Compositions and methods for DNA library preparation

Abstract

The present disclosure relates generally to compositions for next generation sequencing and DNA library preparation, and systems and methods for generating DNA libraries using the compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 492,109, filed on March 24, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present application generally relates to compositions for next generation sequencing, DNA library preparation, and systems and methods for generating DNA library preparations using the compositions. Background Art

[0004] Current protocols for DNA library preparation and next generation sequencing technologies often employ a sample preparation process that converts DNA or RNA into libraries of fragmented, sequenceable templates.

[0005] A library comprising polynucleotides is usually prepared in any suitable manner to attach oligonucleotide adapters to the target polynucleotides. Sequencing may result in determining all or part of the sequence of the target polynucleotide. By using transposase-mediated fragmentation and labeling, the number of steps involved in converting nucleic acids into adapter-modified templates in a solution prepared for forming clusters and sequencing on a substrate can be reduced or even minimized in some cases. This process is called "tag fragmentation", which involves fragmenting and adapter labeling of nucleic acids by a transpososome complex, which includes a transposase complexed with an adapter including a transposon end sequence, as described in, for example, US20180245069.

[0006] Sample preparation methods typically involve multiple steps, material transfers, and expensive instrumentation to achieve DNA fragmentation and DNA indexing, and thus can be time consuming and inefficient. Summary of the invention

[0007] The present disclosure provides benefits and advantages to previous workflows for next generation sequencing by reducing the number of steps required to perform a library preparation workflow. The present disclosure provides compositions comprising a series of lyophilized materials (e.g., microspheres or beads) containing library preparation reagents, which release their contents under predetermined release conditions so that DNA preparation reactions can occur in a single container or in a minimum number of containers, thereby minimizing DNA losses during addition or removal of reagents and transfer of samples to different sample containers. Lyophilization of reagents and the ability to combine multiple lyophilized microspheres into a single container provide improved storage and stability of reagents, and result in fewer processing steps and an improved user experience.

[0008] The present invention provides a composition comprising:

[0009] a first lyophilized material (e.g., microspheres, beads, or cake) comprising one or more tag fragmentation reagents,

[0010] a plurality of first particles, the plurality of first particles comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents, and

[0011] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

[0012] The present invention also provides a composition comprising:

[0013] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0014] A plurality of first particles, the plurality of first particles comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning reagents; and a second shell, the second shell being located within the first core and surrounding the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-ligation and PCR.

[0015] Also contemplated herein is a composition comprising:

[0016] a first lyophilized material comprising one or more tag fragmentation reagents,

[0017] a plurality of first particles, the plurality of first particles comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising a sugar or plastic, the sugar or plastic comprising one or more tag fragmentation poisoning agents, optionally wherein the one or more tag fragmentation poisoning agents are coated on the surface of the non-lyophilized microspheres or beads and dried, and

[0018] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising sugar or plastic, the sugar or plastic comprising one or more extension-link and PCR reagents, optionally wherein the one or more extension-link and PCR reagents are coated on the surface of the non-lyophilized microspheres or beads and dried.

[0019] The present invention also provides a composition comprising:

[0020] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0021] A plurality of first particles, comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising sugar or plastic, the sugar or plastic comprising one or more tag fragmentation poisoning reagents, optionally wherein the one or more tag fragmentation poisoning reagents are coated on the surface of the non-lyophilized microspheres or beads and dried; and a second shell, the second shell being located within the first core and surrounding the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising sugar or plastic, the sugar or plastic comprising one or more extension-link and PCR reagents, optionally wherein the one or more extension-link and PCR reagents are coated on the surface of the non-lyophilized microspheres or beads and dried.

[0022] The present invention provides a composition comprising:

[0023] a first lyophilized material (e.g., microspheres, beads, or cake) comprising one or more tag fragmentation reagents,

[0024] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and

[0025] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

[0026] The present invention also provides a composition comprising:

[0027] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0028] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and

[0029] A second shell is located within the first core and surrounds the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

[0030] Also contemplated herein is a composition comprising:

[0031] a first lyophilized material comprising one or more tag fragmentation reagents,

[0032] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising sugar or plastic, the sugar or plastic comprising one or more tag fragmentation poisoning agents, optionally wherein the one or more tag fragmentation poisoning agents are coated on the surface of the non-lyophilized microspheres or beads and dried; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and

[0033] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising sugar or plastic, the sugar or plastic comprising one or more extension-link and PCR reagents, optionally wherein the one or more extension-link and PCR reagents are coated on the surface of the non-lyophilized microspheres or beads and dried.

[0034] The present invention also provides a composition comprising:

[0035] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0036] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more non-lyophilized microspheres or beads, the microspheres or beads comprising sugar or plastic, the sugar or plastic comprising one or more tag fragmentation poisoning agents, optionally wherein the one or more tag fragmentation poisoning agents are coated on the surface of the non-lyophilized microspheres or beads and dried; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and

[0037] A second shell, which is located within the first core and surrounds the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more non-lyophilized microspheres or beads, which comprise sugars or plastics, which comprise one or more extension-link and PCR reagents, optionally wherein the one or more extension-link and PCR reagents are coated on the surface of the non-lyophilized microspheres or beads and dried.

[0038] In various embodiments, the lyophilized material is a lyophilized microsphere, a lyophilized bead or a lyophilized cake. In various embodiments, the lyophilized cake comprises a plurality of stacked lyophilized cakes.

[0039] In various embodiments, the polymer is a hydrophilic or water-soluble polymer. In various embodiments, the polymer is a hydrophobic or water-insoluble polymer. In various embodiments, the water-insoluble polymer undergoes a transition from hydrophobic to hydrophilic or from solid to liquid upon triggering of the triggering release condition.

[0040] In various implementations, the first trigger-release condition and the second trigger-release condition are different.

[0041] In various implementations, the first, second and / or third trigger release conditions are different. In various implementations, the first, second and / or third trigger release conditions are the same.

[0042] In various embodiments, the triggered release condition is a time release condition, a temperature release condition, or a pH release condition.

[0043] In various embodiments, the one or more tag fragmentation reagents are bead-linked transposomes (BLTs), transposases, primers, buffers, divalent cations, magnesium sulfate, Tris buffers, cobalt buffers, and / or lyophilized reagents.

[0044] In various implementations, the tag fragmentation reagent comprises a bead-attached transposome, a primer tag, cobalt chloride, and trehalose. In various implementations, the tag fragmentation reagent comprises a bead-attached transposome, magnesium sulfate, Tris buffer, and trehalose.

[0045] In various specific implementations, the one or more tag fragmentation poisoning agents are chelating agents, deoxynucleotide triphosphates (dNTPs), sodium dodecyl sulfate (SDS), Tris buffer, potassium buffer, ammonium salts and / or lyophilized reagents.

[0046] In various embodiments, the tag fragmentation poisoning reagent comprises Tris buffer, potassium chloride, ammonium sulfate, cyclen tetrahydrochloride, alpha-cyclodextrin, dNTPs and trehalose. In various embodiments, the tag fragmentation poisoning reagent comprises potassium chloride, ammonium sulfate, SDS, dNTPs and trehalose.

[0047] In various embodiments, the extension-ligation and PCR reagents include alpha-cyclodextrin, magnesium sulfate, one or more polymerases, ampligase, NAD+, betaine, surfactants, and / or lyophilized reagents.

[0048] In various embodiments, the extension-link and PCR reagents include magnesium sulfate, Q5 without glycerol hot start, tTaq 608 polymerase, ampligase, NAD+, betaine, Tween-20, and trehalose. In various embodiments, the extension-link and PCR reagents include Q5 without glycerol hot start, tTaq 608 polymerase, ampligase, NAD+, betaine, alpha-cyclodextrin, Tween-20, and trehalose.

[0049] In various embodiments, the first and / or second shell comprises a temperature-triggered release polymer and / or a time-release polymer. In various embodiments, the first and / or second shell comprises a temperature-triggered release polymer, a time-release polymer and / or a functional additive. In various embodiments, the first and / or second shell comprises an antistatic agent, a defoamer, a plasticizer, one or more upper critical solution temperature (UCST) polymers, one or more lower critical solution temperature (LCST) polymers, hydroxypropyl methylcellulose (HPMC), or a combination thereof.

[0050] In various specific implementations, the first and / or second shell comprises Efka 6783, Makon 17R4, magnesium stearate, UCST polymer 1, UCST polymer 2, LCST polymer, hydroxypropyl methylcellulose (HPMC), or a combination thereof. UCST polymer 1 may also be referred to herein as the first UCST polymer, and UCST polymer 2 may also be referred to herein as the second UCST polymer.

[0051] In various implementations, the first and / or second shell comprises Efka 6783, Makon 17R4, polyethylene glycol (PEG) stearate, UCST polymer 1, UCST polymer 2, LCST polymer, hydroxypropyl methylcellulose (HPMC), or a combination thereof.

[0052] In various implementations, the first shell comprises a UCST / LCST polymer and one or more functional additives, such as an anti-sticking agent, a defoaming agent, a plasticizer, an antistatic agent, or a combination thereof. In various implementations, the first shell comprises Efka6783, Makon 17R4, magnesium stearate, and UCST polymer 1.

[0053] In various implementations, the first shell comprises a UCST / LCST polymer and one or more functional additives, such as an anti-sticking agent, a defoaming agent, a plasticizer, an antistatic agent, or a combination thereof. In various implementations, the first shell comprises Efka6783, Makon 17R4, stearic acid PEG ester, and UCST polymer 1.

[0054] In various specific implementations, when the particle comprises a first inner shell and a first outer shell, the first inner shell comprises a hydrophobic polymer. In various embodiments, the first inner shell comprises cellulose acetate (CA), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), ethyl cellulose (EC), methyl ethyl cellulose (MEC), polyvinyl acetate, fatty acids, fatty acid esters, or combinations thereof. In various embodiments, the first inner shell comprises cellulose acetate.

[0055] In various specific implementations, when the particle comprises a first inner shell and a first outer shell, the first outer shell comprises a hydrophilic polymer. In various embodiments, the first outer shell comprises carboxymethyl cellulose (CMC), carboxymethyl ethyl cellulose (CMEC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), ethyl hydroxyethyl cellulose (EHEC), pullulan, polyvinyl pyrrolidone, polyvinyl alcohol, copolymers of vinyl pyrrolidone or vinyl alcohol, polyacrylamide, copolymers of polyacrylic acid, ethyl acrylic acid or methacrylic acid and other acrylic acid derivatives such as butyl methacrylate, methyl methacrylate, ethyl methacrylate, ethyl acrylate, methacrylate (2-dimethylaminoethyl) ester and methacrylate chloride (trimethylaminoethyl) ester homopolymers and copolymers, P (NIPAAm-co-nBu) LCST 15 ° C or a combination thereof. In various implementations, the first shell comprises hydroxypropyl methylcellulose (HPMC) or P(NIPAAm-co-nBu) LCST 15° C. In various implementations, the first shell comprises hydroxypropyl methylcellulose (HPMC).

[0056] In various specific implementations, the second shell comprises hydroxypropyl methylcellulose (HPMC), UCST / LCST polymer and one or more functional additives, such as anti-adherent, defoamer, plasticizer, antistatic agent or a combination thereof. In various specific implementations, the second shell comprises Efka 6783, Makon 17R4, magnesium stearate, UCST polymer 2 and hydroxypropyl methylcellulose (HPMC).

[0057] In various specific implementations, the first release condition is a temperature release condition. In various specific implementations, the temperature of the first release condition is above about 45° C. Exemplary temperature-triggered release polymers are UCST polymers such as P (acrylonitrile-co-acrylamide), P (N-acryloyl glycinamide-co-styrene) and LCST polymers such as P (N-isopropylacrylamide) derivatives.

[0058] In various specific implementations, the second release condition is a time release condition.Exemplary time-release polymers are hydroxypropylmethylcellulose (HPMC), poly(vinyl pyrrolidone-co-vinyl acetate), and cellulose acetate.

[0059] In various implementations, the first or second release condition is a pH-dependent release condition.Exemplary pH-dependent release polymers include polymers that dissolve at different pH levels (eg, between about pH 4.5 and about pH 9, or about pH 6 to about pH 8.5). Exemplary polymers include, but are not limited to, polymers soluble at or below pH 5: cellulose acetate, timellitate, cellulose acetate phthalate, PVA phthalate, HPMC phthalate; chitosan (soluble below pH 6); polymers soluble between pH 5 and pH 6 or above: Kollicoat MAE 100, Kollicoat 30D, Eudragit L100, Eudragit L100-55, Eudragit L30D-55, Eudragit L12.5, polymers soluble between about pH 6 and about pH 7: Eudragit L100 / S100 1:1, Eudragit L / S12.5, Eudragit S100, Eudragit FS30D, Eudragit S12.5, shellac; carboxymethyl cellulose (soluble at pH 6 or below); 7.4 or above) or rennet casein (soluble at around pH 9).

[0060] In various implementations, the composition is stable when stored at between about 4°C and about 37°C.

[0061] The present disclosure also contemplates one or more containers for preparing a DNA library comprising the compositions described herein.

[0062] In various specific implementations, the container includes:

[0063] a first lyophilized material comprising one or more tag fragmentation reagents,

[0064] a plurality of first particles, the plurality of first particles comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents, and

[0065] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more extension-link and PCR reagents.

[0066] In various specific implementations, the container includes:

[0067] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0068] A plurality of first particles, the plurality of first particles comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents.

[0069] In various specific implementations, the container includes:

[0070] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more extension-link and PCR reagents.

[0071] In various specific implementations, the container includes:

[0072] a first lyophilized material comprising one or more tag fragmentation reagents,

[0073] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and

[0074] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

[0075] In various specific implementations, the container includes:

[0076] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0077] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and

[0078] A second shell is located within the first core and surrounds the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

[0079] In various specific implementations, the container includes:

[0080] a first lyophilized material comprising one or more tag fragmentation reagents, and

[0081] A plurality of first particles, the plurality of first particles comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism.

[0082] In various specific implementations, the container includes:

[0083] A plurality of second particles, the plurality of second particles comprising a second shell, the second shell surrounding a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more extension-link and PCR reagents.

[0084] In various embodiments, the container is a PCR tube, a vial, a microtube, a flow cell, a multiwell plate (eg, a 96-well or 384-well plate), a glass tube, a transwell membrane / mesh insert, a cartridge, or a microfluidic chip.

[0085] Also provided are kits comprising one or more compositions or containers as described herein and instructions for use. In one embodiment, the kit comprises a container comprising a first lyophilized material and first particles and a separate container comprising a plurality of second particles.

[0086] The present disclosure contemplates a method of preparing a DNA library, the method comprising:

[0087] adding a sample comprising DNA to a container comprising a composition as described herein, wherein the addition of the sample triggers dissolution of the first lyophilized material thereby releasing one or more tag fragmentation reagents into the container thereby initiating a tag fragmentation reaction;

[0088] allowing the tag fragmentation reaction to continue under conditions and for a sufficient period of time to complete tag fragmentation of the DNA in the sample;

[0089] initiating a first triggered release condition of the first shell, wherein the first triggered release condition of the first shell causes the one or more tag fragmentation poisoning agents of the first core to be released into the container to induce a tag fragmentation poisoning reaction; and

[0090] The tag fragmentation poisoning reaction is allowed to proceed under conditions and for a sufficient period of time to initiate a second triggered release condition of the second shell, wherein one or more extension-link and PCR reagents are released from the second core, wherein the extension-link and PCR reagents perform gap filling and PCR amplification.

[0091] Also provided is a method for preparing a DNA library, the method comprising:

[0092] adding a sample comprising DNA to a container comprising a composition as described herein, wherein the addition of the sample triggers dissolution of the first lyophilized material thereby releasing one or more tag fragmentation reagents into the container thereby initiating a tag fragmentation reaction;

[0093] allowing the tag fragmentation reaction to continue under conditions and for a sufficient period of time to at least substantially complete tag fragmentation of the DNA in the sample;

[0094] initiating a second trigger release condition for the first outer shell, which second trigger release condition subsequently initiates a first trigger release condition for the first inner shell, wherein the first trigger release condition of the first inner shell causes the one or more tag fragmentation poisoning agents of the first core to be released into the container to initiate a tag fragmentation poisoning reaction; and,

[0095] The tag fragmentation poisoning reaction is allowed to proceed under conditions and for a sufficient period of time to initiate a second triggered release condition of the second shell, wherein one or more extension-link and PCR reagents are released from the second core, wherein the extension-link and PCR reagents perform gap filling and PCR amplification.

[0096] In various embodiments, the method further comprises isolating the DNA and sequencing the DNA library.

[0097] The present disclosure also contemplates a method of preparing a DNA library, the method comprising:

[0098] adding a sample comprising DNA to a system as described herein, wherein the addition of the sample triggers the dissolution of the first lyophilized material thereby releasing one or more tag fragmentation reagents into the container thereby initiating a tag fragmentation reaction;

[0099] allowing the tag fragmentation reaction to continue under conditions and for a sufficient period of time to complete tag fragmentation of the DNA in the sample;

[0100] initiating a first triggered release condition of the first shell, wherein the first triggered release condition of the first shell causes the one or more tag fragmentation poisoning agents of the first core to be released into the container to induce a tag fragmentation poisoning reaction; and

[0101] The tag fragmentation poisoning reaction is allowed to proceed under conditions and for a sufficient period of time to initiate a second triggered release condition of the second shell, wherein one or more extension-link and PCR reagents are released from the second core, wherein the extension-link and PCR reagents perform gap filling and PCR amplification.

[0102] In various embodiments, the method further comprises isolating the DNA and sequencing the DNA library.

[0103] In various embodiments, the tag fragmentation reaction is carried out at about 37°C to about 41°C for about 3 minutes to about 15 minutes, or about 5 minutes to about 10 minutes. In various embodiments, the tag fragmentation reaction is carried out at about 37°C to about 41°C for about 5 minutes.

[0104] In various implementations, the first release condition is a temperature release condition. In various implementations, the temperature of the first temperature release condition is above about 45°C.

[0105] In various embodiments, the tag fragmentation poisoning reaction is carried out at about 55° C. to about 65° C. for about 1 to about 5 minutes. In various embodiments, the tag fragmentation poisoning reaction is carried out at about 60° C. to about 65° C. for about 1 to about 5 minutes.

[0106] In various implementations, the second release condition is a time release condition. In various implementations, the time release condition is about 1 to about 3 minutes at about 50°C.

[0107] In various embodiments, the first, second or third release condition is a pH-dependent release condition. In various embodiments, the pH-dependent release condition is about pH 4.5 to about pH 9 or about pH 6 to about pH 8.5. Exemplary pH-dependent release polymers include polymers that dissolve at or below pH 5: cellulose acetate, trimellitate, cellulose acetate phthalate, PVA phthalate, HPMC phthalate; chitosan (soluble below pH 6); polymers that dissolve between pH 5 and pH 6 or above: Kollicoat MAE 100, Kollicoat 30D, Eudragit L100, Eudragit L100-55, Eudragit L30D-55, Eudragit L12.5, polymers that dissolve between about pH 6 and about pH 7: Eudragit L100 / S100 1:1, Eudragit L / S12.5, Eudragit S100, Eudragit FS30D, Eudragit S12.5, shellac; carboxymethyl cellulose (soluble at pH 6 or below); 7.4 or above) or rennet casein (soluble at around pH 9).

[0108] In various implementations, the one or more extension-ligation and PCR reagents are released from the second core at about 50° C., wherein the extension-ligation and PCR reagents perform extension, ligation gap filling, and PCR amplification.

[0109] It should be understood that each feature or implementation or combination described herein is a non-limiting, illustrative example of any aspect of the present invention, and is therefore meant to be combined with any other feature or implementation or combination described herein. For example, where features are described using language such as "one implementation," "various implementations," "some implementations," "certain implementations," "additional implementations," "particular exemplary implementations," and / or "another implementation," each of these types of implementations is a non-limiting example of a feature that is intended to be combined with any other feature or combination of features described herein, without necessarily listing every possible combination.

[0110] These features or combinations of features are applicable to any aspect of the present invention. When examples of values ​​falling within a range are disclosed, any of these examples are considered possible endpoints of the range, any and all numerical values ​​between such endpoints are considered, and any and all combinations of upper and lower limits are considered possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1Shown is a comparison of DNA yields following tag fragmentation reactions using liquid or lyophilization buffer.

[0112] Figure 2 Shown is a comparison of DNA yields after tag fragmentation poisoning reactions using liquid or lyophilization buffer.

[0113] Figure 3 Shown is a comparison of DNA yields following PCR reactions using liquid or lyophilized buffer.

[0114] Figure 4 An optional workflow is depicted for completing the initial steps in DNA library preparation using a lyophilized microsphere composition.

[0115] Figure 5 Shown is the amount of DNA produced by a PCR reaction when lyophilized microspheres containing magnesium are encapsulated in wax.

[0116] Figure 6 The following is a workflow of DNA library preparation according to one specific implementation of the present disclosure. DETAILED DESCRIPTION

[0117] The present disclosure relates to an improved workflow for preparing a DNA library by reducing the number of interactive steps or contact points involved in the DNA library preparation process, such as from tag fragmentation, Tn5 removal, gap filling and PCR amplification. The workflow includes using a series of multiple lyophilized microspheres containing reagents for completing DNA library preparation. The use of multiple microspheres encapsulating reagents in different orders enables most workflow steps to be completed in a single container, thereby minimizing sample loss during processing. In addition, the use of lyophilized microspheres reduces the liquid volume of the reagents used, thereby allowing the volume of the sample used for each reaction to be increased.

[0118] The compositions, systems, and methods for DNA library preparation described herein can also be used within the workflows of other DNA library preparation methods, such as spatial indexing, sequencing by synthesis (SBS), and other next-generation sequencing (NGS) technologies.

[0119] It should be understood that certain aspects, modes, specific implementations, variations and features of the present disclosure are described below in various levels of detail in order to provide a substantial understanding of the present technology. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as those generally understood by those of ordinary skill in the art. The use of the term "including" and other forms is not restrictive. The use of the term "having" and other forms is not restrictive. As used in the present disclosure, whether in a transitional phrase or in the body of the claims, the terms "comprise(s)" and "comprising" will be interpreted as having an open meaning. That is, the term should be interpreted synonymously with the phrase "at least having" or "at least including".

[0120] The term "about" (particularly with respect to a given amount) is intended to encompass a deviation of ±5%.

[0121] As used herein, the terms “includes,” “including,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion such that a process, method, product defined by a method, or composition of matter that includes, contains, or contains an element or list of elements includes not only those elements, but may include other elements not expressly listed or inherent to such process, method, product defined by a method, or composition of matter.

[0122] The terms "substantially," "approximately," "about," "relatively," or other such similar terms that may be used throughout this disclosure, including the claims, are used to describe and illustrate small fluctuations from a reference or parameter, for example, due to changes in processing. Such small fluctuations also include zero fluctuations from a reference or parameter. For example, a fluctuation may refer to less than or equal to ±10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0123] The terms "connected," "contacted," and / or "coupled" include a variety of arrangements and components. These arrangements and techniques include, but are not limited to: (1) the direct engagement of one component with another component without intervening components (i.e., the components are in direct physical contact); and (2) the engagement of one component with another component with one or more components in between, provided that the one component is "connected to" or "contacted with" or "coupled to" the other component to some extent in operational communication (e.g., electrical, fluidic, physical, optical communication, etc.) with the other component (optionally with one or more additional components in between). Some components that are in direct physical contact with each other may or may not be in electrical and / or fluidic contact with each other. In addition, two components that are electrically connected, electrically coupled, optically connected, optically coupled, fluidically connected, or fluidically coupled may or may not be in direct physical contact, and one or more other components may be positioned between the two connected components.

[0124] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA made from nucleotide analogs. As used herein, the term also encompasses cDNA, i.e., complementary DNA or copy DNA produced by an RNA template, for example, by the action of a reverse transcriptase.

[0125] Nucleic acids include naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleic acids typically have a backbone comprising a phosphodiester bond. Analog structures can have alternative backbone bonds, including any backbone bond in a variety of backbone bonds known in the art, such as peptide nucleic acids (PNA) or locked nucleic acids (LNA). Naturally occurring nucleic acids typically have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0126] Nucleic acids may contain any of a variety of analogs of these sugar moieties known in the art. Nucleic acids may include natural or non-natural bases. Natural deoxyribonucleic acids may have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, and ribonucleic acids may have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-natural bases that may be included in nucleic acids are known in the art.

[0127] As described herein, the term nucleotide may include natural nucleotides and analogs thereof, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and other molecules referred to as nucleotides. As described herein, "nucleotides" may include nitrogenous heterocyclic bases, sugars, and one or more phosphate groups. Nucleotides may be monomeric units of nucleic acid sequences, such as to identify subunits present in DNA or RNA chains. Nucleotides may also include molecules that are not necessarily present in polymers, such as molecules that can be incorporated into polynucleotides by polymerases in a template-dependent manner. Nucleotides may include nucleoside units that have, for example, 0, 1, 2, 3 or more phosphate groups on 5' carbon. Nucleoside tetraphosphates, nucleoside pentaphosphates, and nucleoside hexaphosphates may be useful, and nucleotides having more than 6 phosphate groups (such as 7, 8, 9, 10 or more phosphate groups) on 5' carbon may also be useful. 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.

[0128] Non-natural nucleotides include nucleotide analogs, such as those that are not present in natural biological systems or are not substantially incorporated into polynucleotides by polymerases in their natural environment (e.g., in non-recombinant cells expressing polymerases). Non-natural nucleotides include those that are incorporated into polynucleotide chains by polymerases at a rate that is significantly faster or slower than the rate at which another nucleotide (such as a natural nucleotide that has base pairing with the same Watson-Crick complementary base) is incorporated into the chain by the polymerase. For example, when compared to the incorporation rate of natural nucleotides, non-natural nucleotides can be incorporated at a rate that is at least 2 times different, 5 times different, 10 times different, 25 times different, 50 times different, 100 times different, 1000 times different, 10000 times different, or more times different. Non-natural nucleotides can be further extended after incorporation into polynucleotides. Examples include nucleotide analogs with a 3' hydroxyl group or nucleotide analogs with a reversible terminator portion at the 3' position, which can be removed to allow further extension of the polynucleotide into which the nucleotide analog is incorporated. Examples of reversible terminator moieties are described in, for example, U.S. Patent Nos. 7,427,673, 7,414,116, and 7,057,026, and WO 91 / 06678 and WO 07 / 123744, each of which is hereby incorporated by reference in its entirety. It should be understood that in some examples, nucleotide analogs having a 3' terminator moiety or lacking a 3' hydroxyl group (such as dideoxynucleotide analogs) can be used under conditions where the polynucleotide into which the nucleotide analog has been incorporated is not further extended. In some examples, the nucleotide may not include a reversible terminator moiety, or the nucleotide will not include an irreversible terminator moiety, or the nucleotide will not include any terminator moiety at all.

[0129] The present disclosure contemplates the inclusion of fluorescently labeled nucleotides (or any other detection label) that can be used in any of the methods disclosed herein, either incorporated into or associated with a larger molecular structure or conjugate.

[0130] Fluorescent labels can include compounds selected from any known fluorescent substances, such as rhodamine or cyanine. Fluorescent labels as disclosed herein can be attached to any position on the nucleotide base and can optionally include a joint. The function of the joint is generally to help the chemical attachment of the fluorescent label to the nucleotide. In a specific implementation, the resulting analog can still be subjected to Watson-Crick base pairing. The joint group can be used to covalently attach the dye to a nucleoside or nucleotide. The joint portion can have a sufficient length to connect the nucleotide to the compound so that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by the nucleic acid replicator. Therefore, the joint can also include a spacer unit. The spacer distance is, for example, the distance of the nucleotide base from the cleavage site or the label.

[0131] As used herein, the term "substrate" is intended to represent a solid carrier or support structure. The term includes any material that can be used as a solid or semi-solid basis for forming features, such as a hole for depositing a biopolymer (including nucleic acids, polypeptides and / or other polymers). For example, the substrate as provided herein is modified, or can be modified to adapt to the attachment of biopolymers by a variety of methods well known to those skilled in the art. Exemplary types of substrate materials include glass, modified glass, functionalized glass, inorganic glass, microspheres (including inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or optical fiber bundles, various polymers other than those illustrated above, and porous microtiter plates. Exemplary plastics of specific types include acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane and Teflon TM Specific types of exemplary silica-based materials include silicon and various forms of modified silicon.

[0132] In some embodiments, the solid support comprises one or more surfaces capable of contacting a reagent, bead or analyte. The surface may be substantially flat or planar. Alternatively, the surface may be rounded or contoured. Exemplary contours that may be included on the surface are holes (e.g., micropores or nanopores), depressions, columns, ridges, channels, etc. Exemplary materials that may be used as surfaces include: glass, such as modified or functionalized glass; plastics, such as acrylic acid, polystyrene or copolymers of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or Teflon; polysaccharides or cross-linked polysaccharides, such as agarose or agarose gel; nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon, carbon fiber; metal; inorganic glass; fiber bundles, or a variety of other polymers. A single material or a mixture of several different materials may form a surface that can be used in certain examples. In some examples, the surface includes holes (e.g., micropores or nanopores). In some aspects, the surface comprises an array of pores (e.g., micropores or nanopores) on a glass, silicon, plastic, or other suitable solid support having a patterned covalently attached gel such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM, see, e.g., U.S. Patent Application Publication No. 2014 / 0079923A1, which is incorporated herein by reference). In some examples, the support structure may include one or more layers.

[0133] As used herein, "derivative" or "analog" means a synthetic nucleotide or nucleoside derivative with a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed in, for example, Bücher, NUCLEOTIDE ANALOGS (John Wiley & Son, 1980) and Uhlmann et al., "Antisense Oligonucleotides: A New Therapeutic Principle," Chemical Reviews 90: 543-584 (1990), and these two documents are incorporated by reference in their entirety. Nucleotide analogs may also include modified phosphodiester bonds, including phosphorothioate bonds, phosphorodithioate bonds, alkylphosphonate bonds, aniline phosphate bonds, and phosphoramidate bonds. "Derivatives," "analogs," and "modified" as used herein may be used interchangeably, and are covered by terms "nucleotide" and "nucleoside" as described herein.

[0134] As used herein, the term "plurality" is intended to represent a population of two or more different members. The plurality of numbers can range in size from small, medium, large to extremely large. The size of a small plurality of numbers can range from, for example, a few members to tens of members. A medium-sized plurality of numbers can range from, for example, tens of members to about 100 members or hundreds of members. A large plurality of numbers can range from, for example, about hundreds of members to about 1000 members, to thousands of members, and up to tens of thousands of members. An extremely large plurality of numbers can range from, for example, tens of thousands of members to about hundreds of thousands, one million, several million, tens of millions, and up to or exceeding hundreds of millions of members. Therefore, a plurality of sizes measured in terms of the number of members can range from two to well over 100 million members, as well as all sizes between and exceeding the above exemplary ranges. Exemplary features of numbers within a microarray include 1.28 cm 2 A plurality of about 500,000 or more discrete features within a nucleic acid. Exemplary nucleic acid plurality numbers include, for example, about 1×10 5 pcs, 5×10 5 and 1×10 6 The term "polynucleotide" refers to a population of one or more different nucleic acid species. Thus, the definition of the term is intended to include all integer values ​​greater than two. The upper limit of the plurality of numbers can be set, for example, by the theoretical diversity of the oligonucleotide sequences in the nucleic acid sample.

[0135] As used herein, the term "double-stranded" when used to refer to a nucleic acid molecule means that substantially all of the nucleotides in the nucleic acid molecule are hydrogen-bonded to complementary nucleotides. A partially double-stranded nucleic acid may account for at least 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95% of the nucleotides that are hydrogen-bonded to complementary nucleotides.

[0136] As used herein, the term "single-stranded" when used in reference to a nucleic acid molecule means that substantially no nucleotides in the nucleic acid molecule are hydrogen bonded to complementary nucleotides.

[0137] As used herein, when used in conjunction with polynucleotides, the term "complementary" is intended to mean a polynucleotide comprising a nucleotide sequence that can selectively anneal to an identification region of a target polynucleotide under certain conditions. As used herein, the term "substantially complementary" and grammatical equivalents are intended to mean a polynucleotide comprising a nucleotide sequence that includes an identification region that can specifically anneal to a target polynucleotide under certain conditions. Annealing refers to the nucleotide base pairing interaction of one nucleic acid with another nucleic acid, which results in the formation of a duplex, triplex or other higher ordered structures. The primary interaction is typically with nucleotide base specificity through Watson-Crick and Hoogsteen type hydrogen bonds, for example, A:T, A:U and G:C. In some specific implementations, base stacking and hydrophobic interactions may also contribute to duplex stability. Conditions for annealing polynucleotides to complementary or substantially complementary regions of target nucleic acids are well known in the art, for example, as described in Nucleic Acid Hybridization, A Practical Approach, edited by Hames and Higgins, IRL Press, Washington, DC (1985), and Wetmur and Davidson, Mol. Biol. 31:349 (1968). Annealing conditions will depend on the specific application and can be routinely determined by those skilled in the art without undue experimentation.

[0138] As used herein, the term "hybridization" refers to a process in which two single-stranded polynucleotides are non-covalently bound to form a stable double-stranded polynucleotide. The resulting double-stranded polynucleotide is a "hybrid" or "duplex". Hybridization conditions will typically include a salt concentration of less than about 1M, more typically less than about 500mM and may be less than about 200mM. Hybridization buffers include buffered saline solutions, such as 5% SSPE, or other such buffers known in the art. The hybridization temperature may be as low as 5°C, but is typically greater than 22°C, and more typically greater than about 30°C, and typically exceeds 37°C. Hybridization is typically performed under stringent conditions, i.e., conditions under which the probe will hybridize to its target subsequence but will not hybridize to other non-complementary sequences. Stringent conditions are sequence-dependent and are different in different situations, and can be routinely determined by those skilled in the art.

[0139] As used herein, the term "dNTP" refers to deoxynucleoside triphosphates. Purine bases (Pu) include adenine (A), guanine (G) and their derivatives and analogs. Pyrimidine bases (Py) include cytosine (C), thymine (T), uracil (U) and their derivatives and analogs.

[0140] As used herein, the terms "ligation", "ligating" and their grammatical equivalents are intended to mean the formation of a covalent bond or linkage between the ends of two or more nucleic acids (e.g., oligonucleotides and / or polynucleotides). The nature of the bond or linkage can vary widely, and the linkage can be performed enzymatically or chemically. As used herein, the linkage is typically performed enzymatically to form a phosphodiester linkage between the 5' carbon terminal nucleotide of an oligonucleotide and the 3' carbon of another nucleotide. The term "ligation" also encompasses the non-enzymatic formation of a phosphodiester bond, and the formation of a non-phosphodiester covalent bond (such as a phosphorothioate bond, a disulfide bond, etc.) between the ends of an oligonucleotide.

[0141] As used herein, the term "each," when used in reference to a collection of items, is intended to identify a single item in the collection, but does not necessarily refer to every item in the collection unless the context clearly indicates otherwise.

[0142] As used herein, when used to refer to nucleic acids, the term "extension" is intended to mean the addition of at least one nucleotide or oligonucleotide to a nucleic acid. In a specific implementation, one or more nucleotides can be added to the 3' end of a nucleic acid, for example, by polymerase catalysis (e.g., DNA polymerase, RNA polymerase, or reverse transcriptase). Chemical or enzymatic methods can be used to add one or more nucleotides to the 3' or 5' end of a nucleic acid. One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (e.g., ligase catalysis) methods. Nucleic acids can be extended in a template-guided manner, whereby the extension product is complementary to the template nucleic acid, which hybridizes to the extended nucleic acid.

[0143] As used herein, the terms "tagmentation", "labeled fragments" or "tagmenting" refer to the conversion of nucleic acids (e.g., DNA) into adaptor-modified templates in solution ready for cluster formation and sequencing by using transposase-mediated fragmentation and tagging. The method generally involves fragmentation of nucleic acids and adaptor tagging by a transposome complex comprising a transposase complexed with a recognition sequence and an adaptor comprising a transposon end sequence. Tag fragmentation results in simultaneous fragmentation of the nucleic acid and attachment of the adaptor to the 5' end of the double-stranded DNA. After removal of the transposase, additional sequences are added to the ends of the adapted fragments by extension-ligation and PCR.

[0144] "Transposase" means an enzyme that can form a functional complex with a composition comprising a transposon end (e.g., a transposon, a transposon end, a transposon end composition), and, for example, in an in vitro transposition reaction, catalyzes the insertion or transposition of a composition containing a transposon end into a double-stranded target nucleic acid incubated therewith. Transposases as described herein may also include integrases from retrotransposons and retroviruses. Transposases, transpososomes, and transposome complexes are generally known to those skilled in the art, as shown in the disclosure of U.S. Patent Publication No. 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety. Although the specific implementations described herein relate to Tn5 transposases and / or highly active Tn5 transposases, it should be understood that any transposition system capable of inserting transposon ends into 5'-tags with sufficient efficiency and fragmenting target nucleic acids for their intended purpose can be used in the present invention. In certain embodiments, the preferred transposition system is capable of inserting the transposon ends into the 5'-tags and fragmenting the target nucleic acid in a random or nearly random manner.

[0145] As used herein, the term "transposition reaction" refers to a reaction in which one or more transposons are inserted into a target nucleic acid, for example, at a random site or a nearly random site. The essential components in the transposition reaction are a transposase and a DNA oligonucleotide (combined into a transposome before the tag fragmentation reaction), which exhibits the nucleotide sequence of the transposon, including the transposon sequence transferred and its complementary sequence (non-transferred transposon end sequence) and other components required to form a functional transposition or transposome complex. The DNA oligonucleotide may also contain additional sequences (e.g., adapter or primer sequences) as needed or desired. In some specific implementations, the methods provided herein are exemplified by the following: using a transposition complex formed by a highly active Tn5 transposase and a Tn5-type transposon end (Goryshin and Reznikoff, 1998, J. Biol. Chem., 273: 7367) or by a MuA transposase and a Mu transposon end comprising R1 and R2 terminal sequences (Mizuuchi, 1983, Cell, 35: 785; Savilahti et al., 1995, EMBO J., 14: 4893). However, any transposition system capable of inserting transposon ends into 5'-tags in a random or nearly random manner with sufficient efficiency and fragmenting target DNA for its intended purpose can be used in the present invention.

[0146] Examples of transposition systems known in the art that can be used in the methods of the present invention include, but are not limited to, Staphylococcus aureus Tn552 (Colegio et al., 2001, J Bacterid., 183:2384-2388; Kirby et al., 2002, Mol Microbiol, 43:173-186), TyI (Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-3772 and International Patent Application No. WO 95 / 23875), transposon Tn7 (Craig, 1996, Science. 271:1512; Craig, 1996, Review in: Curr Top Microbiol Immunol, 204:27-48), Tn10 and IS10 (Kleckner et al., 1996, Curr Top Microbiol Immunol, 204: 49-82), Mariner transposase (Lampe et al., 1996, EMBO J., 15: 5470-5479), Tci (Plasterk, 1996, Curr Top Microbiol Immunol, 204: 125-43), P factor (Gloor, 2004, Methods Mol Biol, 260: 97-114), TnJ (Ichikawa and Ohtsubo, 1990, J Biol Chem. 265: 18829-18832), bacterial insertion sequence (Ohtsubo and Sekine, 1996, Curr. Top. Microbiol. Immunol. 204: 1-26), retrovirus (Brown et al., 1989, Proc Natl Acad Sci USA, 86: 2525-2529), and yeast retrotransposons (Boeke and Corces, 1989, Annu Rev Microbiol. 43: 403-34). The method for inserting the transposon end into the target sequence can be performed in vitro using any suitable transposition system, for which a suitable in vitro transposition system is available or can be developed based on knowledge in the art. Generally speaking, a suitable in vitro transposition system for use in the methods provided herein uses at least a transposase and a transposon end of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity, and the transposase forms a functional complex with the transposon end, and the functional complex has a corresponding transposase that can catalyze a transposition reaction. Suitable transposase transposon end sequences that can be used in the present invention include, but are not limited to, wild-type, derivative, or mutant transposon end sequences that form a complex with a transposase selected from a wild-type, derivative, or mutant transposase.

[0147] As used herein, the term "transposome complex" refers to a transposase that is non-covalently bound to a double-stranded nucleic acid. For example, the complex can be a transposase that is pre-incubated with a double-stranded transposon DNA under conditions that support the formation of a non-covalent complex. The double-stranded transposon DNA may include, but is not limited to, Tn5 DNA, a portion of Tn5 DNA (e.g., a Tn5 recognition site), a transposon end composition, a mixture of transposon end compositions, or other double-stranded DNAs that can interact with a transposase (such as a highly active Tn5 transposase).

[0148] The compositions, systems, and methods described herein include particles having a shell surrounding a core, and the core can comprise one or more lyophilized microspheres (ie, the composition can comprise encapsulated lyophilized microspheres) or lyophilized material.

[0149] As used herein, "encapsulate", "encapsulated" and "encapsulation" include enclosing one or more materials or substances as described herein, such as microspheres. Microencapsulation as described herein refers to embedding at least one component (e.g., an active agent) into at least one other material (e.g., a shell material). Encapsulation according to the present disclosure includes, but is not limited to, bulk encapsulation, macroencapsulation, microencapsulation, nanoencapsulation, single molecule and ion encapsulation. According to the present disclosure, the compositions, systems and methods described herein have many benefits, including, for example, increasing the stability of microspheres, using macroencapsulation to allow multiple batches of cartridges, and using microencapsulation to allow simplified workflows and reduced reagent well numbers. The compositions, systems and methods described herein use encapsulation of particles that would otherwise react to pH changes to stabilize these buffers and improve SBS performance. The compositions, systems and methods described herein also use encapsulation to reduce the risk of static charge, which would otherwise cause difficulties in the distribution and dry mixing of microspheres during manufacturing.

[0150] As used herein, "microspheres" include spherical particles that include a shell and a core and have a diameter of 0.1 μm to 1,000 μm. For example, the microspheres may have a diameter of about 0.1 μm, 0.5 μm, 1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, or any diameter between about 0.1 μm and about 1,000 μm. In a specific implementation, the encapsulated microspheres have a diameter between about 100 μm and 1000 μm.

[0151] Microspheres may refer to lyophilized particles comprising an agent and / or an active ingredient. In certain embodiments, the microspheres may comprise a polymer shell (e.g., a biodegradable polymer and / or a water-soluble polymer), and optionally a core located inside the shell. Microspheres according to the present disclosure include microspheres prepared by conventional techniques known to those skilled in the art. For example, microspheres may be prepared by freezing a liquid into frozen pellets, followed by placing the frozen microspheres in a dryer (e.g., a rotary dryer).

[0152] As described herein, a "shell" includes a composition surrounding a core. In one embodiment, the shell includes the outer layer of the microsphere and, or in an alternative embodiment, includes the outer layer of the macrosphere. In one embodiment, the shell comprises a shell material, for example, selected from the group consisting of carrageenan, agarose, poloxamer, shellac, trehalose, paraffin, fatty acids (myristic acid, palmitic acid), fatty acid esters (Span 60, stearic acid PEG40 ester), gelatin, hydroxypropyl methylcellulose (HPMC), cellulose acetate, pullulan, oxygen scavengers, alginate, chitosan, starch film, benzoxaborole-poly (vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinyl pyrrolidone (PVP), poly (vinyl pyrrolidone-co-vinyl acetate), polyvinyl alcohol (PVA), poly (vinyl alcohol-graft-PEG), UCST polymer, LCST polymer, or any combination thereof.

[0153] Water-soluble (hydrophilic) polymers include carboxymethyl cellulose (CMC), carboxymethyl ethyl cellulose (CMEC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT) and ethyl hydroxyethyl cellulose (EHEC), pullulan, polyvinyl pyrrolidone, polyvinyl alcohol, copolymers of vinyl pyrrolidone or vinyl alcohol, polyacrylamide, polyacrylic acid, ethyl acrylic acid or methacrylic acid ( The water-soluble polymers include copolymers of poly(N-isopropylacrylamide)-co-poly(acrylic acid) n-butyl P(NIPAAm-co-nBu), P(NIPAAm-co-nBu) having a lower critical solution temperature (LCST) of 15°C.

[0154] The hydrophobic polymers include, but are not limited to, ethyl cellulose (EC), methyl ethyl cellulose (MEC), cellulose acetate (CA), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), polyvinyl acetate, fatty acids, fatty acid esters, or combinations thereof.

[0155] As used herein, polymers for use in shells or coatings may include other shell materials such as waxes (e.g., paraffin), fatty acids and fatty acid esters that can release agents at higher temperatures (above the melting point). Such other materials such as waxes, fatty acids and fatty acid esters can be used as natural materials or in combination with other polymers (e.g., cellulose acetate) for encapsulation.

[0156] The shell herein may comprise a temperature-triggered release polymer and / or a time-delayed release polymer, and optionally a functional additive. In various specific implementations, the shell as contemplated herein comprises one or more of an antistatic agent, a defoamer, a plasticizer, one or more UCST polymers, one or more LCST polymers, hydroxypropyl methylcellulose (HPMC), or a combination thereof.

[0157] Antistatic agents include ethoxylated PEI, VA64, IR, HEPES, MOPS and tetraalkylammonium chloride IO, IO 6786, Larostat 902A, sodium lauryl sulfate, sodium oleate, stearic acid, magnesium stearate, tribasic sodium citrate dehydrate, sodium L-ascorbate, 60. 60 Polyethyleneimine, Lauric acid diethanolamide, FC370, FC550, monostearate triglyceride, sebacate piperidinyl ester, vitamin E / tocopherol, trioleyl glycerol hydrophobic agent l, polyaniline, coumarin 6, Tween 17R4. (See, e.g., US20220331770, which is incorporated herein by reference).

[0158] Exemplary plasticizers include PEG, triethyl citrate (Citroflex 2), triacetin (triacetin), acetyl triethyl citrate (Citrofin A2), Carbowax 400 (polyethylene glycol 400), Makon 17R4, diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. See, for example, WO 2017 / 100861.

[0159] Defoaming agents include, but are not limited to, 13-sorbitan sesquioleate, silicones, fats, oils, waxes, fatty acids or esters, alcohols, sulfates, sulfonates, fatty acids, soaps, nitrogen-containing compounds, phosphates, polyglycols, sulfides, thio compounds, siloxanes, halogenated and inorganic compounds, ethylene oxide propylene oxide copolymers, Makon 17R4, polyether polyols such as Foamblast 882 (Emerald Foam Control), Erol DF 204K (Ouvrie PMC), DG436 (ODG Industries, Inc.), and KFO 880 (KABO Chemicals, Inc). See, for example, U.S. Pat. No. 8,076,113.

[0160] Anti-adherent agents include, but are not limited to, magnesium stearate, glyceryl monostearate, silicone oxide, titanium oxide, talc, or PEG stearate.

[0161] The shells herein may comprise a pH triggered release polymer. Exemplary pH dependent release polymers for use in the shell include polymers that dissolve at different pH levels (eg, between about pH 4.5 to about pH 9, or about pH 6 to about pH 8.5). Exemplary polymers include, but are not limited to, polymers soluble at pH 5 or below: cellulose acetate, timellitate, cellulose acetate phthalate, PVA phthalate, HPMC phthalate; chitosan (soluble below pH 6); polymers soluble between pH 5 and pH 6 or above: Kollicoat MAE 100, Kollicoat 30D, Eudragit L100, Eudragit L100-55, Eudragit L30D-55, Eudragit L12.5, polymers soluble between about pH 6 and about pH 7: Eudragit L100 / S100 1:1, Eudragit L / S12.5, Eudragit S100, Eudragit FS30D, Eudragit S12.5, shellac; carboxymethyl cellulose (soluble at pH 5 or below); 7.4 or above) or rennet casein (soluble at around pH 9).

[0162] The amount of shell material comprises any amount that is suitable for producing the shell result of expectation for example.In a specific implementation, shell material exists with the amount between about 1 % by weight and about 100 % by weight of shell.For example, shell material can exist with about 1 % by weight, 2 % by weight, 3 % by weight, 4 % by weight, 5 % by weight, 6 % by weight, 7 % by weight, 8 % by weight, 9 % by weight, 10 % by weight, 15 % by weight, 20 % by weight, 25 % by weight, 30 % by weight, 35 % by weight, 40 % by weight, 45 % by weight, 50 % by weight, 60 % by weight, 70 % by weight, 80 % by weight, 90 % by weight, 100 % by weight or any amount between shell.In a specific implementation, shell material exists with the amount between about 10 % by weight and about 90 % by weight of shell, or about 10 % by weight and about 80 % by weight, or about 10 % by weight and about 70 % by weight, or about 10 % by weight and about 60 % by weight, or about 10 % by weight and about 50 % by weight.

[0163] As described herein, the shell may include one layer or multiple layers of different composition. For example, the shell may include one, two, three, four, five, six, seven, eight, nine, ten, or more than ten layers. Each layer may include the same or different materials as the other layers present in the shell.

[0164] In various embodiments, the thickness of the shell is between about 1 μM and 50 μM, between about 1 μM and about 30 μM, between about 1 μM and about 20 μM, between about 5 μM and about 20 μM, between about 3 μM and about 10 μM, or between about 4 μM and about 6 μM, such as about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM. , about 8μM, about 9μM, about 10μM, about 11μM, about 12μM, about 13μM, about 14μM, about 15μM, about 16μM, about 17μM, about 18μM, about 19μM, about 20μM, about 21μM, about 22μM, about 23μM, about 24μM, about 25μM, about 30μM, about 35μM, about 40μM, about 45μM, or about 50μM.

[0165] In a specific implementation, the shell as described herein may include a shell additive. The shell additive may be present in an amount between about 0.01% w / w of the shell and about 99% w / w of the shell. In a specific implementation, the shell additive is present in an amount between about 10% w / w and about 90% w / w of the shell. In a specific implementation, the shell additive is present in an amount between about 10% w / w and about 40% w / w. In a specific implementation, the shell additive is a moisture-proof material present in an amount not exceeding 90% w / w of the shell. In a specific implementation, the shell additive is present in an amount of at least 10% w / w concentration of the shell. For example, in a specific implementation, the shell additive may be present in an amount between 0.1% w / w of the shell and about 15.0% w / w of the shell. For example, the shell additive may be present at about 0.01% w / w, 0.05% w / w, 0.1% w / w, 0.5% w / w, 1.0% w / w, 1.5% w / w, 2.0% w / w, 2.5% w / w, 3.0% w / w, 3.5% w / w, 4.0% w / w, 4.5% w / w, 5.0% w / w, 5.5% w / w, 6.0% w / w, 6.5% w / w, 7.0% w / w, The shell additive may be present in an amount of 7.5% w / w, 8.0% w / w, 8.5% w / w, 9.0% w / w, 9.5% w / w, 10.0% w / w, 10.5% w / w, 11.0% w / w, 11.5% w / w, 12.0% w / w, 12.5% ​​w / w, 13.0% w / w, 13.5% w / w, 14.0% w / w, 14.5% w / w, 15% w / w, or any amount therebetween. The amount of shell additive may be adjusted to accommodate a particular agent or combination of agents, or to accommodate a particular microsphere composition.

[0166] Exemplary shell additives include one or more of the following: polymers, copolymers, block copolymers, second polyvinyl alcohol (PVA), ammonium salts, conductivity promoters, stearic acid derivatives, oleic acid derivatives, lauric acid derivatives, polyether compounds, amino acids, tocopheryl acetate, piperidinium sebacate, sodium salts, buffers, chelating agents, imidazolium salts, polyaniline, or any combination thereof. In a specific implementation, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In a specific implementation, the stearic acid derivative or oleic acid derivative is selected from magnesium stearate, stearic acid PEG ester, stearic acid triglyceride, 60. 60. Triolein, 80, or any combination thereof. In some embodiments, the amino acid is selected from one or more of the following: leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In some embodiments, the sodium salt is selected from one or more of the following: sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In various embodiments, the buffer is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polyquaternary ammonium or (copolymer of vinyl pyrrolidone and quaternized vinyl imidazole) or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof. In various embodiments, the shell additive is magnesium stearate or stearic acid polyethylene glycol (PEG) ester.

[0167] As used herein, a "core" or "core region" includes any material within the surrounding shell. In various embodiments, the core comprises one or more lyophilized microspheres. In various embodiments, the core comprises lyophilized beads. In various embodiments, the core comprises beads made of non-lyophilized sugar or plastic, optionally wherein the reagent is coated on the surface of the non-lyophilized microspheres or beads and dried.

[0168] Lyophilization according to the present disclosure includes methods according to conventional techniques known to those skilled in the art. Lyophilization is also referred to herein as freeze drying. In the present disclosure, the term "lyophilize" or "lyophilizate" will be used as an equivalent term for "lyophilized," "lyophilisate," or "freeze dried," e.g., with respect to compositions, systems, or methods described herein.

[0169] Lyophilizable preparations can be reconstituted into solutions, suspensions, emulsions or any other form suitable for administration or use. Lyophilizable preparations 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 reconstitution volume of the lyophilized preparation. Preferably, the final reconstitution volume of the lyophilized preparation is less than the total liquid volume before lyophilization. Lyophilization methods are known to those of ordinary skill in the art and typically include sublimation of water from the frozen preparation under controlled conditions.

[0170] Freeze-dried preparations can be stored at a wide range of temperatures. Freeze-dried preparations can be stored below 25 ° C, for example, refrigerated at 2 ° C to 8 ° C, or stored at room temperature (e.g., about 25 ° C). Freeze-dried preparations are stored below about 25 ° C, more preferably at about 4 ° C to 20 ° C, about 4 ° C or less, about -20 ° C or less, about -40 ° C, about -70 ° C, or about -80 ° C. The stability of freeze-dried preparations can be measured in a variety of ways known in the art, for example, by the visual appearance of microspheres and / or agglomerates and / or by moisture content. The compositions of the present disclosure can also withstand temperature surges that may occur during transportation, for example, up to 70 ° C, for a short period of time.

[0171] The lyophilized formulation is typically rehydrated (interchangeably referred to herein as "reconstitution") by adding an aqueous solution to dissolve the lyophilized formulation. A variety of aqueous solutions can be used to reconstitute the lyophilized formulation, including water, saline, or another electrolyte or non-electrolyte diluent. The lyophilized formulation can be rehydrated with a solution comprising water (e.g., USP WFI or water for injection) or bacteriostatic water (e.g., USP WFI containing 0.9% benzyl alcohol). However, solutions comprising additives, buffers, excipients, and / or carriers can also be used and are described herein.

[0172] Freeze-dried or lyophilized preparations are usually prepared from liquids, i.e., solutions, suspensions, emulsions, etc. Therefore, the liquid to be subjected to freeze drying or lyophilization may preferably contain all the components desired in the final reconstituted liquid preparation. Therefore, when rehydrated or reconstituted, the freeze-dried or lyophilized preparation will produce the desired liquid preparation upon reconstitution. The core additive and / or shell additive, when present, may be incorporated into the reagent when the composition described herein is rehydrated.

[0173] In one embodiment, the core includes, but is not limited to, one or more reagents, such as one or more enzymes, salts, surfactants, buffers, enzyme inhibitors, primers, nucleotides, organic permeates, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof.

[0174] As used herein, the term "reagent" describes a single agent or a mixture of two or more agents that can be used to react with a sample, interact with a sample, dilute a sample, or add to a sample, and may include agents for nucleic acid reactions, including, for example, buffers, chemicals, enzymes, polymerases, primers (including primers less than 60 base pairs in size), template nucleic acids, nucleotides, labels, dyes, or nucleases. In certain embodiments, reagents as described herein may include enzymes such as polymerases, ligases, recombinases, or transposases; binding partners such as antibodies, epitopes, 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 analyzing biological fluids. According to some embodiments disclosed herein, reagents may include one or more beads, especially magnetic beads, according to specific workflows and / or downstream applications.

[0175] In a specific implementation, the reagent according to the present disclosure is a polymerase. The polymerase according to the present disclosure may include any polymerase that can tolerate the incorporation of phosphate-labeled nucleotides. Examples of polymerases that may be useful according to the present disclosure include, but are not limited to, Q5 polymerase, tTaq608, 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, polβ and polγ. Polymerases designed to have specific properties can be used.

[0176] Primers as disclosed herein include nucleic acid molecules that can hybridize with a target sequence of interest. In various implementations, primers can be used as substrates to which nucleotides can be polymerized by a polymerase. However, in some examples, primers can be incorporated into a synthetic nucleic acid chain and provide a site to which another primer can hybridize to initiate the synthesis of a new chain complementary to the synthetic nucleic acid molecule. Primers can include any combination of nucleotides or their analogs. In some implementations, primers are single-stranded oligonucleotides or polynucleotides.

[0177] Non-limiting examples of nucleic acid molecules that can be encapsulated within microspheres include those described above, for example, DNA, such as genomic or cDNA; RNA, such as mRNA, sRNA or rRNA; or a hybrid of DNA and RNA. The nucleic acid molecules may also contain labeled nucleotides.

[0178] The term "salt" 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 pharmaceutically acceptable non-toxic acids, including inorganic acids and organic acids. Salts that can be used as reagents herein include NaCl, KCl, MgCl 2 and / or MgSO 4 .

[0179] Any surfactant known to those skilled in the art can be used as the reagent in the microsphere. The surfactant can be nonionic or ionic (particularly cationic or anionic) or can be zwitterionic. Examples of suitable surfactants include, but are not limited to, polyacrylate surfactants, silicone surfactants and / or other commercially available surfactants or detergents. Examples of cationic surfactants are cetyl dimethyl ammonium acetamide, octadecyl-dimethyl ammonium acetamide, tetradecyl-dimethyl ammonium acetamide, dodecyl-dimethyl ammonium acetamide, cetyl trimethyl ammonium, octadecyl-trimethyl ammonium, tetradecyl-trimethyl ammonium, dodecyl-trimethyl ammonium, dimethyl di (octadecyl) ammonium, di (octadecyl) dimethyl ammonium and mixtures thereof. Suitable sources of these cations of cationic surfactants include, but are not limited to, alkyl trimethyl ammonium salts: such as cetyl trimethyl ammonium bromide (CTAB) or cetyl trimethyl ammonium chloride (CTAC); cetyl pyridinium chloride (CPC); dimethyl dioctadecyl ammonium chloride; dioctadecyl dimethyl ammonium bromide (DODAB); cetyl dimethyl acetamide ammonium bromide; or other cationic surfactants, again, including lipids. Alternatively, the surfactant may be benzyl hexadecyl dimethyl ammonium chloride (BHDC). The core may comprise an anionic surfactant containing an anionic functional group at one end, such as sulfate, sulfonate, phosphate, and carboxylate functional groups. An example of an anionic surfactant is sodium dodecyl sulfate. The core may comprise a neutral surfactant, such as polyethylene glycol lauryl ether.

[0180] The microspheres may further or in the alternative include enzyme inhibitors, molecular probes, crowding agents, organic osmolytes, cyclodextrins, adenosine triphosphate (ATP), ethylenediaminetetraacetic acid (EDTA), creatine kinase, phosphocreatine, palladium, lipoic acid, hexaethylene glycol, trihydroxypropanephosphine, sodium ascorbate, or any combination thereof. Enzyme inhibitors as described herein include any molecule that binds to an enzyme and reduces its activity, such as proteinase K or sodium dodecyl sulfate. Molecular probes as described herein include, for example, digoxin, 8-anilinonaphthalene-1-sulfonic acid ("ANS"), porphyrins, BODIPY, cyanines, or any combination thereof. Crowding agents as described herein include any crowding agent known to those skilled in the art. Examples include, but are not limited to, polyethylene glycol, ficoll, dextran, and serum albumin.

[0181] In various specific implementations, the one or more tag fragmentation reagents comprise bead-linked transposomes (BLTs), primers, probes, transposases, ligases, buffers, divalent cations, Tris buffers, cobalt buffers, and / or lyophilized reagents. In various specific implementations, the one or more tag fragmentation reagents comprise bead-linked transposomes (BLTs), transposases, primers, buffers, Tris buffers, cobalt buffers, and / or lyophilized reagents. In various specific implementations, the tag fragmentation reagents comprise bead-linked transposomes, primers, cobalt chloride, and trehalose. In various specific implementations, the tag fragmentation reagents comprise bead-linked transposomes, magnesium sulfate, Tris buffer, and trehalose.

[0182] In various specific implementations, the one or more tag fragmentation poisoning reagents include chelators, salts, primers, polymerases (e.g., Q5, tTaq), coenzymes (e.g., NAD+), deoxynucleotide triphosphates (dNTPs), Tris buffer, potassium buffer, ammonium salts, and / or lyophilized reagents. In various specific implementations, the tag fragmentation poisoning reagents include potassium chloride, ammonium sulfate, cyclopentane tetrahydrochloride, α-cyclodextrin, hydroxypropyl-β-cyclodextrin (HPBCD), dNTPs, and / or trehalose. In various specific implementations, the tag fragmentation poisoning reagents include Tris buffer, potassium chloride, ammonium sulfate, cyclopentane tetrahydrochloride, α-cyclodextrin, dNTPs, and / or trehalose. In various specific implementations, the tag fragmentation poisoning reagents include Tris buffer, potassium chloride, sodium dodecyl sulfate, dNTPs, and trehalose. In various specific implementations, the tag fragmentation poisoning reagents include potassium chloride, ammonium sulfate, SDS, dNTPs, and trehalose.

[0183] In various embodiments, the extension-link and PCR reagents include one or more of a magnesium buffer, a polymerase (e.g., Q5, tTaq), a coenzyme (e.g., NAD+), a deoxynucleotide triphosphate (dNTP), a ligase, a primer, a probe, a divalent cation, a Tris buffer, an ammonium salt, betaine, a surfactant, or a lyophilized reagent. In various embodiments, the extension-link and PCR reagents include one or more of magnesium sulfate, Q5 without glycerol hot start, tTaq 608 polymerase, ampligase, NAD+, betaine, Tween-20, and / or trehalose. In various embodiments, the extension-link and PCR reagents include α-cyclodextrin, Q5 without glycerol hot start, tTaq 608 polymerase, ampligase, NAD+, betaine, Tween-20, and / or trehalose. In various embodiments, the extension-ligation and PCR reagents include glycerol-free hot start Q5, tTaq 608 polymerase, ampligase, NAD+, betaine, alpha-cyclodextrin, Tween-20, and trehalose.

[0184] Those skilled in the art of sequencing technology will appreciate that there are additional reagents not expressly described herein that may be used in the compositions, systems, and methods of the present disclosure.

[0185] In a specific implementation, the core as described herein may further comprise one or more additives. The one or more additives in the core improve the ability to control the release of one or more lyophilized microspheres. In a specific implementation, the additive is selected from one or more sugars, amino acids, polymers, mesoporous silica, quaternary amines, lyophilization agents, or any combination thereof.

[0186] Lyophilizing agents (which may also be referred to as excipients or cryoprotectants, lyoprotectants or cryoprotectants) generally help preserve the structure of proteins, liposome bilayers and other substances during freezing. Lyoprotectants stabilize these substances during drying, especially freeze drying. In freeze drying, lyoprotectants may also be considered cryoprotectants, so the term "cryoprotectant" used herein may also include lyoprotectants or lyoprotectants. Examples of lyoprotectants include polyhydroxy compounds such as sugars (monosaccharides, disaccharides and polysaccharides), trehalose and sucrose, and polyols such as glycerol, mannitol, sorbitol and their derivatives, which are natural lyoprotectants.

[0187] In one embodiment, the cryoprotectant and / or lyoprotectant is selected from the group consisting of trehalose, sucrose, mannitol, maltose, maltodextrin, dextran, cyclodextrin, inulin, raffinose, sorbitol, inositol, glucose and mannose. In one embodiment, the cryoprotectant is trehalose. Trehalose, also known as α, α-trehalose; aD-pyranoglucopyranosyl-(1→1)-aD-pyranoglucoside, mycose or tremalose, is a natural α-linked disaccharide formed by an α, α-1, 1-glucoside bond between two α-glucose units. Trehalose can exist in the form of anhydrous or dihydrate.

[0188] In a specific implementation, when the additive comprises a lyophilized reagent, it can be sugar, wherein the sugar is selected from trehalose, mannitol, cyclodextrin, dextran, sucrose or any combination thereof. In another specific implementation, when the additive comprises an amino acid, the amino acid has a hydrophobic side chain. In another specific implementation, when the additive comprises a polymer, the polymer is selected from polyvinyl pyrrolidone, polyvinyl alcohol or a combination thereof. In some specific implementations, the additive can be, for example, one or more copolymers, ionic liquids or any combination thereof. The additive can be added in any amount suitable for producing a desired effect, for example, between about 0.1 % by weight to about 50 % by weight of the core. In one embodiment, the concentration of the additive in the core is about 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or any amount therebetween.

[0189] The composition (i.e., comprising freeze-dried microspheres or beads, whether encapsulated or not encapsulated) can be suitable for covering one or more reagents and suitable for use in the preparation of a library for sequencing. Any suitable size or volume. In a specific implementation, the composition has a reagent volume between about 0.1 μL and about 50 μL in the core region. For example, the composition (i.e., comprising freeze-dried microspheres that are encapsulated or not encapsulated) can have an active reagent volume of about 0.1 μL, 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, or any volume between about 0.1 μL and about 50 μL. In a specific implementation, the active reagent volume is between about 10 μL and about 40 μL. The composition (i.e., encapsulated or unencapsulated lyophilized microspheres) can have, for example, a diameter of about 2 μm to about 120 μm, such as, for example, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm or 120 μm, or a diameter within a range defined by any two of the above values.

[0190] The compositions described herein may include additional agents in the shell of the microspheres. In one embodiment, the encapsulated microspheres include agents or additives in the microsphere shells. The agents in the shells may include, for example, any of the aforementioned agents or additives. In one embodiment, the shells do not contain nucleic acid molecules, for example, the shells do not contain DNA. In one embodiment, the shells contain more than one agent and, or in the alternative, contain more than one additive.

[0191] The compositions described herein (i.e., comprising lyophilized microspheres, beads or cakes, whether encapsulated or not) can be used for multiple sequential co-assays, including sequential lysis, DNA analysis, RNA analysis, protein analysis, tag fragmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, SBS technology, assay for transposase accessible chromatin using sequencing (ATAC-seq), contiguity-preserving transposition sequencing (CPT-seq), or any combination thereof. In a specific implementation, the composition is used to perform multiple co-assay reactions. The compositions, systems and methods described herein can improve sequencing quality, enable one-pot library preparation, and simplify manufacturing. As used herein, the term "one-pot reaction" may also be referred to as a "transfer-free reaction".

[0192] The compositions, systems and methods described herein can be prepared for various stages of sequencing, including but not limited to sample extraction, library preparation, enrichment and sequencing. In the sample extraction composition, the core can include enzymes, salts, surfactants, buffers and any combination thereof. Sample extraction can be performed at a pH of about 7.5, with a reaction volume between about 1 mL and about 5 mL. In the library preparation composition, the freeze-dried microspheres and / or cores can include enzyme inhibitors, salts, primers, enzymes, nucleotides, organic permeates, magnetic beads and any combination thereof. Library preparation can be performed at a pH of about 8, with a reaction volume of about 0.05 mL. In the enrichment composition, the freeze-dried microspheres and / or cores can include nucleotides, molecular probes, enzymes, magnetic beads, crowding agents and any combination thereof. Enrichment can be performed at a pH of about 8.5, with a reaction volume between about 0.1 mL and about 0.2 mL. In the clustering composition, the freeze-dried microspheres and / or cores can include salts, enzymes, one or more nucleotides, small molecules, surfactants, primers and any combination thereof. Clustering can be performed at a pH of about 8.6 with a reaction volume between about 1 mL and about 5 mL. In the sequencing composition, the lyophilized microspheres and / or cores can include labeled nucleotides, fluorophores, surfactants, salts, enzymes, small molecules, and any combination thereof. Sequencing can be performed at a pH of about 7 to about 10 with a reaction volume between about 30 mL and about 100 mL.

[0193] In various embodiments, the lyophilized microspheres and / or shells can be rehydrated at a pH between 1 and 14. In one embodiment, the shell can include one or more shell layers, and each layer can be rehydrated under the same or different conditions. For example, the shell can include multiple layers that are rehydrated under different conditions. In one embodiment, the shell can include two or more layers (e.g., three or more layers) that are released at different pH levels, for example, one layer can be released at pH 5, one layer can be released at pH 5.5, one layer can be released at pH 6, one layer can be released at pH 6.5, one layer can be released at pH 7, one layer can be released at pH 7.5, and / or one layer can be released at pH 8.

[0194] In various embodiments, 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 secretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural effusion, transudate, exudate, gallbladder fluid, bile, urine, gastric juice, intestinal juice, stool samples, liquid containing single or multiple cells, liquid containing organelles, fluidized tissue, fluidized organisms, liquid containing multicellular organisms, biological swabs, and biological washes. Biological samples can include nucleic acids, such as DNA, genomic DNA, RNA, mRNA, or analogs thereof; nucleotides, such as deoxyribonucleotides, ribonucleotides, or analogs thereof, such as analogs having terminator portions, such as those described in Bentley et al., “Accurate Whole Human Genome Sequencing Using Reversible Terminator Chemistry,” Nature 456:53-59 (2008); WO / 2013 / 131962; U.S. Pat. No. 7,057,026; WO / 2008 / 042067; WO / 2013 / 117595; U.S. Pat. Nos. 7,329,492, 7,211,414, 7,315,019, 7,405,281; and U.S. Patent Publication No. 20080108082, all of which are hereby incorporated by reference in their entirety.

[0195] Any suitable method can be used to form the microspheres. Standard microsphere manufacturing techniques will be known to those skilled in the art and include preparing frozen pellets and placing those pellets in a dryer as described herein. A variety of microspheres are contemplated according to the compositions, systems and methods of the present disclosure, and the microspheres include, for example, sustained release, immediate pulse, timed pulse release, organic acid Beads and alkaline buffer Bead microspheres. The compositions, systems, and methods described herein also encompass various types of encapsulation, including but not limited to bulk encapsulation, microencapsulation, nanoencapsulation, single molecule encapsulation, and ion encapsulation.

[0196] Modifications to standard microsphere production can be used to make compositions as described herein. For example, one or more additional feed buffer tanks and one or more suitable nozzles and / or nozzle plates can be added to a standard microsphere production apparatus. Other modifications can be made, particularly to the curing system to produce various types of shells, and include compounds such as hydrocolloids, alginates, and pectins as described herein.

[0197] As used herein, the rehydration (or reconstitution) solution (e.g., a sample with DNA) may include water, deionized water, saline solution, acidic solution, alkaline solution, detergent solution, and / or buffer. In a preferred embodiment, the rehydration solution is water or a buffer. Additional additives as described herein may be provided in the rehydration solution to further improve the control of microsphere release.

[0198] In various specific implementations, the pH in the rehydration solution is between about 6.0 and about 10.0, or between about 7.0 and about 8.0. The pH of the rehydration solution can be, for example, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0 or any amount therebetween. The rehydration time will vary according to composition content and reaction conditions (e.g., reagent, temperature, pH). In various specific implementations, the rehydration time can be between 0.1 second and 10 hours. For example, the rehydration time can be about 0.1 second, 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 amount of time therebetween.

[0199] In some embodiments, the first triggered release condition is effective to release the lyophilized microsphere (e.g., the second lyophilized microsphere as described in the composition or system). The lyophilized microsphere is consistent with the lyophilized microspheres and compositions described herein.

[0200] In some embodiments, the second triggered release condition is effective to release the lyophilized microsphere (e.g., a third lyophilized microsphere as described in the composition or system), wherein the content of the third lyophilized microsphere is different from the content of the second lyophilized microsphere. The third lyophilized microsphere is consistent with the lyophilized microspheres and compositions described herein.

[0201] In some embodiments, the first trigger release condition is effective to release the lyophilized microsphere (e.g., the second lyophilized microsphere as described in the composition or system), and the first trigger release condition is preceded by a second release trigger mechanism that causes the first trigger release condition. The lyophilized microsphere is consistent with the lyophilized microspheres and compositions described herein.

[0202] In some embodiments, the third triggered release condition is effective to release the lyophilized microsphere (e.g., the third lyophilized microsphere as described in the composition or system), wherein the content of the third lyophilized microsphere is different from the content of the second lyophilized microsphere. The third lyophilized microsphere is consistent with the lyophilized microspheres and compositions described herein.

[0203] One way to enable the release of lyophilized reagents in sequence is to trigger the release by temperature, for example, by immersing the particles comprising the first or second shell and filled with microspheres in paraffin or another shell agent as described herein. Similarly, such methods enable the release of time triggering by adding additives (e.g., amino acids) to the rehydration solution. In addition to time and (or in an alternative) temperature or in place of time and temperature, other reaction characteristics can be changed. For example, pH and humidity can be changed to further control the release of one or more encapsulated microspheres and the reagents contained therein.

[0204] In one example, two compositions (e.g., particles) having a shell and a core, respectively, can be in a single container, and those particles can be dissolved with different triggers. In another example, two or more particles can be in the container, and the second particle dissolves after the first particle is released when in contact with a liquid, and then the third particle dissolves after the second capsule is released when in contact with a liquid, and this is repeated for as many particles as there are in a particular container. These implementations can also be triggered by other modifications such as temperature changes (heating or cooling) or pH.

[0205] Provided herein is a system comprising a composition as described herein. The system comprises one or more compositions as described herein and one or more containers, wherein the one or more compositions are placed in one or more containers under conditions of effectively forming a sequential system for preparing a DNA library. In various specific implementations, the system includes one or more containers for accommodating the composition, wherein the one or more containers include PCR tubes, vials, microtubes, flow cells, multi-well plates, glass tubes, cross-chamber membrane / screen inserts, cartridges or microfluidic chips.

[0206] The system may also include a temperature controller or sensor. The temperature controller can be used to change or adjust the temperature of the system to further control the release of the various components of the compositions described herein. For example, the temperature controller can be used to accelerate or slow down the release from the first shell or the second shell. Similarly, the temperature controller can be used to accelerate or slow down the release of the inner core to promote or control the release of one or more reagents. In a specific implementation, the system includes a temperature controller on a container in the system. For example, the temperature controller may include a resistance heater near the wall of the container (such as a cartridge, a tube, a chip or a hole) to provide heat thereto. The temperature controller may also include a temperature sensor. The temperature controller may also include a circuit that starts and stops the heater to keep the hole at a specific temperature.

[0207] Methods for generating DNA libraries

[0208] Also contemplated herein is a method of producing a DNA library preparation, the method comprising providing a composition as described herein, and mixing the composition with a solution under a first trigger condition effective to control the release of an agent from the lyophilized material and / or control the release of one or more lyophilized microspheres or lyophilized beads from the core of the composition.

[0209] The methods contemplated herein are performed according to the specific implementations previously described, particularly with respect to the characteristics of the one or more lyophilized microspheres or beads and their shells, cores and / or encapsulations.

[0210] In various implementations, a method for preparing a DNA library is provided, the method comprising: adding a sample comprising DNA to a container comprising a composition or system as described herein, wherein the addition of the sample triggers the dissolution of a first lyophilized material to release one or more tag fragmentation reagents into the container to trigger a tag fragmentation reaction;

[0211] allowing the tag fragmentation reagent to continue under conditions and for a sufficient period of time to complete tag fragmentation of the DNA in the sample;

[0212] initiating a first triggered release condition, wherein the first triggered release condition of the first shell causes the one or more tag fragmentation poisoning agents of the first core to be released into the container to induce a tag fragmentation poisoning reaction; and

[0213] The tag fragmentation poisoning reaction is allowed to proceed under conditions and for a sufficient period of time to initiate a second triggered release condition of the second shell, wherein one or more extension-link and PCR reagents are released from the second core, wherein the extension-link and PCR reagents undergo gap filling and PCR amplification.

[0214] In various embodiments, the method further comprises isolating the DNA and sequencing the DNA library.

[0215] In various embodiments, the tag fragmentation reaction is performed between 37°C and 42°C, such as at 37°C, 38°C, 39°C, 40°C, 41°C or 42°C.

[0216] In various embodiments, the tag fragmentation reaction is performed for up to 15 minutes, such as 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0217] In various embodiments, the tag fragmentation reaction is performed at between 37°C and 41°C for 5 minutes.

[0218] In various specific implementations, the first release condition is a temperature release condition, a pH release condition or a time release condition. In various specific implementations, the first release condition is a temperature release condition.

[0219] In various specific implementations, the temperature of the first temperature release condition is equal to or higher than about 45°C, such as 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C or 65°C. In various specific implementations, the first release condition is met for up to about 10 minutes, such as about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes or about 10 minutes. In various specific implementations, the condition of the first trigger release condition is about 45°C for about 5 minutes.

[0220] In various specific implementations, the tag fragmentation poisoning reaction is performed between about 45° C. and about 65° C., for example, at about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., or about 65° C. In various specific implementations, the tag fragmentation poisoning reaction is performed between about 60° C. and about 65° C., for example, at about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., or about 65° C.

[0221] In various embodiments, the tag fragmentation poisoning reaction is performed for up to about 10 minutes, such as about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes.

[0222] In various embodiments, the tag fragmentation poisoning reaction is performed at about 60° C. to 65° C. for about 1 minute to 10 minutes.

[0223] In various specific implementations, the second release condition is a temperature release condition, a pH release condition or a time release condition. In various specific implementations, the second release condition is a time release condition.

[0224] In various embodiments, the time release condition is between about 1 minute and about 15 minutes, or between about 5 minutes and about 15 minutes, such as about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes. In various embodiments, the time release condition is between about 1 minute and about 15 minutes, or between about 5 minutes and about 15 minutes at pH and / or between about 45°C and about 60°C. In various embodiments, the time release condition is between about 45°C and about 60°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or about 6 minutes.

[0225] In various implementations, the time release condition is met when the tag fragmentation poisoning reaction is cooled to about 50° C. and maintained for about 1 minute to about 10 minutes.

[0226] In various implementations, the time release conditions are about 1 minute to about 3 minutes at about 50°C.

[0227] Sequencing of the DNA library can be performed using any suitable sequencing technology, and methods for determining the sequence of amplified adapter-target-adapter molecules are known in the art and described in, for example, U.S. Pat. No. 8,053,192, WO2016 / 130704, U.S. Pat. Nos. 8,895,249 and 9,309,502.

[0228] The compositions, systems and methods for DNA library preparation described herein can also be used within the workflow of other DNA library preparation methods, such as spatial indexing, sequencing by synthesis (SBS), and other next generation sequencing (NGS) technologies. The compositions comprising lyophilized material as described herein are adapted to comprise reagents for use in spatial indexing workflows (e.g., tag fragmentation reagents, extension-ligation reagents), and can be added to an array or substrate suitable for performing the desired reaction.

[0229] Exemplary SBS systems and methods are described in U.S. Patent Publication Nos. 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2012 / 0270305, and 2013 / 0260372, U.S. Patent No. 7,057,026, WO 05 / 065814, U.S. Patent Publication Nos. 2005 / 0100900, WO 06 / 064199, and WO 07 / 010,251, U.S. Patent Publication No. 2013 / 0079232.

[0230] For the ligation reaction, a variety of ligases may prove useful in the method. In various embodiments, the polymerase is T4 DNA ligase, T4 RNA ligase 2 (T4Rnl2), SplintR DNA ligase, E. coli DNA ligase, Ampligase, or R2D ligase. In various embodiments, the ligation reaction is performed at between 16°C and 50°C, between 25°C and 45°C, or between 30°C and 42°C. In various embodiments, the ligation reaction is performed at 37°C.

[0231] In various implementations, the separated PCR templates are placed in tubes for DNA library preparation.

[0232] In some implementations, the DNA in the sample undergoes a PCR cleanup reaction. In various implementations, the PCR cleanup includes PCR cleanup using dual solid phase reversible immobilization (dSPRI) to remove small and large DNA fragments, and to remove unused primers and bases.

[0233] Reagent test kit

[0234] Kits and articles are also encompassed herein. Such kits may include carriers, packages or containers separated to receive one or more containers, such as vials, tubes, etc., each container including an element in the independent elements to be used in the methods described herein. Suitable containers include, for example, PCR tubes, vials, microtubes, flow cells, multi-well plates (e.g., 96-well plates or 384-well plates), glass tubes, cross-chamber membrane / screen inserts, cartridges or microfluidic chips. The container may be formed of a variety of materials, such as glass or plastic. For example, the one or more containers may include one or more spatially addressable probes disclosed herein, optionally in a composition or in combination with another agent as disclosed herein. Optionally, the container has a sterile inlet (e.g., the container may be an intravenous infusion bag or a vial with a stopper that can be pierced by a hypodermic needle). Optionally, such kits include an identification description or label or instructions for use thereof in the methods described herein.

[0235] The kit will typically include one or more additional containers, each with one or more of the various materials (such as reagents and / or devices, optionally in concentrated form) that are desirable from a commercial and user standpoint for use with the spatially addressable probes described herein. Non-limiting examples of such materials include, but are not limited to: buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials, and / or tube labels listing the contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0236] The marking may be on or associated with the container. The marking may be on the container where the letters, numbers or other characters forming the marking are attached, molded or etched into the container itself, or may be associated with the container when it is present in a receptacle or carrier that also supports the container, such as in a package insert. The marking may be used to indicate that the contents are to be used for a particular application. The marking may also indicate the direction in which the contents are to be used, such as in the methods described herein.

[0237] In the present disclosure, reference is made to the accompanying drawings which form a part of the present disclosure and in which are shown by way of illustration specific implementations that may be implemented. These embodiments are described in detail to enable those skilled in the art to practice the present disclosure, and it should be understood that other implementations may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the present disclosure. Therefore, the following description of the embodiments should not be construed as limiting.

[0238] The present disclosure can be further illustrated by reference to the following examples.

[0239] Example

[0240] Example 1

[0241] In experiments to determine whether lyophilized microspheres could help reduce the number of steps in DNA library preparation and improve the overall workflow process, reagents in a DNA library preparation protocol were lyophilized and their activities were compared to non-lyophilized reagents in the same workflow.

[0242] A mixture of tag fragmentation reagents including bead-linked transposomes and cobalt chloride was mixed with trehalose and lyophilized to form lyophilized microspheres (Table 1). In a second formulation, bead-linked transposomes, Tris buffer, and magnesium sulfate were mixed with trehalose and lyophilized.

[0243] Table 1. Labeled fragmented microsphere preparations

[0244] Tag Fragment Buffer (2X) concentration unit Bead-linked transposomes 20 u Cobalt chloride 5-10 mM Trehalose 550 mM Total solute content (%) 19.96

[0245] Tag Fragment Buffer (2X) concentration unit Bead-linked transposomes 20 u Tris HCl, pH 7.5 20 mM Magnesium sulfate 4 mM Trehalose 560 mM Total solute content (%) 20.53

[0246] A second set of reagents is mixed in a particle having a shell and a core to form a tag fragmentation poisoning buffer. In one embodiment, the core comprises potassium chloride, ammonium sulfate, cyclamen tetrahydrochloride, dNTPs, and trehalose in approximately 25 μL. In another formulation, the core comprises potassium chloride, ammonium sulfate, SDS, dNTPs, and trehalose.

[0247] Table 2. Labeled fragmented poison microsphere preparations

[0248] Tn5 poison (5X) concentration unit Potassium chloride 375 mM Tris, pH 8.8 475 mM Ammonium sulfate 50 mM Cyclotrimethylenetetrahydrochloride 37.5 mM dNTP 1 mM Trehalose 400 mM Total solute content (%) 41.17

[0249] Tn5 poison (5X) concentration unit Potassium chloride 375 mM Ammonium sulfate 50 mM SDS 0.5 ％ dNTP 1 mM Trehalose 750 mM Total solute content (%) 29.68

[0250] The shell comprises a temperature-triggered release polymer, such as a UCST polymer, i.e., poly(acrylonitrile-co-acrylamide) and poly(N-acryloyl glycinamide-co-styrene) or a LCST polymer, i.e., poly(N-isopropyl acrylamide). Alternatively, the shell comprises a mixture of wax or a time-release polymer (i.e., HPMC), cellulose acetate, and wax (i.e., paraffin, myristic acid). In one embodiment, the shell comprises polymer materials Efka 6783, Makon 17R4, magnesium stearate, and an upper critical solubility temperature (UCST) polymer 1 (e.g., poly(acrylonitrile-co-acrylamide) or poly(N-acryloyl glycinamide-co-styrene). The reagents for the core are lyophilized to obtain lyophilized microspheres, which are then encapsulated in the shell. The shell may also comprise stearic acid PEG ester.

[0251] The third group of reagents used in the extension-link and PCR reactions for DNA library preparation are mixed in a shell and core format to form particles containing freeze-dried microspheres. The extension-link and PCR reagents magnesium sulfate, glycerol-free hot start Q5, tTaq608 polymerase, ampligase, NAD+, betaine, Tween-20, HPBCD, and trehalose are freeze-dried. In another preparation, the extension-link and PCR reagents Tris buffer, glycerol-free hot start Q5, tTaq 608 polymerase, ampligase, NAD+, betaine, Tween-20, αCD, and trehalose are freeze-dried. The freeze-dried microspheres are encapsulated in a double-layer shell, which contains a time-release polymer (i.e., HPMC), cellulose acetate as an inner shell, and a temperature-release polymer (i.e., wax, UCST, or LCST polymer) as an outer shell. Functional additives such as anti-sticking agents, antistatic agents, defoamers and plasticizers can be incorporated into a polymer mixture containing Efka 6783, Makon 17R4, magnesium stearate and UCST polymer 2 (e.g., poly(acrylonitrile-co-acrylamide) or poly(N-acryloylglycinamide-co-styrene) and hypromellose (Table 3).

[0252] Table 3. Extension-ligation and PCR hybrid microsphere preparations

[0253] Extension-ligation and PCR (2X) concentration unit Magnesium sulfate 4 mM NAD+ 1 mM Betaine 2000 mM Twain 20 0.2 ％ Glycerin-free Hot Start Q5 Pol 4 u tTaq 608Pol 1 u Ampligase 0.2 u HPBCD 100 mM Trehalose 100 mM Total solute content (%) 42.52

[0254] Extension-ligation and PCR (2X) concentration unit Tris, pH 8.8 190 mM NAD+ 1 mM Betaine 500 mM Twain 20 0.2 ％ Glycerin-free Hot Start Q5 Pol 4 u tTaq 608Pol 1 u Ampligase 0.2 u α-Cyclodextrin 4.5 ％ Trehalose 336 mM Total solute content (%) 24.43

[0255] Lyophilized microspheres containing tag fragmentation reagents and particles containing tag fragmentation poisoning reagents or extension-ligation and PCR mixtures are placed separately in individual PCR tubes for initial library preparation experiments. In certain embodiments, the stabilizer used in the extraction includes alkyl polyglucoside and / or PEG 6K.

[0256] The sample (50 μL, about 150 ng DNA) containing genomic DNA is inserted into the tube, and the aqueous solution reconstitutes the freeze-dried microspheres containing the tag fragmentation reagent. The tag fragmentation reaction is carried out at 37 ° C for 5 minutes to complete the fragmentation of the genomic DNA and the addition of DNA adapters (such as A14 and B15) (see, for example, US20180245069 incorporated by reference) to the DNA fragments. In order to terminate the tag fragmentation reaction and remove the Tn5 transposon from the fragmented DNA, the temperature is raised to 65 ° C and maintained for 1 minute. The increase in temperature triggers the dissolution of the shell on the poisoned microsphere particles and the release of the tag fragmentation poisoning reagent from the core to the PCR tube. Complete the removal of Tn5, and the DNA sample is ready for extension-linking and PCR. The temperature of the mixture is reduced to 50 ° C, which triggers the dissolution of the shell of the microsphere particles for extension-linking and PCR mixture, and the core releases its contents into the PCR tube for gap filling and linking.

[0257] Comparison of tag fragmentation reactions using liquid or lyophilized reagents showed essentially no difference in reaction outcomes when lyophilized reagents were used for tag fragmentation ( Figure 1 A comparison of the tag fragmentation poisoning reagents when lyophilized or in liquid form indicated that DNA yields may be slightly lower when lyophilization buffer is used ( Figure 2 For PCR mix reactions, lyophilized and liquid forms of the reagents produced similar results ( Figure 3 ).

[0258] Example 2

[0259] When using multiple microsphere release conditions in a single-pot assay, this may benefit from temperature and pH changes to accommodate the different release conditions. The risk of these changes is that by cooling the reaction or changing the pH, the material may become insoluble and precipitate into the reaction mixture and interfere with the process.

[0260] In another workflow design, tag fragmentation is performed at 55°C and poisoning is performed at 60°C to achieve a first shell release condition in the reaction mixture, where the shell is a UCST polymer. To perform the extension and ligation steps, the reaction is reduced to 45°C and a second shell release condition is met, such as time, pH or temperature release, where the second shell is a LCST polymer. If PCR is then performed under PCR cycling conditions (e.g., heating to 98°C and cooling to 55°C), the polymer material can precipitate out of solution.

[0261] To address this issue, an alternative workflow is discussed. Tag fragmentation is performed at a lower temperature (e.g., below 42°C). The temperature is then raised to 60°C to 65°C to perform the tag fragmentation poisoning reaction and release the first shell from the UCST polymer having a temperature release condition of approximately 45°C. The temperature is then lowered to approximately 50°C and maintained for approximately 1 minute to promote the second shell release condition, which is a UCST polymer and delay condition that releases extension-link and PCR reagents into the mixture. The higher temperature of the second release condition minimizes the risk of UCST precipitation because most UCST polymers are soluble above 45°C. In an alternative workflow, the poisoning step is performed at 60°C to 65°C and the UCST trigger condition is set to this time or temperature, and wherein the second shell release is also a UCST having a 45°C release condition, and the reagent is released as the temperature is gradually raised to the desired temperature. Optionally, the second shell also contains a delay polymer to allow the poisoning reaction to proceed to completion and to allow the temperature to cool back to 50°C before the second core extension-link and PCR reagents are released (see Figure 4 ).

[0262] Improvements in reagent freeze drying can be made by minimizing harmful or interfering agents in which active ingredients can be stored. For example, bead-linked transposons are stored in glycerol buffer, and the tag fragmentation reaction mixture may contain 5% dimethylformamide (DMF). However, glycerol interferes with the lyophilization process, and DMF also interferes with the lyophilization process. In order to minimize the impact of glycerol, BLT that is not in glycerol buffer is mixed with cobalt chloride that does not contain DMF for the tag fragmentation reagent mixture. In addition, the tag fragmentation reaction temperature is reduced to between 37°C and 41°C to provide uniform sequencing coverage and promote certain trigger release conditions.

[0263] In an alternative formulation, wax (e.g., paraffin) is used to encapsulate magnesium-containing microspheres and demonstrate passive counteraction of poisons. To test the ability to counteract poisons, tagged fragmented DNA (after removal of Tn5) is added to a PCR mixture lacking magnesium (Mg). Liquid Mg, Mg microspheres, or Mg microspheres encapsulated in wax are added to the mixture. The initial step of the PCR cycling conditions is a 68°C step above the melting temperature of the wax. The large volume of wax in this format increases viscosity and reduces yield, so it is speculated that encapsulation with UCST at the single microsphere level will reduce the amount of material and the associated viscosity increase ( Figure 5 ).

[0264] In alternative COCl-based 2 In the workflow of , a pH increase is observed between the tag fragmentation to Tn5 removal process. The pH increase can be in the range of about pH 6 to about pH 8.5. This natural pH increase is then used as a triggered release of extension-linking and PCR reagents from microspheres having a pH-dependent polymer shell as described herein. In addition, the results demonstrate that in a pH-dependent release system, the second microsphere releases a buffer that changes the pH of the reaction. The change in pH triggers the dissolution of a shell (e.g., a third shell) on a different microsphere in the reaction mixture. This also provides an improved pH preparation for the subsequent PCR reaction.

[0265] The benefit of using lyophilized microspheres in a one-pot workflow system is that the reagents themselves do not require as much liquid volume, leaving more volume in the preparation tube for the sample. Table 4 below shows the amounts of reagents that can be used when lyophilized microspheres are used instead of liquid reagents.

[0266] Table 4. Possible reagents in the reaction

[0267]

[0268] It is expected that those skilled in the art will think of numerous modifications and variations of the present invention as set forth in the above illustrative embodiments.Accordingly, only such limitations as appear in the appended claims should be imposed on the present invention.

Claims

1. A composition comprising: a lyophilized material comprising one or more tag fragmentation reagents, a first particle, the first particle comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents, and A second particle, wherein the second particle comprises a second shell, the second shell surrounds a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, and the one or more third lyophilized microspheres comprise one or more reagents for extension-link and PCR.

2. A composition comprising: A lyophilized material comprising one or more tag fragmentation reagents, and a first particle, the first particle comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents, and A second shell, the second shell is located within the first core and surrounds the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

3. A composition comprising: a first lyophilized material, the first lyophilized material comprising one or more tag fragmentation reagents, and A first particle comprising a first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, wherein the one or more second lyophilized microspheres comprise one or more tag-fragmenting poisoning agents; and a first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and A second particle, wherein the second particle comprises a second shell, the second shell surrounds a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, and the one or more third lyophilized microspheres comprise one or more reagents for extension-link and PCR.

4. A composition comprising: a first lyophilized material, the first lyophilized material comprising one or more tag fragmentation reagents, and a first particle comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag-fragmenting poisoning agents; and a first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism; and a second shell located within the first core and surrounding the second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more reagents for extension-link and PCR.

5. The composition according to any one of the preceding claims, wherein the lyophilized material is selected from the group consisting of lyophilized microspheres, lyophilized beads and lyophilized cakes.

6. The composition according to any one of the preceding claims, wherein the first triggered release condition and the second triggered release condition are different.

7. The composition according to any one of the preceding claims, wherein the triggered release condition is a time release condition, a temperature release condition or a pH release condition.

8. The composition of any of the preceding claims, wherein the one or more tag fragmentation reagents are bead-linked transposomes (BLTs), transposases, primers, buffers, divalent cations, Tris buffers, cobalt buffers, and / or lyophilized reagents.

9. The composition of claim 8, wherein the tag fragmentation reagent comprises a bead-attached transposome, a primer tag, cobalt chloride, and trehalose.

10. The composition of claim 8, wherein the tag fragmentation reagent comprises bead-attached transposome, magnesium sulfate, Tris buffer, and trehalose.

11. The composition according to any one of the preceding claims, wherein the one or more tag fragmentation poisoning agents are chelating agents, deoxynucleotide triphosphates, Tris buffer, potassium buffer, ammonium salts and / or lyophilized agents.

12. The composition according to claim 11, wherein the tag fragmentation poisoning reagent comprises Tris buffer, potassium chloride, ammonium sulfate, cyclamen tetrahydrochloride, α-cyclodextrin, dNTP and trehalose.

13. The composition of claim 11, wherein the tag fragmentation poisoning reagent comprises potassium chloride, ammonium sulfate, SDS, dNTP and trehalose.

14. The composition of any of the preceding claims, wherein the extension-ligation and PCR reagents comprise magnesium sulfate, one or more polymerases, ampligase, NAD+, betaine, a surfactant, and / or lyophilized reagents.

15. The composition of claim 14, wherein the extension-ligation and PCR reagents comprise magnesium sulfate, glycerol-free hot start Q5, tTaq 608 polymerase, ampligase, NAD+, betaine, Tween-20, and trehalose.

16. The composition of claim 14, wherein the extension-ligation and PCR reagents comprise glycerol-free hot start Q5, tTaq 608 polymerase, ampligase, NAD+, betaine, alpha-cyclodextrin, Tween-20, and trehalose.

17. A composition according to any one of the preceding claims, wherein the polymer is a water-soluble or hydrophilic polymer.

18. The composition of claim 17, wherein the hydrophilic polymer is selected from the group consisting of carboxymethyl cellulose (CMC), carboxymethyl ethyl cellulose (CMEC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT) and ethyl hydroxyethyl cellulose (EHEC), pullulan, polyvinyl pyrrolidone, polyvinyl alcohol, copolymers of vinyl pyrrolidone or vinyl alcohol, polyacrylamide, polyacrylic acid, copolymers of ethyl acrylic acid or methacrylic acid, acrylic acid derivatives such as homopolymers and copolymers of butyl methacrylate, methyl methacrylate, ethyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl) methacrylate and (trimethylaminoethyl) chloromethacrylate, or combinations thereof.

19. A composition according to any one of the preceding claims, wherein the polymer is a water-insoluble or hydrophobic polymer.

20. The composition of claim 19, wherein the hydrophobic polymer is selected from the group consisting of ethyl cellulose (EC), methyl ethyl cellulose (MEC), cellulose acetate (CA), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), polyvinyl acetate, fatty acids, fatty acid esters, or combinations thereof.

21. The composition of any of the preceding claims, wherein the first shell or the second shell comprises an antistatic agent, a defoaming agent, an anti-sticking agent, a plasticizer, one or more UCST polymers, one or more LCST polymers, hydroxypropyl methylcellulose, or a combination thereof.

22. The composition of any one of the preceding claims, wherein the first shell comprises Efka 6783, Makon 17R4, polyethylene glycol (PEG) stearate, and UCST polymer 1.

23. The composition of any one of the preceding claims, wherein the second shell comprises Efka 6783, Makon 17R4, polyethylene glycol (PEG) stearate, UCST polymer 2, and hydroxypropyl methylcellulose (HPMC).

24. The composition of any one of the preceding claims, wherein the first release condition is a temperature release condition.

25. The composition of claim 24, wherein the temperature of the first release condition is above about 45°C.

26. A composition according to any one of the preceding claims, wherein the second release condition is a time release condition or a pH dependent condition.

27. The composition of claim 26, wherein the pH triggered release condition is an increase in pH between about pH 6 and about pH 8.

5.

28. The composition of any of the preceding claims, wherein the composition is stable when stored between about 4°C and about 37°C.

29. A system for preparing a DNA library, the system comprising one or more containers comprising the composition of any one of claims 1 to 28.

30. A container comprising a first lyophilized material, the first lyophilized material comprising one or more tag fragmentation reagents, a first particle, the first particle comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents, and A second particle, wherein the second particle comprises a second shell, the second shell surrounds a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more extension-link and PCR reagents.

31. A container, comprising: a first lyophilized material, the first lyophilized material comprising one or more tag fragmentation reagents, and A first particle, comprising a first shell, the first shell surrounding a first core, wherein the first shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents.

32. A container comprising a first lyophilized material, the first lyophilized material comprising one or more tag fragmentation reagents, a first particle, the first particle comprising a first inner shell, the first inner shell surrounding a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, the one or more second lyophilized microspheres comprising one or more tag fragmentation poisoning agents; and a first outer shell, the first outer shell surrounding the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism, and A second particle, wherein the second particle comprises a second shell, the second shell surrounds a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a third triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more extension-link and PCR reagents.

33. A container, comprising: a first lyophilized material, the first lyophilized material comprising one or more tag fragmentation reagents, and A first particle, comprising a first inner shell, which surrounds a first core, wherein the first inner shell comprises a hydrophilic or hydrophobic polymer and has a first triggered release condition, and wherein the first core comprises one or more second lyophilized microspheres, wherein the one or more second lyophilized microspheres comprise one or more tag fragmentation poisoning agents; and a first outer shell, which surrounds the first inner shell, wherein the first outer shell comprises a hydrophilic polymer and the first outer shell has a second triggered release mechanism.

34. The container of any one of claims 30 to 33, wherein the one or more tag fragmentation reagents are bead-linked transposomes (BLTs), transposases, primers, buffers, divalent cations, Tris buffers, cobalt buffers and / or lyophilized reagents.

35. The container of claim 33, wherein the tag fragmentation reagent comprises a bead-attached transposon, a primer tag, cobalt chloride, and trehalose.

36. The container of claim 33, wherein the tag fragmentation reagent comprises bead-attached transposome, magnesium sulfate, Tris buffer, and trehalose.

37. The container according to any one of claims 30 to 36, wherein the one or more tag fragmentation poisoning agents are chelating agents, deoxynucleotide triphosphates, Tris buffer, potassium buffer, ammonium salts and / or lyophilized agents.

38. The container of claim 37, wherein the tag fragmentation poisoning reagent comprises Tris buffer, potassium chloride, ammonium sulfate, cyclamen tetrahydrochloride, alpha-cyclodextrin, dNTPs, and trehalose.

39. The container of claim 37, wherein the tag fragmentation poisoning reagent comprises potassium chloride, ammonium sulfate, SDS, dNTPs, and trehalose.

40. A container, comprising: A second particle, wherein the second particle comprises a second shell, the second shell surrounds a second core, wherein the second shell comprises a hydrophilic or hydrophobic polymer and has a second triggered release condition, and wherein the second core comprises one or more third lyophilized microspheres, the one or more third lyophilized microspheres comprising one or more extension-link and PCR reagents.

41. The container of any one of claims 30 to 40, wherein the extension-ligation and PCR reagents comprise magnesium sulfate, one or more polymerases, ampligase, NAD+, betaine, surfactants and / or lyophilized reagents.

42. The container of claim 41, wherein the extension-ligation and PCR reagents comprise magnesium sulfate, Q5 hot start without glycerol, tTaq 608 polymerase, ampligase, NAD+, betaine, Tween-20, and trehalose.

43. The container of claim 41, wherein the extension-ligation and PCR reagents comprise glycerol-free hot start Q5, tTaq 608 polymerase, ampligase, NAD+, betaine, alpha cyclodextrin, Tween-20, and trehalose.

44. The container of any one of claims 30 to 43, wherein the first shell or the second shell comprises an antistatic agent, a defoaming agent, an anti-sticking agent, a plasticizer, one or more UCST polymers, one or more LCST polymers, hydroxypropyl methylcellulose, or a combination thereof, or a combination thereof.

45. The container of any one of claims 30 to 39 and 41 to 44, wherein the first shell comprises Efka6783, Makon 17R4, polyethylene glycol (PEG) stearate, and UCST polymer 1.

46. ​​The container of any one of claims 30 to 45, wherein the second shell comprises Efka 6783, Makon 17R4, polyethylene glycol (PEG) stearate, UCST polymer 2, and hydroxypropyl methylcellulose (HPMC).

47. The container of any one of claims 30 to 39 and 41 to 46, wherein the first release condition is a temperature release condition.

48. The container of claim 47, wherein the temperature of the first release condition is above about 45°C.

49. The container of any one of claims 30 to 49, wherein the second release condition is a time release condition or a pH triggered release condition.

50. The container of claim 49, wherein the pH triggered release condition is an increase in pH between about pH 6 and about pH 8.

5.

51. The container of any one of claims 30 to 50, wherein the lyophilized material, microspheres or particles are stable when stored at between about 4°C and about 37°C.

52. The container of any one of claims 30 to 51, wherein the lyophilized material is lyophilized microspheres, lyophilized beads or lyophilized cakes.

53. The container of any one of claims 31 to 52, wherein the container is a PCR tube, a vial, a microtube, a flow cell, a multiwell plate, a glass tube, a cartridge, or a microfluidic chip.

54. A kit comprising a composition according to any one of claims 1 to 28 or a container according to any one of claims 30 to 53, and instructions for use.

55. A kit comprising the container of claim 31 and the container of claim 40, and instructions for use.

56. A method for preparing a DNA library, the method comprising: adding a sample comprising DNA to a container comprising the composition of any one of claims 1 to 28, wherein the addition of the sample triggers the dissolution of the first lyophilized material to release the one or more tag fragmentation reagents into the container to trigger a tag fragmentation reaction; allowing the tag fragmentation reaction to continue under conditions and for a sufficient period of time to complete tag fragmentation of the DNA in the sample; initiating the first triggered release condition, wherein the first triggered release condition of the first shell causes the one or more tag fragmentation poisoning agents of the first core to be released into the container to thereby initiate the tag fragmentation poisoning reaction; as well as The tag fragmentation poisoning reaction is allowed to proceed under conditions and for a sufficient period of time to initiate the second triggered release conditions of the second shell, wherein the one or more extension-link and PCR reagents are released from the second core, wherein the extension-link and PCR reagents undergo gap filling and PCR amplification.

57. A method for preparing a DNA library, the method comprising: adding a sample comprising DNA to a container according to any one of claims 30 to 53, wherein the addition of the sample triggers the dissolution of the first lyophilized material to release one or more tag fragmentation reagents into the container to trigger a tag fragmentation reaction; allowing the tag fragmentation reaction to continue under conditions and for a sufficient period of time to complete tag fragmentation of the DNA in the sample; initiating the first triggered release condition, wherein the first triggered release condition of the first shell causes the one or more tag fragmentation poisoning agents of the first core to be released into the container to induce a tag fragmentation poisoning reaction; as well as The tag fragmentation poisoning reaction is allowed to proceed under conditions and for a sufficient period of time to initiate the second triggered release conditions of the second shell, wherein the one or more extension-link and PCR reagents are released from the second core, wherein the extension-link and PCR reagents undergo gap filling and PCR amplification.

58. The method of claim 56 or 57, further comprising isolating the DNA and sequencing the DNA library.

59. The method of any one of claims 56 to 58, wherein the tag fragmentation reaction is performed at about 37°C to about 41°C for about 5 minutes to about 15 minutes.

60. The method of any one of claims 56 to 59, wherein the first release condition is a temperature release condition.

61. The method of claim 60, wherein the temperature of the first temperature release condition is greater than about 45°C.

62. The method of any one of claims 56 to 61, wherein the tag fragmentation poisoning reaction is performed at about 60°C to about 65°C for about 1 minute to about 5 minutes.

63. The method of any one of claims 56 to 62, wherein the second release condition is a time release condition or a pH triggered release condition.

64. The method of claim 63, wherein the time release conditions are about 1 minute to about 3 minutes at about 50°C.

65. The method of claim 63, wherein the pH triggered release condition is an increase in pH between about pH 6 and about pH 8.

5.

66. The method of any one of claims 56 to 65, wherein the one or more extension-ligation and PCR reagents are released from the second core at about 50°C, wherein the extension-ligation and PCR reagents perform extension, ligation, and gap filling.

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