WO2025136701A1

WO2025136701A1 - Passive encapsulated workflows

Info

Publication number: WO2025136701A1
Application number: PCT/US2024/058983
Authority: WO
Inventors: Sébastien RICOULT; Andrew Slatter; David Jones; Rigoberto PANTOJA; Johan Sebastian Basuki; Qingdi ZHU
Original assignee: Illumina Inc
Current assignee: Illumina Inc
Priority date: 2023-12-22
Filing date: 2024-12-06
Publication date: 2025-06-26
Anticipated expiration: 2026-06-22

Abstract

Lyophilized microspheres or comparable form factors containing different biological sample preparation processing reagents are provided in a single well 302, 505, 603, 705. Triggered release of each of the reagents by different trigger mechanisms (such as time, temperature, light, pH, enzymatic or chemical reaction, etc.) allows sample preparation processing steps to be sequentially or otherwise performed within the single well. In some implementations, protein binding with beads and either biotinylation and cleavage or polymerase chain reaction (PCR) and cleanup are sequentially performed in the single well. In other implementations, DNA amplification, fragmentation, and hybridization are sequentially performed. The numbers of touch points, material transfers, reagents, and turnaround times required for sample preparation can be reduced, and the need for cold storage of samples may be reduced or eliminated.

Description

PASSIVE ENCAPSULATED WORKFLOWS

TECHNICAL FIELD

[0001] This disclosure relates generally to deoxyribonucleic acid (DNA) handling systems and processes. More specifically, this disclosure relates to passive encapsulated workflows.

BACKGROUND

[0002] The advent of massively parallel short-read sequencing technologies, also known as Next Generation Sequencing (NGS), has reduced the costs of sequencing DNA by orders of magnitude. Moreover, the very high throughput data acquisition with NGS has allowed for rapid sequencing of complete genomes with unprecedented ease, providing access to increasing amounts of genomic, transcriptomic, and epigenetic data across all fields of biology. NGS-based projects can be roughly divided into the following process elements: sample pre-processing for nucleic acid extraction (NAE); library preparation; and sequencing (data acquisition and/or bioinformatics). These process elements are typically tailored and optimized to a target nucleic acid (RNA or DNA), and a suitable sequencing system is selected.

SUMMARY

[0003] Lyophilized product(s) (such as cake, beads, or microspheres) containing different biological sample preparation processing reagents are provided in a single well. Triggered release of each of the reagents by different trigger mechanisms (such as time, temperature, light, pH, enzymatic or chemical reaction, etc.) allows sample preparation processing steps to be sequentially or otherwise performed within the single well. In some implementations, protein binding with beads and either biotinylation and cleavage or polymerase chain reaction (PCR) and cleanup are sequentially performed in the single well. In other implementations, DNA amplification, fragmentation, and hybridization are sequentially performed. The numbers of touch points, material transfers, reagents, and turnaround times required for sample preparation can be reduced, and the need for cold storage of samples may be reduced or eliminated.

[0004] In a first implementation, a method of preparing biological samples includes providing, in a single well, first particles including a first reagent for a first biological sample preparation processing step, second particles including a second reagent for a second biological sample preparation processing step, and third particles including a third reagent for a third biological sample preparation processing step. Tire first particles release the first reagent in response to a first release trigger mechanism. The second particles release the second reagent in response to a second release trigger mechanism. The third particles release the third reagent in response to a third release trigger mechanism. Release in response to one of the first or second trigger mechanisms may establish conditions for one of the second or third trigger mechanisms (“domino” release). The first, second, and third particles are in a form of lyophilized microspheres or a comparable form factor, and at least the third particles are in a form of one of encapsulated particles, stacked cakes separated by trigger layers, or solid substrate beads coated with an active material and encapsulated with trigger release shell(s). Each encapsulated particle includes an inner core including a lyophilized microsphere and an outer shell. The method also includes sequentially performing the first sample preparation processing step, the second sample preparation processing step, and the third sample preparation processing step in the single well using triggered release of one or both of the first reagent and the second reagent and separately-triggered release of the third reagent from the encapsulated particles.

[0005] In various implementations, the first sample preparation processing step is amplification, the second sample preparation processing step is fragmentation, and the third sample preparation processing step is hybridization.

[0006] In various implementations, the first reagent is an amplification and random primer mixture in the form of particles, the second reagent is a fragmentation mixture in the form of particles with a time delay release, and the third reagent is a hybridization buffer in the form of the encapsulated particles with temperature release.

[0007] In various implementations, the amplification includes whole genome amplification.

[0008] In various implementations, the single well is located on a first plate. The first reagent includes a whole genome amplification reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature. The second reagent includes a fragmentation solution reagent with time delay release. The method also includes adding sample DNA to a well containing DNA denaturant on a second plate. The method further includes adding water and a buffer to produce denatured DNA. The method still further includes robotically transferring the denatured DNA from the well on the second plate to the single well on the first plate. In addition, the method includes heating the single well to release the first reagent and waiting for a period corresponding to the time delay release of the second reagent. The single well contains fragmented DNA following release of the first reagent and the second reagent.

[0009] In various implementations, the first reagent includes a mixture of the whole genome amplification (WGA) reagent and a targeted genome amplification (TGA) reagent in the form of encapsulated particles with temperature release at a second temperature higher than the first temperature. Alternate form factors to encapsulated particles include stacked cakes separated by trigger layers, solid substrate beads (such as polypropylene, dissolvable starch/sugar bead(s) coated with the active material and encapsulated with the trigger release shell(s)). Heating the single well to release the first reagent includes heating the single well to the first temperature to release the WGA reagent and subsequently heating the single well to the second temperature to release the TGA reagent. Alternate trigger mechanisms to elevated temperature include time, low temperature, pH or light, and workflows may be adjusted to enable the triggered releases for each release event.

[0010] In various implementations, the single well is located on a single plate. The single well contains denature reagent in the form of lyophilized microspheres with temperature release at room temperature. The first reagent includes a whole genome amplification reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature. The second reagent includes a fragmentation solution reagent with time delay release. The method also includes adding sample DNA to the single well and adding water and a buffer to the single well at room temperature to produce denatured DNA. The method further includes heating the single well to release the first reagent. In addition, the method includes waiting for a period corresponding to the time delay release of the second reagent. The single well contains fragmented DNA following release of the first reagent and the second reagent.

[0011] In various implementations, denatured genomic DNA is transferred to the single well.

[0012] In various implementations, the single well is located on a first plate. The method also includes adding gDNA to a well containing DNA denaturant in the form of encapsulated particles with time delay release on a second plate and transferring the denatured gDNA from the well on the second plate to the single well on the first plate.

[0013] In various implementations, the DNA denaturant includes a neutralizing reagent core with a sodium hydroxide shell.

[0014] In various implementations, an encapsulation of particles for the third reagent is wax, and the single well is heated to release the third reagent from the encapsulation.

[0015] In various implementations, the first sample preparation processing step is protein binding, the second sample preparation processing step is biotinylation, and the third sample preparation processing step is cleavage.

[0016] In various implementations, the first reagent is stabilized slow off-rate modified aptamer (SOMAmer) beads, the second reagent includes components to biotinylate proteins in the form of particles, and the third reagent is a light, enzymatic, or chemical cleavage mixture in the form of encapsulated particles, stacked cakes separated by trigger layers, or solid substrate beads coated with an active material and encapsulated with trigger release shell(s) with light, enzymatic, or chemical triggered release.

[0017] In various implementations, the first reagent are beads for binding of cleaned analytes, the second reagent includes a polymerase chain reaction (PCR) mixture in the form of particles with a time delay release, and the third reagent is exonuclease shrimp alkaline phosphatase (ExoSAP) in the form of encapsulated particles with temperature release. [0018] In a second implementation, a process for preparing biological samples includes providing a plurality of wells on an integrated reagent plate. Each well contains first particles including an amplification mixture, second particles including a fragmentation mixture, and third particles including a hybridization buffer. The first particles activate the amplification mixture in response to a first mechanism. The second particles activate the fragmentation mixture in response to a second mechanism. The third particles release the hybridization buffer in response to a third mechanism. The first, second, and third particles include lyophilized microspheres, and each of at least the third particles includes an inner core including a lyophilized microsphere and an outer shell. The process includes sequentially performing nucleic acid amplification, fragmentation, and hybridization on a biological sample in the respective well using separate activation of the amplification mixture and the fragmentation mixture and triggered release of the hybridization buffer.

[0019] In various implementations, the amplification mixture includes a whole genome amplification reagent. Each of a first subset of the first particles includes a whole genome amplification inner core including a lyophilized microsphere for the whole genome amplification reagent and a whole genome amplification outer shell. The whole genome amplification outer shell for the first subset of the first particles releases the whole genome amplification reagent for amplification of DNA in response to a first temperature higher than room temperature. Tire process also includes heating each well to the first temperature to release the WGA reagent.

[0020] In various implementations, the amplification mixture further includes a targeted genome amplification reagent. Each of a second subset of the first particles includes a targeted genome amplification inner core including a lyophilized microsphere for the targeted genome amplification reagent and a targeted genome amplification outer shell. The targeted genome amplification outer shell for the second subset of the first particles releases the targeted genome amplification reagent for amplification of the DNA in response to a second temperature higher than the first temperature. The process also includes, subsequent to heating each well to the first temperature, heating each well to the second temperature to release the TGA reagent.

[0021] In various implementations, the fragmentation mixture includes a fragmentation reagent, where each of the second particles includes a fragmentation inner core including a lyophilized microsphere for the fragmentation reagent and a fragmentation outer shell. The fragmentation outer shell for the second particles releases the fragmentation reagent for fragmentation of amplified DNA after a time delay. The process also includes, subsequent to heating each well to the first temperature, allowing time for release of the fragmentation reagent and for fragmentation.

[0022] In var ious implementations, each well further contains fourth particles including a DNA denaturant. The fourth particles activate the DNA denaturant in response to a fourth mechanism. The fourth particles include lyophilized microspheres. The process also includes adding at least sample DNA in an aqueous solution.

[0023] In a third implementation, a process for preparing biological samples includes providing a well containing first particles including protein binding beads, second particles including a biotinylation mixture, and third particles including a light cleavage mixture. Tire protein beads may be targeted to a panel of specific proteins using affinity reagents such as aptamers, for example, SOMAmers. The first particles activate protein binding in response to a first mechanism, the second particles activate the biotinylation mixture in response to a second mechanism, and the third particles release the light cleavage mixture in response to a third mechanism. The first, second, and third particles include lyophilized microspheres, and each of at least the second and third particles includes an inner core including a lyophilized microsphere and an outer shell. The process also includes sequentially performing protein binding, biotinylation, and light cleavage on a biological sample in the well using separate activation of the protein binding beads and triggered release of the biotinylation mixture and the light cleavage mixture.

[0024] In various implementations, the protein binding beads include stabilized slow off-rate modified aptamers (e.g. SOMAmer) beads.

[0025] In various implementations, the second mechanism is a time delay, and the third mechanism is light.

[0026] In a fourth implementation, a system for preparing biological samples includes a container with an opening configured to receive a biological sample. The container is configured to provide, in a single well, a workflow reagent release system including a first reagent for a first sample preparation processing step for the biological sample, a second reagent for a second sample preparation processing step for the biological sample, and a third reagent for a third sample preparation processing step for the biological sample. At least the third reagent is contained within encapsulated particles. The single well is configured such that the first sample preparation processing step, the second sample preparation processing step, and the third sample preparation processing step are sequentially performed in the single well using triggered release of one or both of the first reagent and the second reagent and separately-triggered release of the third reagent from the encapsulated particles.

[0027] In various implementations, the first sample preparation processing step is amplification, the second sample preparation processing step is fragmentation, and the third sample preparation processing step is hybridization.

[0028] In various implementations, the first reagent is an amplification and random primer mixture in the form of particles, the second reagent is a fragmentation mixture in the form of particles with a time delay release, and the third reagent is a hybridization buffer in the form of the encapsulated particles with temperature release.

[0029] In various implementations, the amplification and random primer mixture includes a whole genome amplification reagent.

[0030] In various implementations, the single well is located on a first plate, where the first reagent includes a whole genome amplification reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature. The second reagent includes a fragmentation solution reagent with time delay release. A well on a second plate is configured to receive sample DNA and DNA denaturant. The well on the second plate is also configured to receive water and a buffer to produce denatured DNA. The well on the second plate is further configured to allow the denatured DNA to be robotically transferred from the well on the second plate to the single well on the first plate. The single well is configured to be heated to release the first reagent. After a period corresponding to the time delay release of the second reagent, the single well contains fragmented DNA.

[0031] In various implementations, the first reagent includes a mixture of the whole genome amplification reagent and a targeted genome amplification reagent in the form of encapsulated particles with temperature release at a second temperature higher than the first temperature. The single well is configured to be heated to the first temperature to release the whole genome amplification (WGA) reagent and to be subsequently heated to the second temperature to release the TGA reagent.

[0032] In various implementations, the single well is located on a single plate. The single well contains denature reagent in the form of lyophilized microspheres with temperature release at room temperature. The first reagent includes a whole genome amplification reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature. The second reagent includes a fragmentation solution reagent with time delay release. The single well is configured to receive sample DNA. The single well is also configured to receive water and a buffer at room temperature to produce denatured DNA. The single well is further configured to be heated to release the first reagent. After a period corresponding to the time delay release of the second reagent, the single well contains fragmented DNA following release of the first reagent and the second reagent.

[0033] In various implementations, the single well is configured to receive denatured genomic DNA.

[0034] In various implementations, the single well is located on a first plate. A well on a second plate is configured to receive gDNA and DNA denaturant in the form of encapsulated particles with time delay release. The well on the second plate is configured to allow the denatured gDNA to be transferred from the well on the second plate to the single well on the first plate.

[0035] In various implementations, the DNA denaturant includes a neutralizing reagent core with a sodium hydroxide shell.

[0036] In various implementations, an encapsulation of particles for the third reagent is wax. The single well is configured to be heated to release the third reagent from the encapsulation.

[0037] In various implementations, the first sample preparation processing step is protein binding, the second sample preparation processing step is biotinylation, and the third sample preparation processing step is cleavage.

[0038] In various implementations, the first reagent is stabilized slow off-rate modified aptamer (SOMAmer) beads, the second reagent includes components to biotinylate proteins in the form of particles, and the third reagent is a light cleavage mixture in the form of the encapsulated particles with light triggered release.

[0039] In various implementations, the first reagent are beads for binding of cleaned analytes, the second reagent includes a polymerase chain reaction (PCR) mixture in the form of particles with a time delay release, and the third reagent is exonuclease shrimp alkaline phosphatase (ExoSAP) in the form of the encapsulated particles with temperature release.

[0040] In various implementations, the single well is a container including a radio-frequency identification (RFID) tag. In various implementations, the RFID tag is embedded on the container. The RFID tag may have the capacity to store at least 8 kilobytes of information,

[0041] In various implementations, the container includes an opening for receiving a biological sample including nucleic acids in a cup-shaped receptacle.

[0042] In various implementations, the container includes a heating element and a temperature sensor coupled to the container.

[0043] In various implementations, the container is tamper-proof.

[0044] In various implementations, the container is made from polypropylene or cyclic olefin copolymer. In various implementations, the container is a PCR tube, vial, microtube, flow cell, multiwell plate, glass tube, cartridge or microfluidic chip.

[0045] In a fifth implementation, a composition includes first particles including a first reagent for a first biological sample preparation processing step. The first particles are configured to release the first reagent in response to a first release trigger mechanism. The composition also includes second particles including a second reagent for a second biological sample preparation processing step. The second particles are configured to release the second reagent in response to a second release trigger mechanism for the biological samples. The composition further includes third particles including a third reagent for a third biological sample preparation processing step. The third particles are configured to release the third reagent in response to a third release trigger mechanism. The first, second, and third particles include lyophilized microspheres, and at least the third particles include encapsulated particles. Each encapsulated particle includes an inner core including a lyophilized microsphere and an outer shell. The composition is configured such that the first biological sample preparation processing step, the second biological sample preparation processing step, and the third biological sample preparation processing step are sequentially performed in a single well containing the first, second, and third particles by triggered release of one or both of the first reagent and the second reagent and separately- triggered release of the third reagent from the encapsulated particles.

[0046] In various implementations, the first sample preparation processing step is amplification, the second sample preparation processing step is fragmentation, and the third sample preparation processing step is hybridization.

[0047] In various implementations, the first reagent is an amplification and random primer mixture in the form of particles, the second reagent is a fragmentation mixture in the form of particles with a time delay release, and the third reagent is a hybridization buffer in the form of encapsulated particles with temperature release.

[0048] In various implementations, the amplification and random primer mixture includes a whole genome amplification reagent.

[0049] In various implementations, the single well is located on a first plate. The first reagent includes a whole genome amplification reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature. The second reagent includes a fragmentation solution reagent with time delay release.

[0050] In various implementations, the first reagent includes a mixture of the whole genome amplification reagent and a targeted genome amplification reagent in the form of encapsulated particles with temperature release at a second temperature higher than the first temperature. The single well is configured to be heated to the first temperature to release the WGA reagent and to be subsequently heated to the second temperature to release the TGA reagent.

[0051] In various implementations, the single well is located on a single plate. The single well contains denaturation reagent in the form of lyophilized microspheres with temperature release at room temperature. The first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature. The second reagent includes a fragmentation solution reagent with time delay release.

[0052] In various implementations, the composition includes DNA denaturant including a neutralizing reagent core with a sodium hydroxide shell.

[0053] In various implementations, the first sample preparation processing step is protein binding, the second sample preparation processing step is biotinylation, and the third sample preparation processing step is cleavage.

[0054] In various implementations, the first reagent is stabilized slow off-rate modified aptamer (e.g., SOMAmer) beads, the second reagent includes components to biotinylate proteins in the form of particles, and the third reagent is a light cleavage mixture in the form of encapsulated particles with light triggered release. Alternatively, use of non-light cleavage mechanisms such as an enzyme (e.g., protease that target specific peptide sequence, lipase that target ester linkage, and cellulase/glycoside hydrolase that target saccharide) or chemically-induced cleavage (e.g., pH via an acid or base, reduction-oxidation (redox) via a reducing agent such as dithiothreitol (DTT)/tris(2- carboxyethyl)phosphine (TCEP), and use of a metal catalyst such as palladium (Pd)Ztetrahydropyran (THP) to cleave an allyl ether) may be used to trigger release.

[0055] In various implementations, the first reagent are beads for binding of cleaned analytes, the second reagent includes a polymerase chain reaction (PCR) mixture in the form of particles with a time delay release, and the third reagent is exonuclease shrimp alkaline phosphatase (ExoSAP) in the form of encapsulated particles with temperature release.

[0056] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0057] Unless defined otherwise herein, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art within the context of the disclosure, and in the specific context where each term is used. It will further be understood that common terms and phrases, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined here. However, so that the present disclosure may be more readily understood, before undertaking the DETAILED DESCRIPTION below, it may be advantageous to set forth definitions of certain words and phrases used throughout this patent document. Therefore, certain terms are first defined, and additional definitions are set forth throughout the document.

[0058] The terms “include” and “comprise,” as well as derivatives thereof, mean inclusion without limitation. The term “or” is inclusive, meaning and/or. The phrase “associated with,” as well as derivatives thereof, means to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, have a relationship to or with, or the like.

[0059] As used here, terms and phrases such as “have,” “may have,” “include,” or “may include” a feature (like a number, function, operation, or component such as a part) indicate the existence of the feature and do not exclude the existence of other features. Also, as used here, the phrases “A or B,” “at least one of A and/or B,” or “one or more of A and/or B” may include all possible combinations of A and B. For example, “A or B,” “at least one of A and B,” and “at least one of A or B” may indicate all of (i) including at least one A, (ii) including at least one B, or (iii) including at least one A and at least one B. Further, as used here, the terms “first” and “second” may modify various components regardless of importance and do not limit the components. These terms are only used to distinguish one component from another. For example, a first user device and a second user device may indicate different user devices from each other, regardless of the order or importance of the devices. A first component may be denoted a second component and vice versa without departing from the scope of this disclosure.

[0060] It will be understood that, when an element (such as a first element) is referred to as being (operatively or communicatively) “coupled with/to” or “connected with/to” another element (such as a second element), it can be coupled or connected with/to the other element directly or via a third element. In contrast, it will be understood that, when an element (such as a first element) is referred to as being “directly coupled with/to” or “directly connected with/to” another element (such as a second element), no other element (such as a third element) intervenes between the element and the other element.

[0061] As used here, the phrase “configured (or set) to” may be interchangeably used with the phrases “suitable for,” “having the capacity to,” “designed to,” “adapted to,” “made to,” or “capable of” depending on the circumstances. The phrase “configured (or set) to” does not essentially mean “specifically designed in hardware to.” Rather, the phrase “configured to” may mean that a device can perform an operation together with another device or parts. For example, the phrase “processor configured (or set) to perform A, B, and C” may mean a generic-purpose processor (such as a CPU or application processor) that may perform the operations by executing one or more software programs stored in a memory device or a dedicated processor (such as an embedded processor) for performing the operations.

[0062] The terms and phrases as used here are provided merely to describe some implementations of this disclosure but not to limit the scope of other implementations of this disclosure. It is to be understood that the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. The term “plurality” refers to more than one element. That is, as used herein, the term “plurality” is intended to mean a population of two or more different members. Pluralities can range in size from small, medium, large, to very large. The size of small plurality can range, for example, from a few members to tens of members. Medium sized pluralities can range, for example, from tens of members to about 100 members or hundreds of members. Large pluralities can range, for example, from about hundreds of members to about 1000 members, to thousands of members and up to tens of thousands of members. Very large pluralities can range, for example, from tens of thousands of members to about hundreds of thousands, a million, millions, tens of millions and up to or greater than hundreds of millions of members. Therefore, a plurality can range in size from two to well over one hundred million members as well as all sizes, as measured by the number of members, in between and greater than the above example ranges. An example number of features within a microarray includes a plurality of about 500,000 or more discrete features within 1.28 square centimeters (cm²). Example nucleic acid pluralities include, for example, populations of about IxlO⁵, 5xl0⁵ and IxlO⁶ or more different nucleic acid species. Accordingly, the definition of the term is intended to include all integer values greater than two. An upper limit of a plurality can be set, for example, by the theoretical diversity of nucleotide sequences in a nucleic acid sample.

[0063] The terms “substantially,” “approximately,” “about,” “relatively,” or other such similar terms that may be used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing, from a reference or parameter. Such small fluctuations include a zero fluctuation from the reference or parameter as well. For example, fluctuations can refer to less than or equal to +10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to +0.2%, such as less than or equal to +0.1%, such as less than or equal to +0.05%.

[0064] Definitions for other certain words and phrases may be provided throughout this document. Those of ordinary skill in the ail should understand that in many if not most instances, such definitions apply to prior as well as future uses of such defined words and phrases. In some cases, the terms and phrases defined here may be interpreted to exclude implementations of this disclosure.

[0065] None of the description in this application should be read as implying that any particular element, step, or function is an essential element that must be included in the claim scope. The scope of patented subject matter is defined only by the claims. Moreover, none of the claims is intended to invoke 35 U.S.C. § 112(f) unless the exact words “means for” are followed by a participle. Use of any other term, including without limitation “mechanism,” “module,” “device,” “unit,” “component,” “element,” “member,” “apparatus,” “machine,” “system,” “processor,” or “controller,” within a claim is understood by the Applicant to refer to structures known to those skilled in the relevant ait and is not intended to invoke 35 U.S.C. § 112(f).

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] For a more complete understanding of this disclosure and its advantages, reference is now made to the following description taken in conjunction with the accompanying drawings, in which like reference numerals represent like pa ts:

[0067] FIGURE 1 illustrates an example proteomic workflow in accordance with the present disclosure;

[0068] FIGURE 2 illustrates an example array workflow in accordance with the present disclosure;

[0069] FIGURE 3 illustrates an example one-pot target preparation workflow with lyophilized microsphere plates for leveraging temperature-triggered controlled release in accordance with the present disclosure;

[0070] FIGURE 4 illustrates an example one-pot target preparation workflow with lyo plates and automation for leveraging lyo reagent strips and an integrated reagent plate in accordance with the present disclosure;

[0071] FIGURE 5 illustrates an example process for employing a denature lyo plate and a WGA/fragmentation lyo plate for one-pot amplification and fragmentation in accordance with the present disclosure;

[0072] FIGURE 6 illustrates an example process for employing a single lyo plate for one-pot amplification and fragmentation in accordance with the present disclosure; and

[0073] FIGURE 7 illustrates an example process for employing a denature lyo plate and a WGA/TGA lyo plate for one-pot amplification and fragmentation in accordance with the present disclosure.

DETAILED DESCRIPTION

[0074] FIGURES 1 through 7, described below, and the various implementations used to describe the principles of the present disclosure are by way of illustration only and should not be construed in any way to limit the scope of this disclosure. Those skilled in the art will understand that the principles of the present disclosure may be implemented in any type of suitably arranged device or system.

[0075] As noted above, NGS includes sample pre-processing and library preparation as process elements. These process elements remain lengthy, multi-step, low-throughput processes. Library preparation can be an important or essential process with several aspects that affect the efficiency of NGS. Reliable and standardized implementation and quality control measures of the process are necessary or desirable for these process elements.

[0076] Challenges can be encountered at each of the aforementioned workflow steps, and benefits and advantages may be realized by tackling these challenges to enable high-quality sequencing results. During library preparation, for example, major challenges can be observed: complexity of the protocols; imprecise pipetting; contamination; and cost. Bead-based purification steps in particular, which entail the handling of magnets and magnetic particles by a user, are often error-prone and can result in failure of the library preparation. Moreover, sample contamination is an inherent problem since libraries are usually prepared in parallel. Major sources of contamination can include the preamplifications required for low starting concentrations of nucleic acids. Multiple liquid-handling steps also increase the risk of sample cross-contamination.

[0077] Standard workflows are thus both complex and expensive, requiring expensive laboratory equipment and reagents together with trained personnel, and usually also involving many liquid-handling steps. Therefore, even as the cost of data acquisition (sequencing) continues to decrease, for many large-scale genomic experiments, sample acquisition, sample storage and the requisite cold chain, sample pre-processing, and library preparation for sequencing create a time, cost, and labor bottleneck. The bottleneck represents a severe constraint in resource-limited settings. As current technologies allow for sequencing millions or billions of DNA fragments in parallel at relatively low costs, the scope of data generation is often limited by difficulties in sample preparation rather than sequencing capacity.

[0078] The present disclosure addresses these or other shortcomings by providing workflows that utilize particles, such as those having a core-shell composite or alternate form factors described herein, engineered to deliver and release lyophilized compositions into biological samples for sample or library preparation in a single reaction vessel, such as “one-pot format,” or minimal containers or vessels. This can be useful in a variety of applications, such as next generation DNA sequencing or array-based genotyping. The compositions, systems, and methods described here enable the integration and streamlining of sample or library preparation in a workflow while reducing or eliminating the need for a cold chain for sample storage and transportation. The compositions may include particles including an inner core loaded with lyophilized microspheres of releasable workflow reagent(s) for one-pot sample preparation, where the inner core is encapsulated by an outer, stimuli-responsive polymeric carrier shell that is engineered for triggered release of the lyophilized workflow reagent(s) microspheres into a biological sample in a controlled manner, such as in response to a specific environmental trigger or stimuli.

[0079] Depending on the implementation, the compositions, systems, and methods described in the present disclosure have many benefits. This may include, for example, stabilization of reagents, reducing or eliminating the need for cold transportation and storage, room temperature shipping and storage of reagents and complete assays, protection of the encapsulated lyophilized reagent microspheres against harsh environmental conditions, time-controlled reagent release, simplification of workflows by reducing or eliminating the need to individually pipette microliter quantities of potentially expensive assay reagents, and reduction of the risk of sample contamination. Fewer pipetting steps and less sample handling also help reduce or minimize training requirements, reduce or minimize costs (such as shipping, storage, and training costs), and save time.

[0080] The robustness and reliability of an assay are also improved, and the risk of sample contamination is reduced or minimized. The described compositions, systems, and methods also improve data quality and reliability of results while reducing or minimizing contamination risks, are compatible with downstream applications, reduce transportation costs through the ability to ship without refrigeration, increase shelf life resulting in less reagent waste, and provide batch-to-batch consistency with all samples treated substantially uniformly.

[0081] The present disclosure also relates to preparation of biological samples for sequencing. The term “sample” herein refers to a sample, typically derived from a biological fluid, cell, tissue, organ, or organism containing a nucleic acid or a mixture of nucleic acids containing at least one nucleic acid sequence that is to be sequenced and/or phased and/or containing proteins that can be assayed. Such samples include, but are not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, a fine needle biopsy sample (such as a surgical biopsy, fine needle biopsy, etc.), urine, peritoneal fluid, pleural fluid, tissue explant, organ culture, and any other tissue or cell preparation, or fraction or derivative thereof or isolated therefrom. As used herein, the terms “blood,” “plasma,” and “serum” expressly encompass fractions or processed portions thereof. Similarly, where a sample is taken from a biopsy, swab, smear, etc., the “sample” expressly encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc.

[0082] Although the sample is often taken from a human subject (such as a patient), samples can be taken from any organism having nucleic acid sequences, including, but not limited to, dogs, cats, horses, goats, sheep, cattle, pigs, corn, soy, bacteria, viruses, etc. The sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids, and so forth. Methods of pretreatment may also involve, but are not limited to, filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, lyophilization, concentration, amplification, nucleic acid fragmentation, inactivation of interfering components, the addition of reagents, lysing, etc. If such methods of pretreatment are employed with respect to the sample, such pretreatment methods are typically such that the nucleic acid(s) of interest remain in the test sample, sometimes at a concentration proportional to that in an untreated test sample (such as a sample that is not subjected to any such pretreatment method(s)). Such “treated” or “processed” samples are still considered to be biological “test” samples with respect to the compositions, systems, and methods described herein.

[0083] A sample can be a primary cell culture or culture adapted cell line including, but not limited to, genetically-engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiatable cell lines, transformed cell lines, stem cells, germ cells (such as sperm, oocytes), transformed cell lines, and the like. For example, polynucleotide molecules may be obtained from primary cells, cell lines, freshly-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 can be obtained from any subject or biological source, including, but not limited to, human or non-human animals, including mammals and non-mammals, vertebrates and invertebrates. Biological samples may also be any multicellular organism or single-celled organism, such as eukaryotic (including plants and algae) or prokaryotic organisms, archaeon, microorganisms (such as bacteria, archaea, fungi, protists, and viruses), and aquatic plankton.

[0084] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecules” are used interchangeably and refer to a covalently-linked sequence of nucleotides (such as ribonucleotides for RNA and deoxyribonucleotides for DNA) in which the 3’ position of the pentose of one nucleotide is joined by a phosphodiester group to the 5’ position of the pentose of the next. The nucleotides include sequences of any form of nucleic acid, including, but not limited to, RNA and DNA molecules such as cfDNA molecules. The term “polynucleotide” includes, without limitation, single- and double-stranded polynucleotide. The terms as used herein also encompasses cDNA, that is complementary or copy DNA, produced from an RNA template, such as by the action of reverse transcriptase. In some implementations, the nucleic acid to be analyzed, such as by sequencing through use of the described systems, is immobilized on a substrate (like a substrate within a flow cell or one or more beads upon a substrate such as a flow cell, etc.). The term immobilized as used herein is intended to encompass direct or indirect, covalent, or non-covalent attachment, unless indicated otherwise either explicitly or by context. The analytes (such as nucleic acids) may remain immobilized or attached to the support under conditions in which it is intended to use the support, such as in nucleic acid sequencing applications. In some implementations, the template polynucleotide is one of a plurality of template polynucleotides attached to a substrate. In some implementations, the plurality of template polynucleotides attached to the substrate includes a cluster of copies of a library polynucleotide.

[0085] Nucleic acids include naturally-occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs are capable of hybridizing to a nucleic acid in a sequence-specific fashion or are capable of being used as a template for replication of a particular’ nucleotide sequence. The nucleic acid described herein can be of any length suitable for use in the provided compositions, systems, and methods. For example, target nucleic acids can be at least 10 kilobase (kb), at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 75 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, at least 500 kb, or at least 1000 kb in length or longer.

[0086] The term “Next Generation Sequencing” (NGS) herein refers to sequencing methods that allow for massively parallel sequencing of clonally-amplified molecules and of single nucleic acid molecules. Non-limiting examples of NGS include sequencing-by-synthesis (SBS) using reversible dye terminators and sequencing-by-ligation.

[0087] The term “library” refers to a collection or plurality of nucleic acid template molecules that have a common use or common property, such as a common origin; an example may include when all members of the library come from a single sample. The members of the library may be processed or modified so that their membership in the library is clearly identified. For example, all members of a library may share a common sequence at their 5’ ends and a common sequence at their 3’ ends. Use of the term “library” to refer to a collection or plurality of template molecules should not be taken to imply that the templates making up the library are derived from a particular source or that the “library” has a particular composition. By way of example, use of the term “library” should not be taken to imply that the individual templates within the library must be of different nucleotide sequence or that the templates be related in terms of sequence and/or source.

[0088] Tire terms “address,” “index,” “index sequence,” “unique identifier,” “barcode,” “barcode sequence,” and “tag” are used interchangeably herein unless specified otherwise. The terms refer to a sequence of nucleotides, such as oligonucleotides, that can be used to identify a sequence of interest, such as region of a genome or haplotype. The address, index, index sequence, unique identifier, barcode, barcode sequence, or tag sequence may be exogenously incorporated into the sequence of interest by ligation, extension, or other methods known in the art. The index sequence may also be endogenous to the sequence of interest, such as when a segment in the sequence of interest itself may be used as an index. A nucleotide address, index, index sequence, unique identifier, barcode, barcode sequence, or tag can be a random or a specifically-designed nucleotide sequence. An address, index, index sequence, unique identifier, barcode, barcode sequence, or tag can be of any desired sequence length so long as it is of sufficient length to be a unique nucleotide sequence within a plurality of indices in a population and/or within a plurality of polynucleotides that are being analyzed or interrogated. A nucleotide address, index, index sequence, unique identifier, barcode, barcode sequence, or tag is useful, for example, to be attached to a target polynucleotide to tag or mark a particular species for identifying all members of the tagged species within a population. Accordingly, an index is useful as a barcode where different members of the same molecular species can contain the same index and where different species within a population of different polynucleotides can have different indices.

[0089] As used herein, the term “target nucleic acid” is intended to mean a nucleic acid that is the object of an analysis or action. The analysis or action may include subjecting the nucleic acid to copying, amplification, sequencing, and/or other procedure for nucleic acid interrogation. A target nucleic acid can include nucleotide sequences additional to the target sequence to be analyzed. For example, a target nucleic acid can include one or more adapters, including an adapter that functions as a primer binding site, that flank(s) a target nucleic acid sequence that is to be analyzed. A target nucleic acid hybridized to a capture oligonucleotide or capture primer can contain nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide in such a way that not all of the target nucleic acid is amenable to extension.

[0090] As used herein, the term “substrate” is intended to mean a solid or semi-solid support or support structure. The term includes any material that can serve as a solid or semi-solid foundation for creation of features such as wells for the deposition of biopolymers, including nucleic acids, polypeptides, and/or other polymers. Non-limiting examples of substrates include a bead array, a spotted array, clustered particles arranged on a surface of a chip, a film, a multi-well plate, a cartridge, and a flow cell. A substrate as provided herein is modified or can be modified, for example, to accommodate attachment of biopolymers by a variety of methods well known to those skilled in the art. Example types of substrate materials include glasses, modified glasses, functionalized glasses, inorganic glasses, microspheres (including inert and/or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or optical fiber bundles, a variety of polymers other than those exemplified above, and multi-well microtiter plates. Specific types of example plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, and TEFLON. Specific types of example silica-based materials include silicon and various forms of modified silicon.

[0091] In some implementations, the solid or semi-solid support includes one or more surfaces that are accessible to contact with reagents, beads, or analytes. The surface can be substantially flat or planar-. Alternatively, the surface can be rounded or contoured. Example contours that can be included on a surface are wells (such as microwells or nanowells), depressions, pillar’s, ridges, channels, or the like. Example materials that can be used as a surface include glasses; modified glasses; functionalized glasses; plastics such as acrylic, polystyrene, a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or TEFLON; polysaccharides or cross-linked polysaccharides such as agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials including silicon and modified silicon; carbon-fiber; metals; inorganic glasses; optical fibers or optical fiber bundles; or a variety of other polymers. A single material or mixture of several different materials can form a surface useful in certain examples. In some examples, a surface includes wells (such as microwells or nanowells). In some aspects, the surface includes wells in an array of wells (such as microwells or nanowells) on glass, silicon, plastic, or other suitable solid or semi-solid supports with patterned, covalently-linked gel. In some examples, a support structure can include one or more layers.

[0092] 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 a complementary nucleotide. A partially double stranded nucleic acid can have at least 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95% of its nucleotides hydrogen bonded to a complementary nucleotide. As used herein, the term “single-stranded,” when used in reference to a nucleic acid molecule, means that essentially none of the nucleotides in the nucleic acid molecule are hydrogen- bonded to a complementary nucleotide.

[0093] As used herein, the term “dNTP” refers to deoxynucleoside triphosphates. NTP refers to ribonucleotide triphosphates. The purine bases (Pu) include adenine (A), guanine (G), and derivatives and analogs thereof. The pyrimidine bases (Py) include cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof. Examples of such derivatives or analogs, by way of illustration and not limitation, are those that are modified with a reporter group, biotinylated, amine modified, radiolabeled, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like. The reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, a terbium chelator such as N-(hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.

[0094] As used herein, the term “size selection” means a procedure during which a subpopulation of nucleic acid fragments, the majority of which have a number of nucleotides falling in a defined range, is selected from a population of nucleic acid fragments. Thus, the percentage of nucleic acid fragments having a number of nucleotides falling in the defined range increases.

[0095] As used herein, the term “protease” refers to a protein, polypeptide, or peptide exhibiting the ability to hydrolyze polypeptides or substrates having a polypeptide portion. The protease(s) provided in the present compositions, systems, and methods can be a single protease possessing broad specificity. The present compositions, systems, and methods can use a mixture of various proteases. The proteases provided herein can be heat-labile and thus can be inactivated by heat. In certain implementations, the proteases provided herein can be inactivated at a temperature above about 35° C, 40° C, 45° C, 50° C, 55° C. 60° C, 65° C, 70° C, 75° C, 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 DNase to protect DNA from degradation. The proteases provided herein include, but are not limited to, serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases, and metalloproteases. Typically, aspartic, glutamic and metallo-proteases activate a water molecule, which performs a nucleophilic attack on the peptide bond to hydrolyze that bond. Serine, threonine, and cysteine proteases typically use a nucleophilic residue to perform a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolyzed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme. An example protease used herein includes a serine protease isolated from a recombinant Bacillus strain. Example proteases used herein also include Proteinase K, subtilisin and variants thereof, including alcalase, alcalase 0.6L, alcalase 2.5L, ALK-enzyme, bacillopeptidase A, bacillopeptidase B, Bacillus subtilis alkaline proteinase 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, subtilopeptidase, SP 266, savinase 8.0L, savinase 4.0T, kazusase, protease VIII, opticlean, protin A 3L, savinase, savinase 16.0L, savinase 32.0 L EX, orientase 10B, protease S, serine endopeptidase. In particular- implementations of the compositions, systems, and methods presented herein, a heat-labile protease such as Proteinase K and heat-labile variants thereof can be used.

[0096] As used herein, the term “protease inhibitor” refers to a substance, such as a compound, capable of at least partially reducing the ability of a protease to hydrolyze peptides. Examples of protease inhibitors known in the art that can be used with the present compositions, systems, and methods include, but are not limited to, FOCUS PROTEASEARREST protease inhibitor cocktail, PEFABLOC SC (4-(2-Aminoethyl)-benzolsulfonylfluorid-hydrochloride) (AEBSF) protease inhibitor, Aprotinin protease inhibitor, Bestatin protease inhibitor, Leupeptin protease inhibitor, Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, and tripeptidyl chloromethyl ketones (TCK/TPCK, TLCK, and E-64) protease inhibitors.

[0097] As used herein, the term “tagmentation” refers to the modification of DNA by a transposome complex including transposase enzyme complexed with adaptors including transposon end sequence. Tagmentation results in the simultaneous fragmentation of the DNA and ligation of the adaptors to the 5' ends of both strands of duplex fragments. Additional sequences can be added to the ends of the adapted fragments, such as by PCR, ligation, or any other suitable methodology known to those of skill in the art. As used herein, the term “transposome complex” (TSM) refers to a transposase enzyme non-covalently bound to a double-stranded nucleic acid. For example, the complex can be a transposase enzyme preincubated with double-stranded transposon DNA under conditions that support non-covalent complex formation. Double-stranded transposon DNA can include, without limitation, Tn5 DNA, a portion of Tn5 DNA (such as Tn5 recognition site), a transposon end composition, a mixture of transposon end compositions, or other double-stranded DNAs capable of interacting with a transposase such as the hyperactive Tn5 transposase.

[0098] As used herein, the term “transposition reaction” refers to a reaction where one or more transposons are inserted into target nucleic acids, such as at random sites or almost random sites. Components in a transposition reaction are a transposase and DNA oligonucleotides that exhibit the nucleotide sequences of a transposon, including the transferred transposon sequence and its complement (the non-transferred transposon end sequence) as well as other components used to form a functional transposition or transposome complex. The DNA oligonucleotides can further include additional sequences (such as adaptor or primer sequences) as needed or desired. In some implementations, the compositions, systems, and methods provided herein are exemplified by employing a transposition complex formed by a hyperactive Tn5 transposase and a Tn5-type transposon end. However, any transposition system that is capable of inserting a transposon end in a random or in an almost random manner with sufficient efficiency to 5'- tag and fragment a target DNA for its intended purpose can be used in the present disclosure. Examples of transposition systems known in the art that can be used for the present compositions, systems, and methods include, but are not limited to, Staphylococcus aureus Tn552, bacterial insertion sequences, and retrotransposon of yeast.

[0099] Tire method for inserting a transposon end into a target sequence can be carried out in vitro using any suitable transposon system for which a suitable in vitro transposition system is available or that can be developed based on knowledge in the art. In general, a suitable in vitro transposition system for use in the compositions, systems, and methods provided herein uses, at a minimum, a transposase enzyme of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity and a transposon end with which the transposase forms a functional complex with the respective transposase that is capable of catalyzing the transposition reaction. Suitable transposase transposon end sequences that can be used in the disclosure include, but are not limited to, wild-type, derivative, or mutant transposon end sequences that form a complex with a transposase chosen from among a wildtype, derivative, or mutant form of the transposase.

[0100] As used herein, the term “transposase” refers to an enzyme that is capable of forming a functional complex with a transposon end-containing composition (such as transposons, transposon ends, and transposon end compositions) and catalyzing insertion or transposition of the transposon endcontaining composition into the double-stranded target nucleic acid with which it is incubated, such as in an in vitro transposition reaction. A transposase as presented herein can also include integrases from retrotransposons and retroviruses. Transposases, transposomes and transposome complexes are generally known to those of skill in the art.

[0101] Although many implementations described herein refer to Tn5 transposase and/or hyperactive Tn5 transposase, it will be appreciated that any transposition system that is capable of inserting a transposon end with sufficient efficiency to 5'-tag and fragment a target nucleic acid for its intended purpose can be used in the present disclosure. In particular implementations, a transposition system is capable of inserting the transposon end in a random or in an almost random manner to 5'-tag and fragment the target nucleic acid.

[0102] As used herein, the term “library of tagged nucleic acid fragments” refers to a collection or population of tagged nucleic acid fragments (such as di-tagged nucleic acid fragments) generated from a resource, such as whole genome, where the combination of the tagged nucleic acid fragments in the collection or population exhibits sequences that are qualitatively and/or quantitatively representative of the sequence of the resource from which the tagged nucleic acid fragments were generated, such as whole genome. It is possible that a library of tagged nucleic acid fragments does not contain a tagged nucleic fragment representing every sequence that is exhibited by the resource.

[0103] As used herein, the term “primer” is an oligonucleotide (“oligo”), generally with a free 3'-OH group that can be extended by a nucleic acid polymerase. For a template- dependent polymerase, generally at least the 3 '-portion of the primer oligo is complementary to a portion of a template nucleic acid to which the oligo “binds” (or “complexes,” “anneals,” or “hybridizes”) by hydrogen bonding and other molecular forces to the template to give a primer/template complex for initiation of synthesis by a DNA polymerase and which is extended by the addition of covalently -bonded bases linked at its d'end that are complementary to the template in the process of DNA synthesis. The result is a primer extension product. [0104] As used herein, the term “adaptor” or “adapter” are used interchangeably and can refer to an oligonucleotide that may be attached to the end of a nucleic acid. Adaptor sequences may include, but are not limited to, priming sites, the complement of a priming site, recognition sites for endonucleases, common sequences, and promoters. Adaptors may also incorporate modified nucleotides that modify the properties of the adaptor sequence. For example, phosphorothioate groups may be incorporated in one of the adaptor strands.

[0105] The compositions, systems, and methods described herein include particles having a shell surrounding a core, where the core may include one or more lyophilized microspheres (such as the composition may include an encapsulated lyophilized microsphere). Alternate form factors to encapsulated particles include stacked cakes separated by trigger layers, solid substrate beads (such as polypropylene, dissolvable starch/sugar bead(s) coated with the active material and encapsulated with the trigger release shell(s)). In connection with encapsulated particles, the terms “encapsulate,” “encapsulated,” and “encapsulation” include the enclosing of one or more microspheres as described herein. Microencapsulation as described herein refers to the embedding of at least one ingredient, such as an active agent, into at least one other material, such as a shell material. Triggered release of the active agent include time, elevated temperature, low temperature, pH or light, and workflows may be adjusted to enable the triggered releases for each release event. Encapsulation in accordance with the present disclosure includes, but is not limited to, bulk encapsulation, macroencapsulation, microencapsulation, nanoencapsulation, single molecule encapsulation, and ionic encapsulation. In accordance with the present disclosure, the compositions, systems, and methods described herein have many benefits including, for example, increasing stability of microspheres, use of macroencapsulation to enable multi-run cartridges, and use of microencapsulation to enable simplified workflows and reduced number of reagent wells. The compositions, systems, and methods described herein use encapsulation of particles that would otherwise be responsive to pH changes to stabilize these buffers and increase SBS performance.

[0106] As used herein, the term “microsphere” includes a spherical particle that includes a shell and a core. A microsphere has a diameter of 0.1 micron (pm) to 1,000 pm. For example, a microsphere may have a diameter of about 0.1 pm, 0.5 pm, 1 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 150 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm, or any diameter between about 0.1 pm and about 1,000 pm. In some implementations, an encapsulated microsphere has a diameter between about 100 pm and 1000 pm. Microspheres may refer to lyophilized particles including reagents and/or active ingredients. In certain implementations, microspheres may include a polymer shell, such as one or more biodegradable polymers and/or water- soluble polymers, and optionally an inner core inside the shell. Microspheres in accordance with the present disclosure include those prepared by conventional techniques, which are known to those skilled 1 in the art. For example, microspheres may be prepared by freezing a liquid into frozen pellets, followed by placing frozen microspheres in a dryer, such as a rotational dryer.

[0107] As used herein, the term “shell” includes a composition that surrounds a core. In some implementations, a shell includes an outer layer of a microsphere or an outer layer of a macrosphere. In some implementations, the shell includes, for example, a shell material selected from the group consisting of carrageenan, agarose, poloxamer, shellac, trehalose, paraffin wax, fatty acid (myristic acid, almitic acid), and fatty acid ester such as PEG stearate, gelatin, hydroxypropyl methylcellulose (HPMC), cellulose acetate, fullalin, oxygen scavenger, alginate, chitosan, starch film, benzoxaborole- poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), poly(vinylpyrrolidone- co-vinyl acetate), polyvinyl alcohol (PVA), Poly(vinylalcohol-graft-PEG), one or more upper critical soluble temperature (USCT) polymers, such as poly(acrylamide-co-acrylonitrile), poly(N-acryloyl glycinamide), one or more lower critical soluble temperature (LCST) polymers, such as poly(N- isopropyl acrylamide) and its co-polymer, or any combination thereof.

[0108] As used herein, the terms “core” and “core region” include any material within the surrounding shell. In various implementations, a core includes one or more lyophilized microspheres. In various implementations, a core includes lyophilized beads. In various implementations, a core includes beads made of non-lyophilized sugar or plastic, optionally where a reagent is coated and dried on the surface of the non-lyophilized microspheres or beads.

[0109] 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, polymerase, primers including those having a size of less than 50 base pairs, template nucleic acids, nucleotides, labels, dyes, or nucleases. A reagent as described herein may, in certain implementations, include enzymes such as polymerases, ligases, recombinases, or transposases; binding partners such as antibodies, epitopes, streptavidin, avidin, biotin, lectins, or carbohydrates; or other biochemically-active molecules. Other example reagents include reagents for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids. According to some implementations disclosed herein, a reagent may include one or more beads, such as magnetic beads, depending on specific workflows and/or downstream applications.

[0110] The terms “connect,” “connected,” “contact,” “coupled,” and/or the like are broadly defined herein to encompass a variety of divergent arrangements and assembly techniques. These arrangements and techniques include, but are not limited to, (i) the direct joining of one component and another component with no intervening components therebetween (such as the components are in direct physical contact); and (ii) the joining of one component and another component with one or more components therebetween, provided that the one component being “connected to” or “contacting” or “coupled to” the other component is somehow in operative communication (such as electrically, fluidly, physically, optically, etc.) with the other component (notwithstanding the presence of one or more additional components therebetween). It is to be understood that some components that are in direct physical contact with one another may or may not be in electrical contact and/or fluid contact with one another. Moreover, two components that are electrically connected, electrically coupled, optically connected, optically coupled, fluidly connected, or fluidly coupled may or may not be in direct physical contact, and one or more other components may be positioned therebetween.

[0111] As used herein, “room temperature” refers to temperatures at or between 15 to 25° C, or at or between 17 to 23° C, or at or between 20 to 25° C.

[0112] Particle core-shell composite material

[0113] The present disclosure relates to one or more particles including a core-shell composite materials having (i) an inner core optionally including releasable lyophilized microspheres or lyophilized beads of one or more workflow reagents: and (ii) an outer shell encapsulating the inner core. The outer shell includes one or more layers of a stimuli-sensitive polymer(s), and the outer shell is designed to be stimuli-responsive, where one or more physio-chemical properties change upon the application of different stimuli, releasing the encapsulated lyophilized microspheres into in a specified environment (such as “external environment”), for example, a biological sample. The core-shell composite material may be a macro-sized, a micro-sized, or a nano-sized particle. In some implementations, the core includes, but is not limited to, one or more reagents, such as at least one enzyme, salt, surfactant, buffering agent, enzyme inhibitor, primer, nucleotide, organic osmolite, magnetic bead, molecular probe, crowding agent, small molecule, labelled-nucleotide, a fluorophore, or any combination thereof.

[0114] In some implementations, the core-shell composite may exhibit a total thickness of the shell structure of around 1-25 pm. As particular’ examples, the thickness may be selected from 2.5, 5, 10, 15, 20, or 25 pm, or the thickness may be provided in a range having an upper and lower limit selected from these values. When the outer shell includes more than one shell layer, the shell layers may be independently from 1 to 25 pm thick. In various implementations, the shell is between about 1 pm to 25 pm, between about 1 pm to about 20 pm, between about 5 pm to about 20 pm, between about 3 pm to about 10 pm, or between about 4 pm to about 6 pm, such as about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, or about 25 pm in thickness. The thickness may be advantageously adjusted according to the residence time of the composite material. For example, the shell may be at least 5 pm for a homogeneous coating, which will enable predictable release.

[0115] In some implementations, the core-shell composite material may be substantially spherical in shape with a diameter of about 0.2 pm to about 1 ,000 pm. As particular examples, the coreshell composite material may have an average diameter of about 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, or 1000 pm. In various implementations, the microspheres, with or without coating material, have a diameter from about 300 to 700 pm, from about 350 to 625 pm, or from about 400 to 600 pm. The core-shell composite material may include substantially monodisperse particles, each having substantially the same average diameter. The core-shell material may also include lyophilized microspheres or lyophilized beads having a distribution of average diameters.

[0116] Tire shell, as described herein, may include one layer or a plurality of layers of varying compositions. For example, the shell may include one layer, two layers, three layers, four layers, five layers, six layers, seven layers, eight layers, nine layers, ten layers, or more than ten layers. Each of the layers may include the same or different materials from the other layers that are present in the shell.

[0117] In some implementations, 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-(Vinylacetate-Co-Crotonic Acid) (PVP- co-PVAc), Eudragits, Isoleucine Eudragit RL/RS, Opadry CA, polyester (such as Polylactic-co- glycolic acid (PLGA)), wax, upper critical solution temperature (UCST) polymer, lower critical solution temperature (LSCT) polymer, carrageenan, shellac, paraffin wax, fatty acid, fatty acid ester, gelatin, pullalan, oxygen scavenger, alginate, chitosan, starch film, benzoxaborole-poly(vinyl alcohol) (benzoxaborole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof. In one example, the shell may include, but is not limited to, starch, cellulose, hydrocolloid, alginate, collagen, and any combination thereof. Water soluble (hydrophilic) polymers include ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), carboxymethyl ethylcellulose (CMEC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethylhydroxy ethylcellulose (EHEC), pullulan, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid esters, polyacrylamide, polyacrylic acid, copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT, ROHM AMERICA, INC., Piscataway, N.J.) and other acrylic acid derivatives such as homopolymers and copolymers of butylmethacrylate, methylmethacrylate, ethylmethacrylate, ethylacrylate, (2-dimethylaminoethyl)methacrylate, and (trimethylaminoethyl) methacrylate chloride.

[0118] The amount of shell material includes, for example, any amount suitable to produce a desired shell result. In some implementations, the shell material is present in an amount between about 1 percent by weight (wt%) and about 100 wt% of the shell. For example, the shell material may be present in about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, or 100 wt% of the shell or any amount therebetween. In some implementations, the shell material is present in an amount between about 10 wt% and about 90 wt%, between about 10 wt% and about 80 wt%, between about 10 wt% and about 70 wt%, between about 10 wt% and about 60 25 wt%, or between about 10 wt% and about 50 wt% of the shell.

[0119] The shell as described herein may, in various implementations, include a shell additive. The shell additive may be present in an amount between about 0.01% weight by weight (w/w) of the shell and about 99% w/w of the shell. In some implementations, the shell additive is present in an amount between about 10% w/w and about 90% w/w of the shell. In some implementations, the shell additive is present in an amount between about 10% w/w and about 40% w/w. In some implementations, the shell additive is a moisture barrier material present in an amount no more than 90% w/w of the shell. In some implementations, the shell additive is present in an amount of at least 10% w/w concentration of the shell. For example, the shell additive may, in some implementations, be present in an amount between 0.1% w/w of the shell and about 15.0% w/w of the shell. As particular examples, the shell additive may be present in an amount of about 0.01% w/w, 0.05% w/w, 0.1% w/w, 0.5% w/w, 1.0% w/w, 1.5% w/w, 2.0% w/w, 2.5% w/w, 3.0% w/w, 3.5% w/w, 4.0% w/w, 4.5% w/w, 5.0% w/w, 5.5% w/w, 6.0% w/w, 6.5% w/w, 7.0% w/w, 7.5% w/w, 8.0% w/w, 8.5% w/w, 9.0% w/w, 9.5% w/w, 10.0% w/w, 10.5% w/w, 11.0% w/w, 11.5% w/w, 12.0% w/w, 12.5% w/w, 13.0% w/w, 13.5% w/w, 14.0% w/w, 14.5% w/w, 15% w/w, or any amount therebetween. The amount of the shell additive may be adjusted to accommodate a particular reagent or combination of reagents or to accommodate a particular microsphere composition.

[0120] Example shell additives include, but are not limited to, one or more of a polymer, a copolymer, a block copolymer, an anti-tacking agent (e.g., PEG stearates or Mg stearates, both mentioned below), an anti-static agent, an anti-foaming agent, a plasticizer, a second polyvinyl alcohol (PVA), an ammonium salt, a conductivity promoter, a stearate derivative, an oleate derivative, a laurate derivative, a polyether compound, an amino acid, tocopherol acetate, piperidyl sebacate, sodium salt, a buffer, a chelating agent, imidazolium salt, polyaniline, or any combination thereof. In some implementations, the polyether compound is selected from polyethylene glycol, polypropylene glycol, a block copolymer derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In some implementations, the stearate derivative or oleate derivative is selected from magnesium stearate, PEG stearate, triglycerol stearate. SPAN 60, TWEEN 60, glycerol trioleate, TWEEN 80, or any combination thereof. In some implementations, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In some implementations, the polymer is neutral, cationic, or anionic. In some implementations, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In various implementations, the buffer is Trizma, tris hydrochloride (Tris-HCl), or a combination thereof. In some implementations, the ammonium salt is selected from tetraalkyl ammonium chloride, tris(hydroxyethyl) alkylammonium chloride, or a combination thereof. In some implementations, the imidazolium salt is selected from l-ethyl-3-methyl- imidazolium salt or polyquaternium or LUVIQUAT (copolymer of vinyl pyrrolidone and quaternized vinylimidazole) or a combination thereof. In some implementations, the shell additive includes ammonium salt, copolymer, polyvinyl alcohol graft polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof. In various implementations, the shell additive is magnesium stearate or polyethylene glycol stearate.

[0121] Inner core and lyophilized microspheres

[0122] As described herein, a “core” or “core region” includes any material within the encapsulating shell. A core in accordance with the present disclosure includes one or more lyophilized microspheres or lyophilized beads. The lyophilized microspheres of the present disclosure can include any reagent that is desired for controlled delivery and that can be unitized in substantially small sizes to be amenable to being lyophilized or particularized in size ranges described herein.

[0123] In some implementations, the inner core includes lyophilized reagents that are suitable for use in multiple sequential co-assays including lysis, DNA analysis, RNA analysis, protein analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, SBS technology, assay for transposase accessible chromatic using sequencing (ATAC-seq), contiguitypreserving transposition (CPT-seq), single cell combinatorial indexed sequencing (SCl-seq), single cell genome amplification, or any combination thereof performed sequentially. In some implementations, the composition is used for performing multiple co-assay reactions. The compositions, systems, and methods described herein (such as encapsulation of lyophilized microspheres) may, in some implementations, improve sequencing quality, enable one-pot library prep, and simplify manufacturing. As used herein, the term “one-pot reaction” may also be referred to as “transfer-free reaction,” where no interactions of the user are required. More generally, one pot library preparation can be an additive prep, where reagents are sequentially added to the same tube at different timepoints, or a passive prep that is transfer free. In further implementations, the inner core includes lyophilized reagents that may be prepared for various stages of sequencing including, but not limited to, sample extraction, library preparation, enrichment, clustering, and sequencing.

[0124] Lyophilized spheres including sample preparation reagents

[0125] In some implementations, the lyophilized microspheres include lyophilized lysis solution. A lysis solution enables efficient lysis (such as of cells in a biological sample) to release nucleic acids, effectively protects the released nucleic acids from degradation in the lysate by inhibiting or degrading nucleases, and is compatible with subsequent steps for analysis of the extracted nucleic acids (such as target capture, amplification, detection, and/or sequencing). The components of the lysis buffer can be tailored depending on the types and source of cells, the desired final molecule or structure, and the level of their functionality.

[0126] In some implementations, the lyophilized microspheres include a lysis buffer for DNA extraction from whole blood. Whole blood and blood fractions are a common biological starting sample for DNA extraction, such as in most epidemiologic studies. Compared to other minimally -invasive sources of genomic gDNA (gDNA), such as saliva or buccal cells, gDNA yield from blood or blood fractions is comparatively higher and less fragmented. Whole blood contains red blood cells (RBCs), nucleated white blood cells (WBCs), platelets, and plasma. Genomic DNA is found in the nuclei of WBCs. Unlike the WBCs, mature RBCs are nonnucleated and therefore do not contain DNA. Most DNA extraction procedures from whole blood include a two-step lysis approach.

• Step 1 - Selective lysis and removal of RBCs with minimal effect on WBCs. RBCs contain no DNA and are a potential source of downstream inhibitors. Thus, it can be advantageous to separate them from WBCs prior to DNA isolation. Lysis of WBCs to extract DNA and degrade proteins, followed by DNA recovery and washing, is also contemplated.

• Step 2 - Remove the protein, leaving the DNA supernatant for collection.

[0127] In various implementations, a lyophilized lysis solution 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), chelating reagents (such as EDTA or CDTA), and a lyoprotectant/lyophilization reagent (such as sucrose or trehalose).

[0128] In some implementations, the lyophilized microspheres of the present disclosure provide reagents for a passive, one-step whole blood lysis approach using a lysing buffer mix capable of lysing both WBCs and RBCs cell types in one step. This one-step lysis approach has a number of advantages over the traditional two-step lysis method, including improved DNA yield due to elimination of sample loss incurred in a two-step procedure: single-vessel reaction that eliminates the need for pipetting and lowers the risk of contamination; and reduction in time and reagent cost for additional enzymes such as RNase.

[0129] A first component of the lysis solution is a buffer that maintains the pH of the solution (such as a Tris buffer or any known buffer). For example, the pH of the buffer may be at least about 8, at least about 8.5, or at least about 9 (such as 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 (such as 8.1, 8.3, 8.5, 8.6, 8.8, or 8.9) and may be used at a concentration of 50- 150 millimolar (mM) (such as 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, or 140 mM). In some implementations, a Tris buffer is an appropriate buffer. In some instances, a Tris buffer with a pH of 8.0 and a concentration of 100 mM is used. In some other implementations, a base may be used to adjust the pH of the lysis solution. The base may be one that can raise the pH of the solution to no less than 7 (such as a pH of 7.5, 8, 8.5, or 9.0). In some instances, the base may be an alkali-metal hydroxide. Such alkali-metal hydroxides include, but are not limited to, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0130] In some implementations, the lysis solution includes a broad-spectrum protease for proteolytic lysis. In some implementations, the broad-spectrum proteases include a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamic acid protease, or a metalloprotease. In some implementations, the broad-spectrum protease is a serine protease. In some implementations, the serine protease is Proteinase K. Proteinase K is a stable serine protease that is active under a wide range of pH, temperature, salt, solvent, and detergent concentrations. The activity of Proteinase K peaks in the presence of moderate denaturants, 2-4 molar chaotropic salts, and ionic detergents, which act both to stimulate enzymatic activity and increase substrate accessibility by destabilizing protein secondary structure. At completion, Proteinase K digestion will have reduced polypeptides to small di- and tri-peptides and in the process degraded itself by autodigestion, thus eliminating the vast majority of enzyme added to samples. Proteolysis buffer is a key additive in DNA extraction methods and can be important or critical to DNA isolation from complex biological samples. In sample mixtures, a proteolysis buffer is designed to preserve target nucleic acids, establish optimum conditions for proteolysis, solubilize lipids and microvesicles, break down colloids and particulate matter, and inhibit or prevent precipitation over the course of protease reactions.

[0131] Proteinase K may be present in the lysis buffer at a concentration of about 0.001 milligrams per milliliter (mg/mL) to about 50 mg/mL. For example, the concentration of Proteinase K in the lysis buffer may be about 0.001 mg/mL, about 0.005 mg/mL, about 0.01 mg/mL, about 0.05 mg/mL, about 0.1 mg/mL, about 0.5 mg/mL, about 0.8 mg/mL, about 1 mg/mL, about 1.5 mg/ml, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, or greater than about 10 mg/mL. In certain instances, a suitable Proteinase K solution has a concentration of 20 mg/mL Proteinase K. In some implementations, a suitable lysis solution includes Proteinase K at a concentration of about 0.45 to about 1.8 mg/mL. In some implementations, a suitable lysis solution includes Proteinase K at a concentration of about 10 mg/mL.

[0132] If nucleic acid preparation and tagmentation steps are performed in the same reaction tube, it can be beneficial that the proteases according to the present disclosure can be effectively inactivated without disturbing the next tagmentation step, which typically uses double-stranded DNA. In some implementations, the proteases can be inactivated by increasing temperature prior to the tagmentation step. High temperature can denature double-stranded DNA conformation. Thus, in some implementations, the proteases provided herein can be inactivated at relatively low temperature without denaturing double-stranded DNA. Tn some implementations, one or more proteases are inactivated by increasing temperature to 50° C to 80° C. In some implementations, the one or more proteases are inactivated by increasing temperature to 50° C, 55° C, 60° C, 65° C, or 70° C. In various implementations, the protease is Proteinase K that can be heat inactivated.

[0133] In some implementations, the lysis solution includes a detergent. Detergents can act as both a lysing agent and as an inhibitor of analyte degradation following the lysis of blood cells. Detergents are particularly useful for inhibiting the degradation of nucleic acids. Non-limiting examples of surfactants or detergents that may be used include: non-ionic surfactants including polyoxy ethylene glycol alkyl ethers (sold as BRIJ series detergents including BRIJ 58, BRIJ 52, BRIJ L4 and BRIJ L23), octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, polyoxypropylene glycol alkyl ethers, glucoside alkyl ethers (such as decyl glucoside, lauryl glucoside, octyl glucoside), polyoxyethylene glycol octylphenol ethers (such as Triton X-100), polyoxyethylene glycol alkylphenol ethers (such as nonoxynol-9), glycerol alkyl esters (such as glyceryl laurate), polyoxyethylene glycol sorbitan alkyl esters (such as 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, polyoxyethylene glycol (20) sorbitan tristearate, polyoxyethylene glycol (20) sorbitan monooleate)), sorbitan alkyl esters (such as sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, sorbitan isostearate), cocamide monoethanolamine, cocamide diethanolamine, dodecyldimethylamine oxide, poloxamers including those sold under the PLURONIC, SYNPERONIC and KOLLIPHOR tradenames, and polyethoxylated tallow amine (POEA): anionic surfactants including 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 dodecylbenzene sulfonate, sodium laurate, sodium lauryl ether sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium pareth sulfate, sodium stearate; cationic surfactants including benzalkonium chloride, benzethonium chloride, bronidox, cetrimonium bromide, cetrimonium chloride, distearyldimethylammonium chloride, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride, octenidine dihydrochloride, olaflur, and tetramethylammonium hydroxide; and zwitterionic surfactants including CHAPS detergent, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dipalmitoylphosphatidylcholine, lecithin, hydroxysultaine, and sodium lauroamphoacetate.

[0134] Although anionic, cationic, and zwitterionic detergents may all be used in a lysis solution, the lysis solution of the present disclosure may include at least one anionic surfactant and at least one non-ionic surfactant. In some implementations, the lysis solution contains the anionic surfactant SDS, and the non-ionic surfactant TWEEN 20. In some implementations, the SDS may be present at a concentration of about 0.1% to about 10% weight/volume. For example, suitable SDS concentrations include, but are not limited to, from about 0.1% to about 0.2%, from about 0.2% to about 0.3%, from about 0.3% to about 0.4%, from about 0.4% to about 0.5%, from about 0.5% to about 0.6%, from about 0.6% to about 0.7%, from about 0.7% to about 0.8%, from about 0.8% to about 0.9%, from about 0.9% to about 1%, from about 1% to about 2%, from about 2% to about 3%, from about 3% to about 4%, from about 4% to about 5%, from about 5% to about 6%, from about 6% to about 7%, from about 7% to about 8%, from about 8% to about 9%, and from about 9% to about 10%, as well as combinations of the above ranges, such as about 0.1% to about 0.5%, about 1% to about 2%, about 1% to about 5%, about 3% to about 7%, about 5% to about 9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0135] In some implementations, the TWEEN 20 may be present at a concentration about 0.5% to about 10% weight/volume percent. For example, suitable TWEEN 20 concentrations include, but are not limited to, from about from about 0.5% to about 0.6%, from about 0.6% to about 0.7%, from about 0.7% to about 0.8%, from about 0.8% to about 0.9%, from about 0.9% to about 1%, from about 1% to about 2%, from about 2% to about 3%, from about 3% to about 4%, from about 4% to about 5%, from about 5% to about 6%, from about 6% to about 7%, from about 7% to about 8%, from about 8% to about 9%, and from about 9% to about 10%, as well as combinations of the above ranges, such as about 0.1% to about 0.5%, about 1% to about 2%, about 1% to about 5%, about 3% to about 7%, about 5% to about 9%, about 1%, about 2%, about 3%. about 4%, about 5%. about 6%, about 7%, about 8%, about 9%. or about 10%.

[0136] Alternatively, the concentration of the surfactant can be measured in mg/ml or in grams per liter (g/L). In typical implementations, either surfactant is present at about 1-5 mg/ml, at about 5- 10 mg/ml, at about 10-15 mg/ml, at about 15-25 mg/ml, at about 25-50 mg/ml, at about 50-60 mg/ml, at about 60-70 mg/ml, at about 70-80 mg/ml, and at about 80 to 90 mg/ml, as well as combinations of the above ranges.

[0137] In order to reduce or prevent degradation of nucleic acids, nuclease-free water can be used in the lysis solution. In some implementations, a chelating agent may also be used to inhibit or prevent degradation of contaminating nucleic acid. The use of a chelating agent inhibits or prevents nucleic acid polymers from being degraded to smaller fragments, which may cause additional contamination problems. The chelating agent may be present at a concentration of 1-100 mM (such as 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 a concentration of 1-10 mM (such as 1.5 mM, 2 mM, 3 mM, 4 mM, 6 mM, 7 mM, or 9 mM). In some instances, the chelating agent ethylenediaminetetraacetic acid (EDTA) is used. In other instances, the chelating agent cyclohexanediaminetetraacetic acid (CDTA) is used. [0138] An anti -coagulant, if present in the lysis reagent, is at a concentration sufficient to inhibit clotting of the sample (such as whole blood or red blood cells). By inhibiting clotting, the anticoagulant eliminates the need to centrifuge samples during a process to isolate red blood cells. Example anti-coagulants include EDTA, EDTA-Na2, EGTA, heparin, or citrate. Example concentrations of EDTA in the lysis reagent include from about 0.05 mM to about 15 mM, from about 0.1 mM to about 10 mM, from about 0.5 mM to about 5 mM, about 10 mM, about 2.5 mM, or about 0.1 mM. Example concentrations of EDTA-Na2 in the lysis reagent include from about 0.05 mM to about 15 mM, from about 0.1 mM to about 10 mM, from about 0.5 mM to about 5 mM, about 10 mM, about 2.5 mM, or about 0.1 mM.

[0139] In further implementations, the lysis solution also contains cryoprotective agents (CPAs) or cryoprotectants. Cryoprotectants (which may also be called excipients or cryoprotective agents, lyoprotectants, or lyophilization reagent) contribute to the preservation of the structures of proteins, liposome bilayers, and other substances during freezing in general. Lyoprotectants stabilize these substances during drying, especially freeze-drying. In freeze-drying, lyoprotectant may be also considered as a cryoprotectant, so as used herein the term “cryo-protectant” may also include lyoprotectants. Protective additives can be generally considered to have two types: (i) amorphous glass forming and (ii) eutectic crystallizing salts. Examples of lyoprotectants include polyhydroxy compounds such as sugars (mono-, di-, and polysaccharides), trehalose and sucrose as natural lyoprotectants, and polyalcohols such as glycerol, mannitol, sorbitol, and their derivatives. Both of these groups belong to the first type listed above. In some implementations the cryoprotective and/or lyoprotective agent(s) is selected from the group consisting of trehalose, sucrose, mannitol, maltose, maltodextrin, dextran, inulin, and raffinose. In some implementations the cryoprotectant is trehalose. Trehalose, also known as a,a-trehalose; a-D-glucopyranosyl-( 1— >1 )-a-D-gluco- pyranoside, mycose or tremalose, is a natural alpha-linked disaccharide formed by an a, a-1 ,1 -glucoside bond between two a-glucose units. Trehalose may be present as anhydrous or as dihydrate. In some implementations, the trehalose is D(+)-trehalose dehydrate.

[0140] In some implementations, the trehalose concentration can be measured in mg/ml or in g/L. In typical implementations, trehalose is present at about 5-250 mg/ml. For example, suitable trehalose concentrations include, but are not limited to, from about 5 mg/mL to about 75 mg/mL: from about 50 mg/mL to about 200 mg/mL; from about 75 mg/mL to about 200 mg/mL: from about 100 mg/mL to about 200 mg/mL; from about 25 mg/mL to about 175 mg/mL; from about 50 mg/mL to about 175 mg/mL; from about 75 mg/mL to about 175 mg/mL; from about 100 mg/mL to about 175 mg/mL; from about 25 mg/mL to about 150 mg/mL; from about 50 mg/mL to about 150 mg/mL; from about 75 mg/mL to about 150 mg/mL; from about 100 mg/mL to about 150 mg/mL; from about 25 mg/mL to about 125 mg/mL; from about 50 mg/mL to about 125 mg/mL; from about 75 mg/mL to about 125 mg/mL; from about 25 mg/mL to about 100 mg/mL; from about 125 mg/mL to about 175 mg/mL; from about 125 mg/mL to about 200 mg/mL; from about 5 mg/mL to 200 mg/mL; from about 200 mg/mL to 250 mg/mL; from about 5 mg/mL to 250 mg/mL; from about 75 mg/mL to 250 mg/mL; from about 100 mg/mL to 250 mg/mL; or from about 150 mg/mL to 250 mg/mL. In various implementations, the trehalose is in a concentration of about 150 mg/ml.

[0141] Simplifying proteomic workflow by leveraging encapsulation

[0142] Large-scale proteomics studies are increasingly applied to detect and characterize differential protein expression patterns in health and disease. Proteomics has the potential to provide crucial insights for biomarker discovery and drug development, and proteins are the primary target of nearly all drugs currently in development. However, conventional proteomics assays are constrained by a lack of sensitivity, particularly for low abundance proteins, and an inability to detect proteins over a wide range of concentrations. Proteomic workflows may utilize blood plasma samples. Passive separation may be used to separate and dilute plasma from blood at time of collection. Encapsulation and lyophilized microspheres are implemented to reduce touch points relative to the SomaScan® Assay protocol published by SomaLogic Operating Co., Inc. The compositions, systems, and methods of this disclosure can be used to harness the full potential of current sequencing technologies, provide for lower reagent, sample storage, and shipping costs, and simplify sample preprocessing and library preparation workflows to reduce the number of liquid handling steps and required hands-on time. Among other things, streamlining sample preprocessing and library preparation reduces costs. More important than cost, however, quality of data will also increase by controlling the grade of input material and reducing touchpoints/potential points for user error.

[0143] FIGURE 1 illustrates an example proteomic workflow 100 in accordance with the present disclosure. As shown in FIGURE 1, a timeline 101 for the workflow 100 is drawn to a scale 102 indicated, and a legend 103 identifies how different portions of the workflow 100 (specifically pipette transfers or washes and waiting periods) are indicated. The example proteomic workflow 100 in FIGURE 1 begins within a first tube 104 (also called a well) in which plasma separation is performed in order to extract proteins from a blood plasma sample.

[0144] After a suitable delay following addition of the sample to the first tube 104 to allow separation of the proteins from the blood plasma sample, a robotic transfer 105 of at least a portion of the diluted sample from the first tube 104 to a second tube 106 is performed. The second tube 106 includes three reagents provided for protein binding and selection. One of the three reagents within the second tube 106 includes aptamer beads (e.g., stabilized slow off-rate modified aptamer (SOMAmer) beads) for binding of target proteins within the transferred sample. In some implementations, the aptamer beads are in the form of lyophilized microspheres or a gel. Also, in some cases, release of the aptamer beads may be triggered by addition of a rehydration solution. Protein binding action by the first reagent may be activated or triggered by contact with the proteins in diluted form within the liquid added to the second tube 106 in conjunction with transfer of the sample from the first tube 104 or liquid (such as the rehydration solution and/or an assay buffer) added to the second tube 106 following the transfer. Aptamer beads include beads (e.g., strep avidin beads) with aptamers bound to their surface, for example, via a linker such as a cleavable linker. Aptamers are nucleic acids that bind to molecular targets, such as proteins, with high affinity and specificity. Advancements in aptamer selection and design include Systematic Evolution of Ligands by Exponential enrichment (SELEX). In SELEX, high affinity nucleic acids for different analytes of interest can be isolated from a combinatorial library, permitting high throughput characterization of aptamer-target binding and multiplexed assays for analytes in a complex biological sample. Suitable aptamers and aptamer beads for use are SOMAmers (SomaLogic, Boulder CO). SOMAmers are aptamers that contain modified nucleotides that help facilitate protein binding and contain a linker and binding moiety, e.g., biotin, that allows them to be bound to streptavidin beads.

[0145] A second of the three reagents within the second tube 106 includes a biotinylation mixture for attaching biotin to the proteins. Biotinylation creates a sensitive and specific tag for purification and detection of target proteins. A commercially-available mixture of biotinylation components may be utilized, such as in the form of lyophilized microspheres. In some implementations, activity by the second reagent may be triggered by temperature, such as by the contents of the second tube 106 reaching room temperature. Accordingly, the second tube 106 may be stored at a temperature lower than room temperature until shortly before use and allowed to adjust to room temperature (at least in part) after the sample is transferred to the second tube 106. However, once the sample has been transferred, the second tube 106 and the contents therein may remain at room temperature. In some implementations, annealing of microspheres reduces or avoids any need for cooling the storage environment of the second tube 106 below room temperature, since temperature-triggered release only occurs in a “wet” state (when an aqueous solution has been added), obviating any need for temperature control beforehand.

[0146] The third reagent within the second tube 106 includes a light cleavage mixture with poly-ionic competitors, such as in the form of encapsulated lyophilized microspheres. In some implementations, reagent components in the light cleavage mixture are segregated into at least two different lyophilized microspheres to prevent, reduce, and/or control undesired interactions. A light cleavage mixture may be employed with Pd in the core, where the cleavage mixture might involve segregation of Pd from another reagent within the mixture to reduce the thermosensitivity of the mixed reagent. The shell encapsulating the light cleavage mixture may be light blocking or substantially light blocking to protect light-sensitive components from light degradation.

[0147] Within the second tube 106, the three sample preparation processing steps described above may be sequentially performed without transfer of the sample to another container. Protein binding of proteins within the diluted sample transferred into the second tube 106 occurs first as a result of the sample transfer and/or addition of a liquid (rehydration solution and/or buffer). Biotinylation of proteins within the transferred sample subsequently occurs once the temperature of the second tube 106 and the initial content(s) therein before the sample transfer (such as at least the three reagents) reaches room temperature. Finally, cleavage occurs subsequent to biotinylation by exposure of the second tube 106 and the contents therein to suitable light to trigger the light cleavage reagent released from the encapsulating shell. Alternatively, use of non-light cleavage reagents such as an enzyme (e.g., protease that target specific peptide sequence, lipase that target ester linkage, and cellulase/glycoside hydrolase that target saccharide) or chemically-induced cleavage (e.g., pH via an acid or base, reduction-oxidation (redox) via a reducing agent such as DTT/TCEP, and use of a metal catalyst such as Pd/THP to cleave an allyl ether) may be used to trigger release.

[0148] Once sufficient time has elapsed (following exposure of the second tube 106 to a suitable light source), the contents within the second tube 106 may undergo one or more post-processing operations in order to use the contents within the second tube 106. For example, the contents within the second tube 106 may undergo three washes (which may or may not require heating control) in accordance with the known art to remove reaction contaminants and proteins other than the target protein, thereby producing cleaned analytes. Following another suitable delay, a robotic transfer 107 of at least a portion of the cleaned analytes within the second tube 106 to a third tube 108 from the second tube 106 is performed.

[0149] The third tube 108 includes three sample processing reagents for polymerase chain reaction (PCR) to convert the aptamer beads into libraries. One of the three reagents within the third tube 108 includes capture beads for binding of cleaned analytes, such as in the form of lyophilized microspheres. Release of the capture beads may be triggered by addition of an additional rehydration solution. Protein binding action by the first reagent may be activated or triggered by contact with the cleaned analytes.

[0150] A second of the three reagents within the third tube 108 includes an isothermal PCR mixture for amplifying the captured aptamers. A commercially-available PCR mixture may be utilized, such as in the form of encapsulated lyophilized microspheres. Activity by the second PCR mixture reagent may be triggered by time, such as by rehydration of lyophilized PCR mixture in a timedependent manner. For example, a water-soluble shell with time-delayed dissolution may encapsulate the PCR mixture.

[0151] Tire third reagent within the third tube 108 includes exonuclease shrimp alkaline phosphatase (ExoSAP) to clean-up PCR products before sequencing by enzymatic removal of excess nucleotides and primers from PCR reactions. In some implementations, the ExoSAP is in the form of encapsulated lyophilized microspheres with a temperature (such as room temperature) trigger. As with the second tube 106, the third tube 108 may be stored at a temperature lower than room temperature until shortly before use and allowed to adjust to room temperature (at least in part) after the sample is transferred from the second tube 106 to the third tube 108. However, once the sample has been transferred, the third tube 108 and the contents therein may remain at room temperature.

[0152] Within the third tube 108, the three sample preparation processing steps described above may again be sequentially performed without transfer of the sample to another container. Protein binding of proteins within the sample transferred into the third tube 108 occurs first as a result of the sample transfer and/or addition of a liquid (rehydration solution and/or buffer). PCR amplification of captured aptamers (e.g., SOMAmers) within the transferred sample subsequently occurs once the time associated with delay trigger of the PCR mixture within the third tube 108 has elapsed. Finally, ExoSAP cleanup of the PCR products is triggered by temperature, such as by the third tube 108 reaching room temperature. It may be advantageous to compress the dynamic range of the sample due to large differences in protein abundance. Several approaches may be taken to achieve this, including diluting the sample into different reactions with different SOMAmers on beads to account for the different protein abundances. One implementation of dynamic range compression may be to include a proportion of the SOMAmers for high abundant proteins without the cleavable modification. In this case, SOMAmers bound to high abundant proteins would be retained on the beads after the cleavage step while allowing the lower abundance aptamers to be removed from the beads for detection, and thus limit the signal from those high abundant proteins in the final sequencing library.

[0153] Although FIGURE 1 illustrates one example of a proteomic workflow 100, various changes may be made to FIGURE 1. For example, while the second tube 106 and third tube 108 and their respective contents are described as being used together in the same proteomic workflow 100, this is not necessarily required. The second tube 106 and its contents may be used with or without the third tube 108 and its contents (or vice versa), and each tube 106 or 108 may be used in any suitable workflow.

[0154] Table 1 below compares robotic transfer tip count, plate count, number of reagents (such as stock keeping units or “SKUs”), turnaround time (TAT) in hours, and number of samples per 96 well deck for proteomic for the example proteomic workflow 100 with alternative processes. Note that the proteomic workflow 100 may be utilized without encapsulation of the light cleavage mixture, the PCR mixture, and/or the ExoSAP as described above. As apparent, the encapsulation further reduces the number of necessary tips, plates, and reagents required. With or without encapsulation, the proteomic workflow 100 significantly reduces the amount of equipment and the number of reagents required.

Table 1

[0155] Simplifying array workflow by leveraging encapsulation

[0156] Array workflows may employ passive denaturation carried out with sodium hydroxide (NaOH) and neutralized with encapsulated microspheres. Amplification, fragmentation, and hybridization may be carried out sequentially in the same tube in a passive fashion, such as via triggered release.

[0157] FIGURE 2 illustrates an example array workflow 200 in accordance with the present disclosure. As shown in FIGURE 2, a timeline 201 for the workflow 200 is drawn to a scale 202 indicated, and a legend 203 identifies how different portions of the workflow 200 (specifically pipette transfers and waiting periods) are indicated. The example array workflow 200 in FIGURE 2 begins within a first well 204 on a plate in which DNA denaturant is performed, destroying base pairs to separate the double-stranded helix into two single strands without changing the primary structure of the DNA. In some implementations, the first well 204 is provided with DNA denaturant in the form of encapsulated lyophilized microspheres having a core of neutralizing reagent and a sodium hydroxide shell. In other implementations, the first well 204 is provided with sodium hydroxide in the form of lyophilized microspheres or cake and a neutralizing reagent in the form a lyophilized microspheres. DNA and a rehydration solution are added to the first well 204. After a period sufficient to allow release and action by the DNA denaturant, a robotic transfer 205 of at least a portion of the gDNA in the first well 204 to a second well 206 is performed.

[0158] Prior to that transfer, three reagents are provided in the second well 206 for genome amplification and fragmentation. A first of the three reagents in the second well 206 includes an amplification and random primer mixture, such as in the form of lyophilized microspheres. For example, the mixture may include a reagent for whole genome amplification (WGA) and random primers, short segments of single-stranded DNA used for synthesis and cloning. A second reagent in the second well 206 includes a fragmentation mixture, such as in the form of lyophilized microspheres with a time-triggered release (or an alternative release trigger, such as pH for a material that becomes soluble gradually as pH drops), for enzymatic breaking of intact DNA and long sequences into fragments suitable for hybridization onto an array. A third reagent in the second well 206 includes a hybridization buffer for bonding two complementary single-stranded DNA molecules together to form a double-stranded molecule. In some implementations, the hybridization buffer is in the form of encapsulated lyophilized microspheres with a temperature release trigger. In some implementations, a multi-layered coating imparts a first layer for a pH trigger, soluble after the second denaturation, followed by a second layer that imparts a time delay (i.e., “domino” release).

[0159] Within the second well 206, the three sample preparation processing steps described above may be sequentially performed without transfer of the sample to another container. Amplification of the DNA within the sample transferred into the second well 406 occurs first as a result of the sample transfer and/or addition of a liquid (rehydration solution and/or buffer). Fragmentation of the amplified DNA subsequently occurs after a time delay then occurs, followed by hybridization when the second well 206 and the contents therein reach a temperature (such as room temperature). After a period of time sufficient to allow hybridization to complete, a transfer 207 of the plate including the second well 206 into a system 208 for scanning can occur. In some implementations, hybridization occurs on a surface of an array of silica microbeads housed in etched microwells.

[0160] Although FIGURE 2 illustrates one example of an array workflow 200, various changes may be made to FIGURE 2. For example, the second well 206 and its contents may be used in any suitable workflow.

[0161] FIGURE 3 illustrates an example one-pot target preparation workflow 300 with lyophilized microsphere (“lyo”) plates for leveraging temperature-triggered controlled release in accordance with the present disclosure. As shown in FIGURE 3, the workflow 300 leverages three types of lyo plates in this example implementation, namely a sodium hydroxide lyo plate 301, a WGA/fragmentation lyo plate 302, and a hybridization buffer lyo plate 303. A DNA sample plate 304 is loaded 305 into a robot deck, and a robotic transfer 306 of at least portions of DNA samples are transferred from wells on the DNA sample plate 304 to corresponding wells on the sodium hydroxide lyo plate 301 containing lyophilized microspheres of sodium hydroxide. A rehydration solution is added to the wells containing the DNA samples and the lyophilized microspheres of sodium hydroxide, triggering release of the sodium hydroxide.

[0162] A robotic transfer 307 is performed of at least portions of the contents in the wells of the sodium hydroxide lyo plate 301 to corresponding wells on the WGA/fragmentation lyo plate 302. The wells on the WGA/fragmentation lyo plate 302 are provided with lyophilized microspheres of WGA reagent, which may be separated by wax from lyophilized microspheres of a fragmentation reagent. Addition of content from plate 301 to plate 302 by the automated robot rehydrates and releases the WGA reagent. An on-deck heater/thermocycler (ODTC) may be used to heat the wells to a predetermined temperature for a predetermined time (such as to about 35° C for about 1 hour), during which time amplification occurs. The wells are then heated to a predetermined temperature for a predetermined time (such as to about 60° C for about 5 minutes) to melt the wax barrier. In some cases, mineral oil may be used to separate the wax barrier and release the fragmentation reagent. Examples of candidates for the wax material may include the materials listed in Table 2.

Table 2

A wax barrier can accommodate the amplification reaction time requirement (such as up to about 1 hour or more). If a shorter reaction (such as about 1-10 minutes) can be conducted, a different material may be employed.

[0163] Tire wells are then heated to a predetermined temperature for a predetermined time (such as to about 37° C for about 1 hour), during which time fragmentation occurs. DNA within the wells of the WGA/fragmentation lyo plate 302 is then denatured, chemically or by heating (such as to about 95° C for about 15 minutes). A robotic transfer 308 is performed of at least portions of the contents in the wells of the WGA/fragmentation lyo plate 302 to corresponding wells on the hybridization buffer lyo plate 303. The wells on the hybridization buffer lyo plate 303 are provided with lyophilized microspheres of a hybridization buffer reagent. Addition of a rehydration solution by the automated robot releases the rehydration buffer reagent, and the wells are heated to a predetermined time (such as to about 56° C for about 0.5 hours), during which time hybridization occurs. The DNA in the hybridization buffer may then be collected 309. Notably, the workflow 300 involves only two touch points: setting up the robot deck and collecting the denatured DNA in the hybridization buffer.

[0164] Although FIGURE 3 illustrates one example of a one-pot target preparation workflow 300 with lyophilized microsphere plates for leveraging temperature-triggered controlled release, various changes may be made to FIGURE 3. For example, the plates 301-303 and their contents may be used separately or together in any suitable workflow. Also, while shown as involving separate plates 301-303, the workflow 300 may alternatively proceed using a single integrated reagent plate 310 (such as one with the different reagents organized by column).

[0165] FIGURE 4 illustrates an example target preparation workflow 400 with lyo plates and automation for leveraging lyo reagent strips and an integrated reagent plate in accordance with the present disclosure. As shown in FIGURE 4, the workflow 400 utilizes lyo-reagent strips 401 , where each strip may include one reagent. An integrated reagent plate 402 may be used, such as one with the reagents assembled by column. In this example, the integrated reagent plate 402 includes columns with lyophilized sodium hydroxide (NaOH), columns for a lyophilized accelerated amplification mixture (AAX), columns for a lyophilized multi-sample amplification mixture (MA2), columns for a lyophilized fragmentation reagent (FMS), and columns for a lyophilized resuspension, hybridization, and wash solution (IBX).

[0166] The workflow 400 is a one-pot workflow since all reactions occur in a sample plate 403. Rehydration, liquid transfer, and incubation/reaction cycles 404 can be handled by an automated robot. The sequence for rehydration of the reagents and the sequence for transfer of reagents from the integrated reagent plate 402 to the sample plate 403 are indicated in FIGURE 4. Because an integrated reagent plate 402 with lyo strips is used, less contamination is likely, quality control is facilitated, deck space is saved, and samples may be shipped at ambient temperatures.

[0167] Although FIGURE 4 illustrates one example of a one-pot target preparation workflow 400 with lyo plates and automation for leveraging lyo reagent strips and an integrated reagent plate, various changes may be made to FIGURE 4. For example, the strips 401 and plates 402-403 and their contents may be used separately or together in any suitable workflow.

[0168] FIGURE 5 illustrates an example process 500 for employing a denature lyo plate and a WGA/fragmentation lyo plate for one-pot amplification and fragmentation in accordance with the present disclosure. As shown in FIGURE 5, the process 500 begins with a plate 501 with wells containing lyophilized microspheres of DNA denaturant or a sodium hydroxide solution. Sample DNA and water and/or a buffer are added 502 to the wells, producing denatured DNA 503. The denatured DNA 503 is robotically transferred 504 to wells on a WGA-fragmentation lyo plate 505 in which the wells contain lyophilized microspheres of WGA reagent and lyophilized microspheres of a fragmentation reagent. The wells are heated 506 (such as to about 37° C) for release and incubation of the WGA reagent to produce amplified DNA 507. The process 500 is delayed 508 for a time sufficient to allow release and incubation of the fragmentation reagent, producing fragmented DNA 509. The fragmented DNA is then subjected to purification and hybridization 510.

[0169] FIGURE 6 illustrates an example process 600 for employing a single lyo plate for one- pot amplification and fragmentation in accordance with the present disclosure. As shown in FIGURE 6, the process 600 begins with a plate 601 with wells containing lyophilized microspheres of DNA denaturant, lyophilized microspheres of a WGA reagent, and lyophilized microspheres of a fragmentation reagent. Sample DNA and water and/or a buffer are added 602 to the wells to trigger release of the denature reagent and produce denatured DNA 603. The wells are heated 604 (such as to about 37° C) for release and incubation of the WGA reagent to produce amplified DNA 605. The process 600 is delayed 606 for a time sufficient to allow release and incubation of the fragmentation reagent, producing fragmented DNA 607. The fragmented DNA is then subjected to purification and hybridization 608.

[0170] FIGURE 7 illustrates an example process 700 for employing a denature lyo plate and a WGA/TGA lyo plate for one-pot amplification and fragmentation in accordance with the present disclosure. As shown in FIGURE 7, the process 700 begins with a plate 701 with wells containing lyophilized microspheres of DNA denaturant. Sample DNA and water and/or a buffer are added 702 to the wells, producing denatured DNA 703. The denatured DNA is robotically transferred 704 to wells on a WGA/TGA/fragmentation lyo plate 705 in which the wells contain lyophilized microspheres of WGA reagent, lyophilized microspheres of a targeted genome amplification (TGA) reagent, and lyophilized microspheres of a fragmentation reagent. WGA incubation is allowed to proceed to produce WGA amplified DNA 707. The wells are subjected to thermocycling 708 to release and incubate the TGA reagent, producing WGA amplified and TGA amplified DNA 709. The fragmentation reagent is released 710, producing fragmented DNA 711. The fragmented DNA is subjected to purification and hybridization 712.

[0171] Although FIGURES 5 through 7 illustrate example processes for one-pot amplification and fragmentation, various changes may be made to FIGURES 5 through 7. For example, the plates shown as being used in FIGURES 5 through 7 may be used in any other suitable processes.

[0172] Although this disclosure has been described with reference to various example implementations, various changes and modifications may be suggested to one skilled in the art. It is intended that this disclosure encompass such changes and modifications as fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing biological samples, the method including: providing, in a single well, first particles including a first reagent for a first biological sample preparation processing step, second particles including a second reagent for a second biological sample preparation processing step, and third particles including a third reagent for a third biological sample preparation processing step, wherein the first particles release the first reagent in response to a first release trigger mechanism, the second particles release the second reagent in response to a second release trigger mechanism, and the third particles release the third reagent in response to a third release trigger mechanism, wherein one of the first or second trigger mechanisms and one of the second or third trigger mechanisms may be configured for domino release, wherein the first, second, and third particles are in a form of lyophilized microspheres, and wherein at least the third particles are in a form of one of encapsulated particles, stacked cakes separated by trigger layers, or solid substrate beads coated with an active material and encapsulated with trigger release shell(s), each encapsulated particle including an inner core including a lyophilized microsphere and an outer shell: and sequentially performing the first sample preparation processing step, the second sample preparation processing step, and the third sample preparation processing step in the single well using triggered release of one or both of the first reagent and the second reagent and separately-triggered release of the third reagent from the encapsulated particles.

2. The method of claim 1, wherein the first sample preparation processing step is amplification, the second sample preparation processing step is fragmentation, and the third sample preparation processing step is hybridization.

3. The method of claim 2, wherein the first reagent is an amplification and random primer mixture in the form of particles, the second reagent is a fragmentation mixture in the form of particles with a time delay release, and the third reagent is a hybridization buffer in the form of the encapsulated particles with temperature release.

4. The method of claims 1 or 2, wherein the amplification includes whole genome amplification.

5. The method of any of claims 1 through 3, wherein the single well is located on a first plate, wherein the first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature, and wherein the second reagent includes a fragmentation solution reagent with time delay release, the method further including: adding sample DNA to a well containing DNA denaturant on a second plate; adding water and a buffer to produce denatured DNA; robotically transferring the denatured DNA from the well on the second plate to the single well on the first plate; heating the single well to release the first reagent; and waiting for a period corresponding to the time delay release of the second reagent; and wherein the single well contains fragmented DNA following release of the first reagent and the second reagent.

6. The method of claim 5, wherein the first reagent includes a mixture of the WGA reagent and a targeted genome amplification (TGA) reagent in the form of encapsulated particles with temperature release at a second temperature higher than the first temperature, wherein heating the single well to release the first reagent further includes: heating the single well to the first temperature to release the WGA reagent; and subsequently heating the single well to the second temperature to release the TGA reagent.

7. The method of any of claims 1 through 3, wherein the single well is located on a single plate, wherein the single well contains denature reagent in the form of lyophilized microspheres with temperature release at room temperature, wherein the first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature, and wherein the second reagent includes a fragmentation solution reagent with time delay release, the method further including: adding sample DNA to the single well; adding water and a buffer to the single well at room temperature to produce denatured DNA; heating the single well to release the first reagent; and waiting for a period corresponding to the time delay release of the second reagent; and wherein the single well contains fragmented DNA following release of the first reagent and the second reagent.

8. The method of any of claims 1 through 6, wherein denatured genomic DNA (gDNA) is transferred to the single well.

9. Tire method of claim 8, wherein the single well is located on a first plate, the method further including: adding gDNA to a well containing DNA denaturant in the form of encapsulated particles with time delay release on a second plate; and transferring the denatured gDNA from the well on the second plate to the single well on the first plate.

10. The method of claim 9, wherein the DNA denaturant includes a neutralizing reagent core with a sodium hydroxide shell.

11. The method of any of claims 1 through 10, wherein an encapsulation of particles for the third reagent is wax, and wherein the single well is heated to release the third reagent from the encapsulation.

12. The method of claim 1, wherein the first sample preparation processing step is protein binding, the second sample preparation processing step is biotinylation, and the third sample preparation processing step is cleavage.

13. The method of claim 12, wherein the first reagent is stabilized slow off-rate modified aptamer beads, the second reagent includes components to biotinylate proteins in the form of particles, and the third reagent is a light, enzymatic, or chemical cleavage mixture in the form of one of encapsulated particles, stacked cakes separated by trigger layers, or solid substrate beads coated with an active material and encapsulated with trigger release shell(s) with light, enzymatic, or chemical triggered release.

14. The method of claim 12, wherein the first reagent are beads for binding of cleaned analytes, the second reagent includes a polymerase chain reaction (PCR) mixture in the form of particles with a time delay release, and the third reagent is exonuclease shrimp alkaline phosphatase (ExoSAP) in the form of encapsulated particles with temperature release.

15. A process for preparing biological samples, the process including: providing a plurality of wells on an integrated reagent plate, each well containing first particles including an amplification mixture, second particles including a fragmentation mixture, and third particles including a hybridization buffer, wherein the first particles activate the amplification mixture in response to a first mechanism, the second particles activate the fragmentation mixture in response to a second mechanism, and the third particles release the hybridization buffer in response to a third mechanism, wherein the first, second, and third particles include lyophilized microspheres and each of at least the third particles includes an inner core including a lyophilized microsphere and an outer shell; and sequentially performing nucleic acid amplification, fragmentation, and hybridization on a biological sample in the respective well using separate activation of the amplification mixture and the fragmentation mixture and triggered release of the hybridization buffer.

16. The process of claim 15, wherein the amplification mixture includes a whole genome amplification (WGA) reagent, wherein each of a first subset of the first particles includes a WGA inner core including a lyophilized microsphere for the WGA reagent and a WGA outer shell, and wherein the WGA outer shell for the first subset of the first particles releases the WGA reagent for amplification of DNA in response to a first temperature higher than room temperature, the process further including: heating each well to the first temperature to release the WGA reagent.

17. The process of claim 16. wherein the amplification mixture further includes a targeted genome amplification (TGA) reagent, wherein each of a second subset of the first particles includes a TGA inner core including a lyophilized microsphere for the TGA reagent and a TGA outer shell, and wherein the TGA outer shell for the second subset of the first particles releases the TGA reagent for amplification of the DNA in response to a second temperature higher than the first temperature, the process further including: subsequent to heating each well to the first temperature, heating each well to the second temperature to release the TGA reagent.

18. The process of claim 16 or 17, wherein the fragmentation mixture includes a fragmentation reagent, wherein each of the second particles includes a fragmentation inner core including a lyophilized microsphere for the fragmentation reagent and a fragmentation outer shell, and wherein the fragmentation outer shell for the second particles releases the fragmentation reagent for fragmentation of amplified DNA after a time delay, the process further including: subsequent to heating each well to the first temperature, allowing time for release of the fragmentation reagent and for fragmentation.

19. The process of any of claims 15 through 18, wherein each well further contains fourth particles including a DNA denaturant, wherein the fourth particles activate the DNA denaturant in response to a fourth mechanism, and wherein the fourth particles include lyophilized microspheres, the process further including: adding at least sample DNA in an aqueous solution.

20. A process for preparing biological samples, the process including: providing a well containing first particles including protein binding beads, second particles including a biotinylation mixture, and third particles including a light cleavage mixture, wherein the first particles activate protein binding in response to a first mechanism, the second particles activate the biotinylation mixture in response to a second mechanism, and the third particles release the light cleavage mixture in response to a third mechanism, wherein the first, second, and third particles include lyophilized microspheres and each of at least the second and third particles includes an inner core including a lyophilized microsphere and an outer shell; and sequentially performing protein binding, biotinylation, and light cleavage on a biological sample in the well using separate activation of the protein binding beads and triggered release of the biotinylation mixture and the light cleavage mixture.

21. The process of claim 20, wherein the protein binding beads include stabilized slow off- rate modified aptamer beads.

22. The process of claim 20 or 21, wherein the second mechanism is a time delay and the third mechanism is light.

23. A system for preparing biological samples, the system including: a container with an opening configured to receive a biological sample, the container configured to provide, in a single well, a workflow reagent release system including a first reagent for a first sample preparation processing step for the biological sample, a second reagent for a second sample preparation processing step for the biological sample, and a third reagent for a third sample preparation processing step for the biological sample, at least the third reagent contained within encapsulated particles, wherein the single well is configured such that the first sample preparation processing step, the second sample preparation processing step, and the third sample preparation processing step are sequentially performed in the single well using triggered release of one or both of the first reagent and the second reagent and separately-triggered release of the third reagent from the encapsulated particles.

24. The system of claim 23, wherein the first sample preparation processing step is amplification, the second sample preparation processing step is fragmentation, and the third sample preparation processing step is hybridization.

25. The system of claim 23 or 24, wherein the first reagent is an amplification and random primer mixture in the form of particles, the second reagent is a fragmentation mixture in the form of particles with a time delay release, and the third reagent is a hybridization buffer in the form of the encapsulated particles with temperature release.

26. The system of claim 25, wherein the amplification and random primer mixture includes a whole genome amplification reagent.

27. The system of any of claims 23 through 26, wherein the single well is located on a first plate, wherein the first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature, wherein the second reagent includes a fragmentation solution reagent with time delay release, wherein a well on a second plate is configured to receive sample DNA and DNA denaturant, wherein the well on the second plate is further configured to receive water and a buffer to produce denatured DNA, wherein the well on the second plate is configured to allow the denatured DNA to be robotically transferred from the well on the second plate to the single well on the first plate, wherein the single well is configured to be heated to release the first reagent, and wherein, after a period corresponding to the time delay release of the second reagent, the single well is configured to contain fragmented DNA.

28. The system of claim 27, wherein the first reagent includes a mixture of the WGA reagent and a targeted genome amplification (TGA) reagent in the form of encapsulated particles with temperature release at a second temperature higher than the first temperature, and wherein the single well is configured to be heated to the first temperature to release the WGA reagent and to be subsequently heated to the second temperature to release the TGA reagent.

29. The system of claim 23, wherein the single well is located on a single plate, wherein the single well contains denature reagent in the form of lyophilized microspheres with temperature release at room temperature, wherein the first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature, and wherein the second reagent includes a fragmentation solution reagent with time delay release, wherein the single well is configured to receive sample DNA, wherein the single well is further configured to receive water and a buffer at room temperature to produce denatured DNA, wherein the single well is configured to be heated to release the first reagent, and wherein, after a period corresponding to the time delay release of the second reagent, the single well is configured to contain fragmented DNA following release of the first reagent and the second reagent.

30. The system of any of claims 23 through 29. wherein the single well is configured to receive denatured genomic DNA (gDNA).

31. The system of claim 30, wherein the single well is located on a first plate, wherein a well on a second plate is configured to receive gDNA and DNA denaturant in the form of encapsulated particles with time delay release, and wherein the well on the second plate is configured to allow the denatured gDNA to be transferred from the well on the second plate to the single well on the first plate.

32. The system of claim 31, wherein the DNA denaturant includes a neutralizing reagent core with a sodium hydroxide shell.

33. The system of any of claims 23 through 32, wherein an encapsulation of particles for the third reagent is wax, and wherein the single well is configured to be heated to release the third reagent from the encapsulation.

34. The system of claim 23, wherein the first sample preparation processing step is protein binding, the second sample preparation processing step is biotinylation, and the third sample preparation processing step is cleavage.

35. The system of claim 34, wherein the first reagent is stabilized slow off-rate modified aptamer (SOMAmer) beads, the second reagent includes components to biotinylate proteins in the form of particles, and the third reagent is a light cleavage mixture in the form of the encapsulated particles with light triggered release.

36. The system of claim 35, wherein the first reagent are beads for binding of cleaned analytes, the second reagent includes a polymerase chain reaction (PCR) mixture in the form of particles with a time delay release, and the third reagent is exonuclease shrimp alkaline phosphatase (ExoSAP) in the form of the encapsulated particles with temperature release.

37. A composition, including: first particles including a first reagent, wherein the first particles release the first reagent in response to a first release trigger mechanism; second particles including a second reagent, wherein the second particles release the second reagent in response to a second release trigger mechanism for the biological samples; and third particles including a third reagent, wherein the third particles release the third reagent in response to a third release trigger mechanism, wherein the first, second, and third particles include lyophilized microspheres and at least the third particles include encapsulated particles, each encapsulated particle including an inner core including a lyophilized microsphere and an outer shell, and wherein the first reagent effects first biological sample preparation processing when released in response to the first release trigger mechanism, the second reagent effects second biological sample preparation processing when released in response to the second release trigger mechanism, and the third reagent effects third biological sample preparation processing when released in response to the third release trigger mechanism, wherein the first, second, and third biological sample preparation processing occur sequentially in a single well containing the first, second, and third particles by triggered release of one or both of the first reagent and the second reagent and separately-triggered release of the third reagent from the encapsulated particles.

38. The composition of claim 37, wherein the first sample preparation processing is amplification, the second sample preparation processing is fragmentation, and the third sample preparation processing is hybridization.

39. The composition of claim 37 or 38, wherein the first reagent is an amplification and random primer mixture in the form of particles, the second reagent is a fragmentation mixture in the form of particles with a time delay release, and the third reagent is a hybridization buffer in the form of encapsulated particles with temperature release.

40. The composition of claim 39, wherein the amplification and random primer mixture includes a whole genome amplification reagent.

41. The composition of any of claims 37 through 40, wherein the single well is located on a first plate, wherein the first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature, and wherein the second reagent includes a fragmentation solution reagent with time delay release.

42. The composition of claim 41, wherein the first reagent includes a mixture of the WGA reagent and a targeted genome amplification (TGA) reagent in the form of encapsulated particles with temperature release at a second temperature higher than the first temperature, and wherein the single well is configured to be heated to the first temperature to release the WGA reagent and to be subsequently heated to the second temperature to release the TGA reagent.

43. The composition of claim 42, wherein the single well is located on a single plate, wherein the single well contains denature reagent in the form of lyophilized microspheres with temperature release at room temperature, wherein the first reagent includes a whole genome amplification (WGA) reagent in the form of encapsulated particles with temperature release at a first temperature higher than room temperature, and wherein the second reagent includes a fragmentation solution reagent with time delay release.

44. The composition of any of claims 37 through 43, further including DNA denaturant including a neutralizing reagent core with a sodium hydroxide shell.

45. The composition of claim 37, wherein the first sample preparation processing is protein binding, the second sample preparation processing is biotinylation, and the third sample preparation processing is cleavage.

46. The composition of claim 45, wherein the first reagent is stabilized slow off-rate modified aptamer (SOMAmer) beads, the second reagent includes components to biotinylate proteins in the form of particles, and the third reagent is a light cleavage mixture in the form of encapsulated particles with light triggered release.

47. The composition of claim 46, wherein the first reagent are beads for binding of cleaned analytes, the second reagent includes a polymerase chain reaction (PCR) mixture in the form of particles with a time delay release, and the third reagent is exonuclease shrimp alkaline phosphatase (ExoSAP) in the form of encapsulated particles with temperature release.