Compositions and methods for nucleic acid extraction and library preparation

Abstract

This disclosure provides a system and method for preparing DNA libraries, which includes storage-stable lyophilized microspheres containing DNA library preparation reagents that provide a streamlined workflow for DNA library preparation. This disclosure further provides smart disposable containers for sample collection and transport.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority benefit of U.S. Patent Provisional Application No. 63 / 492,081, filed on March 24, 2023, which is hereby incorporated by reference in its entirety.

[0002] (Field of the Invention) The present disclosure generally relates to compositions, systems, and methods of use thereof in the preparation of nucleic acid libraries, including nucleic acid library preparation reagents. In particular, the present disclosure provides materials adapted to deliver and release lyophilized reagents to a sample for sequential nucleic acid extraction and library preparation in a streamlined process for various downstream applications, including, for example, next - generation DNA sequencing.

Background Art

[0003] The advent of massively parallel short - read sequencing technology, also known as next - generation sequencing (NGS) (Bentley et al., 2008, Nature. 456(7218):53 - 59), has reduced DNA sequencing costs by orders of magnitude. Furthermore, the very high - throughput data acquisition using NGS has made rapid sequencing of complete genomes more accessible than ever, enabling access to increasing amounts of genomic, transcriptomic, and epigenetic data in all fields of biology.

[0004] NGS-based projects can be broadly categorized into the following process elements, which should be tailored and optimized to the target nucleic acid (RNA or DNA) and the selected sequencing system: sample preparation for nucleic acid extraction (NAE), library preparation, sequencing itself / data acquisition, and bioinformatics. Both NAE and library preparation remain long, multi-step, and low-throughput processes. NAE can be broadly divided into four steps, which are configurable depending on the sample and downstream application: (i) cell disruption, (ii) removal of membrane lipids, proteins, and other nucleic acids, (iii) nucleic acid purification / conjugation from bulk, and (iv) nucleic acid concentration. Library preparation is an essential process that involves several aspects that affect the efficiency of NGS. This often involves the following main steps: fragmentation of input DNA, end repair and A-tailing of DNA fragments, ligation of an indexed sequencing adapter, and optional amplification of the ligation product. Furthermore, one or more cleanup steps between steps are useful to purify DNA reaction products from reagents derived from previous reactions. An alternative method is library preparation based on fragmentation (e.g., tagmentation), in which adapters can be added during the fragmentation process, eliminating the need for A-tailing. Reliable and standardized implementation and quality control measures are crucial for all stages of the process.

[0005] Challenges are encountered at each of the aforementioned workflow steps, and addressing these challenges can lead to benefits and advantages, potentially enabling high-quality sequencing results. For example, extracting a sufficient amount of high-quality DNA free from inhibitors of enzymatic reactions occurring downstream in the NGS workflow can vary in complexity depending on the sample type and its storage conditions. During library preparation, three main challenges may be observed: protocol complexity, inaccurate pipetting, contamination, and cost. In particular, bead-based purification steps involving user handling of magnets and magnetic particles are prone to errors and can lead to library preparation failure (Meyer and Kircher, 2010 Cold Spring Harb Protoc. 2010(6):pdb.prot5448). Since libraries are usually prepared in parallel, sample contamination is an inherent problem (Kotrova et al., 2017 Mol Diagn Ther. 21(5):481-492; Salter et al., 2014 BMC Biol. 12:87). The main source of contamination is the pre-amplification required for low starting nucleic acid concentrations (Kotrova et al., 2017. Mol Diagn Ther. 21(5):481-492). Multiple liquid processing steps also increase the risk of cross-contamination of samples.

[0006] Therefore, this standard NGS workflow is complex and expensive, requiring costly laboratory equipment and reagents, trained personnel, and involving numerous liquid handling steps. Consequently, despite the continuing decrease in data acquisition (sequencing) costs, many large-scale genomic experiments are lagging behind in terms of sample acquisition, sample storage and the necessary cold chain, sample preprocessing for sequencing, and library preparation, creating bottlenecks in terms of time, cost, and effort. This poses a severe constraint in resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health research institutions. As a result, a large portion of diverse and multi-ethnic populations continue to be underestimated in health-related genetic research, potentially exacerbating existing disease and healthcare imbalances (Bentley et al., 2019, Ethn Dis., 29(Suppl 1):179-186; Wojcik et al., 2019, Nature 570:514-518).

[0007] To increase participant and researcher diversity in genome research and fully utilize the potential of current sequencing technologies, it is necessary to reduce the costs of reagent, sample storage, and transportation, as well as to simplify sample preparation and library preparation workflows to reduce the number of liquid handling steps and the required execution time. By streamlining sample preparation and library preparation, NGS can be made affordable for laboratories and health systems worldwide. [Overview of the project]

[0008] This specification describes a method for integrating DNA extraction, fragmentation, and indexing to reduce the time and complexity of template preparation, enabling higher throughput and reduced process costs compared to conventional methods.

[0009] This specification provides a system for collecting and preparing nucleic acids from a biological sample for DNA library amplification, the system comprising a container having an opening configured to receive a biological sample, the container comprising a workflow reagent release system, the release system comprising a plurality of first particles, the first particles comprising: a. one or more lyophilized microspheres comprising a lysis buffer and a proteinase; and b. a plurality of first particles, the first particle comprising: i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism; and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second inner core in response to a second release trigger mechanism.

[0010] Furthermore, a system is envisioned for collecting and preparing nucleic acids from biological samples for DNA library amplification, the system comprising a container having an opening configured to receive a biological sample, the container comprising a workflow reagent release system, the release system comprising a. one or more lyophilized microspheres comprising a lysis buffer and a thermally unstable proteinase, and b. a plurality of first particles comprising i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism, and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second inner core in response to a second release trigger mechanism, and a plurality of first particles.

[0011] This specification provides a system for collecting and preparing nucleic acids from a biological sample for DNA library amplification, the system comprising a container having an opening configured to receive a biological sample, the container comprising a workflow reagent release system, the release system comprising a plurality of lyophilized microspheres comprising a lysis buffer and a proteinase, and b) a plurality of first particles comprising i) a first outer shell enclosing a first internal core, the first internal core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first internal core in response to a first release trigger mechanism, and ii) a second outer shell enclosing a second internal core, the second internal core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second internal core in response to a second release trigger mechanism.

[0012] Furthermore, a system is envisioned for collecting and preparing nucleic acids from biological samples for DNA library amplification, the system comprising a container having an opening configured to receive a biological sample, the container comprising a workflow reagent release system, the release system comprising a plurality of lyophilized microspheres comprising a lysis buffer and a thermally unstable proteinase, and b) a plurality of first particles comprising i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism, and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second inner core in response to a second release trigger mechanism, and a plurality of first particles.

[0013] Furthermore, this specification provides a system for collecting and preparing nucleic acids from a biological sample for DNA library amplification, the system comprising a container having an opening configured to receive a biological sample, the container comprising a workflow reagent release system, the release system comprising a lyophilized cake comprising a lysis buffer and a proteinase, and b a plurality of first particles comprising i) a first outer shell enclosing a first internal core, the first internal core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first internal core in response to a first release trigger mechanism, and ii) a second outer shell enclosing a second internal core, the second internal core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second internal core in response to a second release trigger mechanism.

[0014] Furthermore, a system is envisioned for collecting and preparing nucleic acids from biological samples for DNA library amplification, the system comprising a container having an opening configured to receive a biological sample, the container comprising a workflow reagent release system, the release system comprising a lyophilized cake comprising a lysis buffer and a thermally unstable proteinase, and b a plurality of first particles comprising i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism, and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second inner core in response to a second release trigger mechanism, and a plurality of first particles.

[0015] In various embodiments, the first particle optionally includes a third outer shell enclosing a third inner core, the third inner core containing workflow reagents for extension ligation and PCR, and the third outer shell releases the third inner core in response to a third release trigger mechanism.

[0016] In various embodiments, the system further comprises a plurality of second particles, each including a third outer shell enclosing a third internal core, the third internal core containing workflow reagents for extension ligation and PCR, and the third outer shell releasing the third internal core in response to a third release trigger mechanism.

[0017] In various embodiments, the first and second external shells are affected only by the first or second release trigger mechanism, respectively.

[0018] In various embodiments, the first trigger release mechanism involves the biological sample dissolving the freeze-dried microspheres, thereby forming a dissolution solution.

[0019] In various embodiments, the first lyophilized microspheres contain one or more reagents for lysing cells contained in a biological sample. In various embodiments, one or more first lyophilized microspheres contain one or more reagents for lysing cells contained in a biological sample. In various embodiments, one or more reagents for lysing cells are selected from the group consisting of phosphate buffers, salts, surfactants, alcohols, proteases, lysis buffers, lyophilization inhibitors, or combinations thereof.

[0020] In various embodiments, the lyophilized cake contains one or more reagents for lysing cells contained in the biological sample. In various embodiments, the one or more reagents for lysing cells are selected from the group consisting of phosphate buffer, salt, surfactant, alcohol, protease, lysis buffer, lyophilization inhibitor, or a combination thereof. In various embodiments, the proteinase is a broad-spectrum serine protease. In various embodiments, the broad-spectrum serine protease is proteinase K, optionally a thermally unstable proteinase K.

[0021] In various embodiments, the surfactant is sodium dodecyl sulfate (SDS).

[0022] In various embodiments, the freeze-dried cake contains sodium dodecyl sulfate (SDS), EDTA, Tween, and proteinase.

[0023] In various embodiments, the first, second, or third release trigger is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof. In some embodiments, the second trigger release mechanism includes a temperature-controlled trigger release or a time-controlled trigger release.

[0024] In various embodiments, the second internal core comprises one or more lyophilized microspheres containing one or more tagmentation reagents. In various embodiments, the one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelator, a primer having a transpososome, a lyophilization inhibitor, and optionally a proteinase K inhibitor. In various embodiments, the SDS chelator is a cyclodextrin (CD) selected from the group consisting of α-CD, β-CD, and γ-CD.

[0025] In various embodiments, the first, second, and / or third outer shell, if present, comprises one or more of polyvinyl alcohol, polyvinyl pyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, Eudragit®, cellulose acetate, ethyl cellulose, UCST and LCST polymers, or any combination thereof.

[0026] In various embodiments, the cryoprotectant is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

[0027] In various embodiments, the biological sample is blood.

[0028] In various embodiments, the nucleic acid is DNA. In various embodiments, the DNA is genomic DNA (gDNA).

[0029] This disclosure provides a method for preparing a nucleic acid library from a biological sample, the method comprising: A. a container having an opening configured to collect a biological sample from a subject and receive the biological sample, the container comprising a workflow reagent release system, the workflow reagent release system comprising: a. a first lyophilized microsphere comprising a lysis buffer and a proteinase; and b. a first particle comprising: i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism; and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the first inner core in response to a second release trigger mechanism A biological sample in a container comprising a first particle comprising a second outer shell which releases a second inner core, wherein the biological sample interacts with a lyophilized lysis buffer in the container, resulting in the release of nucleic acids derived from cells in the biological sample, and allowing the lysis buffer to react for a time sufficient to carry out the lysis of cells in the biological sample; B. Activating a first release trigger mechanism to release a proteinase inhibitor and a surfactant chelating agent from the first inner core, thereby inactivating the lysis reaction (A) after the said time, and allowing the inactivation reaction to proceed for a time sufficient to inactivate the proteinase and chelate the surfactant; C. Activating a second release trigger mechanism to release a reagent for DNA tagging from the second inner core, thereby stopping the inactivation reaction (B) and allowing the tagging reaction to proceed for a time sufficient to tag the nucleic acids derived from the biological sample.

[0030] In various embodiments, the method further includes isolating nucleic acids from (C) and generating a nucleic acid library using a library preparation kit.

[0031] This disclosure provides a method for preparing a nucleic acid library from a biological sample, the method comprising: A. a container having an opening configured to collect a biological sample from a subject and receive the biological sample, the container comprising a workflow reagent release system, the workflow reagent release system comprising: a. a lyophilized cake comprising a lysis buffer and a proteinase, and b. a first particle comprising: i) a first outer shell enclosing a first internal core, the first internal core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, the first outer shell releasing the first internal core in response to a first release trigger mechanism, and ii) a second outer shell enclosing a second internal core, the second internal core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, the second outer shell releasing the second internal core in response to a second release trigger mechanism A biological sample in a container comprising a first particle including a second outer shell that releases an inner core, wherein the biological sample interacts with a lyophilized lysis buffer in the container, resulting in the release of nucleic acids derived from cells in the biological sample, and allowing the lysis buffer to react for a sufficient time to carry out the lysis of cells in the biological sample; B. Activating a first release trigger mechanism to release a proteinase inhibitor and a surfactant chelating agent from the first inner core, thereby inactivating the lysis reaction (A) after the said time, and allowing the inactivation reaction to proceed for a sufficient time to inactivate the proteinase and chelate the surfactant; C. Activating a second release trigger mechanism to release a reagent for DNA tagging from the second inner core, thereby stopping the inactivation reaction (B) and allowing the tagging reaction to proceed for a sufficient time to tag the nucleic acids derived from the biological sample.

[0032] In various embodiments, the method further includes isolating nucleic acids from (C) and generating a nucleic acid library using a library preparation kit.

[0033] In various embodiments, the first and second external shells are affected only by the first or second release trigger mechanism, respectively.

[0034] In various embodiments, the first trigger mechanism is initiated when a biological sample dissolves lyophilized microspheres containing a lysis buffer.

[0035] In various embodiments, the first lyophilized microspheres contain one or more reagents for lysing cells in a biological sample. In various embodiments, one or more first lyophilized microspheres contain one or more reagents for lysing cells in a biological sample. In various embodiments, one or more reagents for lysing cells are selected from the group consisting of phosphate buffers, salts, surfactants, alcohols, proteases, lysis buffers, lyophilization inhibitors, or combinations thereof.

[0036] In various embodiments, the proteinase is a broad-spectrum serine protease. In various embodiments, the broad-spectrum serine protease is proteinase K, optionally a thermally unstable proteinase K.

[0037] In various embodiments, the surfactant is sodium dodecyl sulfate (SDS).

[0038] In various embodiments, the first, second, or third release trigger (if present) is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof. In various embodiments, the second trigger release mechanism includes a temperature-controlled trigger release or a time-controlled trigger release.

[0039] In various embodiments, the second internal core comprises one or more lyophilized microspheres containing one or more tagmentation reagents. In various embodiments, the one or more tagmentation reagents are selected from the group consisting of Tn5 transposase enzymes, one or more transposons, linker sequences, Tn5 2× tagmentation buffer, Mg2+, SDS chelating agents, primers having transpososomes, lyophilization inhibitors, and optionally proteinase inhibitors.

[0040] In various embodiments, the second internal core comprises one or more lyophilized microspheres containing one or more tagmentation reagents. In various embodiments, the one or more tagmentation reagents are selected from the group consisting of Tn5 transposase enzymes, one or more transposons, linker sequences, Tn5 2× tagmentation buffer, Mg2+, SDS chelating agents, primers having transpososomes, lyophilization inhibitors, and optionally proteinase K inhibitors.

[0041] In various embodiments, one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelating agent, a primer having a transposome, and a lyophilization inhibitor. In various embodiments, the heat-unstable proteinase is inhibited by the temperature of the tagmentation reaction. In various embodiments, the temperature is 50°C to 80°C and for, for example, 10 minutes.

[0042] In various embodiments, the SDS chelating agent is a cyclodextrin (CD) selected from the group consisting of α-CD, β-CD, and γ-CD.

[0043] In various embodiments, the tagmentation reaction occurs at approximately 25°C to 55°C. In various embodiments, the tagmentation reaction occurs at approximately 30°C to 50°C, or approximately 37°C to 45°C, or approximately 40°C to 50°C. In various embodiments, the tagmentation reaction occurs at approximately 25°C, approximately 30°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, approximately 40°C, approximately 41°C, approximately 42°C, approximately 43°C, approximately 44°C, approximately 45°C, approximately 46°C, approximately 47°C, approximately 48°C, approximately 49°C, or approximately 50°C.

[0044] In various embodiments, the first, second, and / or third outer shell, if present, comprises one or more of the following: polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, Eudragit®, cellulose acetate, ethylcellulose, UCST and LCST polymers, or any combination thereof.

[0045] In various embodiments, the freeze-drying inhibitor is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

[0046] In various embodiments, the biological sample is blood.

[0047] In various embodiments, nucleic acids are DNA. In various embodiments, DNA is genomic DNA (gDNA).

[0048] In various embodiments, the method comprises (a) contacting a sample with a first lyophilized microsphere containing a lysis reagent that produces a cell lysate, wherein the lysis reagent has one or more proteases, and the cell lysate contains a target nucleic acid. In various embodiments, the release of the tagmentation reagent involves applying at least one transposon terminal composition containing at least one transposase and a transfer chain under conditions in which the target nucleic acid and the transposon terminal composition undergo a rearrangement reaction to produce a mixture, wherein the target nucleic acid is fragmented to produce a plurality of target nucleic acid fragments, and the transfer chain of the transposon terminal composition is ligated to the 5' end of each of the plurality of target nucleic acid fragments to produce a plurality of 5' tagged target nucleic acid fragments.

[0049] In various embodiments, the method comprises (a) contacting a sample with a lyophilized cake containing a lysis reagent that produces a cell lysate, wherein the lysis reagent has one or more proteases, and the cell lysate contains the target nucleic acid. In various embodiments, the release of the tagmentation reagent involves applying at least one transposon terminal composition containing at least one transposase and a transfer chain under conditions in which the target nucleic acid and the transposon terminal composition undergo a rearrangement reaction to produce a mixture, wherein the target nucleic acid is fragmented to produce a plurality of target nucleic acid fragments, and the transfer chain of the transposon terminal composition is ligated to the 5' end of each of the plurality of target nucleic acid fragments to produce a plurality of 5' tagged target nucleic acid fragments.

[0050] In various embodiments, the target nucleic acid is double-stranded DNA. In various embodiments, the target nucleic acid remains double-stranded DNA before application of the transposase and transposon terminus composition.

[0051] Furthermore, this specification provides a container for collecting biological samples, the container comprising a workflow reagent release system described herein, and the container comprising an indicator that changes upon completion of the workflow reagent release system within the container.

[0052] In various embodiments, the container includes a radio-frequency identification (RFID) tag. In various embodiments, the RFID tag is embedded in the container, and optionally, the RFID has a capacity to store at least 8 kilobytes of information.

[0053] In various embodiments, the container includes an opening for receiving a biological sample containing nucleic acids.

[0054] In various embodiments, the container comprises a heating element and a temperature sensor coupled to the container, and an RFID tag on the container stores the temperature history.

[0055] In various embodiments, the container is designed to prevent tampering.

[0056] In various embodiments, the container is made from polypropylene or a cyclic olefin copolymer. In various embodiments, the container is a PCR tube, vial, microtube, flow cell, multiwell plate, glass tube, cartridge, or microfluidic tip.

[0057] Further intended by this disclosure is a method for transporting a sample for the preparation of a nucleic acid library, the method comprising: inserting the sample into a container configured to receive a biological sample, the container comprising a system described herein; sealing the container so that the system initiates the process of sample lysis and nucleic acid library preparation; transporting the sealed container to a nucleic acid sequencing laboratory, the system so that upon arrival at the sequencing laboratory, the system completes sample lysis, tagmentation of nucleic acids in the sample, extension ligation of nucleic acids in the sample and PCR.

[0058] In various embodiments, the container is stored at approximately 4°C to 30°C during the insertion, sealing, and / or transport processes. In various embodiments, the container is stored at approximately 4°C to 8°C, approximately 4°C to 25°C, or approximately 20°C to 30°C during the insertion, sealing, and / or transport processes.

[0059] In various embodiments, the transport method further includes removing the container from the shipment and isolating nucleic acids from the sample in the container. In various embodiments, the container includes an RFID tag. In various embodiments, the container further includes an indicator indicating that library preparation is complete.

[0060] In various embodiments, the biological sample is blood.

[0061] In various embodiments, nucleic acids are DNA. In various embodiments, DNA is genomic DNA (gDNA).

[0062] A composition is also provided, which comprises a plurality of first lyophilized microspheres comprising a lysis buffer and a proteinase, and b a plurality of first particles comprising i) a first outer shell for enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism, and ii) a second outer shell for enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second inner core in response to a second release trigger mechanism.

[0063] Further intended is a composition comprising: a. a plurality of first lyophilized microspheres comprising a lysis buffer and a thermally unstable proteinase; and b. a plurality of first particles comprising: i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent; and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation; and a plurality of first particles.

[0064] A composition is also provided, which comprises a lyophilized cake comprising a lysis buffer and proteinase, and b a plurality of first particles comprising i) a first outer shell for enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres comprising a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releasing the first inner core in response to a first release trigger mechanism, and ii) a second outer shell for enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres comprising one or more reagents for DNA tagmentation, and the second outer shell releasing the second inner core in response to a second release trigger mechanism.

[0065] Further intended is a composition comprising: a. a lyophilized cake containing a lysis buffer and a thermally unstable proteinase; and b. a plurality of first particles comprising: i) a first outer shell enclosing a first inner core, the first inner core comprising one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent; and ii) a second outer shell enclosing a second inner core, the second inner core comprising one or more lyophilized microspheres containing one or more reagents for DNA tagmentation; and a plurality of first particles comprising:

[0066] In various embodiments, the first particle optionally includes a third outer shell enclosing a third inner core, the third inner core containing workflow reagents for extension ligation and PCR, and the third outer shell releases the third inner core in response to a third release trigger mechanism.

[0067] In various embodiments, the composition further comprises a plurality of second particles, each including a third outer shell enclosing a third internal core, the third internal core containing workflow reagents for extension ligation and PCR, and the third outer shell releasing the third internal core in response to a third release trigger mechanism.

[0068] In various embodiments, the first outer shell releases a first inner core in response to a first release trigger mechanism, the second outer shell releases a second inner core in response to a second release trigger mechanism, and, if a third release trigger mechanism is present, the third outer shell releases a third inner core in response to a third release trigger mechanism. In various embodiments, the first trigger release mechanism is activated by a biological sample dissolving the lyophilized microspheres, thereby forming a dissolution solution.

[0069] In various embodiments, the first lyophilized microspheres include one or more reagents for lysing cells. In various embodiments, the one or more reagents for lysing cells are selected from the group consisting of phosphate buffers, salts, surfactants, alcohols, proteases, lysis buffers, lyophilization inhibitors, or combinations thereof.

[0070] In various embodiments, the lyophilized cake contains one or more reagents for lysing cells. In various embodiments, one or more reagents for lysing cells are selected from the group consisting of phosphate buffer, salt, surfactant, protease, lysis buffer, lyophilization inhibitor, or a combination thereof.

[0071] In various embodiments, the proteinase is a broad-spectrum serine protease. In various embodiments, the broad-spectrum serine protease is proteinase K, optionally a thermally unstable proteinase K.

[0072] In various embodiments, the surfactant is sodium dodecyl sulfate (SDS).

[0073] In various embodiments, the freeze-dried cake contains sodium dodecyl sulfate (SDS), EDTA, Tween, and proteinase.

[0074] In various embodiments, the first, second, or third release trigger is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof.

[0075] In various embodiments, the second trigger release mechanism includes temperature-controlled trigger release or time-controlled trigger release.

[0076] In various embodiments, the second internal core comprises a lyophilized microsphere containing one or more tagmentation reagents. In various embodiments, the one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelator, a primer having a transpososome, a lyophilization inhibitor, and optionally a proteinase K inhibitor. In various embodiments, the SDS chelator is a cyclodextrin (CD) selected from the group consisting of α-CD, β-CD, and γ-CD.

[0077] In various embodiments, the first, second, and / or third outer shell, if present, comprises one or more of the following: polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, Eudragit®, cellulose acetate, ethylcellulose, UCST and LCST polymers, or any combination thereof.

[0078] In various embodiments, the freeze-drying inhibitor is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

[0079] Each feature, embodiment, or combination described herein is a non-limiting descriptive example of any aspect of the Invention and is therefore understood to be combinatable with any other feature, embodiment, or combination described herein. For example, where a feature is described using words such as “one embodiment,” “various embodiments,” “several embodiments,” “a certain embodiment,” “further embodiments,” “a particular exemplary embodiment,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combined with any other feature or combination of features described herein, without the need to enumerate all possible combinations.

[0080] Such features or combinations of features apply to any aspect of the present invention. Where examples of values ​​falling within a range are disclosed, any of these examples are intended as possible endpoints of the range, and all possible numerical values ​​between such endpoints are intended, and all possible combinations of upper and lower limits are assumed. [Brief explanation of the drawing]

[0081] The drawings illustrate one or more disclosed embodiments and are useful in illustrating the principles of the disclosed embodiments. However, it should be understood that the drawings are designed for illustrative purposes only and are not designed as a definition of the limitations of the invention. [Figure 1] (Figure below) A schematic diagram of the sample lysis and DNA tagging workflow according to one embodiment of the present disclosure is shown. [Figure 1] (Above diagram) The conventional workflow for tagging and indexing is schematic, which is now integrated into a single simplified workflow along with sample dissolution. [Figure 2] This figure shows a simplified diagram of the steps and conditions of a one-pot workflow according to one embodiment of the present disclosure. [Figure 3] This is a schematic perspective view showing one embodiment of a modular system that can be used to carry out aspects of this disclosure. [Figure 4] This is a schematic diagram of an embodiment of a radio-frequency identification ("RFID") tagged container for collecting and transporting biological samples, the container comprising lyophilized microspheres and a core / shell particle containing lyophilized microspheres of reagents for passive processing of the samples during transport. [Figure 5] This is a schematic diagram of an embodiment of an RFID-tagged container for passively processing a biological sample, comprising the lyophilized microspheres and core / shell reagents of the present disclosure for dissolution, inactivation, and tagmentation of the sample, according to one embodiment of the present disclosure. [Figure 6] This is a schematic diagram of an embodiment of an RFID-tagged container containing the core / shell reagent of the present disclosure for collecting, transporting, and passively processing biological samples, according to one embodiment of the present disclosure for one-pot library preparation. [Figure 7A] This provides characteristics of lyophilized products derived from soluble reagent formulations after freeze-drying. The vial shown in Figure 7A indicates drying below the decay temperature. [Figure 7B] This provides characteristics of lyophilized products derived from dissolving reagent formulations after freeze-drying. The vial shown in Figure 7B represents a lyophilized product freeze-dried at a temperature sufficiently lower than the glass transition temperature (Tg'). [Figure 7C] This provides characteristics of the lyophilized product derived from the dissolving reagent formulation after freeze-drying. Figure 7C is a scatter plot of Tg' versus total solute concentration, showing that the presence or absence of proteinase K in the lyophilized mixture does not have a significant effect on Tg'. [Figure 7D] This provides characteristics of lyophilized products derived from soluble reagent formulations after freeze-drying. Figure 7D shows bright-field microscope images at 1x (top image) and 10x (bottom image) magnification of exemplary core pre-coatings according to one or more embodiments of this disclosure. [Figure 8A]An exemplary test concerning the compatibility of proteinase K with polymer shells is shown. Figure 8A is a schematic diagram of the procedure used in such a test, in which DNA is extracted from a blood sample with 5 μL or 20 μL of coating added using a standard extraction proteinase K / SDS method, the DNA is purified using solid-phase reversible immobilization (SPRI) magnetic beads, and the DNA is quantified. [Figure 8B] This shows an exemplary test regarding the compatibility of proteinase K with polymer shells. Figure 8B is a bar graph showing the quantification of DNA extracted under control conditions (without proteinase K, lane 1, left to right) or with proteinase K (lane 2), or under test conditions. Blood samples were treated with 5 μL or 20 μL of coating material before DNA extraction. The results show that higher coating concentrations minimize interference with coating dissolution (lane 4). [Figure 9A] This is a comprehensive table comparing the inactivation ability by heat or inhibitor inactivation, the presence / absence of glycerol, and the DNA yield at 37°C for each PK candidate evaluated herein. [Figure 9B] This is an exemplary workflow for evaluating the performance of such proteases, comprising the steps of i) contact of a 25 μL blood sample with a lysis buffer containing each of the candidate PKs, ii) cell lysis and DNA release at 37°C for 15 minutes, iii) PK inactivation, iv) purification of the extracted DNA using solid-phase reversible immobilization (SPRI) magnetic beads, and v) measurement of the total DNA yield using a Qubit fluorometer. [Figure 9C] This study demonstrates the effects of different inhibitors on a standard Illumina DNA preparation method based on tagmentation. [Figure 10]This box plot summarizes the distribution of quality metrics for sequencing runs from five DNA extracts prepared using proteinase K. Each subpanel shows the results for one quality metric, listed from bottom to top as follows: (a) insert size, (b) Q30 base%, (c) mapped reads%, (d) coverage, and (e) clusters passing through the filter (%PF). The x-axis represents the proteinase K used for DNA extraction. Each box plot shows the range and distribution of the quality metrics calculated for the sequencing run. The top and bottom of the box represent the interquartile range, and the median represents the median. [Figure 11A] Figure 12A shows a graphical comparison of DNA yield (Figure 11B) and sequencing run quality indicators (Figure 11B) of DNA libraries treated with liquid reagents versus lyophilized reagents according to one or more embodiments of the present disclosure. Figure 11B(I) shows a control library using purified DNA extracted using a standard method (e.g., Qiagen kit) with and without trehalose, to understand the effect of this reagent in the library workflow; Figure 11B(II) shows a sample in which proteinase K was thermally inactivated and treated with liquid and lyophilized reagents during the dissolution step; and Figure 11B(III) shows a library in which proteinase K was irreversibly inhibited using tetrapeptidylchloromethyl ketone (TCK) targeting the active site of proteinase K and treated with liquid and lyophilized reagents during the dissolution step. [Figure 11B]Figure 12A shows a graphical comparison of DNA yield (Figure 11B) and sequencing run quality indicators (Figure 11B) of DNA libraries treated with liquid reagents versus lyophilized reagents according to one or more embodiments of the present disclosure. Figure 11B(I) shows a control library using purified DNA extracted using a standard method (e.g., Qiagen kit) with and without trehalose, to understand the effect of this reagent in the library workflow; Figure 11B(II) shows a sample in which proteinase K was thermally inactivated and treated with liquid and lyophilized reagents during the dissolution step; and Figure 11B(III) shows a library in which proteinase K was irreversibly inhibited using tetrapeptidylchloromethyl ketone (TCK) targeting the active site of proteinase K and treated with liquid and lyophilized reagents during the dissolution step. [Figure 12A] Exemplary studies on the effects of dimethyl sulfoxide (DMSO) on freeze-drying (glass transition temperature (Tg')) and sequencing indicators are shown. Figure 12A is a scatter plot showing the effect of increasing DMSO concentration on Tg' in solutions with 20% or 30% solute content. DMSO lowered Tg', particularly in solutions with low solute content. A 1% DMSO concentration yields a Tg' value above -40°C, making it most suitable for buffer formulations. [Figure 12B] This section presents exemplary studies on the effects of dimethyl sulfoxide (DMSO) on freeze-drying (glass transition temperature (Tg')) and sequencing indices. Figure 12B shows box plots summarizing the distribution of sequencing run quality indices for a control library prepared with TL NEB proteinase K, and three test libraries prepared with Sigma proteinase K and different concentrations of TCK prepared with different concentrations of DMSO. [Figure 13] This provides bar graphs showing comparable sequencing QC indices for libraries prepared using TCK inhibitors and DMSO at final concentrations of 0.7%, 0.25%, and 0.08%. [Figure 14A]A graph is provided showing the sequencing QC index of libraries prepared using liquid reagents with or without cyclodextrin in the tagmentation buffer. Figure 14A compares the performance of liquid tagmentation buffer and lyophilized tagmentation buffer, showing that it can be lyophilized. [Figure 14B] This provides a graph showing sequencing QC indices for libraries prepared using liquid reagents with or without cyclodextrin in the tagmentation buffer. Figure 14B (liquid only) compares the addition of the three reagents as a single mixture to the addition of CD+ tagmentation buffer and subsequent addition of TCK. The results indicate that the three reagents can potentially be lyophilized together. [Figure 15] This specification provides an example method for preparing a DNA library using the compositions or systems described herein. [Figure 16] This specification describes one method for transporting a sample in a container or smart consumable to a facility for sequencing. [Figure 17] This demonstrates the effect of the lysis buffer on killing viruses in blood samples. [Modes for carrying out the invention]

[0082] Current technologies enable the sequencing of millions or billions of DNA fragments in parallel at relatively low cost; therefore, the scope of data generation is often limited by the difficulty of sample preparation rather than sequencing capability. Despite recent advances, efficiently sequencing nucleic acids in situ remains difficult, if possible, and thus requires the extraction of nucleic acids from the material under test and subsequent conversion into a DNA library. Typically, methods for nucleic acid extraction and template library preparation for next-generation sequencing involve multiple steps and transfers of reaction liquid volumes between containers. This makes NGS sample preparation complex and inefficient and increases the risk of cross-contamination of samples because samples are prepared in parallel. Furthermore, molecular loss occurs during both steps of sample preparation, posing a challenge to working with low nucleic acid inputs.

[0083] This disclosure addresses the aforementioned shortcomings by providing particles, for example, containing a core-shell complex, that are engineered to deliver and release a lyophilized composition into a biological sample for passive nucleic acid extraction and library preparation in a single reaction vessel, i.e., a “one-pot format,” or in a minimal container or vessel, for various applications, including next-generation DNA sequencing. The Specified provides compositions and methods that enable the integration and streamlining of nucleic acid extraction and library preparation in a single workflow while eliminating the need for a cold chain for sample storage and transport. These compositions may include particles containing an inner core filled with lyophilized microspheres of a releaseable workflow reagent for one-pot NGS sample preparation, the inner core being encapsulated by an externally stimulated polymer carrier shell engineered for triggered release of the lyophilized workflow reagent microspheres into a biological sample in a controlled manner in response to a specific environmental trigger or stimulus. The compositions and processes described herein provide higher quality and longer gDNA strands compared to conventional methods. High-quality, longer DNA strands enable a higher level of linked long reads in sequencing reactions.

[0084] According to this disclosure, the compositions, systems, and methods described herein offer numerous advantages, such as eliminating the need for cryogenic transport and storage by stabilizing reagents, enabling transport and storage of reagents and completion of assays at room temperature, protecting encapsulated lyophilized reagent microspheres from harsh environmental conditions, time-controlled reagent release, simplifying workflows by eliminating the need to individually pipette microliters of potentially expensive assay reagents, and reducing the risk of sample contamination. Fewer pipetting steps and less sample handling also help minimize training requirements, reduce costs (e.g., transport, storage, and training costs), and save time. Assay robustness and reliability are also improved, along with data quality, and the risk of sample contamination is minimized. This method also improves data quality and result reliability while reducing contamination risk, is compatible with downstream applications such as NGS, reduces transport costs by allowing transport without refrigeration, reduces reagent waste by extending shelf life, supports field applications including remote or inaccessible locations with inadequate infrastructure (e.g., developing countries) without affecting sample or data quality, and provides batch-to-batch consistency in which all samples are processed uniformly.

[0085] term Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art within the context of this disclosure and in the specific context in which each term is used. However, certain terms are defined first in order to make this disclosure more easily understandable. Further definitions are provided throughout this application.

[0086] In the present invention, the singular forms "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. The term "plural" means two or more elements. For example, the term is used herein in reference to several leads for generating islands processed phase by phase using the method disclosed herein.

[0087] The terms “substantially,” “approximately,” “about,” “relatively,” or other similar terms, which may be used throughout this disclosure including the claims, are used to describe and consider small variations from a standard or parameter, such as variations in the process. Such small variations include zero-point variations from the standard or parameter. For example, variations may refer to ±10%, such as ±5%, ±2%, ±1%, ±0.5%, ±0.2%, ±0.1%, ±0.05%, etc.

[0088] As used herein, the term “sample” typically refers to a sample derived from a biological fluid, cell, tissue, organ, or organism containing nucleic acids to be sequenced and / or phased, or a sample derived from a mixture of nucleic acids containing at least one nucleic acid sequence to be sequenced and / or phased. Such samples include, but are not limited to, sputum / oral fluid, amniotic fluid, blood, blood fractions, fine-needle biopsy specimens (e.g., surgical biopsy, needle biopsy, etc.), urine, peritoneal fluid, pleural fluid, tissue explants, organ cultures, and any other tissue or cell preparations thereof, or fractions or derivatives thereof. As used in the present invention, the terms “blood,” “plasma,” and “serum” expressly encompass fractions thereof or processed portions thereof. Similarly, if the sample is taken from a biopsy, swab, smear, etc., “sample” expressly encompasses the processed fraction or portion derived from the biopsy, swab, smear, etc. Samples are often taken from human subjects (e.g., patients), but can be taken from any organism with chromosomes, including but not limited to dogs, cats, horses, goats, sheep, cattle, and pigs. Samples can be used as they are obtained from biological sources, or directly after pretreatment to modify the characteristics of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids, etc. Pretreatment methods may include, but are not limited to, filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, lyophilization, concentration, amplification, nucleic acid fragmentation, inactivation of interfering components, addition of reagents, dissolution, etc. When such pretreatment methods are employed on a sample, such pretreatment methods typically result in the presence of the target nucleic acid(s) in the test sample at concentrations sometimes proportional to those of the untreated test sample (e.g., samples not subjected to any such pretreatment method(s)). Such “treated” or “processed” samples are still considered biological “test” samples with respect to the methods described herein.

[0089] Samples may include, but are not limited to, genetically modified cell lines containing recombinant nucleic acid sequences integrated into chromosomes or episomes, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiateable cell lines, transformed cell lines, stem cells, germ cells (e.g., sperm, oocytes), transformed cell lines, and primary cell cultures or culture-adapted cell lines. For example, polynucleotide molecules may be obtained from primary cells, cell lines, newly isolated cells or tissues, frozen cells or tissues, paraffin-embedded cells or tissues, fixed cells or tissues, and / or laser-dissected cells or tissues. Biological samples may be obtained from any subject or biological source, such as humans or non-human animals (including mammals and non-mammals), vertebrates and invertebrates, and may also be any multicellular or unicellular organisms, such as eukaryotes (including plants and algae) or prokaryotes, archaea, microorganisms (e.g., bacteria, archaea, fungi, protists, viruses), and aquatic plankton.

[0090] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used synonymously and refer to a covalent-like sequence of nucleotides in which the 3' position of one nucleotide pentose is bonded to the 5' position of the next pentose by a phosphodiester group (i.e., ribonucleotides in the case of RNA, and deoxyribonucleotides in the case of DNA). Nucleotides include, but are not limited to, sequences of nucleic acids in any form, including RNA and DNA molecules such as cfDNA molecules. The term “polynucleotide” includes, but is not limited to, single-stranded and double-stranded polynucleotides. As used herein, these terms also encompass complementary cDNA or copy DNA produced from an RNA template, for example, by the action of reverse transcriptase. In one embodiment, for example, the nucleic acid to be analyzed by sequencing using the system described is immobilized on a substrate (e.g., a substrate in a flow cell or one or more beads on a substrate such as a flow cell). As used herein, immobilization is intended to include direct or indirect, covalent or non-covalent attachment unless otherwise explicitly indicated or indicated by context. The analyte (e.g., nucleic acid) may remain immobilized or attached to the support under conditions where the support is intended to be used, such as in nucleic acid sequencing applications. In one embodiment, the template polynucleotide is one of several template polynucleotides attached to the substrate. In one embodiment, the several template polynucleotides attached to the substrate include clusters of copies of library polynucleotides.

[0091] Nucleic acids include naturally occurring nucleic acids or their functional analogs. Particularly useful functional analogs can be hybridized to nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. The nucleic acids described herein may be of any length suitable for use in the provided methods. For example, the target nucleic acid may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 kb in length.

[0092] In this specification, the term "Next Generation Sequencing (NGS)" refers to sequencing methods that enable large-scale parallel sequencing of cloned amplified molecules and single nucleic acid molecules. Non-exclusive examples of NGS include sequencing-by-synthesis (SBS) and sequencing-by-ligation using reversible dye terminators.

[0093] The term “library” refers to a collection of nucleic acid template molecules or multiple nucleic acid template molecules having a common use or common characteristics, such as a common origin, for example, all members of the library originate from a single sample. Members of the library may be processed or modified so that members within the library are clearly distinguishable. For example, all members of the library may share a common sequence at their 5' ends and a common sequence at their 3' ends. The use of the term “library” to refer to a collection of template molecules or multiple template molecules should not be interpreted as implying that the templates constituting the library originate from a particular source or that the “library” has a particular composition. For example, the use of the term “library” should not be interpreted as implying that the individual templates within the library must have different nucleotide sequences or that the templates are related with respect to sequence and / or source.

[0094] The terms “address,” “index,” “index sequence,” “unique identifier,” “barcode,” “barcode sequence,” and “tag” are used interchangeably herein unless otherwise specified. These terms refer to sequences of nucleotides, typically oligonucleotides, that can be used to identify a sequence of interest, such as a genomic or haplotype region. Addresses, indices, index sequences, unique identifiers, barcodes, barcode sequences, or tag sequences may be exogenously incorporated into the sequence of interest by ligation, extension, or other methods known in the art. Index sequences may also be endogenous to the sequence of interest; for example, a segment in the sequence of interest itself may be used as an index. Nucleotide addresses, indices, index sequences, unique identifiers, barcodes, barcode sequences, or tags may be random or specially designed nucleotide sequences. Addresses, indices, index sequences, unique identifiers, barcodes, barcode sequences, or tags may be of any desired sequence length, as long as they are long enough to be a unique nucleotide sequence within multiple indices and / or multiple polynucleotides in the population being analyzed or investigated. Nucleotide addresses, indices, index sequences, unique identifiers, barcodes, barcode sequences, or tags are useful, for example, for attaching to target polynucleotides to tag or mark a particular species in order to identify all members of a tagged species within a population. Therefore, an index is useful as a barcode, where different members of the same molecular species may contain the same index, and different species within different polynucleotide populations may have different indices.

[0095] As used herein, the term “target nucleic acid” is intended to mean the nucleic acid that is the subject of analysis or action. Analysis or action may include subjecting the nucleic acid to copying, amplification, sequencing and / or other procedures for examining the nucleic acid. The target nucleic acid may include additional nucleotide sequences to the target sequence being analyzed. For example, the target nucleic acid may include one or more adapters, which function as primer binding sites, located flank to the target nucleic acid sequence being analyzed. The target nucleic acid hybridized to a capture oligonucleotide or capture primer may include nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide, such that not all of the target nucleic acid is suitable for extension.

[0096] As used herein, the term “substrate” is intended to mean a solid support or support structure. This term includes any material that can serve as a solid or semi-solid base for producing features such as wells for the deposition of biopolymers, including nucleic acids, polypeptides, and / or other polymers. Non-limiting examples of substrates include bead arrays, spot arrays, clustered particles arranged on the surface of a chip, films, multiwell plates, cartridges, and flow cells. The substrates provided herein are modified or can be modified to adapt to the deposition of biopolymers, for example, by various methods well known to those skilled in the art. Exemplary types of substrate materials include glass, modified glass, functionalized glass, inorganic glass, microspheres containing inert particles 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 exemplified above, and multiwell microtiter plates. Specific examples of plastics include acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon®. Specific examples of silica-based materials include silicon and various forms of modified silicon.

[0097] In some embodiments, the solid support includes one or more surfaces that readily come into contact with reagents, beads, or analytes. The surfaces may be substantially flat or planar. Alternatively, the surfaces may be rounded or contoured. Exemplary contours that may be included on the surface may include wells (e.g., microwells or nanowells), depressions, columns, ridges, channels, etc. Examples of materials that can be used as surfaces include glass, such as modified or functionalized glass; plastics, e.g., acrylic, polystyrene, or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or TEFLON; polysaccharides or crosslinked polysaccharides, e.g., agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials including silicon and modified silicon; carbon fibers; metals; inorganic glass; optical fiber bundles, or various other polymers. A single material or a mixture of several different materials can form a surface useful in the present invention. In some examples, the surface includes wells (e.g., microwells or nanowells). In some embodiments, the surface includes wells in an array of wells (e.g., microwells or nanowells) on a glass, silicon, plastic, or other suitable solid support containing a patterned, covalently linked gel such as poly(N-(5-azidoacetamidylpentyl)acrylamide-coacrylamide) (PAZAM, see, for example, U.S. Patent Application Publication 2014 / 0079923(A1), incorporated herein by reference). In some examples, the support structure may include one or more layers.

[0098] As used herein, the term “multiple” is intended to mean a group of two or more distinct members. A multiple can range in size from small, medium, large to very large. A small multiple might range, for example, from a few members to several dozen members. A medium multiple might range, for example, from several dozen members to about 100 or several hundred members. A large multiple might range, for example, from about several hundred members to about 1,000 members, several thousand members, and tens of thousands of members. A very large multiple might range, for example, from tens of thousands of members to about several hundred thousand, several million, tens of millions, or hundreds of millions or more members. Therefore, a multiple can range in size from 200 million to over 100 million, as well as all sizes measured by the number of members between and above the exemplary ranges described above. An exemplary number of features in a microarray is 1.28 cm². 2 It contains more than 500,000 distinct features. Exemplary nucleic acids include, for example, approximately 1 × 10⁻⁶ 5 , 5×10 5 and 1 × 10 6 Or it includes a population of more than 2 different nucleic acid species. Therefore, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit for multiple values ​​can be set, for example, by the theoretical diversity of nucleotide sequences in a nucleic acid sample.

[0099] As used herein, the term “double-stranded,” when used in reference 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 have at least 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95% of its nucleotides hydrogen-bonded to complementary nucleotides.

[0100] As used herein, the term “single-stranded,” when used in reference to nucleic acid molecules, means that none of the nucleotides in the nucleic acid molecule are essentially hydrogen-bonded to a complementary nucleotide.

[0101] As used herein, the term "dNTP" refers to deoxynucleoside triphosphates. NTP refers to ribonucleotide triphosphates. Purine bases (Pu) include adenine (A), guanine (G), and their derivatives and analogues. Pyrimidine bases (Py) include cytosine (C), thymine (T), uracil (U), and their derivatives and analogues. Examples of such derivatives or analogues, but not limited to, are those modified with reporter groups, biotinylated, amine-modified, radiolabeled, alkylated, and also include phosphorothioates, phosphates, and ring-atom-modified derivatives. Reporter groups may be fluorescent groups such as fluorescein, chemiluminescent groups such as luminol, or terbium chelators such as N-(hydroxyethyl)ethylenediaminetriacetic acid, which can be detected by delayed fluorescence.

[0102] As used herein, the term “size selection” means a procedure that increases the proportion of nucleic acid fragments having a specified number of bases by selecting from a population of nucleic acid fragments a subpopulation of which the majority of the nucleic acid fragments have a specified number of bases.

[0103] As used herein, the term “protease” refers to a protein, polypeptide, or peptide that exhibits the ability to hydrolyze polypeptides or substrates having a polypeptide moiety. The protease provided in this method may be a single protease with broad specificity. The method may use a mixture of various proteases. The proteases provided herein may be thermally unstable (i.e., thermally unstable) and therefore can be inactivated by heat. In some embodiments, the proteases provided herein may be inactivated at temperatures of about 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, above 80°C, or above about 85°C. The proteases provided herein can digest chromatin proteins and other DNA-binding proteins to release naked genomic DNA, and can also digest endogenous DNases to protect DNA from degradation. Proteases provided herein include, but are not limited to, serine proteases, threonine proteases, cysteine ​​proteases, aspartate proteases, glutamate proteases, and metalloproteases. Typically, aspartate, glutamate, and metalloproteases activate water molecules that hydrolyze peptide bonds through nucleophilic attack. Serine, threonine, and cysteine ​​proteases typically perform nucleophilic attack using nucleophilic residues, covalently bonding the protease to a substrate protein and releasing the first half of the product. This covalently bonded acyl-enzyme intermediate is then hydrolyzed by activated water, releasing the second half of the product and regenerating the free enzyme, thereby completing the catalytic action. Exemplary proteases used herein include serine proteases isolated from recombinant Bacillus strains.Exemplary proteases used herein include proteinase K, subtilisin and their variants, e.g., alcalase, alcalase 0.6L, alcalase 2.5L, ALK-enzyme, bacillopeptidase A, bacillopeptidase B, Bacillus subtilis alkaline protease, bioprase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, genenase I, esperase, maxatase, thermoase PC 10, protease XXVII, thermoase, superase, subtilisin Carlsberg, subtilisin DY, subtilin peptidase, SP 266, savinase 8.0L, savinase Examples include 4.0T, kazusase, protease VIII, opticlean, protin A 3L, savinase, savinase 16.0L, savinase 32.0L EX, orientase 10B, protease S, and serine endopeptidase. In certain embodiments of the methods and compositions presented herein, heat-unstable proteases such as proteinase K and its heat-unstable variants may be used.

[0104] As used herein, the term “protease inhibitor” refers to a substance, e.g., a compound, that can at least partially reduce the ability of a protease to hydrolyze a peptide. Examples of protease inhibitors known in the art that can be used in this method include, but are not limited to, the FOCUS® PROTEASEARREST® protease inhibitor cocktail, the PEFABLOC® SC (4-(2-aminoethyl)-benzenesulfonylfluoride-hydrochloride) (AEBSF) protease inhibitor, the aprotinin protease inhibitor, the bestatin protease inhibitor, the leupeptin protease inhibitor, the phenylmethylsulfonylfluoride (PMSF) protease inhibitor, and the tripeptidylchloromethylketone (TCK / TPCK, TLCK, and E-64) protease inhibitor.

[0105] As used herein, the term “tagmentation” refers to the modification of DNA by a transposome complex comprising a transposase enzyme complexed with an adapter containing a transposon terminal sequence. Tagmentation results in the simultaneous fragmentation of DNA and ligation of the adapter to the 5' ends of both strands of the double fragment. Further sequences can be added to the ends of the compatible fragment by, for example, PCR, ligation, or any other suitable method known to those skilled in the art. As used herein, the term “transposome complex (TSM)” refers to a transposase enzyme that non-covalently binds to a double-stranded nucleic acid. For example, the complex may be a transposase enzyme pre-incubated with double-stranded transposon DNA under conditions that support non-covalent complex formation. 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 terminal composition, a mixture of transposon terminal compositions, or other double-stranded DNA that can interact with a transposase such as a hyperactive Tn5 transposase.

[0106] 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 or nearly random site. Essential components of a transposition reaction are a transposase and DNA oligonucleotides that exhibit the nucleotide sequence of the transposon, including the transferred transposon sequence and its complement (the untransferred transposon terminal sequence), as well as other components necessary for functional transposition or the formation of a transposomal complex. The DNA oligonucleotides may further include additional sequences (e.g., adapter or primer sequences) as needed or desired. In some embodiments, the methods provided herein are exemplified by using a transposition complex formed by a hyperactive Tn5 transposase and a Tn5-type transposon terminus (Goryshin and Reznikoff, 1998, J. Biol. Chem., 273:7367), or by a MuA transposase containing R1 and R2 terminal sequences and a Mu transposon terminus (Mizuuchi, 1983, Cell, 35:785; Savilahti et al., 1995, EMBO J., 14:4893). However, any transposition system capable of inserting transposon terminus in a random or near-random manner with sufficient efficiency to 5'-tag and fragment target DNA for the intended purpose may be used in the present invention.Examples of transposition systems known in the art that can be used in the method of the present invention include Staphylococcus aureus Tn552 (Colegio et al., 2001, J Bacterid., 183:2384-8; Kirby et al., 2002, MoI Microbiol, 43:173-86), TyI (Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72 and International Patent Application No. 95 / 23875), transposon Tn7 (Craig, 1996, Science. 271:1512, Craig, 1996, Review in: Curr Top Microbiol Immunol, 204:27-48), TnIO and ISLO (Kleckner et al., 1996, Curr Top Microbiol Immunol, 204:49-82), Mariner transposase (Lampe et al., 1996, EMBO J., 15:5470-9), Tci (Plasterk, 1996, Curr Top Microbiol Immunol, 204:125-43), P element (Gloor, 2004, Methods MoI Biol, 260:97-114), TnJ (Ichikawa and Ohtsubo, 1990, J Biol Chem. 265:18829-32), bacterial insertion sequences (Ohtsubo and Sekine, 1996, Curr.Top.Microbiol.Immunol. 204:1-26), retroviruses (Brown et al., 1989, Proc Natl Acad Sci USA, 86:2525-9), and yeast retrotransposons (Boeke and Examples include, but are not limited to, Corces (1989, Annu Rev Microbiol. 43:403-34). Methods for inserting transposon ends into target sequences can be carried out in vitro using any suitable transposon system that is available or can be developed based on knowledge in the art, provided that a suitable in vitro transposition system is available.Generally, an in vitro transposition system suitable for use in the methods provided herein is one which includes, at a minimum, a transposase enzyme of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity, and a transposon terminal that forms a functional complex with the respective transposase capable of catalyzing the transposition reaction. Suitable transposase transposon terminal sequences that may be used in the present invention include, but are not limited to, wild-type, derivative, or mutant transposon terminal sequences that form a complex with a transposase selected from the wild-type, derivative, or mutant of the transposase.

[0107] As used herein, the term “transposase” refers to an enzyme that forms a functional complex comprising a transposon end-containing composition (e.g., a transposon, a transposon end, or a transposon end composition) and is capable of catalyzing, for example, the insertion or rearrangement of the transposon end-containing composition into a double-stranded target nucleic acid to which it is incubated in an in vitro rearrangement reaction. Transposases presented herein may also include integrases from retrotransposons and retroviruses. Transposases, transpososomes, and transposomal complexes are generally known to those skilled in the art, as exemplified by the disclosure in U.S. Patent Application Publication No. 2010 / 0120098. While many embodiments described herein refer to Tn5 transposases and / or hyperactivated Tn5 transposases, it will be understood that any rearrangement system capable of inserting a transposon end with sufficient efficiency to 5' tag and fragment a target nucleic acid for the intended purpose may be used in the present invention. In certain embodiments, the transposition system can randomly or nearly randomly insert transposon ends to fragment target nucleic acids by tagging them with a 5' tag.

[0108] As used herein, the term “library of tagged nucleic acid fragments” refers to a collection or population of tagged nucleic acid fragments (e.g., ditagged nucleic acid fragments) generated from a resource, such as a whole genome, where combinations of tagged nucleic acid fragments in the collection or population represent sequences qualitatively and / or quantitatively representing the sequence of the resource from which the tagged nucleic acid fragments were generated, such as the whole genome. A library of tagged nucleic acid fragments may not contain tagged nucleic acid fragments representing all sequences represented by the resource.

[0109] As used herein, the term “primer” generally refers to an oligonucleotide ("oligo") having a free 3'-OH group that can be extended by a nucleic acid polymerase. For template-dependent polymerases, generally, at least the 3' portion of the primer oligo is complementary to a portion of the template nucleic acid, in which case the oligo “binds” (or “complexes,” “anneals,” or “hybridizes”) to the template by hydrogen bonds and other molecular forces, giving a primer / template complex for the initiation of synthesis by DNA polymerase, which is extended by the addition of a covalently bonded base to its 3' end, which is complementary to the template in the process of DNA synthesis. The result is a primer extension product.

[0110] As used herein, the terms “adaptor” and “adapter” are interchangeable and may refer to oligonucleotides that can be bound to the ends of nucleic acids. Adapter sequences may include, for example, a priming site, a complement to the priming site, an endonuclease recognition site, a common sequence, and a promoter. Adapters may also incorporate modified nucleotides that modify the characteristics of the adapter sequence. For example, a phosphorothioate group may be incorporated into one of the adapter chains.

[0111] The compositions, systems, and methods described herein include particles having a shell surrounding a core, the core may include one or more lyophilized microspheres (i.e., the compositions may include encapsulated lyophilized microspheres).

[0112] Where used herein, “encapsulate,” “encapsulated,” and “encapsulation” include the encapsulation of one or more microspheres as described herein. Microencapsulation as described herein refers to embedding at least one component, e.g., an activator, in at least one other material, e.g., a shell material. Encapsulation as described herein includes, but is not limited to, bulk encapsulation, macroencapsulation, microencapsulation, nanoencapsulation, single molecule, and ion encapsulation. According to this disclosure, the compositions, systems, and methods described herein have many advantages, including, for example, increased microsphere stability, the use of macroencapsulation to enable multi-run cartridges, and the use of microencapsulation to enable simplified workflows and a reduction in the number of reagent wells. The compositions, systems, and methods described herein stabilize these buffers using encapsulation of particles that are otherwise responsive to pH changes, thereby improving SBS performance.

[0113] As used herein, “microsphere” includes a shell and a core and comprises spherical particles having a diameter of 0.1 μm to 1,000 μm. For example, microspheres may have diameters 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 from about 0.1 μm to about 1,000 μm. In one embodiment, encapsulated microspheres have a diameter of about 100 μm to 1000 μm.

[0114] Microspheres may refer to lyophilized particles containing reagents and / or active ingredients. In some embodiments, microspheres may include a polymer shell, e.g., a biodegradable polymer and / or a water-soluble polymer, and optionally an internal core inside the shell. Microspheres according to this disclosure include those prepared by the prior art known to those skilled in the art. For example, microspheres can be prepared by freezing a liquid into a freeze pellet, and then placing the freeze microspheres in a dryer, e.g., a rotary dryer.

[0115] Where used herein, “shell” includes a composition surrounding the core. In one embodiment, the shell includes an outer layer of a microsphere, or alternatively, an outer layer of a macrosphere. In one embodiment, the shell comprises a shell material selected from the group consisting of, for example, carrageenan, agarose, poloxamer, shellac, trehalose, paraffin wax, fatty acids (myristic acid, almitic acid), and fatty acid esters, i.e., PEG stearate, gelatin, hydroxypropyl methylcellulose (HPMC), cellulose acetate, fullalin, oxygen absorbers, alginates, chitosan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, polyvinylpyrrolidone (PVP), poly(vinylpyrrolidone-co-vinyl acetate), polyvinyl alcohol (PVA), poly(vinyl alcohol-graft-PEG), one or more upper critical dissolution temperature (USCT) polymers, e.g., poly(acrylic-co-acrylonitrile), poly(N-acryloylglycinamide), one or more lower critical dissolution temperature (LCST) polymers, e.g., poly(N-isopropylacrylic) and its copolymers, or any combination thereof.

[0116] Where used herein, “core” or “core region” includes any material within the surrounding shell. In various embodiments, the core includes one or more lyophilized microspheres. In various embodiments, the core includes lyophilized beads. In various embodiments, the core includes beads made of non-lyophilized sugar or plastic, and optionally, reagents are coated onto the surface of the non-lyophilized microspheres or beads and dried. In various embodiments, the core includes one or more lyophilized beads or one or more lyophilized microspheres.

[0117] As used herein, the term “reagent” describes a single agent or a mixture of two or more agents useful for reacting with, interacting with, diluting, or adding to a sample, and may include agents used in nucleic acid reactions, such as buffers, chemicals, enzymes, polymerases, primers including those having a size of less than 50 base pairs, template nucleic acids, nucleotides, labels, dyes, or nucleases. Reagents described herein may, in certain embodiments, include enzymes such as polymerases, ligases, recombinases, or transposases, antibodies, epitopes, binding partners such as streptavidin, avidin, biotin, lectins, or carbohydrates, or other biochemically active molecules. Other exemplary reagents include reagents for biochemical protocols such as nucleic acid amplification protocols, affinity-based assay protocols, enzyme assay protocols, sequencing protocols, and / or protocols for the analysis of biological fluids. According to some embodiments disclosed herein, reagents may include one or more beads, particularly magnetic beads, depending on the specific workflow and / or downstream application.

[0118] The terms “connect,” “connected,” “contact,” and “coupled” are defined broadly herein to encompass a variety of different arrangement and assembly techniques. These arrangements and techniques include, but are not limited to, (1) directly joining one component to another without any intervening components between them (i.e., the components are in direct physical contact), and (2) connecting one component to another with one or more components between them, provided that one component that is “connected,” “contacted,” or “coupled” to the other component is in some operational communication (e.g., electrical, fluid, physical, optical, etc.) with the other component (despite the presence of one or more additional components between them). It should be understood that some components that are in direct physical contact with each other may or may not be in electrical and / or fluid contact with each other. Furthermore, two electrically connected, electrically coupled, optically connected, optically coupled, fluidically connected, or fluidly coupled components may or may not be in direct physical contact, and one or more other components may be positioned between them.

[0119] Particle core-shell composite materials This disclosure relates to one or more particles comprising a core-shell composite material, the core-shell composite material comprising: a) an internal core optionally comprising one or more releaseable lyophilized microspheres or lyophilized beads of workflow reagents; and b) an external shell enclosing such internal core, the external shell comprising one or more irritation-sensitive polymer layers, the external shell being designed to be irritation-responsive, such that its physicochemical properties change upon application of different stimuli, and releasing the encapsulated lyophilized microspheres into a specific environment (i.e., the “external environment”), e.g., into a biological sample.

[0120] The core-shell composite material may consist of macro-sized, micro-sized, or nano-sized particles.

[0121] 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 osmolites, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof.

[0122] The core-shell composite may exhibit a total shell structure thickness of approximately 1 to 25 μm. In embodiments, the thickness may be selected from 2.5, 5, 10, 15, 20, or 25 μm, or the thickness may be provided within a range having upper and lower limits selected from these values. If the outer shell includes two or more shell layers, such layers may independently have a thickness of 1 to 25 μm. In various embodiments, the shell has a thickness of approximately 1 μM to 25 μM, approximately 1 μM to approximately 20 μM, approximately 5 μM to approximately 20 μM, approximately 3 μM to approximately 10 μM, or approximately 4 μM to approximately 6 μM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 16 μM, approximately 17 μM, approximately 18 μM, approximately 19 μM, approximately 20 μM, approximately 21 μM, approximately 22 μM, approximately 23 μM, approximately 24 μM, or approximately 25 μM.

[0123] The thickness can be advantageously adjusted according to the residence time of the composite material. For example, the shell may be at least 5 μm thick for a homogeneous coating that allows for predictable release.

[0124] The core-shell composite material may have a substantially spherical shape with a diameter of about 0.2 μm to about 1,000 μm. The core-shell composite material may have an average diameter of about 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, and 1,000 μm. In various embodiments, microspheres with or without a coating material have a diameter of about 300 to 700 μm, about 350 to 625 μm, or about 400 to 600 μm. The core-shell composite material may also contain substantially monodisperse particles, each having substantially the same average diameter. The core-shell material may also contain lyophilized microspheres or lyophilized beads having an average diameter distribution.

[0125] The shell may consist of one layer or multiple layers of varying compositions, as described herein. For example, the shell may consist of one, two, three, four, five, six, seven, eight, nine, ten, or more than ten layers. Each layer may contain the same or different materials as the other layers present in the shell.

[0126] The core-shell composite material may include a shell material selected from the group consisting of hydroxypropyl methylcellulose (HPMC), cellulose acetate, polyethylene glycol, poly-(vinylpyrrolidone)-poly-(vinyl acetate-cocrotonic acid) (PVP-co-PVAc), Eudragit, isoleucine Eudragit RL / RS, Opadry CA, polyester (i.e., polylactic acid-coglycolic acid (PLGA)), wax, UCST polymer, LSCT polymer, carrageenan, shellac, paraffin wax, fatty acids, fatty acid esters, gelatin, pullulan, oxygen absorbers, alginic acid, chitosan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof. In one example, the shell may include, but is not limited to, starch, cellulose, hydrophilic colloids, alginate, collagen, and any combination thereof.Examples of water-soluble (hydrophilic) polymers include ethylcellulose (EC), methylethylcellulose (MEC), carboxymethylcellulose (CMC), carboxymethylethylcellulose (CMEC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), cellulose phthalate acetate (CAP), cellulose trimellitate acetate (CAT), hydroxypropylmethylcellulose (HPMC), hydroxypropylmethylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose succinate acetate (HPMCAS), hydroxypropylmethylcellulose trimellitate acetate (HPMCAT), and ethyl hydroxyethylcellulose (EHEC), pullulan, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid esters, polyacrylamide, polyacrylic acid, ethacrylic acid, or methacrylic acid copolymers (EUDRAGIT®, Rohm Examples include homopolymers and copolymers of acrylic acid derivatives (America, Inc., Piscataway, NJ), as well as other acrylic acid derivatives, such as butyl methacrylate, methyl methacrylate, ethyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl) methacrylate, and (trimethylaminoethyl) methacrylate chloride.

[0127] The amount of shell material includes, for example, any amount suitable for producing the desired shell result. In one embodiment, the shell material is present in an amount of about 1% to about 100% by weight of the shell. For example, the shell material may be present in an amount of about 1%, 2%, 20%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between. In one embodiment, the shell material is present in an amount of about 10% to about 90% by weight of the shell, or about 10% to about 80% by weight, or about 10% to about 70% by weight, or about 10% to about 60% by weight, or about 10% to about 50% by weight.

[0128] The shells described herein may, in various embodiments, include shell additives. The shell additives may be present in amounts ranging from about 0.01% w / w to about 99% w / w of the shell. In one embodiment, the shell additives may be present in amounts ranging from about 10% w / w to about 90% w / w of the shell. In one embodiment, the shell additives may be present in amounts ranging from about 10% w / w to about 40% w / w. In one embodiment, the shell additives are moisture barrier material present in amounts less than or equal to 90% w / w of the shell. In one embodiment, the shell additives may be present in amounts at least 10% w / w of the shell. For example, in one embodiment, the shell additives may be present in amounts ranging from 0.1% w / w to about 15.0% w / w of the shell. For example, shell additives are approximately 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, and 7.5% w / w. The amount of shell additive may be present in amounts of %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 in between. The amount of shell additive may be adjusted to suit a particular reagent or combination of reagents, or to suit a particular microsphere composition.

[0129] Examples of shell additives include, but are not limited to, one or more of the following: polymers, copolymers, block copolymers, antistatic agents, defoamers, plasticizers, second polyvinyl alcohol (PVA), ammonium salts, conductivity enhancers, stearic acid derivatives, oleic acid derivatives, lauric acid derivatives, polyether compounds, amino acids, tocopherol acetate, piperidyl sebacate, sodium salts, buffers, chelating agents, imidazolium salts, polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from block copolymers derived from polyethylene glycol, polypropylene glycol, ethylene oxide (EO), and propylene oxide (PO), or any combination thereof. In one embodiment, the stearic acid derivative or oleic acid derivative is selected from magnesium stearate, PEG stearate, triglycerol stearate, Span® 60, Tween® 60, glycerol trioleate, Tween® 80, or any combination thereof. In some embodiments, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In some embodiments, the sodium salt is selected from one or more of 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, polyquaternium, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazole), or a combination thereof. In one embodiment, the shell additive includes an ammonium salt, copolymer, polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.In various embodiments, the shell additive is magnesium stearate or polyethylene glycol stearate.

[0130] Internal core and freeze-dried microspheres Where used herein, “core” or “core region” includes any material within the encapsulating shell.

[0131] The core according to this disclosure comprises one or more lyophilized microspheres or lyophilized beads.

[0132] The lyophilized microspheres of this disclosure may comprise any reagent that is desired for controlled delivery and can be unitized to substantially small sizes suitable for lyophilization or micronization within the size range described herein.

[0133] In some embodiments, the internal core contains lyophilized reagents suitable for use in multiple sequential simultaneous assays, including lysis, DNA analysis, RNA analysis, protein analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, SBS technology, transposase-reachable chromatin region analysis by sequencing (ATAC-seq), continuous preserved transposition (CPT-seq), single-cell combinatorial index sequencing (SCI-seq), or single-cell genome amplification, or any combination thereof performed sequentially. In one embodiment, the composition is used to carry out multiple simultaneous assay reactions. The compositions, systems, and methods described herein (e.g., encapsulation of lyophilized microspheres) may, in one embodiment, 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 “transfer-free reaction.”

[0134] In further embodiments, the internal core may include lyophilized reagents that can be prepared for various stages of sequencing, including but not limited to sample extraction, library preparation, concentration, clustering, and sequencing.

[0135] Lyophilized sphere containing sample preparation reagents In one embodiment, the lyophilized microspheres comprise a lyophilized lysis solution. In another embodiment, the lyophilized cake comprises a lyophilized lysis solution. The lysis solution enables efficient dissolution for the release of nucleic acids (e.g., cells in a biological sample), effectively protects the released nucleic acids from degradation in the lysate by inhibiting or degrading nucleases, and is suitable for subsequent steps for the analysis of the extracted nucleic acids, such as target capture, amplification, detection, and / or sequencing. The components of the lysis buffer can be adjusted according to the cell type and source, the desired final molecule or structure, and their level of functionality.

[0136] In one embodiment, the lyophilized microspheres contain a lysis buffer for DNA extraction from whole blood. In another embodiment, the lyophilized cake contains a lysis buffer for DNA extraction from whole blood. Whole blood and blood fractions are common biological starting materials for DNA extraction in most epidemiological studies, for example. Compared to other less invasive sources of genomic gDNA (gDNA), such as saliva or oral cells, gDNA yields from blood or blood fractions are relatively high and less fragmented (Koshy et al., Mol Biol Rep. 44(1):97-108, 2017). Whole blood contains red blood cells (RBCs), nucleated leukocytes (WBCs), platelets, and plasma. Genomic DNA is found in the nuclei of WBCs. Unlike WBCs, mature RBCs are anucleated and therefore do not contain DNA. Most DNA extraction procedures from whole blood involve a two-step lysis approach, with step 1 being selective lysis with minimal impact on WBCs and removal of RBCs. RBCs do not contain DNA and are a potential source of downstream inhibitors. Therefore, it may be advantageous to separate them from WBCs before DNA isolation. Lysis of WBCs for DNA extraction and protein degradation, followed by DNA recovery and washing, is also planned.

[0137] In various embodiments, the lyophilized dissolution of the present disclosure contains a buffer (such as Tris-HCl), a broad-spectrum protease (such as proteinase K), an amphiphilic reagent (such as a detergent or surfactant, or a mixture thereof), a chelating agent (such as EDTA or CDTA), and a lyophilization inhibitor / lyophilization reagent (such as sucrose or trehalose).

[0138] In one embodiment, the lyophilized microspheres of the present disclosure provide a reagent for a passive one-step whole blood lysis approach using a lysis buffer mixture capable of lysing both WBC and RBC cell types in a single step. This one-step lysis approach has many advantages over traditional two-step lysis methods, such as improved DNA yield due to the elimination of sample loss that occurs in the two-step procedure, a single-container reaction that eliminates the need for pipetting, which also reduces the risk of contamination, saves time, and lowers the reagent cost for additional enzymes (e.g., RNases).

[0139] The first component of the dissolution solution is a buffer (e.g., Tris buffer or any known buffer) that maintains the pH of the solution. For example, the pH of the buffer may be at least about 8, at least about 8.5, or even more at least about 9 (e.g., 8.1, 8.4, 8.6, 8.7, 8.9, 9.1, or 9.5). The buffer may have a pKa of at least about 8 (e.g., 8.1, 8.3, 8.5, 8.6, 8.8, or 8.9) and may be used at a concentration of 50–150 mM (e.g., 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, or 140 mM). In some embodiments, Tris buffer is a suitable buffer. In some examples, Tris buffer with a pH of 8.0 and a concentration of 100 mM is used. In some other embodiments, a base may be used to adjust the pH of the dissolution solution. The base may be one that can raise the pH of the solution to 7 or higher (for example, pH 7.5, 8, 8.5, or 9.0). In some examples, the base may be an alkali metal hydroxide. Examples of such alkali metal hydroxides include, but are not limited to, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0140] In some embodiments, the dissolution solution contains a broad-spectrum protease for proteolytic lysis. In some embodiments, the broad-spectrum protease includes serine proteases, threonine proteases, cysteine ​​proteases, aspartate proteases, glutamate proteases, or metalloproteases. In some embodiments, the broad-spectrum protease is a serine protease. In some embodiments, the serine protease is proteinase K. Proteinase K is a stable serine protease that is active over a wide range of pH, temperature, salt, solvent, and surfactant concentrations. The activity of proteinase K peaks in the presence of moderate denaturants, 2-4 moles of chaotropic salts, and ionic surfactants, which act to stimulate enzymatic activity and increase substrate accessibility by destabilizing the secondary structure of the protein. Upon completion, proteinase K digestion breaks down polypeptides into smaller dipeptides and tripeptides. In this process, the enzymes themselves are broken down by autodigestion, thus eliminating most of the enzymes added to the sample. Proteolytic buffers are important additives in DNA extraction methods and are crucial for the isolation of DNA from complex biological samples. In the sample mixture, the proteolytic buffer is designed to preserve the target nucleic acid, establish optimal conditions for proteolysis, solubilize lipids and microvesicles, degrade colloids and particulate matter, and inhibit or prevent precipitation throughout the protease reaction.

[0141] Proteinase K can be present in the lysis buffer at concentrations ranging from approximately 0.001 mg / mL to approximately 10 mg / mL. For example, the concentration of proteinase K in the lysis buffer may be approximately 0.001 mg / mL, approximately 0.005 mg / mL, approximately 0.01 mg / mL, approximately 0.05 mg / mL, approximately 0.1 mg / mL, approximately 0.5 mg / mL, approximately 1 mg / mL, approximately 1.5 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, or greater than approximately 10 mg / mL. In some cases, a suitable proteinase K solution has a proteinase K concentration of 20 mg / mL. In some embodiments, a suitable lysis solution contains proteinase K at concentrations ranging from approximately 0.45 to approximately 1.8 mg / mL. In some embodiments, the appropriate dissolving solution contains proteinase K at a concentration of approximately 0.8 mg / mL.

[0142] When nucleic acid preparation and tagmentation steps are performed in the same reaction tube, it may be beneficial that proteases according to the method of the present invention can be effectively inactivated without interfering with the subsequent tagmentation step, which typically uses double-stranded DNA. In some embodiments, the protease can be inactivated by raising the temperature before the tagmentation step. High temperatures can denature the conformation of double-stranded DNA. Therefore, in some embodiments, the proteases provided herein can be inactivated at relatively low temperatures without denature the double-stranded DNA. In some embodiments, one or more proteases are inactivated by raising the temperature to 50°C to 80°C. In some embodiments, one or more proteases are inactivated by raising the temperature to 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In various embodiments, the protease is proteinase K, which can be thermally inactivated.

[0143] In some embodiments, the dissolution solution contains a surfactant. The surfactant can act both as a solvent and as an inhibitor of analytic degradation after lysation of blood cells. Surfactants are particularly useful for inhibiting nucleic acid degradation. Non-limiting examples of surfactants or detergents that can be used include polyoxyethylene glycol alkyl ethers (marketed as Brij® series detergents such as Brij® 58, Brij® 52, Brij® L4 and Brij® L23), octaethylene glycol monododecyl ethers, pentaethylene glycol monododecyl ethers, polyoxypropylene glycol alkyl ethers, glucoside alkyl ethers (e.g., decyl glucoside, lauryl glucoside, octyl glucoside), and polyoxyethylene glycol octylphenol ethers (e.g., Triton X-100), polyoxyethylene glycol alkylphenol ether (e.g., nonoxynol-9), glycerol alkyl ester (e.g., glyceryl laurate), polyoxyethylene glycol sorbitan alkyl ester (e.g., polyoxyethylene glycol (20) sorbitan monolaurate (TWEEN® 20), polyoxyethylene glycol (40) sorbitan monolaurate (TWEEN® 40), polyoxyethylene glycol (20) sorbitan monopalmitate, polyoxyethylene glycol (20) sorbitan monostearate, polyoxyethylene glycol (4) sorbitan monostearate Poloxamers, including those sold under the trade names Pluronic®, Synperonic®, and Kolliphor®, are also included.Furthermore, nonionic surfactants such as polyethoxylated taloamine (POEA); anionic surfactants such as ammonium lauryl sulfate, ammonium perfluorononanoate, docusate, perfluorobutanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, potassium lauryl sulfate, sodium alkyl sulfate, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate, sodium laurate, sodium lauryl ether sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium pareth sulfate, and sodium stearate. Examples of surfactants include cationic surfactants such as benzalkonium chloride, benzethonium chloride, bronidox, cetrimonium bromide, cetrimonium chloride, distearyldimethylammonium chloride, laurylmethylgluceth-10 hydroxypropyldimonium chloride, octenidine dihydrochloride, olafleur, and tetramethylammonium hydroxide; and amphoteric surfactants such as CHAPS detergent, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dipalmitoylphosphatidylcholine, lecithin, hydroxysultaine, and sodium lauroamphoacetate.

[0144] Anionic, cationic, and amphoteric surfactants can all be used in the dissolution solution, but the dissolution solution of this disclosure comprises at least one anionic surfactant and at least one nonionic surfactant. In one embodiment, the dissolution solution contains SDS, which is an anionic surfactant, and TWEEN® 20, which is a nonionic surfactant.

[0145] In one embodiment, SDS can be present at concentrations of approximately 0.1% to 10% (weight / volume). For example, appropriate SDS concentrations are approximately 0.1% to 0.2%, 0.2% to 0.3%, 0.3% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, and 5% to 6%. Examples include, but are not limited to, percentages of 1%, approximately 6% to 7%, approximately 7% to 8%, approximately 8% to 9%, and approximately 9% to 10%, as well as combinations of the above ranges, such as approximately 0.1% to 0.5%, approximately 1% to 2%, approximately 1% to 5%, approximately 3% to 7%, approximately 5% to 9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, or approximately 10%.

[0146] In one embodiment, TWEEN® 20 may be present at a concentration of approximately 0.5% to approximately 10% (weight / volume percentage). For example, appropriate TWEEN® 20 concentrations include, but are not limited to, approximately 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, and 9% to 10%, as well as combinations of the above ranges, such as approximately 0.1% to 0.5%, 1% to 2%, 1% to 5%, 3% to 7%, 5% to 9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0147] Alternatively, the concentration of the surfactant is measured in mg / mL or g / L. In typical embodiments, any of the surfactants are present in concentrations of approximately 1–5 mg / mL, approximately 5–10 mg / mL, approximately 10–15 mg / mL, approximately 15–25 mg / mL, approximately 25–50 mg / mL, approximately 50–60 mg / mL, approximately 60–70 mg / mL, approximately 70–80 mg / mL, and approximately 80–90 mg / mL, as well as combinations of the above ranges.

[0148] To reduce or prevent the degradation of nucleic acids, water without nucleases is used in the dissolution solution. In some embodiments, chelating agents can also be used to inhibit or prevent the degradation of contaminating nucleic acids. The use of chelating agents inhibits or prevents the nucleic acid polymer from breaking down into smaller fragments that could cause further contamination problems. Chelating agents may be present at concentrations of 1 to 100 mM (e.g., 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 35 mM, 45 mM, 50 mM, 65 mM, 75 mM, 85 mM, or 95 mM) or at concentrations of 1 to 10 mM (e.g., 1.5 mM, 2 mM, 3 mM, 4 mM, 6 mM, 7 mM, or 9 mM). In some examples, ethylenediaminetetraacetic acid (EDTA) is used as a chelating agent. In other examples, the chelating agent cyclohexane-N,N,N',N'tetraacetic acid (CDTA) is used.

[0149] The anticoagulant, when present in the dissolving reagent, is at a concentration sufficient to inhibit coagulation of the sample (e.g., whole blood or red blood cells). By inhibiting coagulation, the anticoagulant eliminates the need to centrifuge the sample during the method for isolating red blood cells. Examples of anticoagulants include EDTA, EDTA-Na2, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), heparin, or citrate. Typical concentrations of EDTA in the dissolving reagent include approximately 0.05 mM to 15 mM, approximately 0.1 mM to 10 mM, approximately 0.5 mM to 5 mM, approximately 10 mM, approximately 2.5 mM, or approximately 0.1 mM. Typical concentrations of EDTA-Na2 in the dissolving reagent include approximately 0.05 mM to 15 mM, approximately 0.1 mM to 10 mM, approximately 0.5 mM to 5 mM, approximately 10 mM, approximately 2.5 mM, or approximately 0.1 mM.

[0150] In further embodiments, the dissolution also contains a cryoprotectant (CPA) or freeze-drying inhibitor. Freeze-drying inhibitors (also called excipients or cryoprotectants, freeze-drying inhibitors or freeze-drying reagents) generally contribute to the preservation of the structure of proteins, liposome bilayers, and other substances during freezing. Freeze-drying inhibitors stabilize these substances during drying, particularly freeze-drying. In freeze-drying, freeze-drying inhibitors can also be considered freeze-drying inhibitors, and therefore, as used herein, the term “freeze-drying inhibitor” may also include freeze-drying inhibitors. Protective additives can generally be considered to have two types: (i) amorphous glass-forming salts and (ii) eutectic crystallizing salts. Examples of freeze-drying inhibitors include sugars (monosaccharides, disaccharides, and polysaccharides) as natural freeze-drying inhibitors, polyhydroxy compounds such as trehalose and sucrose, and polyalcohols such as glycerol, mannitol, sorbitol, and their derivatives. All of these belong to type (i).

[0151] In one embodiment, the antifreeze and / or freeze-drying inhibitor is selected from the group consisting of trehalose, sucrose, mannitol, maltose, maltodextrin, dextran, inulin, and raffinose. In one embodiment, the antifreeze is trehalose. Trehalose, also known as α,α-trehalose, aD-glucopyranosyl-(1→1)-aD-glucopyranoside, micose, or tremalose, is a naturally occurring α-linked disaccharide formed by an a,α-1,1-glucosidic bond between two α-glucose units. Trehalose can exist as anhydrous or dihydrate. In one embodiment, trehalose is D(+)-trehalose anhydrous.

[0152] Trehalose concentration can be measured in mg / mL or g / L. In typical embodiments, trehalose is present at approximately 5 to 250 mg / mL. For example, appropriate trehalose concentrations are approximately 5 mg / mL to 75 mg / mL, 50 mg / mL to 200 mg / mL, 75 mg / mL to 200 mg / mL, 100 mg / mL to 200 mg / mL, 25 mg / mL to 175 mg / mL, 50 mg / mL to 175 mg / mL, 75 mg / mL to 175 mg / mL, 100 mg / mL to 175 mg / mL, 25 mg / mL to 150 mg / mL, 50 mg / mL to 150 mg / mL, 75 mg / mL to 150 mg / mL, and 100 mg / mL to 150 mg / mL. Examples of trehalose concentrations include, but are not limited to, approximately 25 mg / mL to 125 mg / mL, approximately 50 mg / mL to 125 mg / mL, approximately 75 mg / mL to 125 mg / mL, approximately 25 mg / mL to 100 mg / mL, approximately 125 mg / mL to 175 mg / mL, approximately 125 mg / mL to 200 mg / mL, approximately 5 mg / mL to 200 mg / mL, approximately 200 mg / mL to 250 mg / mL, approximately 5 mg / mL to 250 mg / mL, approximately 75 mg / mL to 250 mg / mL, approximately 100 mg / mL to 250 mg / mL, or approximately 150 mg / mL to 250 mg / mL. In various embodiments, the concentration of trehalose is approximately 150 mg / mL.

[0153] In various embodiments, the method further includes a step of de-escalating the sample to reduce or eliminate active viruses in the sample. De-escalation methods include, but are not limited to, guanidinium blockade, thermal inactivation using a lysis buffer (e.g., about 55°C to about 80°C), addition of a surfactant (e.g., Triton X-100, SDS, Tween 20, Brij, ChAPS), addition of a chelating agent (e.g., EDTA), or addition of a degrading enzyme (e.g., proteinase K).

[0154] Table 1 shows examples of lysis buffers intended in this specification.

[0155] [Table 1]

[0156] Tag maintenance solution In one embodiment, the lyophilized sphere comprises a lyophilized tagmentation solution suitable for carrying out an in vitro rearrangement reaction as described herein, for example, a transposase, a DNA oligonucleotide exhibiting the nucleotide sequence of a transposon, components necessary for functional rearrangement or the formation of a transposomal complex, an adapter and / or primer sequence.

[0157] In some embodiments, the Disclosure provides a nucleic acid fragmentation reaction mixture comprising (i) a plurality of transposases, (ii) a polynucleotide containing a first transposon terminal sequence, (iii) a polynucleotide containing a second transposon terminal sequence, (iv) a target nucleic acid molecule, and (v) an activated cation.

[0158] In some embodiments, the first transposon terminal sequence is capable of binding to multiple transposases. In some embodiments, the first transposon terminal sequence contains at least one modification, including damage such as a nick, a gap, a depurine site, or a depyrimidine site.

[0159] In some embodiments, the second transposon terminal sequence is capable of binding to multiple transposases.

[0160] In some embodiments, the second transposon terminal sequence contains at least one modification, including damage such as a nick, gap, depurine site, or depyrimidine site.

[0161] In some embodiments, the first and second transposon terminal sequences contain at least one modification, including damage such as a nick, a gap, a depurine site, or a depyrimidine site.

[0162] In some embodiments, the first or second transposon terminal sequence lacks modification, including damage such as nicks, gaps, depurine sites, or depyrimidine sites.

[0163] In some embodiments, the first and second transposon terminal sequences may be identical or different.

[0164] In some embodiments, the nucleic acid fragmentation reaction mixture comprises (i) a plurality of transposases, (ii) a polynucleotide containing a first transposon terminal sequence, wherein the first transposon terminal sequence is binding to the plurality of transposases and contains at least one modification, including damage such as a nick, gap, depurine site, or depyrimidine site, (iii) a polynucleotide containing a second transposon terminal sequence, wherein the second transposon terminal sequence is binding to the plurality of transposases and contains at least one modification, including damage such as a nick, gap, depurine site, or depyrimidine site, (iv) a target nucleic acid molecule, and (v) an activating cation (e.g., magnesium or manganese). In some embodiments, the target nucleic acid molecule comprises a target DNA molecule.

[0165] In some embodiments, the RNA fragmentation reaction mixture further comprises a buffer (e.g., Tris-acetic acid), a proteinase K inhibitor, an SDS chelating agent (e.g., α- or β-cyclodextrin), and an activating cation. The activating cation includes any cation required by the transposase to catalyze the rearrangement reaction (e.g., magnesium-magnesium acetate).

[0166] In various embodiments, one or more tagmentation reagents include bead-linked transposomes (BLTs), proteinase K inhibitors, random hexamers, primers, probes, transposases, buffers, divalent cations, Tris buffer, cobalt buffer, and / or lyophilized reagents.

[0167] Any buffer suitable for the transposase used can be used in the method of the present invention, but it is preferable to use a buffer particularly suitable for the efficient enzymatic reaction of the transposase used. In this regard, buffers containing dimethylformamide are particularly preferred for use in the method of the present invention, especially during the transposase reaction. Furthermore, buffers containing alternative buffer systems including TAPS, tris-acetic acid, or similar systems can be used. In addition, crowding reagents such as polyethylene glycol (PEG) are useful for increasing the tagmentation efficiency of very small amounts of DNA. The conditions for the tagmentation reaction are described in Picelli et al. (2014) Genome Res. 24:2033-2040.

[0168] The transposase intended in the context of this invention is transposase (Tnp) Tn5. Tn5 is a member of the RNase superfamily of proteins, which includes retroviral integrases. Tn5 can be found in Shewanella and Escherichia bacteria. Transposons encode antibiotic resistance to kanamycin and other aminoglycoside antibiotics. Tn5 and other transposases are particularly inactive. Since DNA transposition events are inherently mutagenic, low transposase activity is beneficial in eliminating transposing elements by reducing the risk of causing lethal mutations in the host. One reason why Tn5 is so inactive is that its N and C termini are located relatively close to each other and tend to inhibit one another. This has been elucidated by the characterization of several mutations that result in hyperactive forms of the transposase. One such mutation, L372P, is a mutation at amino acid 372 in the Tn5 transposase. This amino acid is generally the leucine residue in the center of the α-helix. When this leucine is substituted with a proline residue, the α-helix is ​​disrupted, a conformational change is introduced to the C-terminal domain, and it is separated from the N-terminal domain to a sufficient extent to promote higher protein activity. Therefore, it is preferable to use a modified transposase that is more active than the natural Tn5 transposase. Furthermore, it is particularly preferable that the transposase used in the method of the present invention is loaded with an oligonucleotide to be inserted into the target nucleic acid, especially the target DNA.

[0169] Therefore, the use of hyperactive Tn5 transposase and Tn5-type transposase recognition sites (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or MuA transposase and Mu transposase recognition sites containing RI and R2 terminal sequences is intended (Mizuuchi, K., Cell, 35:785, 1983; Savilahti, H, et al, EMBO J., 14:4893, 1995).Further examples of rearrangement systems that can be used in the method of the present invention include Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol., 183:2384-8, 2001; Kirby C et al, Mol. Microbiol, 43:173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res., 22:3765-72, 1994, and International Publication No. 95 / 23875), transposon Tn7 (Craig, NL, Science. 271:1512, 1996; Craig, NL, Review in: Curr Top Microbiol Immunol, 204:27-48, 1996), Tn / O and IS 10 (Kleckner N, et al, Curr Top Microbiol Immunol, 204:49-82, 1996), Mariner transposase (Lampe DJ, et al, EMBO J., 15:5470-9, 1996), Tel (Plasterk RH, Curr. Topics Microbiol. Immunol., 204:125-43, 1996), P element (Gloor, GB, Methods Mol. Biol, 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:18829-32, 1990), bacterial insertion sequence (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retrovirus (Brown, et al, Proc Natl Acad Sci Examples include USA, 86:2525-9, 1989, and yeast retrotransposons (Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989).Further examples include genetically modified versions of IS5, TnIO, Tn903, IS911, and other transposase family enzymes (Zhang et al, (2009) PLoS Genet. 5:e1000689. Epub 2009 Oct. 16, Wilson C. et al (2007) J. Microbiol. Methods). Examples include those described in 71:332-5) and U.S. Patents No. 5,925,545, No. 5,965,443, No. 6,437,109, No. 6,159,736, No. 6,406,896, No. 7,083,980, No. 7,316,903, No. 7,608,434, No. 6,294,385, No. 7,067,644, No. 7,527,966, and International Publication No. 2012103545, all of which are specifically incorporated herein by reference to their entirety.

[0170] Transposase enzymes catalyze the insertion of nucleic acids, particularly DNA, into target nucleic acids, especially target DNA. The target nucleic acid for insertion, particularly target DNA, is contained in isolated chromatin bound by a chromatin binder. The transposase used in the methods of this disclosure is loaded with an oligonucleotide to be inserted into the target nucleic acid, particularly target DNA. The complex of the transposase and the oligonucleotide is also called a transposome. In various embodiments, the transposome is a heterodimer containing two different oligonucleotides for insertion. In this regard, the oligonucleotide loaded into the transposase comprises multiple sequences. In particular, the oligonucleotide comprises at least a first sequence and a second sequence. The first sequence is used to load the oligonucleotide into the transposase. An exemplary sequence for loading an oligonucleotide into a transposase is shown in U.S. Patent Application Publication No. 2010 / 0120098. The second sequence includes a linker sequence used for primer binding during amplification, particularly during PCR amplification. Therefore, oligonucleotides containing the first and second sequences are inserted into target nucleic acids, particularly target DNA, by transposase enzymes. The oligonucleotide may further contain a sequence containing a barcode sequence. The barcode sequence may be a random sequence or a defined sequence. In this regard, the term “random sequence” according to the present invention should be understood as a sequence of nucleotides having an independent and equal probability that each position is any nucleotide. A random nucleotide may be any nucleotide in any order, e.g., G, A, C, T, U, or a chemical analog thereof, where G is understood to represent guanylate nucleotide, A is adenylate nucleotide, T is thymidylate nucleotide, C is cytidylate nucleotide, and U is uracillate nucleotide. Those skilled in the art will understand that known oligonucleotide synthesis methods can inherently result in an uneven presentation of nucleotides G, A, C, T, or U. For example, synthesis may result in an over-presentation of nucleotides such as G in a randomized DNA sequence. This can result in a reduction in the number of unique random sequences, as would be expected based on the equal presentation of nucleotides.The target nucleic acid, particularly the oligonucleotide for insertion into DNA, may further include a sequencing adapter, such as an adapter suitable for nanopore sequencing or Roche 454 sequencing. Furthermore, the oligonucleotide may also include a biotin tag sequence. The oligonucleotide loaded into the transposase preferably includes such first and second sequences as well as a barcode sequence for indexing. Incorporation of the barcode sequence during the transposase reaction enables the unique identification of each nucleic acid fragment, particularly DNA fragment, during sequencing analysis and / or mapping of molecular interactions.

[0171] The time required for the transposase used to efficiently incorporate nucleic acids, particularly DNA, into target nucleic acids, particularly target DNA, can vary depending on various parameters such as buffer components and temperature. Therefore, various incubation times may be tested / applied before an optimal incubation time is found. In this regard, optimal refers to the optimal time considering the overall efficiency and / or time required to carry out the method of the present invention. While various incubation times do not necessarily correlate with the efficient incorporation of the nucleic acid, particularly DNA, into the target nucleic acid, particularly target DNA, incubation times of less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than 1 minute are preferred. Furthermore, parameters such as temperature and volume may be modified for the best yield. In this regard, the recommended incubation temperature for Tn5 transposase is approximately 37°C. Therefore, the method described herein includes the step of adding the transposase and then incubating for tagmentation at approximately 37°C for optionally about 1 to 5 minutes. However, alternative reaction temperatures may also be used; for example, temperatures above approximately 16°C and below approximately 55°C are used to maintain sample integrity and transposase efficiency.

[0172] A method for preparing a sequencing library may further include an amplification step for incorporating the adapter sequence. Amplification is carried out as described below. The adapter sequence varies depending on the sequencing method used after the preparation of the sequencing library. For example, when Illumina sequencing is used, the i5 and i7 ends may be bound to a nucleic acid fragment. This can also be achieved by a transposase reaction in which an oligonucleotide loaded onto a transposase enzyme contains the sequencing-compatible adapter sequence.

[0173] Primers suitable for use in this method contain a sequence that can hybridize to a second sequence contained in an oligonucleotide contained in the transposome used in the method of the present invention. Furthermore, the primer may contain a sequence used for sequencing. In various embodiments, specific primers are used that are compatible with the sequencing method subsequently used. In this regard, Illumina sequencing as one sequencing method is compatible with primers that introduce flow cell ends and can hybridize to flow cells required for cluster amplification. In this regard, the primer may introduce i5 and i7 ends for Illumina sequencing. Furthermore, the primer may introduce barcodes for multiplexing. In particular, barcodes included in the primer sequence may be used as unique molecular identifiers for identifying PCR replicas and / or as defined barcodes for combining multiple experiments in a single sequencing run.

[0174] [Table 2]

[0175] Trigger release mechanism The polymer shell is configured to withstand fracture or decomposition over a period of time, allowing the delivery of the core to be delayed as desired. The polymer imparts chemical and / or mechanical properties to the particles such that the contents of the core, e.g., lyophilized microspheres or beads, can be substantially released only at a desired time after delivery. For example, the polymer or polymer composite can be configured to decompose under one or more conditions (two trigger mechanisms), and the contents in the core can be released from the particles when the shell decomposes at least partially. In one or more embodiments, decomposition can proceed via one or more of the following: thermal decomposition, oxidative decomposition, chemical decomposition, photodecomposition, pressure-dependent decomposition, ultrasonic decomposition, and mechanical decomposition.

[0176] To control decomposition, the shell may be formed to include one or more chemical functionalities. In one non-limiting example, the shell may include polymers that decompose thermally (e.g., in a desired high or low temperature range), such as polyesters, polyurethanes, polyamides, poly(dialkylsiloxanes), and polycarbonates. In one non-limiting example, the vehicle may contain thermally unstable groups, such as azo compounds, that decompose at a specified temperature. The shell may be configured so that such thermal decomposition proceeds at temperatures of about 40°C or higher, about 50°C or higher, about 60°C or higher, about 70°C or higher, or about 80°C or higher. In an alternative embodiment, the shell may include a polymer that is solubilized at a trigger temperature, e.g., below 15°C.

[0177] In one or more embodiments, the shell may be configured to remain substantially intact at the point of delivery. However, the shell may be further configured to release reagents within the core, such as lyophilized microspheres or beads, over time. In a non-limiting example, the shell may be configured so that the release of the core is delayed for a specific period of time. In one or more embodiments, core release may not occur substantially under standard conditions (e.g., down to the lowest temperature, e.g., up to about 20°C, 25°C, 30°C, 35°C, 40°C, up to about 50°C, or up to about 60°C), but release may be induced when such standard conditions are exceeded. In a non-limiting example, the release of core components from the particles can be delayed as desired by changing the polymer crosslinking density, hydrophobic / hydrophilic balance, particle size, shell thickness and thermal properties, and / or ionic properties. Other methodologies may also be utilized to provide delayed release of components in the core.

[0178] In some embodiments, the delayed release of the core component can be measured from the time the particles are prepared, from the time of the first delivery of the particles (e.g., contact between the particles and the aqueous solution), or from the time the particles first encounter the conditions of the desired delivery site (e.g., the conditions for the tagmentation reaction). The delayed release can be for about 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, or about 45 minutes or more. In each case, the maximum time of delayed release depends on the time it takes for the dissolution reaction to occur.

[0179] In one or more embodiments, the Disclosure may relate the properties of compositions and systems to the conditions under which they are provided. More specifically, compositions and systems may exhibit a first set of features and / or functions under a first set of conditions, and a second set of features and / or functions under a second set of conditions. The first set of conditions (which may be referred to as “standard conditions”) may be the conditions under which particles are prepared and / or stored, and the second set of conditions may include the conditions present at the location where particles are delivered. The first set of conditions may be, for example, near room temperature and atmospheric pressure. The second set of conditions may be, for example, the conditions encountered in a dissolution reaction or tagmentation reaction. As described above, the release of reagents from particles may depend on the conditions encountered by the particles. Specifically, particle decomposition may not be substantially present under the first set of conditions but may be present under the second set of conditions. Similarly, diffusion may not be substantially present under the first set of conditions but may be present under the second set of conditions. Therefore, the second set of conditions can be characterized as the conditions under which the emission of microspheres can proceed.

[0180] In some embodiments, the conditions under which core release may occur may be particularly related to temperature. For example, release may be provided at temperatures of about 50°C or higher, about 60°C or higher, about 70°C or higher, or about 80°C or higher. In some embodiments, such temperatures may have an upper limit that coincides with the average maximum temperature of a DNA library preparation reaction as described herein. In some embodiments, release may be provided at temperatures of about 15°C or lower.

[0181] As a further example, the conditions under which release may occur may be particularly related to pH. In particular, core release may occur when particles are subjected to a pH change (increase or decrease) of at least about 1, at least about 2, or at least about 4. The pH change may be a change of about 1 to about 12, about 1.5 to about 10, or about 2 to about 8.

[0182] A second set of conditions under which content release may occur may encompass any one of the above conditions within the above range. A second set of conditions under which content release may occur may encompass two or more of the above conditions within the above range. For example, core release may occur based on any one of the above temperature, pH range, and salt concentration. In some embodiments, content release may occur when particles are subjected to any of the following combinations of the above conditions, e.g., temperature and pH, and temperature and salinity.

[0183] Freeze drying Any material, particularly storage stability bioassay reagents, can be lyophilized using methods known in the art. Typically, the pre-lyophilization formulation further contains appropriately selected excipients or other components, such as stabilizers, buffers, fillers, and surfactants, to inhibit or prevent the degradation (e.g., protein aggregation, deamidation, and / or oxidation) of the compound of interest during lyophilization and storage. The lyophilization formulation may contain one or more additional components, including lyophilization inhibitors or stabilizers, buffers, fillers, isotonic agents, and surfactants.

[0184] After the target substance and any additional components are mixed together, the formulation is freeze-dried. Freeze-drying generally involves three main stages: freezing, primary drying, and secondary drying. Freezing is necessary to convert water into ice or some amorphous formulation components into crystalline form. Primary drying is the process of removing ice from the freeze product by direct sublimation at low pressure and low temperature. Secondary drying is the process of removing bound water from the product matrix by utilizing the diffusion of residual water onto the evaporation surface. The product temperature during secondary drying is usually higher than during primary drying.

[0185] A method of freeze-drying is described, for example, in Bjelosevic et al. (International Journal of Pharmaceutics, 576:119029, 2020), which is incorporated herein by reference.

[0186] The quality of freeze-drying can be evaluated by measuring the glass transition temperature (Tg') and / or eutectic temperature of the maximally freeze-concentrated fraction. The glass transition temperature is the temperature at which an amorphous polymer changes from a hard / glassy state to a soft / leather-like state, or vice versa. Tg is directly related to the strength and capabilities of the desired material. Generally, the product temperature should be several degrees lower than Tc and / or Tg' to avoid collapse. The eutectic temperature is the lowest melting temperature that the solution can achieve.

[0187] The rehydration (or reconstitution) solution, for example, a sample containing DNA, may, as used herein, include water, deionized water, physiological saline, acidic solution, basic solution, surfactant solution, and / or buffer. In preferred embodiments, the rehydration solution is water or a buffer. Additional additives described herein can be provided in the rehydration solution to further improve control over microsphere release.

[0188] In various embodiments, the pH of the rehydration solution is about 6.0 to about 10.0, or about 7.0 to about 8.0. The pH of the rehydration solution may 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 in between. The rehydration time varies depending on the composition content and reaction conditions (e.g., reagents, temperature, pH). In various embodiments, the rehydration time may be 0.1 seconds to 10 hours. For example, the rehydration time may be about 0.1 seconds, 1 second, 10 seconds, 30 seconds, 45 seconds, 60 seconds, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 5 hours, 8 hours, 10 hours, or any time in between.

[0189] The freeze-dried material can be analyzed for its physical appearance and, after reconstruction, can be evaluated for its effectiveness in producing a good quality library and in other characteristic verification tests. Exemplary visual cake-appearance dissolved and tagmented freeze-dried materials of this disclosure are provided in Figure 7.

[0190] Lyophilization can be carried out in containers such as tubes, bags, bottles, trays, vials (e.g., glass vials), syringes, or any other suitable containers. The containers may be disposable. Lyophilization can also be carried out on a large or small scale. In some cases, it may be desirable to lyophilize the protein preparation in the container in which the protein reconstitution takes place to avoid the transfer step. In this case, the container may be, for example, a 3, 4, 5, 10, 20, 50, or 100 cc vial.

[0191] Generally, freeze-drying results in freeze-dried formulations with a moisture content of less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, less than approximately 1%, and less than approximately 0.5%.

[0192] Furthermore, means for stabilizing the sample during sample collection and upon completion of the dissolution and library preparation processes are also considered. In various embodiments, for example, a sample containing gDNA or other DNA is stabilized with PEG6K, alkyl polyglucosides, salts, chaotropic agents (e.g., GITC, urea), proteinase inhibitors, antioxidants (β-mercaptoethanol or dithiothreitol (DTT)), or buffers for pH stabilization. The stabilizer may be in the same container as the lyophilized cake or lyophilized microsphere, or in a different container or microsphere as described herein.

[0193] In alternative embodiments, the sample is stabilized as a dried blood spot on a surface, which can be eluted in a container such as a tube, multiwell plate, or other container as described herein.

[0194] Smart consumables One aspect of this disclosure envisions consumables for performing molecular analysis workflows that are “smart,” i.e., possess automated recognition data capture (AIDC) capabilities (“AIDC-enabled”). In one embodiment, the molecular analysis workflow is a next-generation sequencing workflow. As disclosed herein, an exemplary NGS workflow may include cell lysis and DNA extraction, optionally isothermal PCR amplification steps, library preparation / tagmentation, sequencing, imaging, and data analysis.

[0195] As used herein, “smart” refers to equipment, devices, materials or items, components and / or parts connected to other equipment, devices, materials or items, components and / or parts as part of a larger network and / or network cloud. Typically, smart equipment, devices, materials or items, components and / or parts can connect to other smart equipment, devices, materials or items, components and / or parts via different wireless protocols or data transmissions, such as Bluetooth®, NFC, Wi-Fi, LiFi, 3G, etc., which can operate to some degree interactively and autonomously. As used herein, “network cloud,” “cloud,” or “the cloud” refers to a private, public, or semi-public space that exists between data transmission endpoints. Generally, data being transmitted enters the network cloud from one endpoint using standard protocols and shares the space within the network cloud with other data transmissions. Often, data can also leave the network cloud, where it may be encapsulated, transformed, and transferred in many ways in the same format in which it entered the network cloud.

[0196] In some embodiments, the AIDC method used in either or both of the smart reaction substrate / holder and / or smart reagent container is a smart label. In some embodiments, the AIDC method used in either or both of the reaction substrate / holder and / or reagent container is a radio frequency identification (RFID) tag. Thus, in some embodiments, the system includes a reaction substrate / holder having a smart label or RFID tag, and / or a reagent container having a smart label or RFID tag. In some embodiments, if the system includes both a reaction substrate / holder having a smart label or RFID tag, e.g., a reaction plate, and one or more reagent containers having smart labels or RFID tags, the reaction substrate / holder smart label / RFID tag and the reagent container smart label / RFID tag can store and share information about the molecular analysis system together or collectively. For example, system information may be stored or shared about the samples, reagents, assays, users, and / or workflows used in the NGS workflow.

[0197] As used herein, “RFID tag” refers to a component such as a chip that stores digital data and / or information. In some embodiments, the tag comprises an integrated circuit, an antenna, and protective material that holds these components together and protects them from various environmental conditions. The protective material is application-dependent, and RFID tags may be available in various shapes and sizes. The integrated circuit may store data that can be communicated (e.g., transmitted or received) by radio frequencies transmitted by the antenna. The integrated circuit and antenna circuit may be printed on a chip. The RFID tag may be read by an RFID reader using an antenna that transmits radio frequencies to query the RFID tag. As used herein, the term “RFID reader” includes an RFID device that can read information from and / or write information to an RFID tag. In some embodiments, the RFID tag may be read-only or read / write, and the information associated with the RFID tag may be hardcoded to the RFID tag at the time of manufacture or at some point later, or the RFID tag may contain information that is written to the RFID tag throughout its lifespan. In some embodiments, the RFID tag is a “passive RFID tag,” which has no energy source of its own but transmits a signal in response to a signal from a reader. In other embodiments, the RFID tag is an “active RFID tag,” which has its own power source, such as a battery. A “writable RFID tag” is an RFID tag that has a memory area that can be written to by an RFID writer. A “smart label” is similar to an RFID tag and can incorporate both RFID technology and barcode technology. In some embodiments, the smart label is made from an adhesive label with an embedded RFID tag and may also contain a barcode and / or other information.Some examples of RFID tags are described in U.S. Patent Nos. 6,147,662, 6,917,291, 5,949,049, 6,652,812, 6,112,152, and U.S. Patent Application Publication 2003 / 0183683, all of which are incorporated herein by reference in their entirety with respect to their disclosures in the design and use of RFID tags, chips, labels, or devices, RFID readers, and RFID systems.

[0198] In some embodiments, the reaction substrate or reaction holder disclosed herein may include, but are not limited to, chambers, channels, cards, arrays, containers, slides, or plates. In various embodiments, the reaction substrate or reaction holder may have multiple reaction sites. Some examples of reaction substrates or reaction holders having multiple reaction sites may include, but are not limited to, multi-well plates, e.g., standard microtiter 96-well, 384-well plates, or microcards, or substantially planar supports, e.g., slides, and open arrays or arrays. In some embodiments, the reaction substrate or reaction holder may be made from glass or plastic or any other suitable material obvious to those skilled in the art. In various embodiments of the reaction substrate or reaction holder, the reaction sites may include depressions, notches, ridges, and combinations thereof, patterned in regular or irregular arrangements formed on the surface of the reaction substrate or reaction holder.

[0199] In various embodiments, one or more reagent containers as disclosed herein may include, but are not limited to, containers, bottles, tubes, vials, wells, or chambers, or any combination thereof. In some embodiments, reagent containers may be made of glass or plastic or any other suitable material obvious to those skilled in the art. Reagent containers may be of any size or dimensions, and may differ from one reagent container to another within the same system.

[0200] The smart consumables disclosed herein may include, for example, one or more smart reaction substrates or reaction holders (e.g., reaction plates or reaction arrays) and / or one or more smart reagent containers. Each smart reaction substrate or reaction holder may include a reaction substrate or reaction holder RFID tag. Similarly, each reagent container may have a reagent container RFID tag. Working together or separately, the reaction substrate or reaction holder RFID tags and the reagent container RFID tags can store and share various data or information. Both the reaction substrate or reaction holder RFID tags and the reagent container RFID tags can transmit and / or receive information. An RFID tag reader can read information stored on an RFID tag. An RFID writer can write (or rewrite) information on an RFID tag.

[0201] As used herein, the term “Information” means data that can be electronically stored in an RFID tag and can be retrieved for use as machine-readable or human-readable data for processing biological reagents and / or carriers.

[0202] In some embodiments, the smart consumable includes pre-spotted reagents. As used herein, “pre-spotted” means a reaction substrate or reaction holder that contains reagents that have been added to (or pre-loaded) by the manufacturer of the reaction substrate or reaction holder and are not directly added to the reaction substrate or reaction holder by the user. A pre-spotted reaction substrate or reaction holder may also be considered ready for immediate use. As used herein, “ready for immediate use” may mean that the number of additional reagents that need to be added to the reagent to initiate the reaction is limited, or that no additional reagents are required, or that only liquids such as water or buffer and / or test samples need to be added to the reagent to initiate the reaction.

[0203] The RFID tags in the RFID-tagged container can store data regarding the characteristics of the reagents in the container, the identification of the reagents applied to or released into the blood sample to initiate the reaction, the volume of the blood sample applied to the container, the date and / or time the blood sample is applied to the container, the identification of the next reagent used in the NGS workflow, and / or the interaction between pre-spotted reagents and added blood samples in the workflow.

[0204] In one embodiment, the smart consumable is an RFID-tagged container pre-spotted with microspheres of the lysis reagent and core / shell particles of the tagmentation reagent for an NGS workflow. In one embodiment, the RFID-tagged container is used to collect a biological sample and perform DNA library preparation. In another embodiment, the NGS workflow begins with the user adding a test sample.

[0205] In one embodiment, a container for collecting a sample contains a plurality of lyophilized microspheres for carrying out a DNA library preparation reaction. The container may also contain a lysis buffer in the lid or cap of a smart consumable having an RFID tag. The lyophilized microspheres may be encapsulated within coated particles. In one embodiment, the uncoated lyophilized microspheres in the container contain a lysis reagent. In another embodiment, the coated particles containing the lyophilized microspheres in the container contain a tagmentation reaction reagent. See Figure 4.

[0206] In another embodiment, the container for collecting the sample contains a lyophilized cake for carrying out the DNA library preparation reaction. The container may also contain a lysis buffer in the lid or cap of a smart consumable having an RFID tag. In one embodiment, the uncoated lyophilized microspheres in the container contain a lysis reagent. In another embodiment, the coated particles containing lyophilized microspheres in the container contain a tagmentation reaction reagent.

[0207] In another embodiment, the container for taking the sample includes freeze-dried beads, coated solid beads, or stacked freeze-dried cakes.

[0208] In alternative embodiments, the smart consumable can leverage elapsed time when transporting the sample to a sequencing laboratory to perform lysis and tagmentation reactions. In one embodiment, the container includes a timestamp or other indicator to notify the consumer when the desired reaction, e.g., tagmentation, gap-filling / extension ligation, or PCR synthesis of the first (fist) strand, is complete. In one embodiment, the container for collecting the sample contains multiple lyophilized microspheres for performing DNA library preparation reactions. The container may also contain lysis buffer within the lid or cap of the smart consumable having RFID. The lyophilized microspheres may be encapsulated within coated particles. In one embodiment, the uncoated lyophilized microspheres in the container contain the lysis reagent. In another embodiment, the coated particles containing the lyophilized microspheres in the container contain the tagmentation reaction reagent. See Figure 5.

[0209] In various embodiments, the container or smart consumable is stored at approximately 4°C to 30°C during the insertion, sealing, and / or transport processes. In various embodiments, the container or smart consumable is stored at approximately 4°C to 8°C, approximately 4°C to 25°C, or approximately 20°C to 30°C during the insertion, sealing, and / or transport processes.

[0210] In another embodiment, the smart consumable container includes a device that enables complete DNA library preparation. In one embodiment, the container includes a heating element that enables library preparation (gap filling, indexing, and denaturation) in transit, and a timestamp or other indicator that notifies the consumer when the desired reaction, e.g., tagmentation, gap filling / extension ligation, or PCR synthesis of the first strand, is complete. In one embodiment, the container for collecting a sample includes a plurality of lyophilized microspheres for carrying out the DNA library preparation reaction. The container may also contain a lysis buffer within the lid or cap of the smart consumable having RFID. The lyophilized microspheres may be encapsulated within coated particles. In one embodiment, the uncoated lyophilized microspheres in the container contain a lysis reagent. In another embodiment, the coated particles containing the lyophilized microspheres in the container contain a tagmentation reaction reagent. In a further embodiment, the container includes additional coated particles that encapsulate the lyophilized microspheres. See Figure 6.

[0211] In one embodiment, the RFID-tagged container may further include a heating element, temperature sensor, optical sensor, and / or motion sensor directly or indirectly coupled to the reagent, container, or RFID tag, the RFID tag storing the temperature history, exposure history, and motion detection history of the reaction substrate or reaction holder. In some embodiments, the RFID-tagged container further includes a barcode. Exemplary RFID-tagged containers are described in International Publication No. 2006081612(A1), U.S. Patent Application Publication No. 20170336428(A1), and International Publication No. 2009076011(A1), which are incorporated herein by reference.

[0212] In one embodiment, the heating mechanism provides at least one temperature zone having a predetermined temperature for thermal inactivation of proteinase K and / or as a thermal trigger release mechanism for the core / shell particles of the disclosure. In a further embodiment, the heating mechanism provides a temperature of about 65°C. Examples of suitable heating mechanisms include, for example, the sodium acetate method, the use of a self-heating hot pot utilizing a supersaturated solution of a metal salt, particularly an aqueous solution of sodium acetate (CH3COONa), which releases heat when the solute crystallizes into sodium acetate trihydrate (CH3COONa·3H2O) due to external mechanical stimulation applied for initial nucleation of sodium acetate crystals, such as strong shaking of the solution or impact with a sharp tip.

[0213] In various embodiments, RFID tags are used to manage patient-specific information associated with a sample throughout the entire process of sample collection, preparation, and analysis, enabling sample identification and tracking. Optionally, the RFID-tagged container further includes a temperature, colorimetric, or fluorescent indicator that responds to the appropriate chemical species in a completed reaction by producing an observable change, thereby alerting the user that the reaction is complete. In one embodiment, the colorimetric indicator is incorporated into the encapsulated particle and released after digestion, along with an enzyme acting as a trigger. In another embodiment, the indicator may be incorporated into the shell to react with the blood lysate. In yet another embodiment, the indicator may be immobilized on the wall of the RFID-tagged container to react to any of the following: changes in environmental conditions, release of intracellular components, or release of components from the encapsulated core.

[0214] The data relating to the characteristics of reagents, blood samples, and RFID-tagged containers may include one or more of the following: ID number, expiration date, part number, barcode, lot number, part type, storage temperature and / or storage temperature range, reagent concentration, reagent shape factor, recommended reagent concentration and / or volume for use in the workflow, configuration for liquid transfer support, sales order number, reagent name, assay name, assay location of the reagent used on the reaction holder, assay ID, proposed or required protocol for the NGS workflow, sample name, master mix name, internet link or address (url), reaction and / or reagent volume, test sample name, analysis settings for molecular analysis, sample type, molecular analysis type, and instrument execution protocol. The data relating to the characteristics of reagents, blood samples, and RFID-tagged containers may be written to and / or rewritten on the reaction substrate or reaction holder RFID tag and / or reagent container RFID tag. The reaction substrate or reaction holder RFID tag and / or reagent container RFID tag may have a capacity to store at least 8 kilobytes of information.

[0215] In another embodiment, the RFID container has tamper-evident features to minimize the risk of contamination, which is widespread in alternative sampling systems for samples such as blood and semen. In one embodiment, such a tamper-evident container is sealed using a built-in tamper-evident and tamper-detection adhesive. Such an adhesive may contain a light or gas activator that indicates tampering.

[0216] In various embodiments, tests are provided to indicate that the dissolution of the sample has progressed to completion. In various embodiments, the test may be a lateral flow test, a colorimetric test, or a temperature sensor. In certain embodiments, the test is performed inside the container. In other embodiments, the test is performed outside the container.

[0217] A system comprising the compositions described herein is provided herein. The system comprises one or more compositions described herein and one or more containers, wherein one or more compositions are placed in one or more containers under conditions effective for forming a continuous system for preparing a DNA library. In various embodiments, the system comprises one or more containers for holding the compositions, and one or more containers include PCR tubes, vials, microtubes, flow cells, multiwell plates, glass tubes, transwell membrane / mesh inserts, cartridges, or microfluidic tips.

[0218] In various embodiments, a composition, container, kit, or method comprises a lyophilized cake in combination with one or more lyophilized microspheres described herein. In various embodiments, the lyophilized cake is contained in a multiwell plate, microtube, cartridge, or other container described herein. In various embodiments, the lyophilized cake is rehydrated with a rehydration solution. In various embodiments, the rehydration solution is a lysis buffer and, optionally, a proteinase, as described herein. Methods for preparing lyophilized materials such as cakes or microspheres for use alone or in combination in a system or container for DNA sample processing are disclosed in U.S. Patent Application Publication 2022 / 0331770, incorporated herein by reference.

[0219] The system may further include a temperature controller or a sensor. The temperature controller can be used to change or adjust the temperature of the system to further control the release of various components of the compositions described herein. For example, the temperature controller can be used to accelerate or decelerate the release from a first or second shell. Similarly, the temperature controller can be used to accelerate or decelerate the release of an inner core to promote or control the release of one or more reagents. In one embodiment, the system comprises a temperature controller on a container in the system. For example, the temperature controller may include a resistance heater adjacent to the walls of the container, e.g., a cartridge, tube, tip, or well, to supply heat to these. The temperature controller may also include a temperature sensor. The temperature controller may also include a circuit to activate and deactivate a heater to maintain the well at a specified temperature.

[0220] kit In another embodiment, the Disclosure provides a kit comprising a container for holding a lyophilized formulation, wherein the formulation can be reconstituted within about 15 minutes, and instructions for reconstituting the lyophilized mixture with a diluent to produce a reconstituted liquid formulation. In some embodiments of this aspect of the Invention, the kit includes instructions for reconstituting the lyophilized formulation with a diluent. The kit may further include a second container containing a diluent.

[0221] The kit may include one or more containers (such as vials, ampoules, containers, tubes, etc.) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing the core / shell lyophilized composition of the present invention in an appropriate dose for DNA extraction and library preparation. The kit may further include instructions for use (e.g., in the form of a leaflet or instruction manual) and means for collecting the biological sample of the present disclosure (e.g., a syringe). [Examples]

[0222] The following embodiments are intended to illustrate and not to limit the scope of the present disclosure as set forth in the appended claims.

[0223] Example 1 - Materials and General Method material Poly(lactic-glycolic acid) (PLGA), DMSO, trehalose anhydride, sucrose, corn starch, α-cyclodextrin, magnesium acetate, tetrapeptidyl chloromethyl ketone (TCK, proteinase inhibitor, catalog number 539470), tris-acetic acid, Tween 20, EDTA, SDS, and tris-HCl polyethylene glycol (PEG) 1500 and 4000 were purchased from Sigma (Sigma Chemical Company, St. Louis, MO) (Thermo Fisher Scientific, Waltham, MA, USA). PEFABLOC® SC-protease inhibitor (catalog number 11429868001) was purchased from MILLIPORE® (Millipore Corp., Bedford, MA). Thermally unstable (TL) NEB proteinase K and non-thermally unstable proteinase K were purchased from New England Biolabs (catalog numbers P8111S and P8107, respectively, NEB, Ipswich, MA). Proteocut K was purchased from Biocatalysts (catalog number PK909L, Cardiff, Wales). Proteinase K and DNA-IQ® paramagnetic particles were purchased from Promega (catalog numbers V3021 and DC6701, respectively, Promega, Madison, WI). Proteinase K was purchased from Sigma (catalog number P4850, Sigma Aldrich, St. Louis, MO). Proteinase K was purchased from Roche (catalog number 3 115 836, Roche Diagnostics GmbH). Proteinase K was purchased from Zymo Research (catalog number D3001-2-20, Zymo Research Corp.). The protease was purchased from Qiagen (catalog number 19157, QIAGEN GmbH, Hilden, Germany).

[0224] Determination of Tg' value of antifreeze solution The glass transition temperature (Tg') of the fraction obtained by maximally freezing and concentrating a 10% w / v antifreeze was determined in a 0.1% PVA aqueous solution using differential scanning calorimetry (DSC). Measurements were performed using a DSC4000 Perkin-Elmer calorimeter under a nitrogen atmosphere, employing a cycle of cooling the solution from +20°C to -70°C at 2°C / min, followed by a reheating cycle of returning to +20°C at 2°C / min. The instrument was calibrated for temperature and heat flow using a two-point calibration method with indium and zinc reference samples at a scanning speed of 10°C / min before measuring the test samples.

[0225] Example 2 - Evaluation of the suitability of various proteinase Ks used in DNA library preparation. To produce high-quality lyophilized microspheres for sample preparation, it is beneficial that the reagents function very well in several respects. The reagents must effectively isolate pure DNA samples from various sample types, and the DNA yield must be as high as possible. They must be lyophilized (lyo-compatible), e.g., free of glycerol. They must be compatible with other components in the DNA extraction process and be thermally unstable (e.g., at 70-80°C) for easy inactivation before the tagmentation process, or efficiently inactivated by a protease inhibitor. They must be user-friendly, meaning the process should not be too cumbersome, the components should not be toxic, and they should be easily disposed of. Figure 9A shows the characteristics of various proteinase K enzymes evaluated herein.

[0226] The proteases from the four suppliers, suppliers A-D, are lyophilized because they do not contain glycerol buffer (Figure 9A). Furthermore, they are all inhibited by PK inhibitors (TCKs) that show minimal interference with the tagmentation reaction (Figure 9C).

[0227] Example 3 - Sigma PK demonstrated the best results in the “one-pot” lyophilization assay of this disclosure. Blood samples were collected from human subjects. Cells were lysed, and DNA was released from 25 μL of sample using a "one-pot" core / shell reagent containing NEB proteinase K and four other proteinase K (i.e., suppliers A-D) that were found to be most compatible with the "one-pot" assay. The blood lysates were loaded into a custom NovaSeq S1 flow cell (ILLUMINA®, San Diego, CA, USA), and library preparation was performed on the flow cell according to the manufacturer's instructions.

[0228] Insert Size: Insert sizes for different libraries were calculated based on read alignment to the reference genome. The average insert size of libraries prepared using TL NEB from suppliers C-D ranged from 362 to 381 bp. Libraries from suppliers A-B had average insert sizes of 430 and 446 bp, respectively. All candidate PKs resulted in insert sizes well over 300 bp, enabling the optimal use of 151 × 151 paired-end reads.

[0229] Base calling accuracy, measured by the Phred-like quality score (Q score) %Q30, is a common metric used to evaluate the accuracy of a sequencing run. A higher quality score indicates a lower probability of individual bases being miscalled. Currently, the Q30 score represents a 1 / 1000 chance of incorrect base identification (Ewing, B., & Green, P., 1998, Genome Research, 8(3):186-194; Ewing et al., 1998 Genome Res, 8(3):175-85.) and is the de facto standard for measuring the accuracy of NGS reads. Q30 is equivalent to a probability of 1 in 1000 incorrect base calls. This means that the base calling accuracy (i.e., the probability of a correct base call) is 99.9%. A base calling accuracy as low as 99% (Q20) results in a step with a 1 in 100 chance of incorrect base calling, meaning that an error may be included in every 100 base pairs of sequencing reads. When sequencing quality reaches Q30, virtually all reads are complete, free from errors and ambiguity. All five libraries in this experiment passed this criterion, with the lowest score coming from supplier A's PK library (77%), while the other four libraries had comparable %≧Q30 scores ranging from 85% to 89% (Figure 11).

[0230] Coverage: Coverage depth refers to the average number of sequencing reads that align to or "cover" each base in the sequenced sample. The Lander / Waterman formula is a method for calculating coverage (C) based on read length (L), number of reads (N), and haploid genome length (G): C = LN / G. Libraries generated using TL NEB and supplier B's PK had a high and comparable genome coverage of 48×. Libraries generated using supplier A, C, and D's PK had lower genome coverage of 34×, 23×, and 15×, respectively.

[0231] Mapping Read Percentage: The mapping read percentage refers to the percentage of reads that are aligned to the reference genome. All libraries had >96% of all reads mapped to the reference genome, but libraries produced using supplier A's protease showed a slightly lower mapping rate of 86% and also exhibited the greatest variability. For very good libraries, the mapping read percentage should exceed 90%, and for good libraries, it should exceed 80%.

[0232] Passing filter (%PF): The Passing filter (PF) is an index used to describe clusters that pass the chastity threshold and is used for further processing and analysis of sequencing data. %PF calculation involves applying a chastity filter to each cluster. "Chastity" is defined as the brightest base intensity divided by the sum of the brightest base intensity and the second brightest base intensity. A cluster "passes the filter" if one or fewer base calls have a chastity value of less than 0.6 in the first 25 cycles. This filtering process removes the least reliable clusters from the image analysis results. Therefore, a higher %pass filter (%PF) result indicates an increased yield of usable sequencing data. In this study, supplier B's PK library consistently showed good results with a 46% %PF, comparable to the TL NEB PK control library (Figure 10). Supplier D's PK run (n=2) had a %PF of 33%, indicating that this PK could be an alternative, although more runs may be beneficial for a definitive evaluation (Figure 9). Suppliers A and D's PK libraries had low %PF values ​​(15% and 25%, respectively), highlighting potential issues with library preparation (Figure 9).

[0233] Overall, this test identified supplier B's PK as the best-performing PK compared to the control TL NEB (Figure 10).

[0234] Example 4 - Effect of coating on freeze-drying and trigger coating The advantage of this method is that multiple lyophilized microspheres or particle-encapsulated lyophilized microspheres are present in the sample collection container, potentially reducing the number of steps used to complete the DNA library preparation workflow.

[0235] In one embodiment, a coating polymer can be incorporated that provides a desired time-delayed release from the particles of freeze-dried microspheres. For example, for a 2-minute delayed release (time delay related to shell thickness), the component may be HPMC, hydroxyethyl / propylcellulose, polyethylene glycol, PVP-co-PVAc, Eudragit, isoleucine, Eudragit RL / RS, or Opadry CA, polyester (i.e., PLGA). For a 15-minute time-triggered release, the material may be methyl / ethylcellulose, cellulose acetate (CA), and / or PLGA. Other release triggers may be temperature-triggered, for example, using proteinase K (Prot K) inactivation at 55°C, and potential materials may be waxes, fatty acids, fatty acid esters, or upper critical dissolution temperature (USCT) polymers, such as poly(acrylamide-co-acrylonitrile), poly(N-acryloylglycinamide), and poly(N-isopropylacrylamide) copolymers.

[0236] During the dissolution phase of the reaction, there is no release trigger. However, to initiate the tagmentation reaction, a 15-minute time-delay release trigger or a temperature release trigger is used to release the tagmentation lyophilized microspheres into the reaction vessel.

[0237] Next, we hypothesized that it would be possible to coat the surface of microspheres with an enzyme, and that this would be a coating material used for PK-compatible microspheres. The particle coating material was prepared using VA64, Protect, Efka, Makon, ethanol, H2O) + PK. The coatings were tested with a functional assay, i.e., a standard extract capable of evaluating PK activity. Figure 8 shows that the DNA yield was similar to the control when 5 μL and 20 μL of the coating material were added.

[0238] Example 5 - Lyophilization and the lyophilized formulation do not reduce the activity of Sigma PK. The effectiveness of lyophilization using Sigma's PK enzyme and the composition of the lyophilized formulation were tested by comparing the DNA yield from the liquid formulation or from extracts containing lyophilized Sigma PK immediately after lyophilization / reconstitution. Since there was no statistically significant difference in DNA extraction yield using the liquid or lyophilized Sigma PK formulations, both formulations essentially extracted DNA equally well (Figure 11A). Furthermore, libraries prepared from DNA extracted using lyophilized PK lysis buffer from supplier B showed comparable sequencing performance to libraries prepared from DNA extracted using the liquid PK formulation from supplier B (Figure 11B). The effect of trehalose (used in the lyophilized formulation) was tested by adding this reagent to a control sample (NA12878) extracted using a standard method (e.g., Qiagen QIAmp DNA blood Mini Kit). There was no significant effect on the sequencing performance of this library (Figure 11B I). PK thermal inactivation was also tested by sequencing libraries prepared using the control liquid formulation, the reconstituted lyophilized PK formulation from supplier B, and PK thermal inactivation. The use of different formulations did not significantly affect the sequencing performance of the two libraries when heat was used for inactivation (Figure 11B II). However, the presence of trehalose appears to interfere with the TCK inactivation of Sigma PK (Figure 11B III). It is hypothesized that combinations of different reagents, such as trehalose with novel excipients (e.g., PVP 40K, dextran 40K), may resolve this issue.

[0239] Example 6 - Evaluation of the suitability of tagmentation and lyophilization reagents by testing their effects on lyophilization and sequencing indices. Cyclodextrins form complexes with hydrophobic and amphiphilic molecules such as surfactants. α-Cyclodextrins are often used in tagmentation buffers in combination with proteinase K inhibitors such as TCK, chelating SDS derived from the lysis buffer. However, TCK inhibitors are more soluble in organic solvents, such as 100% DMSO. Unlike aqueous samples, organic solvents are difficult to freeze, and dilution before freezing is beneficial. Furthermore, due to the physical properties of DMSO, there are conflicting reports regarding its effectiveness for freeze-drying. On the one hand, DMSO's high boiling point (189°C) and low vapor pressure at room temperature (0.08 kPa at 25°C) make it unsuitable for evaporation, while its high freezing point (18.4°C) means it can be efficiently sublimated (Jakubowska et al., 2022. J Drug Deliv Sci Technol, 74:103528). In addition, DMSO is a polar organic compound, and it lowers the freezing point of water by distorting its hydrogen bonds, thereby inhibiting ice formation. As a result, lyophilization of DMSO / water mixtures can be particularly difficult depending on the composition, as the eutectic point of this organic solvent at 50-70% content is approximately -70 to -60°C (Jakubowska et al., 2022. J Drug Deliv Sci Technol, 74:103528).

[0240] [Table 3]

[0241] [Table 4]

[0242] To evaluate the effect of DMSO on the lyophilization of tagmentation buffers and to determine the optimal concentration of DMSO to use, five tagmentation buffers with different DMSO concentrations were formulated (Buffers 1-5, Table 4A), and their Tg' were measured by differential scanning calorimetry (DSC). A Tg' of >-40°C is desirable, as this is a common primary drying temperature in a typical lyophilization process. This test showed that a 1% DMSO concentration yielded a Tg' of >-40°C, making it the most suitable concentration for the formulation. The sequencing indices of three test libraries prepared using Sigma proteinase K and different TCK and DMSO concentrations—0.08 mg / mL TCK & 1.7% DMSO, 0.08 mg / mL TCK & 0.25% DMSO, and 0.07 mg / mL TCK & 0.2% DMSO—were comparable to the indices of the control experiment prepared using TL NEB PK (Table 4B, Figure 12B). This means that the DMSO% for the final 2× formulation can be reduced to 0.4%, which allows for lyophilization of the sample buffer. Since 0.7% and 0.2% DMSO concentrations showed the same performance, this indicates that the DMSO concentration can be further reduced. Buffer 4 (Table 4B) is an exemplary tagmentation buffer formulation with a lower DMSO concentration. Sequence runs of libraries prepared from buffers containing TCK:0.7%DMSO, TCK:0.25%DMSO, and TCK:0.08%DMSO all had equivalent quality indicators (Figure 13). The differences in insert size observed here were due to the batch of TSM used, not the TCK.

[0243] Cyclodextrin and TCK can be included in tagmentation buffer: To evaluate the suitability of the tagmentation buffer for sequencing performance, the sequencing index of libraries prepared using lyophilized tagmentation buffer versus liquid tagmentation buffer (without cyclodextrin) was compared. Lyophilization of the buffer did not significantly affect the sequencing index, and therefore, it is compatible with the formulation (Figure 14A). To evaluate the suitability of TCK for the tagmentation buffer's sequencing performance, the sequencing index of libraries prepared using three components (CD, tag buffer components, and TCK) was evaluated. Figure 14B shows what appears to be compatible when the reagents are lyophilized together.

[0244] Example 7 - Effect of proteinase K on de-escalation of blood samples Next, we determined whether a lysis buffer containing PK could inactivate viruses that could contaminate blood samples. 25 μL of blood containing vaccinia virus was collected in 225 μL of lysis buffer containing PK and incubated at 20°C, 55°C, or 70°C for 15 minutes. After 5 days, the level of virus in the samples was determined. The results showed that vaccinia virus in blood was inactivated by >99% in lysis buffer alone and completely inactivated in lysis buffer at >55°C for 15 minutes (Figure 17).

[0245] The present invention has been described with reference to specific examples and embodiments. However, this application is intended to include modifications and substitutions that are apparent to those skilled in the art and can be made without departing from the spirit and scope of the claims. All references cited herein are incorporated by reference.

Claims

1. A system comprising a container having an opening for receiving a biological sample, wherein the container comprises a workflow reagent dispensing system, and the workflow reagent dispensing system is A plurality of first lyophilized microspheres comprising a lysis buffer and proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A system comprising a plurality of first particles, each comprising a second outer shell enclosing a second inner core, the second outer shell containing one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, the second outer shell releasing the second inner core in response to a second release trigger mechanism.

2. A system comprising a container having an opening for receiving a biological sample, wherein the container comprises a workflow reagent dispensing system, and the workflow reagent dispensing system is A plurality of first lyophilized microspheres comprising a lysis buffer and a thermally unstable proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A system comprising a plurality of first particles, each comprising a second outer shell enclosing a second inner core, the second outer shell containing one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, the second outer shell releasing the second inner core in response to a second release trigger mechanism.

3. A system comprising a container having an opening for receiving a biological sample, wherein the container comprises a workflow reagent dispensing system, and the workflow reagent dispensing system is A lyophilized cake containing a lysis buffer and proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A system comprising a plurality of first particles, each comprising a second outer shell enclosing a second inner core, the second outer shell containing one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, the second outer shell releasing the second inner core in response to a second release trigger mechanism.

4. A system comprising a container having an opening for receiving a biological sample, wherein the container comprises a workflow reagent dispensing system, and the workflow reagent dispensing system is A lyophilized cake containing a lysis buffer and a heat-unstable proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A system comprising a plurality of first particles, each comprising a second outer shell enclosing a second inner core, the second outer shell containing one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, the second outer shell releasing the second inner core in response to a second release trigger mechanism.

5. The system according to any one of claims 1 to 4, wherein the first particle comprises a third outer shell enclosing a third inner core, the third inner core comprising workflow reagents for extension ligation and PCR, and the third outer shell releases the third inner core in response to a third release trigger mechanism.

6. The system according to any one of claims 1 to 4, further comprising a plurality of second particles, each comprising a third outer shell enclosing a third internal core, wherein the third internal core comprises workflow reagents for extension ligation and PCR, and the third outer shell releases the third internal core in response to a third release trigger mechanism.

7. The system according to any one of claims 1 to 6, wherein the first external shell and the second external shell are each affected only by the first release trigger mechanism or the second release trigger mechanism.

8. The system according to any one of claims 1 to 7, wherein the first trigger release mechanism is the dissolution of the lyophilized microspheres by the biological sample, thereby forming a dissolution solution.

9. The system according to any one of claims 1, 2, or 5 to 8, wherein the first freeze-dried microsphere comprises one or more reagents for lysing cells contained in the biological sample.

10. The system according to any one of claims 3 to 8, wherein the freeze-dried cake comprises one or more reagents for lysing cells contained in the biological sample.

11. The system according to claim 9 or 10, wherein one or more reagents for lysing cells are selected from the group consisting of phosphate buffer, salt, surfactant, alcohol, protease, lysis buffer, freeze-drying inhibitor, or a combination thereof.

12. The system according to claim 11, wherein the proteinase is a broad-spectrum serine protease.

13. The system according to claim 12, wherein the broad-spectrum serine protease is proteinase K, and optionally thermally unstable proteinase K.

14. The system according to claim 11, wherein the surfactant is sodium dodecyl sulfate (SDS).

15. The system according to any one of claims 1 to 14, wherein the first release trigger, the second release trigger, or the third release trigger is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof.

16. The system according to any one of claims 1 to 15, wherein the second trigger release mechanism includes temperature-controlled trigger release or time-controlled trigger release.

17. The system according to any one of claims 1 to 16, wherein the second internal core comprises a lyophilized microsphere containing one or more tagmentation reagents.

18. The system according to claim 17, wherein the one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelating agent, a primer having a transpososome, a lyophilization inhibitor, and optionally a proteinase K inhibitor.

19. The system according to claim 18, wherein the SDS chelating agent is a CD selected from the group consisting of α-cyclodextrin (CD), β-CD, and γ-CD.

20. The system according to any one of claims 1 to 19, wherein the first outer shell, the second outer shell, and / or the third outer shell, if present, comprises one or more of the following: polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, Eudragit®, cellulose acetate, ethylcellulose, UCST and LCST polymers, or any combination thereof.

21. The system according to any one of claims 11 to 20, wherein the freeze-drying inhibitor is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

22. The system according to any one of claims 1 to 21, wherein the biological sample is blood.

23. The system according to any one of claims 1 to 22, wherein the nucleic acid is DNA.

24. The system according to claim 23, wherein the DNA is genomic DNA (gDNA).

25. A composition, A plurality of first lyophilized microspheres comprising a lysis buffer and proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A composition comprising a plurality of first particles, each comprising a second outer shell encapsulating a second inner core, the second outer shell comprising one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, wherein the second outer shell releases the second inner core in response to a second release trigger mechanism.

26. A composition, A plurality of first lyophilized microspheres comprising a lysis buffer and a thermally unstable proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, A composition comprising a plurality of first particles, each comprising a second outer shell that encapsulates a second inner core containing one or more lyophilized microspheres containing one or more reagents for DNA tagmentation.

27. A composition, A lyophilized cake containing a lysis buffer and proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A composition comprising a plurality of first particles, each comprising a second outer shell encapsulating a second inner core, the second outer shell comprising one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, wherein the second outer shell releases the second inner core in response to a second release trigger mechanism.

28. A composition, A lyophilized cake containing a lysis buffer and a heat-unstable proteinase, Multiple first particles, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, A composition comprising a plurality of first particles, each comprising a second outer shell that encapsulates a second inner core containing one or more lyophilized microspheres containing one or more reagents for DNA tagmentation.

29. The composition according to any one of claims 25 to 28, wherein the first particle comprises a third outer shell enclosing a third inner core, the third inner core comprising a workflow reagent for extension ligation and PCR, and the third outer shell releases the third inner core in response to a third release trigger mechanism.

30. The composition according to any one of claims 25 to 28, further comprising a plurality of second particles including a third outer shell enclosing a third internal core, wherein the third internal core comprises a workflow reagent for extension ligation and PCR, and the third outer shell releases the third internal core in response to a third release trigger mechanism.

31. The composition according to any one of claims 25 to 30, wherein the first outer shell releases the first inner core in response to a first release trigger mechanism, the second outer shell releases the second inner core in response to a second release trigger mechanism, and if a third release trigger mechanism is present, the third outer shell releases the third inner core in response to a third release trigger mechanism.

32. The composition according to any one of claims 25 to 31, wherein the first trigger release mechanism is activated by the biological sample dissolving one or more freeze-dried microspheres, thereby forming a dissolution solution.

33. The composition according to any one of claims 25, 26, or 29-32, wherein the first freeze-dried microsphere comprises one or more reagents for lysing cells.

34. The composition according to any one of claims 27 to 32, wherein the freeze-dried cake comprises one or more reagents for lysing cells.

35. The composition according to any one of claims 25 to 34, wherein one or more reagents for lysing cells are selected from the group consisting of phosphate buffer, salt, surfactant, alcohol, protease, lysis buffer, freeze-drying inhibitor, or a combination thereof.

36. The composition according to claim 35, wherein the proteinase is a broad-spectrum serine protease.

37. The composition according to claim 36, wherein the broad-spectrum serine protease is proteinase K, and optionally thermally unstable proteinase K.

38. The composition according to claim 37, wherein the surfactant is sodium dodecyl sulfate (SDS).

39. The composition according to any one of claims 25 to 38, wherein the first release trigger, the second release trigger, or the third release trigger is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof.

40. The composition according to any one of claims 25 to 39, wherein the second trigger release mechanism includes temperature-controlled trigger release or time-controlled trigger release.

41. The composition according to any one of claims 25 to 40, wherein the second internal core comprises one or more lyophilized microspheres containing one or more tagmentation reagents.

42. The composition according to claim 41, wherein the one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelating agent, a primer having a transpososome, a lyophilization inhibitor, and optionally a proteinase K inhibitor.

43. The composition according to claim 42, wherein the SDS chelating agent is a CD selected from the group consisting of α-cyclodextrin (CD), β-CD, and γ-CD.

44. The composition according to any one of claims 25 to 43, wherein the first outer shell, the second outer shell, and / or the third outer shell, if present, comprises one or more of the following: polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, Eudragit®, cellulose acetate, ethylcellulose, UCST and LCST polymers, or any combination thereof.

45. The composition according to any one of claims 35 to 44, wherein the freeze-drying inhibitor is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

46. It is a method, A. A biological sample is collected from the subject, and the container has an opening, the container includes a workflow reagent release system, The aforementioned workflow reagent release system is a. A first lyophilized microsphere comprising a lysis buffer and proteinase, b. The first particle, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A biological sample in a container comprising: a first particle comprising: a second outer shell enclosing a second inner core comprising one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, wherein the second outer shell releases the second inner core in response to a second release trigger mechanism; The biological sample interacts with the freeze-dried lysis buffer in the container, resulting in the release of nucleic acids derived from cells in the biological sample. The lysis buffer is reacted with the biological sample for a sufficient amount of time to lyse the cells in the biological sample, and the biological sample is placed in the buffer. B. By activating the first release trigger mechanism to release the proteinase inhibitor and surfactant chelating agent from the first internal core, the dissolution reaction (A) is inactivated after the time, and the inactivation reaction is allowed to proceed for a sufficient time to inactivate the proteinase and chelate the surfactant. C. A method comprising activating the second release trigger mechanism to release the reagent for DNA tagging from the second internal core, thereby stopping the inactivation reaction (B), and allowing the tagging reaction to proceed for a sufficient time to tag the nucleic acid derived from the biological sample.

47. It is a method, A. A biological sample is collected from the subject, and the container has an opening, the container includes a workflow reagent release system, The aforementioned workflow reagent release system is a. A lyophilized cake containing a lysis buffer and proteinase, b. The first particle, A first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A biological sample in a container comprising: a first particle comprising: a second outer shell enclosing a second inner core comprising one or more lyophilized microspheres containing one or more reagents for DNA tagmentation, wherein the second outer shell releases the second inner core in response to a second release trigger mechanism; The biological sample interacts with the freeze-dried lysis buffer in the container, resulting in the release of nucleic acids derived from cells in the biological sample. The lysis buffer is reacted with the biological sample for a sufficient amount of time to lyse the cells in the biological sample, and the biological sample is placed in the buffer. B. By activating the first release trigger mechanism to release the proteinase inhibitor and surfactant chelating agent from the first internal core, the dissolution reaction (A) is inactivated after the time, and the inactivation reaction is allowed to proceed for a sufficient time to inactivate the proteinase and chelate the surfactant. C. A method comprising activating the second release trigger mechanism to release the reagent for DNA tagging from the second internal core, thereby stopping the inactivation reaction (B), and allowing the tagging reaction to proceed for a sufficient time to tag the nucleic acid derived from the biological sample.

48. The method according to claim 46 or 47, further comprising isolating the nucleic acid from (C) and generating a nucleic acid library using a library preparation kit.

49. The method according to any one of claims 46 to 48, wherein the first external shell and the second external shell are each affected only with respect to the first release trigger mechanism or the second release trigger mechanism.

50. The method according to any one of claims 46 to 49, wherein the first trigger mechanism is initiated when the biological sample dissolves the lyophilized microsphere containing the lysis buffer.

51. The method according to any one of claims 46 to 50, wherein the first freeze-dried microsphere comprises one or more reagents for lysing cells in the biological sample.

52. The method according to any one of claims 46 to 50, wherein the freeze-dried cake comprises one or more reagents for lysing cells in the biological sample.

53. The method according to claim 51 or 52, wherein the one or more reagents for lysing cells are selected from the group consisting of phosphate buffer, salt, surfactant, alcohol, protease, lysis buffer, freeze-drying inhibitor, or a combination thereof.

54. The method according to claim 53, wherein the proteinase is a broad-spectrum serine protease.

55. The method according to claim 54, wherein the broad-spectrum serine protease is proteinase K, and optionally thermally unstable proteinase K.

56. The method according to claim 54, wherein the surfactant is sodium dodecyl sulfate (SDS).

57. The method according to any one of claims 46 to 56, wherein the first release trigger, the second release trigger, or the third release trigger is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof.

58. The method according to any one of claims 46 to 57, wherein the second trigger release mechanism includes temperature-controlled trigger release or time-controlled trigger release.

59. The method according to any one of claims 46 to 58, wherein the second internal core comprises one or more lyophilized microspheres containing one or more tagmentation reagents.

60. The method according to claim 59, wherein the one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelating agent, a primer having a transpososome, a lyophilization inhibitor, and optionally a proteinase K inhibitor.

61. The method according to claim 60, wherein the SDS chelating agent is a CD selected from the group consisting of α-cyclodextrin (CD), β-CD, and γ-CD.

62. The method according to claims 46 to 61, wherein the tagmentation reaction occurs at 25°C to 55°C.

63. The method according to any one of claims 46 to 62, wherein the first outer shell, the second outer shell, and / or the third outer shell, if present, comprises one or more of the following: polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, Eudragit®, cellulose acetate, ethylcellulose, UCST and LCST polymers, or any combination thereof.

64. The method according to any one of claims 46 to 63, wherein the freeze-drying inhibitor is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

65. The method according to any one of claims 46 to 64, wherein the biological sample is blood.

66. The method according to any one of claims 46 to 65, wherein the nucleic acid is DNA.

67. The method according to claim 66, wherein the DNA is genomic DNA (gDNA).

68. The method according to any one of claims 46 to 67, wherein the sample is brought into contact with a first lyophilized microsphere containing a lysis reagent for producing a cell lysate, the lysis reagent having one or more proteases, and the cell lysate containing a target nucleic acid.

69. The release of the tagmentation reagent is achieved by applying at least one transposon terminal composition containing at least one transposase and a transfer chain under conditions in which the target nucleic acid and the transposon terminal composition undergo a rearrangement reaction to produce a mixture. The target nucleic acid is fragmented to generate multiple target nucleic acid fragments, The method according to any one of claims 46 to 68, wherein the transfer chain of the transposon terminal composition is ligated to the 5' end of each of the plurality of target nucleic acid fragments to generate a plurality of 5'-tagged target nucleic acid fragments.

70. The method according to any one of claims 46 to 69, wherein the target nucleic acid is double-stranded DNA, and the target nucleic acid remains double-stranded DNA before application of the transposase and transposon terminal composition.

71. A container for collecting a biological sample, wherein the container includes a workflow reagent dispensing system according to any one of claims 1 to 24, and the container is equipped with an indicator that changes upon completion of the workflow reagent dispensing system within the container.

72. The container according to claim 71, wherein the container includes a radio-frequency identification (RFID) tag.

73. The container according to claim 71 or 72, wherein the RFID tag is embedded in the container, and the RFID has a capacity to store at least 8 kilobytes of information.

74. A container according to any one of claims 71 to 73, comprising an opening for receiving a biological sample containing nucleic acids.

75. The container according to claims 71 to 74, further comprising a heating element and a temperature sensor coupled to the container, wherein the RFID tag of the container stores a temperature history.

76. The container according to any one of claims 71 to 75, wherein the container is tamper-proof.

77. The container according to any one of claims 71 to 76, wherein the container is made from polypropylene or a cyclic olefin copolymer.

78. The container according to any one of claims 71 to 77, wherein the container is a PCR tube, vial, microtube, flow cell, multiwell plate, glass tube, cartridge, or microfluidic tip.

79. A method for transporting samples for the preparation of a nucleic acid library, The sample is to be inserted into a container, the container comprising the system described in any one of claims 1 to 24. The container is sealed so that the system can begin the process of sample dissolution and nucleic acid library preparation. A method comprising transporting the sealed container to a nucleic acid sequencing laboratory, and, upon arrival at the sequencing laboratory, the system enabling the system to complete the lysis of the sample, tagmentation of the nucleic acids in the sample, extension ligation of the nucleic acids in the sample, and PCR.

80. The method according to claim 79, wherein the container contains the composition according to any one of claims 22 to 39 before the sample is inserted into the container.

81. The method according to claim 79 or 80, wherein the container is stored at 4°C to 30°C when inserting the sample into the container, when sealing the container, and / or when transporting the sealed container to a laboratory for nucleic acid sequencing.

82. The method according to any one of claims 79 to 81, further comprising removing the container from the shipment and isolating the nucleic acid from the sample in the container.

83. The method according to any one of claims 79 to 82, wherein the container includes an RFID tag.

84. The method according to any one of claims 79 to 83, wherein the container further includes an indicator that the library preparation has been completed.

85. The method according to any one of claims 79 to 84, wherein the biological sample is blood.

86. The method according to any one of claims 79 to 85, wherein the nucleic acid is DNA.

87. The method according to claim 86, wherein the DNA is genomic DNA (gDNA).

88. It is a method, A container, wherein the container includes a workflow reagent dispensing system, and the workflow reagent dispensing system is A first lyophilized microsphere containing a lysis buffer and proteinase, A first particle having a first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A sample in a container comprising: a second particle having a second outer shell enclosing a second inner core, the second outer shell releasing the second inner core in response to a second release trigger mechanism; and a second particle having one or more lyophilized microspheres containing one or more reagents for DNA tagmentation. The sample interacts with the lysis buffer in the container, resulting in the release of nucleic acids derived from cells in the biological sample, and the sample is positioned accordingly. By activating the first release trigger mechanism, the proteinase inhibitor and the surfactant chelating agent are released from the first internal core, A method comprising activating the second release trigger mechanism to release one or more reagents for DNA tagging from the second internal core.

89. It is a method, A container, wherein the container includes a workflow reagent dispensing system, and the workflow reagent dispensing system is A lyophilized cake containing a lysis buffer and proteinase, A first particle having a first outer shell enclosing a first inner core, wherein the first inner core comprises one or more lyophilized microspheres containing a proteinase inhibitor and a surfactant chelating agent, and the first outer shell releases the first inner core in response to a first release trigger mechanism, A sample in a container comprising: a second particle having a second outer shell enclosing a second inner core, the second outer shell releasing the second inner core in response to a second release trigger mechanism; and a second particle having one or more lyophilized microspheres containing one or more reagents for DNA tagmentation. The sample interacts with the lysis buffer in the container, resulting in the release of nucleic acids derived from cells in the biological sample, and the sample is positioned accordingly. By activating the first release trigger mechanism, the proteinase inhibitor and the surfactant chelating agent are released from the first internal core, A method comprising activating the second release trigger mechanism to release one or more reagents for DNA tagging from the second internal core.

90. The method according to claim 88 or 89, further comprising isolating the nucleic acid and generating a nucleic acid library using a library preparation kit.

91. The method according to any one of claims 88 to 90, wherein the first external shell and the second external shell are each affected only with respect to the first release trigger mechanism or the second release trigger mechanism.

92. The method according to any one of claims 88 to 91, wherein the first trigger mechanism is initiated when the biological sample dissolves the lyophilized microsphere containing the lysis buffer.

93. The method according to any one of claims 88 to 92, wherein the first freeze-dried microsphere comprises one or more reagents for lysing cells in the biological sample.

94. The method according to any one of claims 88 to 92, wherein the freeze-dried cake comprises one or more reagents for lysing cells in the biological sample.

95. The method according to claim 93 or 94, wherein the one or more reagents for lysing cells are selected from the group consisting of phosphate buffer, salt, surfactant, alcohol, protease, lysis buffer, freeze-drying inhibitor, or a combination thereof.

96. The method according to claim 95, wherein the proteinase is a broad-spectrum serine protease.

97. The method according to claim 96, wherein the broad-spectrum serine protease is proteinase K, and optionally thermally unstable proteinase K.

98. The method according to claim 95, wherein the surfactant chelating agent is sodium dodecyl sulfate (SDS).

99. The method according to any one of claims 88 to 98, wherein the first release trigger mechanism and / or the second release trigger mechanism is a temperature-controlled release mechanism, a pH-controlled release mechanism, a time-controlled release mechanism, a position-controlled release mechanism, or any combination thereof.

100. The method according to any one of claims 88 to 99, wherein the second release trigger mechanism includes temperature-controlled trigger release or time-controlled trigger release.

101. The method according to any one of claims 88 to 100, wherein the second internal core comprises a lyophilized microsphere containing one or more tagmentation reagents.

102. The method according to claim 101, wherein the one or more tagmentation reagents are selected from the group consisting of a Tn5 transposase enzyme, one or more transposons, a linker sequence, a Tn5 2× tagmentation buffer, Mg2+, an SDS chelating agent, a primer having a transpososome, a lyophilization inhibitor, and optionally a proteinase K inhibitor.

103. The method according to claim 102, wherein the SDS chelating agent is a CD selected from the group consisting of α-cyclodextrin (CD), β-CD, and γ-CD.

104. The method according to claims 88 to 103, wherein the release of one or more reagents for DNA tagmentation brings about a tagmentation reaction, and the tagmentation reaction occurs at 25°C to 55°C.

105. The method according to any one of claims 88 to 104, wherein the first outer shell and / or the second outer shell comprises one or more of the following: polyvinyl alcohol, polyvinylpyrrolidone (PVP), carrageenan, gelatin, hydroxypropyl methylcellulose (HPMC), pullulan, starch film, benzoxabolol-poly(vinyl alcohol) (benzoxabolol-PVA), pectin, Eudragit®, cellulose acetate, ethylcellulose, UCST and LCST polymers, or any combination thereof.

106. The method according to any one of claims 95 to 98 and 102 to 103, wherein the freeze-drying inhibitor is selected from the group consisting of mannitol, sorbitol, inositol, sucrose, glucose, mannose, and trehalose.

107. The method according to any one of claims 88 to 106, wherein the sample is blood.

108. The method according to any one of claims 88 to 107, wherein the nucleic acid is DNA.

109. The method according to claim 108, wherein the DNA is genomic DNA (gDNA).

110. The method according to any one of claims 88 to 109, further comprising placing the sample in a container to rehydrate the one or more freeze-dried microspheres containing the lysis buffer and the proteinase.

111. Releasing one or more reagents for DNA tagmentation involves applying at least one transposon terminal composition containing at least one transposase and a transfer chain under conditions in which the target nucleic acid and the transposon terminal composition undergo a rearrangement reaction to produce a mixture. The target nucleic acid is fragmented to generate multiple target nucleic acid fragments, The method according to any one of claims 88 to 110, wherein the transfer chain of the transposon terminal composition is ligated to the 5' end of each of the plurality of target nucleic acid fragments to generate a plurality of 5'-tagged target nucleic acid fragments.

112. The method according to claim 111, wherein the target nucleic acid is double-stranded DNA, and the target nucleic acid remains double-stranded DNA before application of the transposase and transposon terminal composition.

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