between about 100 micrometers and 1,000 micrometers. In one embodiment, one or both of the shell width or the inner compartment width is greater than 1,000 micrometers. In one embodiment, the shell width is the same as the inner compartment width. As described herein, the "inner compartment" (interchangeably referred to as the "core" or "core region") includes any material within the surrounding shell. The inner compartment according to the present disclosure contains one or more reagents.

[0123] As used herein, the term "reagent" describes an agent or a mixture of two or more agents useful for reacting with, interacting with, diluting or adding to a sample, and can 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.

[0124] In one embodiment, the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or combinations thereof. In one embodiment, the one or more reagents are selected from one or more of enzymes, salts, detergents, buffers, enzyme inhibitors, primers, nucleotides, organic osmolytes, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, or any combination thereof. In some embodiments, the reagents may further or alternatively include lysozyme, proteinase K, random hexamers, transposases (e.g., Tn5), primers (e.g., P5 and P7 adapter sequences), ligases, catalytic enzymes, deoxynucleotide triphosphates, buffers, or divalent cations. The reagents may further or alternatively include, for example, bead-linked transposomes (BLT), Tris pH7, MgCl2, Mg acetate, Mg sulfate, indexed primers, Q5 polymerase, Bst3.0, Tris pH9, dNTPs, NaCl, betaine, or any combination thereof. The 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.

[0125] In one embodiment, the reagent according to the present disclosure is a polymerase. As used herein, the term "polymerase" is intended to be consistent with its use in the art and includes, for example, an enzyme that uses a nucleic acid as a template strand to produce a complementary copy of a nucleic acid molecule. Typically, a DNA polymerase binds to the template strand and then moves downstream along the template strand, sequentially adding nucleotides to the free hydroxyl group at the 3' end of the growing nucleic acid strand. DNA polymerases typically synthesize complementary DNA molecules from DNA templates, and RNA polymerases typically synthesize RNA molecules from DNA templates (transcription). Polymerases can use short RNA or DNA strands (called primers) to initiate strand growth. Some polymerases can displace the strand upstream of the site where they add a base to the strand. Such polymerases can also be said to be strand displacing, which is said to have the activity of removing the complementary strand from the template strand that is read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, the large fragment of Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or sequencing grade T7 exo-polymerase. Some polymerases can degrade the strand in front of them and effectively replace it with the growing strand behind them (5' exonuclease activity). Some polymerases have an activity that can degrade the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0126] 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, and combinations thereof. Polymerases engineered to have specific properties may also be used. In one embodiment, the polymerase may be useful for sequencing ("sequencing polymerase"). In one embodiment, the reagent comprises a polymerase, such as Pol812, 129 DNA polymerase, Taq polymerase, Bsu polymerase, or any combination thereof.

[0127] The primers disclosed herein comprise a nucleic acid molecule that can hybridize to a target sequence of interest. In some embodiments, a primer may function as a substrate onto which nucleotides can be polymerized by a polymerase. However, in some instances, a 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. A primer can comprise any combination of nucleotides or analogs thereof. In one embodiment, a primer is a single-stranded oligonucleotide or polynucleotide.

[0128] Non-limiting examples of nucleic acid molecules that may be included in the above composition include DNA, such as genomic or cDNA; RNA, such as mRNA, sRNA, or rRNA; or a hybrid of DNA and RNA. The composition may further include a labeled nucleotide.

[0129] 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.

[0130] Any surfactant known to those skilled in the art may also be included in the composition, especially if the composition is lyophilized. The surfactant may be polyionic, nonionic or ionic (e.g. cationic or anionic), or zwitterionic. Surfactants described herein include Tween-20, Tween 80, CHAPS, or other detergents such as Brij-L23, Pluronic-F127, or combinations thereof. Examples of suitable surfactants include, but are not limited to, polyacrylate surfactants, silicone surfactants, and / or other commercially available surfactants or detergents. The compositions described herein may include anionic surfactants that contain anionic functional groups at one end, such as sulfate, sulfonate, phosphate, and carboxylate functional groups. The reagent may include a neutral surfactant, for example, polyethelene glycol lauryl ether.

[0131] Sample preparation reagents described herein can include, for example, lysis buffer, proteinase K (PK1), purification beads (PB), resuspension buffer (RSB) and ethanol (EtOH). Library preparation reagents described herein can include, for example, end repair mix, A-tailing mix, ligation mix, unique molecular identifier (UMI), stop ligation buffer, and tag buffer, nicotinamide-adenine dinucleotide (NAD). + , ligase, index, beads, SDS, switching oligo, dNTPs and buffer.

[0132] The composition may further or alternatively include an enzyme inhibitor, a molecular probe, a crowding agent, an organic osmolyte, a cyclodextrin, an adenosine triphosphate (ATP), an ethylenediaminetetraacetic acid (EDTA), a creatine kinase, a creatine phosphate, a palladium, a lipoic acid, a hexaethylene glycol, a trihydroxypropanephosphine, a sodium ascorbate, or any combination thereof. The enzyme inhibitors described herein include any molecule that binds to an enzyme and reduces its activity. The molecular probes described herein include, for example, digoxigenin, 8-anilinonaphthalene-1-sulfonic acid ("ANS"), a porphyrin, a BODIPY, a cyanine, or any combination thereof. The crowding agents described herein include any crowding agents known to those skilled in the art. Examples include, but are not limited to, polyethylene glycol, ficoll, dextran, and serum albumin. In one embodiment, the composition comprises about 5% by weight, about 4% by weight, 5% by weight, about 3% by weight, 5% by weight, about 2% by weight, about 1% by weight, less than about 1% by weight of a crowding agent, for example, about 0.001% by weight, about 0.001% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, less than about 1% by weight, or any amount or range therebetween of an additional compound. In one embodiment, the crowding agent is not present in the composition in a measurable content.

[0133] Those skilled in the art of sequencing technology will recognize that there are additional reagents that may be useful in the compositions, methods, kits, cartridges and systems of the present disclosure that are not explicitly described herein.

[0134] The compositions described herein may further comprise water purification compounds. The water purification compounds described herein include any compounds that can be used to purify water, such as compounds that remove or inactivate undesirable chemicals from water to prepare such water for sequencing applications. The water purification compounds described herein allow the use of atmospheric water capture technology to reduce cartridge size for sequencing applications, as well as reducing environmental impact by reducing or eliminating water transported with or within the cartridge, since water is collected on the device. The water purification compounds described herein solve the water quality problems that would otherwise be associated with atmospheric water capture. Similarly, the water purification compounds described herein may allow the use of other non-purified water sources, such as, for example, municipal water sources, groundwater, and reclaimed or reused water sources.

[0135] For example, the water purification compound, including tablets, may be incorporated into a continuous workflow, which may take multiple forms in one embodiment. For example, in one embodiment, a large single water purification tablet may be used in the tank where the atmospheric capture water is stored. Alternatively, a small water purification tablet containing one or more reagents and sized proportionally to the amount of liquid that the capsule utilizes for rehydration may be incorporated into each composition. In one embodiment, the water purification compound includes sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof. In one embodiment, the water purification compound is present in a location between the shell and the internal compartment. Alternatively, the water purification compound may be on the shell or part of the shell in other embodiments.

[0136] Sodium dichloroisocyanurate (NaDCC) and other water purification compounds may be incompatible with reagents such as those used in sequencing applications or commonly used in sequencing applications. The mechanism of action of NaDCC is to produce hypochlorous acid, which is lethal to microorganisms by inhibiting DNA replication, causing oxidation, causing protein aggregation, and generally causing enzyme / protein inactivation. The compositions, methods, cartridges and systems described herein address this issue by using the delayed release aspect of the design described herein. In one embodiment, water is added to a cartridge containing a reagent with a water purification compound attached, the water purification compound dissolves, water purification occurs via hypochlorous acid release that is harmful to microorganisms, the hypochlorous acid is stopped, a composition containing one or more reagents is released, one or more reagents dissolve, and the reagent mixture is ready for use.

[0137] The compositions, methods, cartridges and systems described herein can include, within an internal compartment, one or more dry reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0138] The compositions and reagents described herein can include dried reagents and can optionally be lyophilized, for example, as lyophilized microspheres. In one embodiment, the composition comprises a cake, bead, or powder. In another embodiment, the composition can be a microsphere, a cake, or a combination thereof.

[0139] When the composition is in the form of a cake or beads (e.g., microspheres), the composition may exhibit mechanical rigidity. As used herein, the "mechanical rigidity" of a bulk composition (e.g., cake or beads) refers to a bulk composition that exhibits a mass loss of up to 5%, more preferably up to 1%, even more preferably up to 0.5%, and most preferably up to 0.1% from the bulk composition after the bulk composition is subjected to mechanical stress, such as vibration or impact stress. Maintaining the mechanical rigidity of the bulk composition helps reduce or prevent loss of lyophilized material during transportation. For example, if the cake or beads lack mechanical stability, incomplete rehydration may occur, resulting in loss of efficiency in sequencing reactions. Incomplete rehydration may be caused by unpredictable position of the lyophilized material, where lyophilized fragments or run-off powder may be located beyond the line of rehydration.

[0140] As used herein, "microspheres" include spherical particles or beads having a diameter of 0.1 μm to 25,000 μm. For example, microspheres can have a diameter of about 0.1 μm, 0.5 μm, 1 μm, 10 μm, 20 μm, 25 μm, 30 μm, 35 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, 10,000 μm, 25,000 μm, or any diameter between about 0.1 μm and about 25,000 μm. In one embodiment, the microsphere has a diameter of about 100 μm to about 1000 μm. In one embodiment, the microsphere has a cross section of about 0.1 mm to about 25 mm. In one embodiment, the microsphere has a cross section of about 0.1 mm to about 1 mm. In one embodiment, the composition has a cross section of greater than about 1 mm. In one embodiment, the composition has a diameter of 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 between about 0.1 mm and about 1,000 mm.

[0141] In one embodiment, the microspheres are spherical, ellipsoidal or toroidal in shape. Microspheres are generally composed of an outer polymer layer and may include one or more of the shell components described herein. Microspheres may include, for example, biodegradable polymers. Microspheres according to the present disclosure include those prepared by conventional techniques known to those of skill 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, for example, a heat-dried oven, or a tray freeze dryer, such as a conventional tray dryer, or a rotary dryer. In the present disclosure, the term "lyophilize" or "lyophilizate" is used as an equivalent term to "lyophilised", "lyophilisate" or "freeze-dried", for example, with respect to the compositions, methods, cartridges and systems described herein. Microencapsulation as described herein includes coating of individual microspheres or particles in one or more powders.

[0142] Macrospheres according to the present disclosure include those prepared by conventional techniques known to those skilled in the art. The compositions, methods, cartridges and systems described herein may include a single microsphere or may include multiple lyophilized microspheres, thereby forming a macrosphere. For example, the compositions described herein may include anywhere between 1 and over 1,000,000 microspheres. In one embodiment, the composition includes 1 microsphere, or less than 25 microspheres, or less than 50 microspheres, or less than 75 microspheres, or less than 100 microspheres or less than 500 microspheres, or any number of microspheres from about 1 to about 1,000,000. In one embodiment, for example, in the macrospheres, the compositions and / or reagents are different. The macroencapsulation described herein includes coating of multiple microspheres or particles in one or more powders. In one embodiment, one or more macrospheres described herein may be coated for timed release.

[0143] In one embodiment, the inner compartment comprises a plurality of microspheres containing a plurality of reagents. In another embodiment, the inner compartment comprises a plurality of microspheres containing one reagent. As described herein, each of the plurality of microspheres can contain a plurality of reagents. Alternatively, the plurality of microspheres can collectively contain a plurality of reagents.

[0144] Lyophilizable formulations can be reconstituted into solutions, suspensions, emulsions, or any other form suitable for administration or use.Lyophilizable formulations are usually first prepared as liquids, 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.The final reconstituted volume of the lyophilized formulation can be less than, greater than, or equal to the total liquid volume before lyophilization.

[0145] The lyophilized formulations can be stored at a wide range of temperatures. The lyophilized formulations can be refrigerated below 25°C, e.g., between 2 and 8°C, or stored at room temperature (e.g., about 25°C). The lyophilized formulations can be stored at about 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or any temperature between 37°C and -80°C. For example, the compositions can be stored at about 15°C to about 37°C, below about 25°C, 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 the visual appearance of the composition and / or cake, and / or by the moisture content. The compositions of the present disclosure can also withstand temperature excursions that may occur during shipping, e.g., up to 70°C. The compositions, methods, cartridges and systems described herein, in one embodiment, exhibit stability when stored for a period of time, e.g., 10 days, 14 days, 20 days, 26 days, 30 days, 60 days, 100 days, 200 days, 300 days, 365 days, or longer, e.g., when stored at a temperature of 37°C.

[0146] 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. In certain circumstances, it may be preferable for the lyophilized compositions described herein to be 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.

[0147] A freeze-dried or lyophilized formulation is usually prepared from a liquid, i.e., from a solution, suspension, emulsion, etc. Thus, the liquid to be freeze-dried or lyophilized may contain all the components desired in the final reconstituted liquid formulation. Alternatively, the liquid to be freeze-dried may contain a single reagent, which then, once freeze-dried, may be dry-formulated with one or more additional freeze-dried reagents, such that these reagents are mixed together upon rehydration to form the reconstituted liquid formulation. Thus, one freeze-dried material may be rehydrated, or two or more freeze-dried materials may be rehydrated together. As a result, upon rehydration or reconstitution, the freeze-dried or lyophilized formulation becomes the desired liquid formulation upon reconstitution.

[0148] In one embodiment, the compositions described herein, when lyophilized, comprise a water content of less than about 10% by weight. For example, the water content may be less than about 9.5% by weight, less than about 9% by weight, less than about 8.5% by weight, less than about 8% by weight, less than about 7.5% by weight, less than about 7% by weight, less than about 6.5% by weight, less than about 6% by weight, less than about 5.5% by weight, less than about 5% by weight of water, less than about 4.5% by weight, less than about 4% by weight, less than about 3.5% by weight, less than about 3% by weight, less than about 2.5% by weight, less than about 2% by weight, less than about 1.5% by weight, less than about 1% by weight, less than about 0.5% by weight, less than about 0.1% by weight of water, or any amount therebetween. In one embodiment, there is no measurable content of water in the lyophilized composition.

[0149] The composition 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 a core region of about 0.1 μL to about 500 μL. For example, the composition 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, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 125 μl, 150 μl, 175 μl, 200 μl, 225 μl, 250 μl, 275 μl, 300 μl, 325 μl, 350 μl, 375 μl, 400 μl, 425 μl, 450 μl, 475 μl, 500 μl, or any volume between about 0.1 μl and about 500 μl. For example, the active reagent volume may be about 10 μL to about 400 μL, about 100 μL to about 500 μL, about 200 μL to about 500 μL, about 300 μL to about 500 μL, about 400 μL to about 500 μL, about 0.1 μL to about 100 μL, or about 0.1 μL to about 500 μL.

[0150] The compositions described herein may include additional reagents in the shell. In one embodiment, the composition includes a reagent or additive in the shell. 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.

[0151] 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), continuous conservative 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, methods, cartridges, and systems described herein can, in one embodiment, improve sequencing quality, enable one-pot library preparation, and simplify manufacture and use. As used herein, the term "one-pot reaction" can also be referred to as "transfer-free reaction."

[0152] The compositions, methods, cartridges and systems described herein can be prepared for various stages of sequencing, including, but not limited to, sample extraction, library preparation, enrichment, clustering and sequencing. The compositions can 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, e.g., SBS technology.

[0153] 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 organelles, mobilized tissue, mobilized organisms, fluids containing multicellular organisms, biological swabs, and biological washes. A biological sample can include nucleic acids, such as DNA, genomic DNA, RNA, mRNA, or analogs thereof, nucleotides, such as deoxyribonucleotides, ribonucleotides, or analogs thereof, such as 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), and WO 2013 / 131962, which are incorporated by reference in their entireties.

[0154] A second aspect relates to a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents.

[0155] This aspect may conform to the aspects already described.

[0156] In one embodiment, the first shell dissolves when the composition is exposed to a first release condition. In one embodiment, the second shell prevents the release of one or more reagents when the composition is exposed to a first release condition. In another embodiment, the second shell dissolves when exposed to a second release condition. In one embodiment, the first shell is an outer shell. In one embodiment, the second shell is an inner shell.

[0157] In one embodiment, the first shell dissolves when the first shell is exposed to at least one additional first shell release condition, one or more of the at least one additional first shell release condition being different from the first release condition. In another embodiment, the second shell prevents the release of one or more reagents when the second shell is exposed to at least one additional first shell release condition. In one embodiment, the second shell releases one or more reagents when the second shell is exposed to at least one additional second shell release condition, one or more of the at least one additional second shell release condition being different from the second release condition. The additional first shell release condition and the additional second shell release condition may be in addition to or instead of the first and second release conditions.

[0158] In one embodiment, the first shell has a first shell width and the second shell has a second shell width, the first shell width being different from the second shell width. In another embodiment, the first shell width is from about 1 micrometer to about 1,000 micrometers. In yet another embodiment, the second shell width is from about 1 micrometer to about 1,000 micrometers.

[0159] In one embodiment, the first shell comprises a water soluble compound, hi another embodiment, the first shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof, as described in the previous aspects.

[0160] In one embodiment, the second shell comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof. In another embodiment, the one or more reagents are lyophilized. In one embodiment, the second shell comprises a plurality of microspheres comprising a plurality of reagents. In another embodiment, the second shell comprises a plurality of microspheres comprising a single reagent.

[0161] A third aspect relates to a composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound.

[0162] This aspect may conform to the aspects already described.

[0163] In one embodiment, the water purification compound is present at a location between a dissolvable first shell and a dissolvable second shell. In one embodiment, the first shell is the outer shell. In one embodiment, the second shell is the inner shell.

[0164] A fourth aspect relates to a method of controlling the release of one or more reagents, the method comprising providing a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, exposing the composition to a first release condition to release the internal compartment, and exposing the internal compartment to a second release condition to release the one or more reagents, the first release condition being different from the second release condition.

[0165] This embodiment may be implemented in accordance with the embodiments already described.

[0166] In one embodiment, the first release condition comprises a pH of about 1.0 to about 10.0. In another embodiment, the second release condition comprises a pH of about 1.0 to about 10.0. For example, the first release condition or the second release condition may comprise a pH of less than 3. Alternatively, the first release condition or the second condition may comprise a pH of greater than 5 or 7 or 8, depending on the materials used. In one embodiment, the second release condition is effective to release a plurality of reagents, the content of at least one reagent being different from the content of at least one other reagent. In one embodiment, exposing the shell to the first release condition and exposing the internal compartment to the second release condition occur sequentially.

[0167] In one embodiment, the pH of the rehydration solution is about 1.0 to about 10.0. The pH of the rehydration solution can be, for example, about 1.0, about 2.0, about 3.0, about 4.0, about 5.0, about 6.0, 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 will vary depending on the composition contents and reaction conditions (e.g., reagents, temperature, pH) as described herein. In one embodiment, the rehydration time can be from 0.1 seconds to 10 hours. For example, the rehydration time may 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 in between.

[0168] The rehydration (or reconstitution) solution used herein may include water, deionized water, saline solution, acidic solution, basic solution, detergent solution and / or buffer solution, and may follow the rehydration solutions described above. In one embodiment, the rehydration solution is water, ethanolamine, or a combination thereof. 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.

[0169] In one embodiment, the method further comprises using the rehydrated composition for sequencing by synthesis process. In another embodiment, the method further comprises exposing the rehydrated composition to a sequencing primer, where incorporation of one or more modified nucleotides in the sequencing primer generates an extended sequencing primer. In another embodiment, the method further comprises applying the rehydrated composition to a solid support comprising a nucleotide cluster, where the nucleotide cluster comprises a target polynucleotide.

[0170] A fifth aspect relates to a method of controlling the release of one or more reagents, the method comprising providing a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents, exposing the composition to first release conditions to dissolve the first shell, and exposing the composition to second release conditions to release the second shell, the first release conditions being different from the second release conditions.

[0171] This embodiment may be implemented in accordance with the embodiments already described.

[0172] A sixth aspect relates to a method of controlling the release of one or more reagents, the method comprising providing a composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound, exposing the composition to a first release condition to dissolve the water purification compound, exposing the composition to a second condition to dissolve the first shell, and exposing the composition to a third release condition to dissolve the second shell, the first release condition being different from the second release condition.

[0173] This embodiment may be implemented in accordance with the embodiments already described.

[0174] In one embodiment, the first shell is an outer shell, hi another embodiment, the second shell is an inner shell.

[0175] A seventh aspect relates to a method comprising providing a capsule in a well at a first temperature, providing a liquid having a temperature in the well, increasing the temperature of the liquid to a second temperature, decreasing the temperature of the liquid from the second temperature to a third temperature, and releasing one or more reagents from the capsule.

[0176] This embodiment may be implemented in accordance with the embodiments already described.

[0177] In one embodiment, the capsule comprises a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0178] In one embodiment, the first temperature is different from the third temperature, hi another embodiment, the first temperature is the same as the third temperature.

[0179] An eighth aspect relates to a method comprising dissolving an outer shell of a capsule in a well at a first temperature, where the well contains a liquid and the capsule contains an outer shell, a water purification compound, an inner shell and one or more reagents, and where dissolving the outer shell of the capsule releases the water purification compound, and raising the temperature of the well to a second temperature, dissolving the inner shell, thereby releasing the one or more reagents.

[0180] This embodiment may be implemented in accordance with the embodiments already described.

[0181] In one embodiment, dissolving the outer shell of the capsule in the well comprises flowing a liquid into the well. In another embodiment, dissolving the inner shell comprises increasing the pH of the liquid to above 7.0. In another embodiment, dissolving the inner shell comprises lowering the pH of the liquid to below 7.0. In yet another embodiment, the inner shell is dissolved by a second temperature. In another embodiment, the inner shell dissolves after a minimum time. In one embodiment, the minimum time is 5 minutes.

[0182] A ninth embodiment relates to a cartridge comprising a reagent reservoir, the reagent reservoir comprising a composition, the composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0183] This aspect may conform to the aspects already described.

[0184] In one embodiment, the first release condition is exposure to a liquid.

[0185] In one embodiment, the second release condition is exposure to a temperature greater than about 25°C.

[0186] Exemplary cartridges and configurations are described, for example, in U.S. Patent No. 8,637,242, which is incorporated by reference in its entirety. Exemplary flow cells are described, for example, in U.S. Patent No. 8,241,573, which is incorporated by reference in its entirety.

[0187] Additionally or alternatively, the cartridge may include separate reservoirs and fluidic systems used to perform the amplification method and to perform the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and also determining the sequence of the nucleic acids include the MiSeq™ platform (Illumina, Inc., San Diego, Calif.) and devices described in U.S. Patent No. 8,951,781, which is incorporated herein by reference in its entirety.

[0188] A tenth embodiment relates to a cartridge comprising a reagent reservoir, the reagent reservoir comprising a composition, the composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents.

[0189] This aspect may be in accordance with the aspects already described. In one embodiment, the first shell is an outer shell. In one embodiment, the second shell is an inner shell.

[0190] An eleventh embodiment relates to a system for controlling the release of one or more reagents, the system comprising a well, a composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition, and a liquid.

[0191] This aspect may conform to the aspects already described.

[0192] The liquid described herein may be present in a well, or alternatively, the composition may be present in a well. In one embodiment, the liquid is present in a well. In another embodiment, the composition is present in a well.

[0193] The system may further include a temperature controller or sensor. The temperature controller may be used to change or adjust the temperature of the system to further control the release of various components of the compositions described herein. For example, the temperature controller may be used to accelerate or slow down the release of the shell or the dissolvable outer shell. Similarly, the temperature controller may be used to accelerate or slow down the release of the inner compartment or the dissolvable inner shell to facilitate or control the release of one or more reagents. In one embodiment, the system includes a temperature controller on the well. For example, the temperature controller may include a resistive heater adjacent to the wall of the well to provide heat to the well. The temperature controller may also include a temperature sensor. The temperature controller may also include circuitry to turn the heater on and off to maintain the well at a specified temperature.

[0194] A twelfth aspect relates to a system for controlling the release of one or more reagents. The system comprises a well, a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents, and a liquid.

[0195] This aspect may conform to the aspects already described.

[0196] In one embodiment, the first shell is an outer shell, hi one embodiment, the second shell is an inner shell.

[0197] A thirteenth embodiment relates to a method, comprising: flowing a liquid having a temperature into a well, the well comprising a capsule, the capsule comprising a first shell surrounding a water purification compound and a second shell surrounding one or more reagents, the first shell releasing the water purification compound when exposed to a first release condition, the second shell releasing the one or more reagents when exposed to a second release condition, the first release condition being different from the second release condition, the water purification compound substantially or completely decomposing when exposed to a decomposition condition, exposing the first shell to the first release condition, thereby releasing the water purification compound, exposing the water purification compound to the decomposition condition, thereby substantially or completely decomposing the water purification compound, and exposing the second shell to the second release condition, thereby releasing the one or more reagents.

[0198] This embodiment may be implemented in accordance with the embodiments already described.

[0199] In one embodiment, the first release condition is exposure to a liquid. In another embodiment, the decomposition condition is an elevated temperature of the liquid. In one embodiment, the elevated temperature is about 25° C. or higher. In one embodiment, the decomposition condition is the same as the second release condition. In one embodiment, flowing a liquid, exposing the first shell to a first release condition, and exposing the water purification compound to a decomposition condition are performed sequentially. In another embodiment, flowing a liquid, exposing the first shell to a first release condition, exposing the water purification compound to a decomposition condition, and exposing the second shell to a second release condition are performed sequentially.

[0200] 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 being 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 being part of the inventive subject matter disclosed herein.

[0201] In this disclosure, reference is made to the accompanying drawings, which form a part hereof, and which show, by way of illustration, specific embodiments that 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 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.

[0202] The present disclosure may be further illustrated by reference to the following examples. EXAMPLES

[0203] The following examples are intended to illustrate, but not to limit the scope of the disclosure, which is set forth in the appended claims.

[0204] Example 1 - Water purification compound for reagents. The use of water-soluble film casings to encapsulate reagents can add value and shorten and simplify the workflow. This can be utilized to further simplify both the workflow and the overall sequencer operation by encapsulating different reagents, e.g., sequencing reagents, in different films with different dissolution times and release triggers. The sequencer / cartridge architecture may move toward just one common well with capsules of all sequencing reagents inside, rather than individual wells for each reagent. A number of approaches can be employed to differentiate the dissolution profiles of the various reagents, including film composition, film thickness, release triggers (pH, light, temperature, time), and capsule design. This sequential release capsule technology can be combined with atmospheric water capture technology to further reduce cartridge size and further reduce the environmental impact in terms of eliminating water (and associated packaging) for global shipping. However, this would mandate addressing potential water quality issues associated with atmospheric water capture. In addition to on-site level management (i.e., by columns, filters), water purification tablets can be incorporated into the continuous workflow. This can take the form of either groundwater purification tablets in the tank where the atmospheric capture water is stored, or mini-tablets incorporated within each reagent capsule, sized proportionally to the amount of liquid the capsule will rehydrate as shown in FIG. 1.

[0205] FIG. 1 illustrates a water purification compound in a continuous workflow according to selected embodiments of the present disclosure. A water purification compound 10 is placed in an atmospheric water tank 11. Reagent capsules 12A-12F are placed in a well 13. Water is added to the well from the atmospheric water tank, as shown at 14. A first reagent capsule dissolves faster than the other reagent capsules, thereby dissolving the first reagent first, as shown at 15. In some embodiments, mixing may be performed. The dissolved first reagent is then aspirated, as shown at 16. More water is added to the well, as shown at 17, dissolving the second reagent capsule. The second capsule may dissolve in water more slowly than the first reagent capsule, or may dissolve under different release conditions, such as upon exposure to light. Once the second capsule is completely dissolved, it may be aspirated, as shown at 18. Subsequent reagent capsules may be dissolved and aspirated, as shown at 19, for example, upon exposing the other reagent capsules to different release conditions.

[0206] Light may be used as at least one of the release conditions. For example, exposure of the capsule to light may cause dissolution of the capsule (or the shell of the capsule). In some embodiments, the release condition may be any or all wavelengths of light. In other embodiments, the release condition may be a specific wavelength or range of wavelengths. For example, a first release condition may include exposure to a first wavelength range and a second release condition may include exposure to a second wavelength range, where the first and second wavelength ranges do not overlap.

[0207] A chemical that may be used for purification in the compositions, methods, cartridges and systems described herein is sodium dichloroisocyanurate. It has a melting temperature of 255°C and therefore can be incorporated into the molding / manufacturing of capsules as shown in Figure 2. An empty block mold may be provided (20). A sodium dichloroisocyanurate (NaDCC) tablet is placed in the block mold 21. A first water-soluble film is placed over the block and tablet (22) and then thermoformed (23). A reagent is then filled into the mold on the first water-soluble film 24. Another water-soluble film, which may be the same or different material as the first water-soluble film, is placed over the reagent and thermoformed (25). The resulting reagent capsule can then be removed from the block mold (26).

[0208] The size of the water tablet corresponds to the rehydration volume / volume of the final reagent mixture, as shown in FIG.

[0209] Example 2 - Addressing compatibility of water purification compounds with reagents. However, there are issues regarding the compatibility of sodium dichloroisocyanurate (NaDCC) tablets with the reagent. The mechanism of action of NaDCC is to generate hypochlorous acid. This acid is lethal to microorganisms by inhibiting DNA replication, causing oxidation, causing protein aggregation, and generally causing enzyme / protein inactivation. This presents an obvious incompatibility for the reagent and workflow, given the reliance on enzymes. This can be addressed by fine-tuning the water purification compound (e.g., NaDCC) to reach a point where the microorganisms are killed but the enzymes are unaffected. However, a more attractive approach is to use a delayed release aspect of the capsule design. In at least one such approach, water is added to a cartridge containing a reagent with a water purification tablet (e.g., NaDCC) attached, the water purification occurs by dissolving the water purification tablet and releasing hypochlorous acid to harm the microorganisms, the hypochlorous acid is stopped (e.g., substantially or completely decomposed or inactivated), the capsule containing the reagent is opened, the reagent begins to dissolve, and the reagent mixture is ready for use.

[0210] Hypochlorous acid is sensitive to or decomposed by many things. For example, "HOCl is unstable to ultraviolet (UV) light, sunlight, contact with air, and high temperatures (≧25° C.)." Ishihara et al., "Stability of Weakly Acidic Hypochlorous Acid Solution with Microbicidal Activity," Biocontrol Science 22(4):223-227, abstract (2017), incorporated herein by reference in its entirety. The presence of various organic compounds and inorganic ions causes rapid consumption of HOCl by oxidation reactions. Using pure water and cold water that is free of contaminating compounds such as proteins and carbohydrates, the HOCl solution and ClO -The residual chlorine levels in both solutions are significantly reduced. The retention rate [R] of HOCl levels appears to require formulation with pure water containing organic and inorganic compounds and ions at the lowest possible levels. These weaknesses can be exploited to develop the aforementioned system. The workflow of the present disclosure is illustrated in FIG. 4. A capsule containing the reagent microspheres with water purification tablets is placed in the well 40. Upon rehydration, the tablet begins to dissolve immediately, but the capsule with the reagent microspheres dissolves at a slower rate (41). As the tablet dissolves, the active ingredient hypochlorous acid kills or disables microorganisms in the water 42, 43. Other water purification compounds may also be provided, for example in the tablet, to help remove or neutralize other harmful chemicals or other substances. The hypochlorous acid is stopped by raising the temperature of the water in the well, for example to above 25° C. (44). After the hypochlorous acid is stopped, the capsule (film or shell) is dissolved and the reagent microspheres are released (45). Dissolution of the capsules can occur after a period of time after exposure to water, or alternatively, can dissolve when exposed to another release condition, such as, for example, light, a particular wavelength or range of wavelengths of light, a pH above or below a particular threshold, or a temperature above or below a particular threshold. After the reagent microspheres have been dissolved and homogenized (e.g., by mixing or diffusion), the reagent can be ready for use (46).

[0211] Example 3 - Composition and capsule design. The capsule design can enable workflows such as that shown in Figure 5. Packaging and utilizing sequencing reagents in this manner allows for easy scaling of dosage. Not only can the number of capsules for each be simply increased (i.e., if one capsule is used for one run, then two runs use two capsules), but large capsules can be made for high throughput customers who currently pool many small (e.g., library) prep kits to run individually into a pool large enough for the Hamilton robot to run 96 samples simultaneously.

[0212] The above setup allows for reuse of the same cartridge. A user may manually refill the cartridge by dropping a refill capsule into the common well before starting a run. Alternatively, a robot or machine may dispense the appropriate refill capsule into the common well before starting a run. The capsule design also provides improvements in manufacturing and packaging, where dispensing is easier due to the pre-dosed format.

[0213] An example of a composition described herein is shown in FIG. 6 having a shell 100 and an internal compartment 102. As specified herein, the shell 100 may be referred to herein as a first shell or a dissolvable first shell or an outer shell. The internal compartment 102 includes at least one reagent. As specified herein, the internal compartment 102 may be referred to herein as a second shell or a dissolvable second shell and may include an internal shell. The shell 100 may, for example, release the internal compartment 102 when the shell is exposed to a first release condition. The internal compartment 102 may, for example, release one or more reagents located inside the internal compartment 102. 7, the present compositions may include multiple compositions or may be used in conjunction with one or more additional compositions including shell 100 (e.g., 100a, 100b, 100c, etc.) and inner compartment 102 (e.g., 102a, 102b, 102c) that include different, the same, or substantially the same reagents. Additionally, outer shell 100 (e.g., 100a, 100b, 100c, etc.) and inner compartment 102 (e.g., 102a, 102b, 102c) may each respond to different, the same, or substantially similar release conditions.

[0214] An example of the release of the compositions described herein is shown in FIG. 8. In FIG. 8, a shell 100 surrounds an internal compartment 102, which contains a plurality of reagents 104. The plurality of reagents may be different types of reagents and may be dry or substantially dry (e.g., lyophilized) as shown in FIG. 8. When the composition is placed under a first release condition, it may dissolve the shell 100 and release the internal compartment 102. When the composition is placed under a second release condition, it may dissolve the internal compartment 102 and release one or more reagents 104. The first release condition may release the first reagent into the surrounding liquid environment. For example, after the first reagent is released, the second release condition may release the second reagent, as demonstrated in FIG. 8. In their current non-encapsulated format, it is unlikely that microspheres can be administered on-board due to static effects and the resulting impact on dosing accuracy. The use of a water-soluble film means allows for on-board metering as the microspheres are encapsulated and static effects are minimized. An exemplary form of microsphere useful in the present disclosure may be lyophilized and is shown in Figures 9 and 10.

[0215] 11 is a flow chart illustrating one embodiment described herein of a method for controlling the release of one or more reagents. The method includes providing a composition including a shell surrounding an internal compartment, the internal compartment including one or more reagents (111). The method further includes exposing the composition to a first release condition to release the internal compartment (112). The method further includes exposing the internal compartment to a second release condition to release the one or more reagents, the first release condition being different from the second release condition (113).

[0216] 12 is a flow chart illustrating one embodiment described herein of a method for controlling the release of one or more reagents. The method includes a composition including a dissolvable first shell and a dissolvable second shell that includes one or more reagents (121). The method further includes exposing the composition to first release conditions to dissolve the first shell (122). The method further includes exposing the composition to second release conditions to dissolve the second shell, the first release conditions being different from the second release conditions (123).

[0217] 13 is a flow chart illustrating one embodiment described herein of a method for controlling the release of one or more reagents. The method includes a composition including a dissolvable first shell, a dissolvable second shell including one or more reagents, and a water purification compound (131). The method further includes exposing the composition to a first release condition to release the water purification compound (132). The method further includes exposing the composition to a second condition to dissolve the first shell, and exposing the composition to a third release condition to dissolve the second shell, the first release condition being different from the second release condition (133).

[0218] 14 is a flow chart illustrating one embodiment of a method described herein. The method includes providing a capsule in a well at a first temperature (141). The method further includes providing a liquid having a temperature in the well 142. The method further includes increasing the temperature of the liquid to a second temperature (143). The method further includes decreasing the temperature of the liquid from the second temperature to a third temperature (144). The method further includes releasing one or more reagents from the capsule (145).

[0219] 15 is a flow chart illustrating one embodiment of a method described herein. The method includes dissolving an outer shell of a capsule in a well at a first temperature, where the well contains a liquid, the capsule contains an outer shell, a water purification compound, an inner shell, and one or more reagents, and dissolving the outer shell of the capsule releases the water purification compound (151). The method further includes increasing the temperature of the well to a second temperature (152). The method further includes dissolving the inner shell, thereby releasing the one or more reagents (153).

[0220] FIG. 16 is a flow chart illustrating one embodiment of a method described herein. The method includes flowing a liquid having a temperature into a well, the well including a capsule, the capsule including a first shell surrounding a water purification compound and a second shell surrounding one or more reagents, the first shell releasing the water purification compound when exposed to a first release condition, the second shell releasing the one or more reagents when exposed to a second release condition, the first release condition being different from the second release condition, and the water purification compound substantially or completely decomposing when exposed to a decomposition condition (161). The method further includes exposing the first shell to a first release condition, whereby the water purification compound is released (162). The method further includes exposing the water purification compound to a decomposition condition, whereby the water purification compound is substantially or completely decomposed (163). The method further includes exposing the second shell condition to a second release condition, thereby releasing the one or more reagents (164).

[0221] 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.

[0222] Implementation Various non-limiting embodiments of the present disclosure are described in the following text. [Embodiment A] A composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0223] [Embodiment B] A composition according to embodiment [A] above or any other embodiment of the present disclosure, wherein the inner compartment prevents release of the one or more reagents when the shell is exposed to the first release condition.

[0224] [Embodiment C] A composition according to embodiment [A] or [B] above, or any other embodiment of the present disclosure, wherein the first release condition occurs before the second release condition.

[0225] [Embodiment D] A composition according to any one of embodiments [A]-[C] above, or any other embodiment of the present disclosure, wherein the second release condition occurs after the first release condition.

[0226] [Embodiment E] A composition according to any one of embodiments [A]-[D] above, or any other embodiment of the present disclosure, wherein the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0227] [Embodiment F] A composition according to any one of embodiments [A]-[E] above, or any other embodiment of the present disclosure, wherein the second release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0228] [Embodiment G] A composition according to any one of embodiments [A]-[F] above, or any other embodiment of the present disclosure, wherein either or both of the first and second release conditions include a temperature change.

[0229] [Embodiment H] A composition according to any one of embodiments [A] to [G] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature greater than about 25°C.

[0230] [Embodiment I] A composition according to any one of the above embodiments [A] to [H], or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature of about 25°C or less.

[0231] [Embodiment J] A composition according to any one of embodiments [A]-[I] above, or any other embodiment of the present disclosure, wherein the shell releases the internal compartment when the shell is exposed to at least one additional shell-release condition, and one or more of the at least one additional shell-release condition is different from the first release condition.

[0232] [Embodiment K] A composition according to any one of embodiments [A]-[J] above, or any other embodiment of the present disclosure, wherein the internal compartment prevents release of the one or more reagents when the shell is exposed to at least one additional shell-release condition.

[0233] [Embodiment L] A composition according to any one of embodiments [A]-[K] above, or any other embodiment of the present disclosure, wherein the inner compartment releases the one or more reagents when the inner compartment is exposed to at least one additional inner compartment release condition, and one or more of the at least one additional inner compartment release condition is different from the second release condition.

[0234] [Embodiment M] A composition according to any one of embodiments [A]-[L] above, or any other embodiment of the present disclosure, wherein the shell has a shell width and the inner compartment has an inner compartment width, and the shell width is different from the inner compartment width.

[0235] [Embodiment N] A composition according to any one of embodiments [A] to [M] above, or any other embodiment of the present disclosure, wherein the shell width is from about 1 micrometer to about 1,000 micrometers.

[0236] [Embodiment O] A composition according to any one of embodiments [A] to [N] above, or any other embodiment of the present disclosure, wherein the internal compartment width is from about 1 micrometer to about 1,000 micrometers.

[0237] [Embodiment P] A composition according to any one of embodiments [A]-[O] above, or any other embodiment of the present disclosure, wherein the shell comprises a water-soluble compound.

[0238] [Embodiment Q] A composition according to any one of embodiments [A]-[P] above, or any other embodiment of the present disclosure, wherein the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0239] [Embodiment R] A composition according to any one of embodiments [A] to [Q] above, or any other embodiment of the present disclosure, wherein the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof.

[0240] [Embodiment S] A composition according to any one of embodiments [A] to [R] above, or any other embodiment of the present disclosure, wherein the one or more reagents are selected from one or more of an enzyme, a salt, a surfactant, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof.

[0241] [Embodiment T] A composition according to any one of embodiments [A]-[S] above, or any other embodiment of the present disclosure, further comprising a water purification compound.

[0242] [Embodiment U] A composition according to any one of embodiments [A]-[T] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0243] [Embodiment V] A composition according to any one of embodiments [A]-[U] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0244] [Embodiment W] A composition according to any one of embodiments [A] to [V] above, or any other embodiment of the present disclosure, wherein the one or more reagents are lyophilized.

[0245] [Embodiment X] A composition according to any one of embodiments [A]-[W] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing a plurality of reagents.

[0246] [Embodiment Y] A composition according to any one of embodiments [A] to [X] above, or any other embodiment of the present disclosure, wherein the inner compartment comprises a plurality of microspheres containing one type of reagent.

[0247] [Embodiment Z] A composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents.

[0248] [Embodiment AA] A composition according to embodiment [Z] above, or any other embodiment of the present disclosure, wherein the first shell is an outer shell.

[0249] [Embodiment AB] A composition according to embodiment [Z] or [AA] above, or any other embodiment of the present disclosure, wherein the second shell is an inner shell.

[0250] [Embodiment AC] A composition according to any one of embodiments [Z]-[AB] above, or any other embodiment of the present disclosure, wherein the first shell dissolves when the composition is exposed to a first release condition.

[0251] [Embodiment AD] A composition according to any one of embodiments [Z]-[AC] above, or any other embodiment of the present disclosure, wherein the second shell prevents release of the one or more reagents when the composition is exposed to the first release condition.

[0252] [Embodiment AE] A composition according to any one of embodiments [Z]-[AD] above, or any other embodiment of the present disclosure, wherein the second shell dissolves when exposed to second release conditions.

[0253] [Embodiment AF] A composition according to any one of embodiments [Z]-[AE] above, or any other embodiment of the present disclosure, wherein the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0254] [Embodiment AG] A composition according to any one of embodiments [Z]-[AF] above, or any other embodiment of the present disclosure, wherein the second release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0255] [Embodiment AH] A composition according to any one of embodiments [Z]-[AG] above, or any other embodiment of the present disclosure, wherein either or both of the first and second release conditions include a temperature change.

[0256] [Embodiment AI] A composition according to any one of embodiments [Z] to [AH] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature greater than about 25°C.

[0257] [Embodiment AJ] A composition according to any one of embodiments [Z] to [AI] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature of about 25° C. or less.

[0258] [Embodiment AK] A composition according to any one of embodiments [Z]-[AJ] above, or any other embodiment of the present disclosure, wherein the first shell dissolves when the first shell is exposed to at least one additional first shell release condition, and one or more of the at least one additional first shell release condition is different from the first release condition.

[0259] [Embodiment AL] A composition according to any one of embodiments [Z]-[AK] above, or any other embodiment of the present disclosure, wherein the second shell prevents release of the one or more reagents when the second shell is exposed to at least one additional first shell-release condition.

[0260] [Embodiment AM] A composition according to any one of embodiments [Z]-[AL] above, or any other embodiment of the present disclosure, wherein the second shell releases the one or more reagents when the second shell is exposed to at least one additional second shell release condition, and one or more of the at least one additional second shell release condition is different from the second release condition.

[0261] [Embodiment AN] A composition according to any one of embodiments [Z] to [AM] above, or a composition according to any other embodiment of the present disclosure, wherein the first shell has a first shell width and the second shell has a second shell width, and the first shell width is different from the second shell width.

[0262] [Embodiment AO] A composition according to any one of embodiments [Z] to [AN] above, or any other embodiment of the present disclosure, wherein the first shell width is from about 1 micrometer to about 1,000 micrometers.

[0263] [Embodiment AP] A composition according to any one of embodiments [Z] to [AO] above, or any other embodiment of the present disclosure, wherein the second shell width is from about 1 micrometer to about 1,000 micrometers.

[0264] [Embodiment AQ] A composition according to any one of embodiments [Z] to [AP] above, or any other embodiment of the present disclosure, wherein the first shell comprises a water-soluble compound.

[0265] [Embodiment AR] A composition according to any one of embodiments [Z]-[AQ] above, or any other embodiment of the present disclosure, wherein the first shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0266] [Embodiment AS] A composition according to any one of embodiments [Z] to [AR] above, or any other embodiment of the present disclosure, wherein the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or combinations thereof.

[0267] [Embodiment AT] A composition according to any one of embodiments [Z] to [AS] above, or a composition according to any other embodiment of the present disclosure, wherein the one or more reagents are selected from one or more of an enzyme, a salt, a surfactant, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof.

[0268] [Embodiment AU] A composition according to any one of embodiments [Z]-[AT] above, or any other embodiment of the present disclosure, further comprising a water purification compound.

[0269] [Embodiment AV] A composition according to any one of embodiments [Z]-[AU] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0270] [Embodiment AW] A composition according to any one of embodiments [Z]-[AV] above, or any other embodiment of the present disclosure, wherein the second shell comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0271] [Embodiment AX] A composition according to any one of embodiments [Z] to [AW] above, or any other embodiment of the present disclosure, wherein the one or more reagents are lyophilized.

[0272] [Embodiment AY] A composition according to any one of embodiments [Z]-[AX] above, or any other embodiment of the present disclosure, wherein the second shell comprises a plurality of microspheres containing a plurality of reagents.

[0273] [Embodiments AZ] A composition according to any one of the above embodiments [Z] to [AY], or any other embodiment of the present disclosure, wherein the second shell comprises a plurality of microspheres containing one type of reagent.

[0274] [Embodiment BA] A composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound.

[0275] [Embodiment BB] A composition according to embodiment [BA] above, or any other embodiment of the present disclosure, wherein the water purification compound is located between the dissolvable first shell and the dissolvable second shell.

[0276] [Embodiment BC] A composition according to embodiment [BA] or [BB] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0277] [Embodiment BD] A composition according to any one of embodiments [BA]-[BC] above, or any other embodiment of the present disclosure, wherein the first shell is an outer shell.

[0278] [Embodiment BE] A composition according to any one of embodiments [BA]-[BD] above, or any other embodiment of the present disclosure, wherein the second shell is an inner shell.

[0279] [Embodiment BF] A method for controlling the release of one or more reagents, the method comprising: providing a composition comprising a shell surrounding an internal compartment, the internal compartment containing one or more reagents; exposing the composition to a first release condition to release the internal compartment; and exposing the internal compartment to a second release condition to release the one or more reagents, the first release condition being different from the second release condition.

[0280] [Embodiment BG] A method according to embodiment [BF] above or any other embodiment of the present disclosure, wherein the inner compartment prevents release of the one or more reagents when the shell is exposed to the first release condition.

[0281] [Embodiment BH] A method according to embodiment [BF] or [BG] above, or any other embodiment of the present disclosure, wherein the first release condition occurs before the second release condition.

[0282] [Embodiment BI] A method according to any one of embodiments [BF]-[BH], wherein the second release condition occurs after the first release condition.

[0283] [Embodiment BJ] A method according to any one of embodiments [BF]-[BI] above, or any other embodiment of the present disclosure, wherein the first release condition includes a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0284] [Embodiment BK] A method according to any one of embodiments [BF]-[BJ] above, or any other embodiment of the present disclosure, wherein the second release condition includes a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0285] [Embodiment BL] A method according to any one of embodiments [BF]-[BK] above, or any other embodiment of the present disclosure, wherein either or both of the first and second release conditions include a temperature change.

[0286] [Embodiment BM] A ​​method according to any one of embodiments [BF]-[BL] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature greater than about 25°C.

[0287] [Embodiment BN] A method according to any one of embodiments [BF]-[BM] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature of about 25°C or less.

[0288] [Embodiment BO] A method according to any one of embodiments [BF]-[BN] above, or any other embodiment of the present disclosure, wherein the first release condition comprises a pH of about 1.0 to about 10.0.

[0289] [Embodiment BP] A method according to any one of embodiments [BF] to [BO] above, or any other embodiment of the present disclosure, wherein the second release condition comprises a pH of about 1.0 to about 10.0.

[0290] [Embodiment BQ] A method according to any one of embodiments [BF] to [BP], wherein the second release conditions are effective to release multiple reagents and the content of at least one reagent is different from the content of at least one other reagent.

[0291] [Embodiment BR] A method according to any one of embodiments [BF]-[BQ] above, or any other embodiment of the present disclosure, wherein exposing the shell to the first release conditions and exposing the internal compartment to the second release conditions occur sequentially.

[0292] [Embodiment BS] A method according to any one of embodiments [BF]-[BR] above, or any other embodiment of the present disclosure, wherein the shell releases the internal compartment when the shell is exposed to at least one additional shell release condition, and one or more of the at least one additional shell release condition is different from the first release condition.

[0293] [Embodiment BT] A method according to any one of embodiments [BF]-[BS] above, or any other embodiment of the present disclosure, wherein the internal compartment prevents release of the one or more reagents when the shell is exposed to at least one additional shell-release condition.

[0294] [Embodiment BU] A method according to any one of embodiments [BF]-[BT] above, or any other embodiment of the present disclosure, wherein the inner compartment releases the one or more reagents when the inner compartment is exposed to at least one additional inner compartment release condition, and one or more of the at least one additional inner compartment release condition is different from the second release condition.

[0295] [Embodiment BV] A method according to any one of embodiments [BF]-[BU] above, or any other embodiment of the present disclosure, wherein the shell has a shell width and the inner compartment has an inner compartment width, and the shell width is different from the inner compartment width.

[0296] [Embodiment BW] A method according to any one of embodiments [BF] to [BV] above, or any other embodiment of the present disclosure, wherein the shell width is from about 1 micrometer to about 1,000 micrometers.

[0297] [Embodiment BX] A method according to any one of embodiments [BF] to [BW] above, or any other embodiment of the present disclosure, wherein the internal compartment width is from about 1 micrometer to about 1,000 micrometers.

[0298] [Embodiment BY] A method according to any one of embodiments [BF]-[BX] above, or any other embodiment of the present disclosure, wherein the shell comprises a water-soluble compound.

[0299] [Embodiment BZ] The method according to any one of embodiments [BF]-[BY] above, or any other embodiment of the present disclosure, wherein the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0300] [Embodiment CA] A method according to any one of embodiments [BF] to [BZ] above, or any other embodiment of the present disclosure, wherein the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof.

[0301] [Embodiment CB] A method according to any one of embodiments [BF] to [CA] above, or any other embodiment of the present disclosure, wherein the one or more reagents are selected from one or more of an enzyme, a salt, a surfactant, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof.

[0302] [Embodiment CC] A method according to any one of embodiments [BF]-[CB] above, or any other embodiment of the present disclosure, further comprising providing a water purification compound.

[0303] [Embodiment CD] The method according to any one of embodiments [BF]-[CC] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0304] [Embodiment CE] A method according to any one of embodiments [BF]-[CD] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0305] [Embodiment CF] A method according to any one of embodiments [BF]-[CE] above, or any other embodiment of the present disclosure, wherein the one or more reagents are lyophilized.

[0306] [Embodiment CG] A method according to any one of embodiments [BF]-[CF] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing a plurality of reagents.

[0307] [Embodiment CH] A method according to any one of embodiments [BF] to [CG] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing one type of reagent.

[0308] [Embodiment CI] A method for controlling the release of one or more reagents, the method comprising providing a composition comprising a dissolvable first shell and a dissolvable second shell comprising one or more reagents, exposing the composition to first release conditions to dissolve the first shell, and exposing the composition to second release conditions to release the second shell, wherein the first release conditions are different from the second release conditions.

[0309] [Embodiment CJ] A method according to embodiment [CI] above, or any other embodiment of the present disclosure, wherein the second shell prevents release of the one or more reagents when the composition is exposed to the first release conditions.

[0310] [Embodiment CK] A method according to embodiment [CI] or [CJ] above, or any other embodiment of the present disclosure, wherein the first release condition comprises a temperature controlled release condition, a pH controlled release condition, a time controlled release condition, a position controlled release condition, or any combination thereof.

[0311] [Embodiment CL] A method according to any one of embodiments [CI]-[CK] above, or any other embodiment of the present disclosure, wherein the second release condition comprises a temperature-controlled release condition, a pH-controlled release condition, a time-controlled release condition, a position-controlled release condition, or any combination thereof.

[0312] [Embodiment CM] A method according to any one of embodiments [CI]-[CL] above, or any other embodiment of the present disclosure, wherein either or both of the first and second release conditions include a temperature change.

[0313] [Embodiment CN] A method according to any one of embodiments [CI]-[CM] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature greater than about 25°C.

[0314] [Embodiment CO] A method according to any one of embodiments [CI]-[CN] above, or any other embodiment of the present disclosure, wherein the change in temperature is to a temperature of about 25° C. or less.

[0315] [Embodiment CP] A method according to any one of embodiments [CI]-[CO] above, or any other embodiment of the present disclosure, wherein the first shell dissolves when the first shell is exposed to at least one additional first shell release condition, and one or more of the at least one additional first shell release condition is different from the first release condition.

[0316] [Embodiment CQ] A method according to any one of embodiments [CI]-[CP] above, or any other embodiment of the present disclosure, wherein the second shell prevents release of the one or more reagents when the second shell is exposed to at least one additional second shell-release condition.

[0317] [Embodiment CR] A method according to any one of embodiments [CI]-[CQ] above, or a method according to other embodiments of the present disclosure, wherein the second shell releases the one or more reagents when the second shell is exposed to at least one additional second shell release condition, and one or more of the at least one additional second shell release condition is different from the second release condition.

[0318] [Embodiment CS] A method according to any one of embodiments [CI]-[CR] above, or any other embodiment of the present disclosure, wherein the first shell has a first shell width and the second shell has a second shell width, and the first shell width is different from the second shell width.

[0319] [Embodiment CT] A method according to any one of embodiments [CI] to [CS] above, or any other embodiment of the present disclosure, wherein the first shell width is from about 1 micrometer to about 1,000 micrometers.

[0320] [Embodiment CU] A method according to any one of embodiments [CI] to [CT] above, or any other embodiment of the present disclosure, wherein the second shell width is from about 1 micrometer to about 1,000 micrometers.

[0321] [Embodiment CV] A method according to any one of embodiments [CI]-[CU] above, or any other embodiment of the present disclosure, wherein the first shell comprises a water-soluble compound.

[0322] [Embodiment CW] The method according to any one of embodiments [CI]-[CV] above, or any other embodiment of the present disclosure, wherein the first shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0323] [Embodiment CX] A method according to any one of embodiments [CI] to [CW] above, or any other embodiment of the present disclosure, wherein the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof.

[0324] [Embodiment CY] A method according to any one of embodiments [CI] to [CX] above, or a method according to any other embodiment of the present disclosure, wherein the one or more reagents are selected from one or more of an enzyme, a salt, a surfactant, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof.

[0325] [Embodiment CZ] A method according to any one of embodiments [CI]-[CY] above, or any other embodiment of the present disclosure, further comprising providing a water purification compound.

[0326] [Embodiment DA] The method according to any one of embodiments [CI]-[CZ] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0327] [Embodiment DB] A method according to any one of embodiments [CI]-[DA] above, or any other embodiment of the present disclosure, wherein the second shell comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0328] [Embodiment DC] A method according to any one of embodiments [CI] to [DB] above, or any other embodiment of the present disclosure, wherein the one or more reagents are lyophilized.

[0329] [Embodiment DD] A method according to any one of embodiments [CI]-[DC] above, or any other embodiment of the present disclosure, wherein the second shell comprises a plurality of microspheres containing a plurality of reagents.

[0330] [Embodiment DE] A method according to any one of embodiments [CI]-[DD] above, or any other embodiment of the present disclosure, wherein the second shell comprises a plurality of microspheres containing one type of reagent.

[0331] [Embodiment DF] A method according to any one of embodiments [CI]-[DE] above, or any other embodiment of the present disclosure, wherein the first shell is an outer shell.

[0332] [Embodiment DG] A method according to any one of embodiments [CI]-[DF] above, or any other embodiment of the present disclosure, wherein the second shell is an inner shell.

[0333] [Embodiment DH] A method for controlling the release of one or more reagents comprising providing a composition comprising a dissolvable first shell, a dissolvable second shell comprising one or more reagents, and a water purification compound, exposing the composition to first release conditions to dissolve the water purification compound, exposing the composition to second conditions to dissolve the first shell, and exposing the composition to third release conditions to dissolve the second shell, wherein the first release conditions are different from the second release conditions.

[0334] [Embodiment DI] The method according to embodiment [DH] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0335] [Embodiment DJ] A method according to embodiment [DH] or [DI] above, or any other embodiment of the present disclosure, wherein the first shell is an outer shell.

[0336] [Embodiment DK] A method according to any one of embodiments [DH] to [DJ] above, or any other embodiment of the present disclosure, wherein the second shell is an inner shell.

[0337] [Embodiment DL] A method comprising providing a capsule in a well at a first temperature, providing a liquid having a temperature in the well, raising the temperature of the liquid to a second temperature, lowering the temperature of the liquid from the second temperature to a third temperature, and releasing one or more reagents from the capsule.

[0338] [Embodiment DM] A method according to embodiment [DL] above, or any other embodiment of the present disclosure, wherein the capsule contains a composition of any one of embodiments [A] to [Y].

[0339] [Embodiment DN] A method according to embodiment [DL] or [DM] above, or a method according to other embodiments of the present disclosure, wherein the capsule comprises any one of the compositions of embodiments [Z] to [AZ].

[0340] [Embodiment DO] A method according to any one of the above embodiments [DL]-[DN], or any other embodiment of the present disclosure, wherein the capsule comprises the composition of embodiments [BA]-[BE].

[0341] [Embodiment DP] A method according to any one of embodiments [DL]-[DO] above, or any other embodiment of the present disclosure, wherein the second temperature is greater than about 25°C.

[0342] [Embodiment DQ] A method according to any one of embodiments [DL] to [DP] above, or any other embodiment of the present disclosure, wherein the third temperature is about 25°C or less.

[0343] [Embodiment DR] A method according to any one of embodiments [DL]-[DQ] above, or any other embodiment of the present disclosure, further comprising providing a water purification compound.

[0344] [Embodiment DS] The method according to any one of embodiments [DL]-[DR] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0345] [Embodiment DT] A method according to any one of embodiments [DL]-[DS] above, or any other embodiment of the present disclosure, wherein the capsule contains a water-soluble compound.

[0346] [Embodiment DU] The method according to any one of embodiments [DL]-[DT], wherein the capsule comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0347] [Embodiment DV] A method according to any one of embodiments [DL] to [DU] above, or any other embodiment of the present disclosure, wherein the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof.

[0348] [Embodiment DW] A method according to any one of embodiments [DL] to [DV] above, or a method according to any other embodiment of the present disclosure, wherein the one or more reagents are selected from one or more of an enzyme, a salt, a surfactant, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof.

[0349] [Embodiment DX] A method according to any one of embodiments [DL] to [DW] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0350] [Embodiment DY] A method according to any one of embodiments [DL] to [DX] above, or any other embodiment of the present disclosure, wherein the one or more reagents are lyophilized.

[0351] [Embodiment DZ] A method according to any one of embodiments [DL] to [DY] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing a plurality of reagents.

[0352] [Embodiment EA] A method according to any one of embodiments [DL]-[DZ] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing one type of reagent.

[0353] [Embodiment EB] A method according to any one of embodiments [DL]-[EA] above, or any other embodiment of the present disclosure, wherein the first temperature is different from the third temperature.

[0354] [Embodiment EC] A method according to any one of embodiments [DL]-[EB] above, or any other embodiment of the present disclosure, wherein the first temperature is the same as the third temperature.

[0355] [Embodiment ED] A method comprising: dissolving an outer shell of a capsule in a well at a first temperature, wherein the well contains a liquid and the capsule contains an outer shell, a water purification compound, an inner shell and one or more reagents, and dissolving the outer shell of the capsule releases the water purification compound; raising the temperature of the well to a second temperature; and dissolving the inner shell, thereby releasing the one or more reagents.

[0356] [Embodiment EE] A method according to embodiment [ED] above, or any other embodiment of the present disclosure, wherein dissolving the outer shell of the capsule in the well comprises flowing a liquid into the well.

[0357] [Embodiment EF] A method according to embodiment [ED] or [EE] above, or any other embodiment of the present disclosure, wherein dissolving the inner shell comprises increasing the pH of the liquid to above 7.0.

[0358] [Embodiment EG] A method according to any one of embodiments [ED]-[EF] above, or any other embodiment of the present disclosure, wherein dissolving the inner shell comprises lowering the pH of the liquid to below 7.0.

[0359] [Embodiment EH] A method according to any one of embodiments [ED]-[EG] above, or any other embodiment of the present disclosure, wherein the inner shell is dissolved by a second temperature.

[0360] [Embodiment EI] A method according to any one of embodiments [ED]-[EH] above, or any other embodiment of the present disclosure, wherein the inner shell is dissolved after a minimum time.

[0361] [Embodiment EJ] A method according to any one of embodiments [ED]-[EI] above, or any other embodiment of the present disclosure, wherein the minimum time is 5 minutes.

[0362] [Embodiment EK] A method according to any one of embodiments [ED]-[EJ] above, or any other embodiment of the present disclosure, wherein the second temperature is greater than about 25°C.

[0363] [Embodiment EL] A method according to any one of embodiments [ED]-[EK] above, or any other embodiment of the present disclosure, further comprising reducing the second temperature to a third temperature.

[0364] [EMBODIMENT EM] The method according to any one of embodiments [ED]-[EL] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0365] [Embodiment EN] A method according to any one of embodiments [ED]-[EM] above, or any other embodiment of the present disclosure, wherein the shell comprises a water-soluble compound.

[0366] [EMBODIMENT EO] The method according to any one of embodiments [ED]-[EN] above, or any other embodiment of the present disclosure, wherein the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof.

[0367] [Embodiment EP] A method according to any one of embodiments [ED]-[EO] above, or any other embodiment of the present disclosure, wherein the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or a combination thereof.

[0368] [Embodiment EQ] A method according to any one of embodiments [ED]-[EP] above, or a method according to any other embodiment of the present disclosure, wherein the one or more reagents are selected from one or more of an enzyme, a salt, a surfactant, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof.

[0369] [Embodiment ER] A method according to any one of embodiments [ED]-[EQ] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises one or more dried reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof.

[0370] [Embodiment ES] A method according to any one of embodiments [ED]-[ER] above, or any other embodiment of the present disclosure, wherein the one or more reagents are lyophilized.

[0371] [Embodiment ET] A method according to any one of embodiments [ED]-[ES] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing a plurality of reagents.

[0372] [Embodiment EU] A method according to any one of embodiments [ED] to [ET] above, or any other embodiment of the present disclosure, wherein the internal compartment comprises a plurality of microspheres containing one type of reagent.

[0373] [Embodiment EV] A cartridge comprising a reagent reservoir, the reagent reservoir comprising a composition, the composition comprising a shell surrounding an internal compartment, the internal compartment comprising one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0374] [Embodiment EW] A cartridge according to embodiment [EV] above, or any other embodiment of the present disclosure, wherein the cartridge contains a water purification compound.

[0375] [Embodiment EX] A cartridge according to embodiment [EW] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0376] [Embodiment EY] A cartridge according to embodiment [EX] above, or any other embodiment of the present disclosure, wherein the first release condition is exposure to a liquid.

[0377] [Embodiment EZ] A cartridge according to embodiment [EY] above, or any other embodiment of the present disclosure, wherein the second release condition is exposure to a temperature greater than about 25°C.

[0378] [Embodiment FA] A cartridge comprising a reagent reservoir, the reagent reservoir containing a composition, the composition comprising a dissolvable first shell and a dissolvable second shell containing one or more reagents.

[0379] [Embodiment FB] A cartridge according to embodiment [FA] above, or any other embodiment of the present disclosure, wherein the cartridge comprises a water purification compound.

[0380] [Embodiment FC] A cartridge according to embodiment [FA] or [FB] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0381] [Embodiment FD] A cartridge according to any one of embodiments [FA]-[FC] above, or any other embodiment of the present disclosure, wherein the first release condition is exposure to a liquid.

[0382] [Embodiment FE] A cartridge according to any one of embodiments [FA]-[FD] above, or any other embodiment of the present disclosure, wherein the second release condition is exposure to a temperature above about 25°C.

[0383] [Embodiment FF] A cartridge according to any one of embodiments [FA]-[FE] above, or any other embodiment of the present disclosure, wherein the first shell is an outer shell.

[0384] [Embodiment FG] A cartridge according to any one of embodiments [FA]-[FF] above, or any other embodiment of the present disclosure, wherein the second shell is an inner shell.

[0385] [Embodiment FH] A system for controlling the release of one or more reagents comprising a composition and a liquid, the composition comprising a well and a shell surrounding an internal compartment, the internal compartment containing one or more reagents, the shell releasing the internal compartment when the shell is exposed to a first release condition, and the internal compartment releasing the one or more reagents when the internal compartment is exposed to a second release condition, the first release condition being different from the second release condition.

[0386] [Embodiment FI] A system according to embodiment [FH] above, or any other embodiment of the present disclosure, wherein the liquid is present in the well.

[0387] [Embodiment FJ] A system according to embodiment [FH] or [FI] above, or any other embodiment of the present disclosure, wherein the composition is present in the well.

[0388] [Embodiment FK] A system according to any one of embodiments [FH] to [FJ] above, or a system according to any other embodiment of the present disclosure, further comprising a temperature controller over the well.

[0389] [Embodiment FL] A system according to any one of embodiments [FH]-[FK] above, or any other embodiment of the present disclosure, further comprising a water purification compound.

[0390] [Embodiment FM] A system according to any one of embodiments [FH]-[FL] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0391] [Embodiment FN] A system for controlling the release of one or more reagents comprising a well, a composition comprising a dissolvable first shell and a dissolvable second shell containing one or more reagents, and a liquid.

[0392] [Embodiment FO] A system according to embodiment [FN] above, or any other embodiment of the present disclosure, wherein the liquid is present in the well.

[0393] [Embodiment FP] A system according to embodiment [FN] or [FO] above, or any other embodiment of the present disclosure, wherein the composition is present in the well.

[0394] [Embodiment FQ] A system according to any one of embodiments [FN] to [FP] above, or a system according to any other embodiment of the present disclosure, further comprising a temperature controller over the well.

[0395] [Embodiment FR] A system according to any one of embodiments [FN]-[FQ] above, or any other embodiment of the present disclosure, further comprising a water purification compound.

[0396] [Embodiment FS] A system according to any one of embodiments [FN]-[FR] above, or any other embodiment of the present disclosure, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

[0397] [Embodiment FT] A system according to any one of embodiments [FN] to [FS] above, or a system according to any other embodiment of the present disclosure, wherein the first shell is an outer shell.

[0398] [Embodiment FU] A system according to any one of embodiments [FN] to [FT] above, or a system according to any other embodiment of the present disclosure, wherein the second shell is an inner shell.

[0399] [Embodiment FV] A method comprising: flowing a liquid having a temperature into a well, the well comprising a capsule, the capsule comprising a first shell surrounding a water purification compound and a second shell surrounding one or more reagents, the first shell releasing the water purification compound when exposed to a first release condition and the second shell releasing the one or more reagents when exposed to a second release condition, the first release condition being different from the second release condition, and the water purification compound substantially or completely decomposing when exposed to a decomposition condition; exposing the first shell to the first release condition, thereby releasing the water purification compound; exposing the water purification compound to the decomposition condition, thereby substantially or completely decomposing the water purification compound; and exposing the second shell to the second release condition, thereby releasing the one or more reagents.

[0400] [Embodiment FW] A method according to embodiment [FV] above, or any other embodiment of the present disclosure, wherein the first release condition is exposure to a liquid.

[0401] [Embodiment FX] A method according to embodiment [FV] or [FW] above, or any other embodiment of the present disclosure, wherein the decomposition conditions are liquid elevated temperature.

[0402] [Embodiment FY] A method according to any one of embodiments [FV]-[FX] above, or any other embodiment of the present disclosure, wherein the elevated temperature is about 25°C or higher.

[0403] [Embodiment FZ] A method according to any one of embodiments [FV]-[FY] above, or any other embodiment of the present disclosure, wherein the decomposition conditions are the same as the second release conditions.

[0404] [Embodiment GA] A method according to any one of embodiments [FV] to [FZ] above, or a method according to any other embodiment of the present disclosure, in which the steps of flowing a liquid, exposing the first shell to first release conditions, and exposing the water purification compound to decomposition conditions are performed in sequence.

[0405] [Embodiment GB] A method according to any one of embodiments [FV] to [GA] above, or any other embodiment of the present disclosure, wherein flowing a liquid, exposing the first shell to first release conditions, exposing the water purification compound to decomposition conditions, and exposing the second shell to second release conditions are performed in sequence.

Claims

1. a shell surrounding an inner compartment, said inner compartment containing one or more reagents; when the shell is exposed to a first release condition, the shell releases the internal compartment; and when the shell is exposed to the first release condition, the internal compartment prevents release of the one or more reagents; A composition wherein said inner compartment releases said one or more reagents when said inner compartment is exposed to a second release condition, said first release condition being different from said second release condition. (i) the shell releases the internal compartment when the shell is exposed to at least one additional shell-release condition, one or more of the at least one additional shell-release condition being different from the first release condition; or (ii) the composition of claim 1, wherein the inner compartment releases the one or more reagents when the inner compartment is exposed to at least one additional inner compartment release condition, and one or more of the at least one additional inner compartment release condition is different from the second release condition.

3. 3. The composition of claim 1 or 2, further comprising a water purification compound, wherein the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, iron sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof.

4. 3. The composition of claim 1 or 2, wherein the shell is a dissolvable first outer shell and the inner component further comprises a dissolvable second inner shell.

5. 1. A method for controlling the release of one or more reagents, comprising: exposing a composition comprising a shell surrounding an inner compartment to a first release condition to release said inner compartment, said inner compartment comprising one or more reagents; exposing said internal compartment to a second release condition to release said one or more reagents, wherein said first release condition is different from said second release condition.

6. 6. The method of claim 5, wherein the interior compartment prevents release of the one or more reagents when the shell is exposed to the first release condition.

7. The method of claim 5 , wherein the first release condition occurs before the second release condition.

8. 10. The composition of claim 1 or the method of claim 5, wherein the first release condition and the second release condition are each independently selected from a temperature-controlled release condition, a pH-controlled release condition, a time-controlled release condition, a position-controlled release condition, or any combination thereof.

9. 10. The composition of claim 1 or the method of claim 5, wherein one or both of the first and second release conditions includes a change in temperature, wherein the change in temperature is to a temperature above about 25°C, or the change in temperature is to a temperature below about 25°C. (i) the first release condition comprises a pH of about 1.0 to about 10.0; and / or the second release condition comprises a pH of about 1.0 to about 10.0; (ii) the second release conditions are effective to release a plurality of reagents, the content of at least one reagent being different from the content of at least one other reagent; (iii) the shell releases the internal compartment when the shell is exposed to at least one additional shell-release condition, one or more of the at least one additional shell-release condition being different from the first release condition, and optionally, the internal compartment prevents release of the one or more reagents when the shell is exposed to the at least one additional shell-release condition; and / or (iv) the internal compartment releases the one or more reagents when exposed to at least one additional internal compartment release condition, wherein one or more of the at least one additional internal compartment release condition is different from the second release condition.

11. 8. The composition of claim 1 or the method of any one of claims 5-7, wherein the shell has a shell width of about 1 micrometer to about 1,000 micrometers and the inner compartment has an inner compartment width of about 1 micrometer to about 1,000 micrometers, and the shell width is different from the inner compartment width.

12. The composition of claim 1 or 2 or the method of any one of claims 5 to 7, wherein the shell comprises a water-soluble compound.

13. (i) the shell comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, or any combination thereof; (ii) the one or more reagents are selected from one or more of an enzyme, a salt, a detergent, a buffer, an enzyme inhibitor, a primer, a nucleotide, an organic osmolyte, a magnetic bead, a molecular probe, a crowding agent, a small molecule, a labeled nucleotide, or any combination thereof; and / or (iii) The composition of claim 1 or 2 or the method of any one of claims 5 to 7, wherein the internal compartment comprises a plurality of microspheres containing a plurality of reagents, or the internal compartment comprises a plurality of microspheres containing one reagent. (i) the one or more reagents are sequencing reagents, sample preparation reagents, library preparation reagents, or any combination thereof; (ii) the internal compartment comprises one or more dry reagents, one or more microspheres, one or more beads, one or more powders, one or more cakes, one or more gels, one or more liquids, or any combination thereof; and / or (iii) The composition of claim 1 or 2 or the method of any one of claims 5 to 7, wherein the one or more reagents are lyophilized.

15. wherein either or both of the shell and the internal compartment further comprise a water purification compound; the water purification compound comprises sodium dichloroisocyanurate, chlorine, chloramine, chlorine dioxide, polyaluminum chloride, aluminum sulfate, ferrous sulfate, hydrogen peroxide, sodium hydroxide bromide, silver nanoparticles, iron, iodine, activated carbon, or any combination thereof; Optionally, the water purification compound substantially or completely decomposes when exposed to decomposition conditions, and further optionally, (i) said decomposition conditions include a change in temperature, and optionally said change in temperature is to a temperature of about 25° C. or greater; or (ii) The method according to any one of claims 5 to 7, wherein the decomposition conditions are the same as the second release conditions.

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