Passive reactions consumables

Abstract

Consumables (100, 300, 600, 900, 1000) for collection and at least partial preparation of biological samples for nucleic acid analysis include an integrated heat source (105, 305, 605, 905) for triggering release of sample processing reagents or components, a filter membrane (306, 606, 906, 1006) and a pump mechanism (307, 607, 907, 1007) for sample extraction, or multiple wells (611-614, 911-912, 1012-1014) for multi-omics processing. These consumables improve the user experience and enable analyte extraction during transit, including by reducing sample degradation during lengthy shipment. The consumables are well-suited for automation of sample processing.

Description

PASSIVE REACTIONS CONSUMABLESTECHNICAL FIELD

[0001] This disclosure relates generally to collection of biological samples for nucleic acid processing. More specifically, this disclosure relates to concurrent biological sample collection and preparation within a single consumable, including for multi-omics.BACKGROUND

[0002] One example of multi-omics is nucleic acid sequencing. Many current sequencing platforms use “sequencing by synthesis” (SBS) technology and fluorescence-based methods for detection. Alternative sequencing methods and improved sample preparation processes and reagents that allow for more cost effective, rapid, and convenient sequencing and nucleic acid detection are desirable as complements to SBS. Current protocols for SBS technology routinely employ a sample preparation process that converts DNA or RNA into a library of fragmented, sequenceable templates. Sample preparation methods often involve multiple steps, material transfers, and expensive instruments to effect fragmentation and are often difficult, tedious, expensive, and inefficient.

[0003] Libraries including polynucleotides are generally prepared in any suitable manner to attach oligonucleotide adapters to target polynucleotides. Sequencing may result in determination of the sequence of the whole or a pail of the target polynucleotides. Sequencing can be carried out using any suitable sequencing technique, and methods for determining the sequence of immobilized and amplified adapter-target-adapter molecules, including strand re-synthesis, are known in the art. SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand. In traditional methods of SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery.

[0004] Tire 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.

[0005] Biological sample collection and analyte extraction (conversion of a raw sample toanalyte(s)) for SBS are collectively a complex, touch point heavy workflow. For example, analyzing cell-free deoxyribonucleic acid (cfDNA), cell-free ribonucleic acid (cfRNA), proteins, and other analytes of clinical interest often benefit from separation of plasma from blood. Separation of plasma from whole blood may be achieved via centrifugation, which uses large external instrumentation, multiple consumables, and significant time, and which also typically involves numerous user touch points. Further, centrifugation normally requires sample transfer, which should be minimized to avoid contaminating genomic DNA (gDNA).SUMMARY

[0006] In a first implementation, an apparatus includes a well within a container configured for holding a biological sample, where the well contains first particles and includes a portion containing a heat source and second particles. The apparatus also includes a seal over an opening into the portion of the well containing the heat source, where removal or rupture of the seal initiates an exothermic reaction by the heat source. The first particles are triggered by addition of the biological sample into the well. The second particles are triggered by the exothermic reaction by the heat source.

[0007] In various forms of the apparatus of the first implementation, a first biological sample preparation processing step may correspond to the first particles and may occur during a first period of time following the addition of the biological sample into the well.

[0008] In various forms of the apparatus of the first implementation, a first biological sample preparation processing step may include histone stripping.

[0009] In various forms of the apparatus of the first implementation, the first particles may include an enzyme.

[0010] In various forms of the apparatus of the first implementation, the enzyme may include pepsin, and the first particles may include sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), polysorbate, and hydrochloric acid (HC1).

[0011] In various forms of the apparatus of the first implementation, a second biological sample preparation processing step may correspond to the second particles and may occur during a second period of time following the initiation of the exothermic reaction by the heat source.

[0012] In various forms of the apparatus of the first implementation, a second biological sample preparation processing step may include nucleic acid capture.

[0013] In various forms of the apparatus of the first implementation, the second particles may include beads.

[0014] In various forms of the apparatus of the first implementation, the second particles may include a-cyclodextrin, 3-(N-morpholino)propanesulfonic (MOPS) acid, guanidine thiocyanate (GTC), and polyethylene glycol (PEG).

[0015] In various forms of the apparatus of the first implementation, a pump mechanism and a filter membrane may be disposed over the portion of the well containing the second particles. The pump mechanism may be configured to drive a portion of plasma in the biological sample through the filter membrane, and the filter membrane may be configured to pass the portion of the plasma from the biological sample into the portion of the well containing the second particles.

[0016] In various forms of the apparatus of the first implementation, the pump mechanism may include a pillar array.

[0017] In various forms of the apparatus of the first implementation, the container may be a cubic container with the pump mechanism and the filter membrane disposed diagonally therein.

[0018] In various forms of the apparatus of the first implementation, a first surface of the container may include a guide for mounting the container on an automation plate, and a second surface of the container may include the opening into the portion of the well containing the heat source and an aspiration hole covered by foil.

[0019] In various forms of the apparatus of the first implementation, the first particles may include a first type of the first particles and a second type of the first particles. The container may include a cylindrical well disposed over the pump mechanism and the filter membrane. The cylindrical well may contain the first type of the first particles. The container may also include a movable plate disposed below the cylindrical well. The movable plate may include a plurality of openings therethrough. The container may further include a drum disposed below the pump mechanism and the filter membrane. The drum may include a plurality of sector-shaped wells, and the heat source may be disposed centrally to the plurality of sector-shaped wells. At least a first of the plurality of sectorshaped wells may contain the second type of the first particles, and at least a second of the plurality of wells may contain the second particles.

[0020] In various forms of the apparatus of the first implementation, the first particles may include an enzyme.

[0021] In various forms of the apparatus of the first implementation, the first type of the first particles may include Proteinase K.

[0022] In various forms of the apparatus of the first implementation, the second type of the first particles may include pepsin.

[0023] In various forms of the apparatus of the first implementation, the second type of the first particles may include a plasma stabilizer.

[0024] In various forms of the apparatus of the first implementation, the second particles may include one or more of a-cyclodextrin, SDS, or a buffer.

[0025] In various forms of the apparatus of the first implementation, the movable plate may be in a first position when the biological sample is collected.

[0026] In various forms of the apparatus of the first implementation, movement of the movable plate from the first position to a second position may align a first of the plurality of openings through the movable plate with at least one of the plurality of sector-shaped wells, allowing passage of the biological sample from the cylindrical well into the at least one of the plurality of sector-shaped wells.

[0027] In various forms of the apparatus of the first implementation, movement of the movable plate from the second position to a third position may align a second of the plurality of openings with an air source, allowing passage of air into contact with the heat source.

[0028] In various forms of the apparatus of the first implementation, the first particles may include a first type of the first particles and a second type of the first particles. The well may include a plurality of biological sample processing wells. The heat source may include a plurality of heat sources disposed between adjacent ones of the plurality of biological sample processing wells. The container may include a collection well disposed over the pump mechanism and the filter membrane. The container may also include a movable plate disposed below the collection well. The movable plate may include a plurality of openings therethrough. At least a first of the plurality of biological sample processing wells may contain the second type of the first particles, and at least a second of the plurality of biological sample processing wells may contain the second particles.

[0029] In various forms of the apparatus of the first implementation, the first of the plurality of biological sample processing wells may contain particles used for extraction of genomic deoxyribonucleic acid (gDNA) from the biological sample. The second of the plurality of biological sample processing wells may contain particles used for extraction of one of cell-free deoxyribonucleic acid (cfDNA). circulating tumor DNA (ctDNA), cell-free fetal DNA (cffDNA), or environmental DNA (eDNA) from the biological sample. A third of the plurality of biological sample processing wells may contain particles used for extraction of one of ribonucleic acid (RNA), cell-free RNA (cfRNA), or circulating tumor RNA (ctRNA) from the biological sample. A fourth of the plurality of biological sample processing wells may contain particles used for extraction of proteins from the biological sample.

[0030] In various forms of the apparatus of the first implementation, the well may include a plurality of biological sample processing wells. The container may include a pump mechanism, a collection well disposed over the pump mechanism, and a filter membrane between the collection well and the pump mechanism and between the well and the pump mechanism. The well may be disposed above the filter membrane.

[0031] In various forms of the apparatus of the first implementation, the pump mechanism may include a movable and compressible sponge below the filter membrane. In a first position, the sponge may be below the collection well and may absorb a filtered portion of the biological sample. Ina second position, the sponge may be below the well such that compression of the sponge pumps the filtered portion of the biological sample into the plurality of biological sample processing wells.

[0032] In various forms of the apparatus of the first implementation, the container may include a capture receptacle over the well. The apparatus may also include a pump mechanism and a filter membrane between the capture receptacle and the well. The pump mechanism may be configured to drive a portion of plasma in the biological sample through the filter membrane, and the filter membrane may be configured to pass the portion of the plasma from the biological sample into the well.

[0033] In various forms, a method of using the any form of the apparatus of the first implementation may include receiving the biological sample within the container. The method may also include, after a predetermined period, removing the seal over the opening to initiate the exothermic reaction. The method may further include removing fluid including an analyte from the well.

[0034] In various forms, the method of using the apparatus of the first implementation may also include removing fluid including a first analyte from a first of a plurality of biological sample processing wells and removing fluid including a second analyte from a second of the plurality of biological sample processing wells. The first analyte may be extracted from the biological sample concurrently with extraction of the second analyte from the biological sample.

[0035] In a second implementation, an apparatus includes a well within a container configured for holding a biological sample. The container holds first particles and second particles that are configured for performance of sequential biological sample preparation processing steps on the biological sample. The apparatus also includes a pump mechanism and a filter membrane. The pump mechanism is configured to drive a portion of plasma in the biological sample through the filter membrane, and the filter membrane is configured to pass the portion of the biological sample into the well.

[0036] In various implementations of the apparatus of the second implementation, the first particles may be disposed within a first portion of the container outside of the well, and the second particles may be disposed within the well.

[0037] In various implementations of the apparatus of the second implementation, a first of the sequential biological sample preparation processing steps may be triggered by rehydration of the first particles by the biological sample, and a second of the sequential biological sample preparation processing steps may be triggered by exposure of the second particles to an elevated temperature.

[0038] In various implementations of the apparatus of the second implementation, the container may also include a heat source proximate to the well, and one of a seal over an opening into the portion of the container containing the heat source or a region for compression of a blister pack including the heat source. Either removal or rupture of the seal or compression of the blister pack may initiate an exothermic reaction by the heat source.

[0039] In various implementations of the apparatus of the second implementation, the container may be a cubic container with the pump mechanism and the filter membrane disposed diagonally therein.

[0040] In various implementations of the apparatus of the second implementation, the container may include a cylindrical collection well disposed over the pump mechanism and the filter membrane. The cylindrical collection well may contain the first particles. The container may also include a movable plate disposed below the cylindrical collection well. The movable plate may include a plurality of openings therethrough. The container may further include a drum disposed below the pump mechanism and the filter membrane. The drum may include a plurality of sector-shaped wells, and at least a first of the plurality of sector-shaped wells may contain the second particles.

[0041] In various implementations of the apparatus of the second implementation, when the movable plate is in a first position, access to the plurality of sector-shaped wells from the cylindrical collection well is blocked while the cylindrical collection well remains accessible for receiving the biological sample.

[0042] In various implementations of the apparatus of the second implementation, movement of the movable plate from the first position to a second position may align a first of the plurality of openings through the movable plate with at least one of the plurality of sector-shaped wells, allowing passage of the portion of plasma in the biological sample into the at least one of the plurality of sectorshaped wells.

[0043] In various implementations of the apparatus of the second implementation, movement of the movable plate from the second position to a third position may align a second of the plurality of openings with an air source, allowing passage of air into contact with a heat source in the container.

[0044] In various implementations of the apparatus of the second implementation, the well may include a plurality of biological sample processing wells. The container may include a collection well disposed over the pump mechanism and the filter membrane. The container may also include a movable plate disposed below the collection well. The movable plate may include a plurality of openings therethrough. At least a first of the plurality of biological sample processing wells may contain the second particles.

[0045] In various implementations of the apparatus of the second implementation, the well may include a plurality of biological sample processing wells. The container may include a collection well disposed over the pump mechanism and the filter membrane. The filter membrane may cover openings into the plurality of biological sample processing wells. The pump mechanism may include a movable and compressible sponge below the filter membrane. In a first position, the sponge may be below the collection well and absorb a filtered portion of the biological sample. In a second position, the sponge may be below the well such that compression of the sponge pumps the filtered portion ofthe biological sample into the plurality of biological sample processing wells. At least a first of the plurality of biological sample processing wells may contain the second particles.

[0046] In various forms, a method of using any form of the apparatus of the second implementation may include receiving the biological sample within the container. The method may also include removing fluid including an analyte from the well.

[0047] In various forms, the method of using the apparatus of the second implementation may also include removing fluid including a first analyte from a first of a plurality of biological sample processing wells and removing fluid including a second analyte from a second of the plurality of biological sample processing wells. The first analyte may be extracted from the biological sample concurrently with extraction of the second analyte from the biological sample.

[0048] In a third implementation, an apparatus includes a container configured for holding a biological sample. The container includes a collection well containing first particles configured for performance of a first biological sample preparation processing step on a biological sample received in the collection well. The container also includes a well including a plurality of biological sample processing wells in fluid communication with the collection well. At least a first of the plurality of biological sample processing wells contains second particles configured for performance of a second biological sample preparation processing step on a portion of the biological sample received from the collection well.

[0049] In various forms of the apparatus of the third implementation, the second particles may include second particles of a first type and second particles of a second type. The second particles of the first type may be configured for biological sample preparation processing targeting a first nucleic acid molecule, and the second particles of the second type may be configured for biological sample preparation processing targeting a second nucleic acid molecule. The first of the plurality of biological sample processing wells may contain the second particles of the first type, and a second of the plurality of biological sample processing wells may contain the second particles of the second type.

[0050] In various forms of the apparatus of the third implementation, the plurality of biological sample processing wells may include the first biological sample processing well containing the second particles of the first type, the second biological sample processing well containing the second particles of the second type, a third biological sample processing well containing the second particles of a third type, and a fourth biological sample processing well containing the second particles of a fourth type. The second particles of the first type may be configured for biological sample preparation processing targeting gDNA. The second particles of the second type may be configured for biological sample preparation processing targeting cfDNA. The second particles of the third type may be configured for biological sample preparation processing targeting RNA. The second particles of the fourth type may be configured for biological sample preparation processing targeting proteins.

[0051] In various forms of the apparatus of the third implementation, the container may include a pump mechanism and a filter membrane between the collection well and the well.

[0052] In various forms of the apparatus of the third implementation, the pump mechanism and the filter membrane may be disposed above the collection well, and the collection well may be cylindrical. The well may be a drum. The plurality of biological sample processing wells may be sectorshaped.

[0053] In various forms of the apparatus of the third implementation, the pump mechanism may include a pillar array.

[0054] In various forms of the apparatus of the third implementation, the apparatus may include a heat source disposed centrally to the plurality of biological sample processing wells.

[0055] In various forms of the apparatus of the third implementation, the apparatus may include a movable plate disposed below the collection well. The movable plate may include a plurality of openings therethrough. When the movable plate is in a first position, access to the plurality of biological sample processing wells from the collection well is blocked while the collection well remains accessible for receiving the biological sample. Movement of the movable plate from the first position to a second position may align a first of the plurality of openings through the movable plate with the plurality of biological sample processing wells, allowing passage of a portion of the biological sample into the plurality of biological sample processing wells.

[0056] In various forms of the apparatus of the third implementation, the filter membrane may be disposed below the collection well and the plurality of biological sample processing wells. The pump mechanism may include a movable and compressible sponge below the filter membrane. In a first position, the sponge may be below the collection well and absorb a filtered portion of the biological sample. In a second position, the sponge may be below the plurality of biological sample processing wells such that compression of the sponge pumps the filtered portion of the biological sample into the plurality of biological sample processing wells.

[0057] In various forms, a method of using any form of the apparatus of the third implementation may include receiving the biological sample within the container. The method may also include removing fluid including an analyte from the well.

[0058] In various forms, the method of using the apparatus of the third implementation may also include the well may include a plurality of biological sample processing wells. The method may also include removing fluid including a first analyte from a first of a plurality of biological sample processing wells and removing fluid including a second analyte from a second of the plurality of biological sample processing wells. The first analyte may be extracted from the biological sample concurrently with extraction of the second analyte from the biological sample.

[0059] In a fourth implementation, a method includes collecting a first biological sample in a collection well of a biological sample container that includes the collection well, a first biological sample processing well, and a second biological sample processing well. The method also includes preparing the biological sample. The method further includes extracting a portion of a first solution in the first biological sample processing well using a first fluid transfer vessel. The method still further includes extracting a portion of a second solution in the second biological sample processing well using a second fluid transfer vessel.

[0060] In various forms of the method of the fourth implementation, preparing the biological sample may be performed using first particles provided in the collection well prior to collection of the biological sample. Extracting a portion of a first solution in the first biological sample processing well may be performed subsequent to the initial biological sample preparation processing step using second particles of a first type provided in the first biological sample processing well prior to the collection of the biological sample. Extracting a portion of the second solution in the second biological sample processing well may be performed subsequent to the initial biological sample preparation processing step using second particles of a second type provided in the second biological sample processing well prior to the collection of the biological sample.

[0061] In various forms of the method of the fourth implementation, the portion of the first solution may be extracted from the biological sample container into a first analyzer for analysis of characteristics of the first nucleic acid molecule. The portion of the second solution may be extracted from the biological sample container into a second analyzer for analysis of characteristics of the second nucleic acid molecule.

[0062] Other technical features may be readily apparent to one skilled in the ait from the following figures, descriptions, and claims.

[0063] 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 ait 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.

[0064] The terms “include" and “include," as well as derivatives thereof, mean inclusion without limitation. The term “or" is inclusive, meaning and / or. The phrase “associated with," as wellas 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.

[0065] 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 pail) 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.

[0066] 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.

[0067] As used here, the phrase “configured for” (or “configured to,” 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.

[0068] 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 thecontext 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, 5xl05and IxlO6or 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.

[0069] 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%.

[0070] Definitions for other certain words and phrases may be provided throughout this document. Those of ordinary skill in the ait 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.

[0071] 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 art and is not intended to invoke 35 U.S.C. § 112(f).BRIEF DESCRIPTION OF THE DRAWINGS

[0072] 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 parts:

[0073] FIGURE 1 illustrates an example consumable for biological sample collection and preparation in accordance with the present disclosure;

[0074] FIGURE 2 illustrates an example process for using the consumable of FIGURE 1 in accordance with the present disclosure;

[0075] FIGURE 3 illustrates an alternative example consumable for biological sample collection and preparation in accordance with the present disclosure;

[0076] FIGURE 4 illustrates an example process for using the consumable of FIGURE 3 in accordance with the present disclosure;

[0077] FIGURE 5 illustrates an example automation plate for use with the consumable of FIGURE 3;

[0078] FIGURES 6 and 6A through 6D collectively illustrate an example consumable for multi-omics biological sample collection and preparation in accordance with the present disclosure;

[0079] FIGURE 7 illustrates an example process for using the consumable of FIGURES 6 and 6A through 6D in accordance with the present disclosure;

[0080] FIGURE 8 illustrates an example automation plate for use with the consumable of FIGURES 6 and 6A through 6D in accordance with the present disclosure;

[0081] FIGURES 9A, 9B and 9C collectively illustrate an alternative example consumable for multi-omics biological sample collection and preparation in accordance with the present disclosure;

[0082] FIGURES 10A, 10B, 10C, 10D, and 10E collectively illustrate an example spongebased consumable for multi-omics biological sample collection and preparation in accordance with the present disclosure; and

[0083] FIGURE 11 conceptually illustrates an example cfDNA extraction from blood using consumables in accordance with the present disclosure.DETAILED DESCRIPTION

[0084] FIGURES 1 through 11, 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.

[0085] 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.

[0086] 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. 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. Repetitive pipetting or repetitive heating may also lead to degradation of nucleic acids.

[0087] 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.

[0088] The present disclosure addresses these or other shortcomings by providing consumables that facilitate nucleic acid analysis. As used herein, “consumable” includes any component that is used up recurrently and may need to be replaced to function as intended. In some cases, consumables may be single-use components requiring disposal subsequent to use. Biological sample collection containers, for instance, are consumables that are often fabricated as sterile glass or plastic tubes configured to facilitate the collection of a predetermined volume of liquid, where the container is or is capable of being sealed subsequent to sample collection. Such biological sample collection containers may include, within the container, predetermined quantities of reagents or other biological sample processing components in the form of coatings, particles, or the like. The reagents or other biological sample processing components may be selected, for example, to stabilize a collected biological sample. The reagents or other biological sample processing components within a biological sample collection container can form part of the same consumable.

[0089] Depending on the implementation, the consumables described in the present disclosurehave many benefits. This may include, for example, stabilization of samples and / or reagents, reducing or eliminating the need for cold transportation and storage, room temperature shipping and storage of samples and / or reagents and complete assays, reducing plastic waste, and resistance of the encapsulated lyophilized reagent microspheres to harsh environmental conditions.

[0090] The present disclosure also relates to consumables involved in preparation of biological samples for processing. 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 or other materials 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.

[0091] Although the sample is often taken from a human subject (such as a patient), samples can come from other sources. For example, 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, environmental DNA (eDNA), 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, ly sing, 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.

[0092] 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.

[0093] 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 cfRNA and cfDNA molecules, circulating tumor RNA (ctRNA) and circulating tumor DNA (ctDNA) molecules, cell-free fetal DNA (cffDNA) molecules, and environmental DNA (eDNA) 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.

[0094] 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.

[0095] 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 reversibledye terminators, sequencing-by-ligation, and nanopore sequencing. The phrase “processing of a biological sample” includes any process relating to nucleic acid analysis, such as (but not limited to) nucleic acid sequencing, genotyping, sample preparation, and library preparation.

[0096] Tire 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.

[0097] The 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.

[0098] 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. Forexample, 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.

[0099] 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.

[0100] 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, pillars, 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 crosslinked 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 fluid dispensing vessel such as a pipette is used to transfer materials into the wells. In some examples, a support structure can include one or more layers.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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 thenhydrolyzed 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.

[0105] 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.

[0106] 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.

[0107] 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 thenucleotide 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.

[0108] The 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 wild-type, derivative, or mutant form of the transposase.

[0109] 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.

[0110] Although many implementations described herein may 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.

[0111] As used herein, the term “library of tagged nucleic acid fragments” refers to a collectionor 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.

[0112] 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 3’- end that are complementary to the template in the process of DNA synthesis. The result is a primer extension product.

[0113] 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.

[0114] Consumables described herein include particles having a coating surrounding a core, where the core may include one or more lyophilized microspheres (such that the composition may include an encapsulated lyophilized microsphere). In connection with encapsulated particles, the terms “encapsulate,” “encapsulated,” and “encapsulation” include the enclosing of one or more microspheres as described herein. In accordance with the present disclosure, the compositions, systems, and methods described herein have many benefits including, for example, increasing stability of microspheres. 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. As used herein in connection with at least lyophilized microspheres, encapsulated lyophilized microspheres, or similar materials, the term “trigger” refers to rehydrating, activating, releasing, or otherwise making the component(s) therein available for interaction with other materials and / or the environment.

[0115] 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, 50L pm, 60 pm, 70 pm, 80 pm, 90 gm, 100 gm, 150 gm, 200 gm, 300 gm, 400 gm, 500 gm, 600 gm, 700 gm, 800 gm, 900 gm, 1000 gm, or any diameter between about 0.1 gm and about 1,000 gm. In some implementations, an encapsulated microsphere has a diameter between about 100 gm and 1000 gm. 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 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.

[0116] Lyophilized reagent beads or “lyo beads’’ (also known as “cryo pellets”) are spherical particles of lyophilized material typically having a diameter larger than 1,000 pm (or 1 millimeter (mm). For example, a lyo bead may have a diameter of about 1.8 mm, 2 mm, 4 mm, or 6 mm, or any diameter in between.

[0117] As used herein, “lyospheres” refers to either lyophilized microspheres or lyophilized reagent beads. Lyospheres are not necessarily spheres or spherical, but instead may be generally spheroid or spheroidal particles — that is, roughly spherical, or round while also being irregular or “lumpy.”

[0118] As used herein, the term “coating” (or “shell”) includes a composition that surrounds a core. In some implementations, a shell includes an outer layer of a microsphere.

[0119] 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 lyospheres.

[0120] 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.

[0121] The terms “connect,” “connected,” “contact,” “coupled,” and / or the like are broadly defined herein to encompass a variety of divergent arrangements and assembly techniques. Thesearrangements 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.

[0122] 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.

[0123] The present disclosure relates to consumables containing one or more particles including a core-coating composite materials having (i) an inner core optionally including releasable lyospheres of one or more workflow reagents; and (ii) an outer coating encapsulating the inner core. The core-coating 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, magnetic bead, molecular probe, crowding agent, small molecule, labelled-nucleotide, a fluorophore, or any combination thereof.

[0124] In some implementations, the core-coating composite may exhibit a total thickness of the coating 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. In various implementations, the coating 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.

[0125] In some implementations, the core-coating 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 core-coating 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 gm, from about 350 to 625 gm, or from about 400 to 600 gm. The core-coating composite material may include substantially monodisperse particles, each having substantially the same average diameter. The core-coating material may also include lyophilized microspheres or lyophilized beads having a distribution of average diameters.

[0126] In some implementations, the core-coating composite material may include a coating material selected from the group consisting of polyethylene glycol, betaine, and a salt.

[0127] The amount of coating material includes, for example, any amount suitable to produce a desired performance. In some implementations, the coating material is present in an amount between about 1 percent by weight (wt%) and about 100 wt% of the coating. For example, the coating 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.

[0128] The coating component as described herein may, in various implementations, include an additive. The coating additive may be present in an amount between about 0.01% weight by weight (w / w) of the coating and about 99% w / w of the coating. In some implementations, the coating additive is present in an amount between about 10% w / w and about 90% w / w of the coating. In some implementations, the coating additive is present in an amount between about 10% w / w and about 40% w / w. In some implementations, the coating additive is a moisture barrier material present in an amount no more than 90% w / w of the coating. In some implementations, the coating additive is present in an amount of at least 10% w / w concentration of the coating. For example, the coating additive may, in some implementations, be present in an amount between 0.1% w / w of the coating and about 15.0% w / w of the coating. As particular examples, the coating 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 coating additive may be adjusted to accommodate a particular reagent or combination of reagents or to accommodate a particular lyosphere composition.

[0129] Example coating additives include, but are not limited to, one or more of a polymer, a copolymer, a block copolymer, an anti-tacking agent (such as PEG stearates or Mg stearates, both mentioned below), an anti-static agent, an anti-foaming agent, a plasticizer, a 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 “Tris”), bistris propane (BTP), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 3-(N-morpholino)- propanesulfonic acid (MOPS), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 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.

[0130] As described herein, a “core” or “core region” includes any material within the encapsulating coating. A core in accordance with the present disclosure includes one or more lyophilized microspheres or lyophilized beads. The lyophilized microspheres or lyophilized beads 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.

[0131] In some implementations, the inner core includes lyophilized reagents that are suitable for use in processing a biological sample using multiple sequential co-assays including lysis, DNA analysis, RNA analysis, protein analysis, metabolite analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, SBS technology, assay for transposase accessible chromatic using sequencing (ATAC-seq), contiguity-preserving transposition (CPT-seq), single cell combinatorial indexed sequencing (SCI-seq), single cell genome amplification, or any combination thereof performed sequentially. In some implementations, the composition is used for performingmultiple co-assay reactions.

[0132] FIGURE 1 illustrates an example consumable 100 for biological sample collection and preparation in accordance with the present disclosure. Among other things, the example consumable 100 may be suitable for using in collecting blood for whole genome sequencing (WGS). As illustrated in FIGURE 1, the consumable 100 includes a cylindrical tube 101 and a lid 102. The tube 101 has a top opening covered by foil 103.

[0133] Inside the tube 101 are a plurality of particles 104 and a heat source 105. In some implementations, the particles 104 may include lyophilized microspheres and encapsulated lyophilized microspheres, which may collectively include one or more of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), polysorbate (e.g., Tween), and an enzyme (e.g., a broadspectrum serine protease such as Proteinase K). For example, the particles 104 may include lyophilized microspheres including two or more of those reagents or components (e.g., the SDS, EDTA, and / or polysorbate) for stabilizing a collected biological sample and encapsulated lyophilized microspheres including a remainder of those reagents or components (e.g., the enzyme) for lysis.

[0134] The heat source 105 may include an inset chamber containing material that spontaneously undergoes an exothermic reaction upon exposure to oxygen. In the example consumable 100, the chamber for the heat source 105 is inset into the bottom of the tube 101 and has an opening to the exterior of the tube 101 that is sealed by foil. In some implementations, the contents of the inset chamber may be sodium acetate or a combination of cellulose, iron, activated carbon, and vermiculite. In some implementations, rather than a sealed opening, the heat source 105 may be in the form of a blister pack containing water and one of sodium hydroxide (NaOH), calcium oxide, or magnesium sulfate (MgSO4). In such implementations, the exothermic reaction is initiated by compression of the blister pack to mix the contents therein. The consumable 100 may include a region for external application of force to effect the necessary compression of the blister pack.

[0135] The lid 102 includes a chamber 106 that may contain water, which may be sealed within the chamber 106 in a manner resulting in release of the water into the tube 101 when the lid 102 is used to close the top opening of the tube 101. The lid 102 also includes a frit structure 107 that may include a frit coated with alpha cyclodextrin powder or two frits with alpha cyclodextrin powder sandwiched therebetween.

[0136] FIGURE 2 illustrates an example process for using the consumable 100 of FIGURE 1 in accordance with the present disclosure. During use, the user first pierces the foil covering the top of the tube 101 and adds about 25 microliters (pL) or other amount of whole blood. The added amount may be about the fluid capacity of the tube 101 or may be indicated by a marking on the tube 101. The user closes the lid 102 on the open top of the tube 101, inverts the closed assembly, and allows the consumable 100 to sit at room temperature (RT) for about 30 minutes or other time period. During thatperiod, some of the particles 104 may be rehydrated by the liquid portion of the biological sample and / or the water within the chamber of the lid 102. After the time period elapses, the user peels the foil seal on the bottom of the consumable 100, exposing the heat source 105 to air and initiating an exothermic reaction. The heat generated by the exothermic reaction may elevate the temperature of the mixture within the consumable 100 to approximately 72°C or other elevated temperature and maintain the mixture at that temperature for at least about 5 minutes or other time period. During that period, encapsulation on a remainder of the particles 104 may melt or dissolve, and those remaining particles 104 may be rehydrated by the liquid in the consumable 100. After the time period elapses, the user pushes the resulting mixture and the lysate therein through the frit structure 107 into a consumable used for testing.

[0137] FIGURE 3 illustrates an alternative example consumable 300 for biological sample collection and preparation in accordance with the present disclosure. Among other things, the example consumable 300 is suitable for using in collecting blood and extraction of cfDNA from the collected sample. As illustrated in FIGURE 3, the consumable 300 includes a generally cubic container 301, which in some cases may have a length, width, and height of approximately 3 centimeters (cm), and includes a fluid transfer port 302 on the top.

[0138] Inside the container 301 are particles 303, particles 304, and a heat source 305. In some implementations, the particles 303 may include lyophilized microspheres including an enzyme such as Pepsin, hydrochloric acid (HC1), and polysorbate such as Tween 20. In other implementations, the particles 303 may include a mixture of two different types of lyophilized microspheres, where a first type includes Pepsin and Tween 20 and a second type includes polyethylene glycol (PEG, such as PEG 300) and guanidine thiocyanate. The particles 304 may include encapsulated lyophilized microspheres configured for temperature-triggered release, such as 3-(N-morpholino)propanesulfonic acid (MOPS), alpha cyclodextrin, beads, and optionally PEG 300 and guanidine thiocyanate (if not present in the particles 303). Release of the components within the particles 303 into a solution may be triggered by rehydration, and release of the components within the particles 304 may be triggered by temperature and rehydration.

[0139] The heat source 305 may include an inset chamber containing material that spontaneously undergoes an exothermic reaction upon exposure to oxygen. In the example consumable 300, the chamber for the heat source 305 is inset into the bottom of the container 301 and has an opening to the exterior sealed by foil. In some implementations, the contents of the inset chamber for the heat source 305 may be sodium acetate or a combination of cellulose, iron, activated carbon, and vermiculite. The interior of the container 301 is divided by a filter 306 and a pump mechanism 307. In some implementations, the filter 306 may include a plasma separation membrane, such as a VIVID plasma separation membrane available from CYTIVA GLOBAL LIFE SCIENCES SOLUTIONSUSA LLC. Also, in some implementations, the pump mechanism 307 may include a micro-pillar array for pumping blood plasma through the filter 306.

[0140] FIGURE 4 illustrates an example process for using the consumable 300 of FIGURE 3 in accordance with the present disclosure. During use, the user adds 6-10 milliliters (mL) or other amount of whole blood through the fluid transfer port 302. The user allows the container 301 to sit at room temperature for about 30-60 minutes or other time period. During that period, particles 303 may be rehydrated by the liquid portion of the biological sample. After the time period elapses, the user peels the foil seal on the bottom of the consumable 300, exposing the heat source 305 to air and initiating an exothermic reaction that may elevate the temperature of the mixture within the consumable 300 to approximately 72°C or other elevated temperature and maintain the mixture at that temperature for at least about 10-30 minutes or other time period. During that period, encapsulation on the particles 304 may melt or dissolve, and those particles 304 may be rehydrated by the liquid in the consumable 300. After the time period elapses, beads or liquid containing those beads, together with cfDNA attached to the beads, may be transferred via the transfer port 302 for testing.

[0141] FIGURE 5 illustrates an example automation plate 500 for use with the example consumable 300 of FIGURE 3 in accordance with the present disclosure. The automation plate 500 includes a plurality of receptacles each configured to hold one of the example consumables 300 in an inverted orientation. Each receptacle includes a hole accommodating the transfer port 302 on the top of the respective consumable 300. In the inverted orientation shown in FIGURE 5, the opening 308 for heat source 305 on each consumable 300 is visible. As discussed above, a foil seal initially covers the opening 308. Removal of that foil seal exposes the opening 308, allowing air to enter the hole into the interior of the consumable 300 and initiate an exothermic reaction. As shown in FIGURE 5, the bottom of each consumable 300 also includes an aspiration hole 309, which may allow a liquid handling robot to aspirate liquid from the respective consumable 300 or a magnetic rod to pull magnetic beads from inside the respective consumable 300.

[0142] FIGURES 6 and 6A through 6D collectively illustrate an example consumable 600 for multi-omics biological sample collection and preparation in accordance with the present disclosure. More specifically, FIGURE 6 is a perspective cutaway view of the consumable 600, FIGURES 6A and 6B illustrate a drum forming a portion of the consumable 600 in FIGURE 6, and FIGURES 6C and 6D are different perspective views of the consumable 600 of FIGURE 6. In some implementations, the example consumable 600 is a multi-analyte sample extractor from blood, enabling workflow passivation and parallelization. For example, the consumable 600 may be configured to extract gDNA, RNA, cfDNA, and protein from blood.

[0143] Tire example consumable 600 includes an upper sample chamber 601 and a lower analyte extraction drum 610. The upper sample well 601 and the lower analyte extraction drum 610are separated by a filter 606, a pump mechanism 607, and a movable plate 608 with an external slider 609. The lower analyte extraction drum 610 includes a centrally disposed region 605 for a of the type described above. The upper sample well 601 has a large opening in the bottom and a fluid transfer port 602 on the top. In some implementations, the upper sample well 601 may hold approximately 10 mL of fluid. The bottom opening of the upper sample well 601 is covered by the filter 606, which may be a plasma separation membrane (such as of the type described above). The pump mechanism 607, which may be a pillar array as described above, is disposed adjacent to the filter 606 and pumps blood plasma through the filter 606. The movable plate 608 is disposed between the upper sample well 601 and the lower analyte extraction drum 610. In some implementations, the example consumable 600 in FIGURES 6 and 6A through 6D may have overall dimensions of about 4 cm height and 3.2 cm diameter.

[0144] As best seen from FIGURES 6A and 6B, the lower analyte extraction drum 610 includes a plurality of sector-shaped analyte extraction wells 611, 612, 613, and 614 for concurrent extraction of different analytes. The lower analyte extraction drum 610 also includes the intercalating region 605 that is central to the sector-shaped analyte extraction wells 611, 612, 613, and 614 and contains the heat source material (e.g., a combination of cellulose, iron, activated carbon, and vermiculite) for an exothermic reaction upon exposure to air. The consumable 600 has an air vent hole between the intercalating region 605 and the exterior of the consumable 600. The movable plate 608 includes holes enabling passage of liquid between wells (e.g., between the upper sample well 601 and one or more of the individual sector-shaped analyte extraction wells 611, 612, 613, and 614) for different actions and for opening the air vent hole. The sector-shaped analyte extraction wells 611, 612, 613, and 614 may contain particles in the form of lyophilized microspheres, encapsulated lyophilized microspheres, or both. Based on the composition of the particles therein, different analytes can be extracted from blood in each of the sector-shaped analyte extraction wells 611, 612, 613, and 614.

[0145] As best seen from FIGURES 6C and 6D, the slider 609 on the example consumable 600 is movable between three different positions. The bottom of the consumable 600 includes foil- sealed analyte extraction openings 621, 622. 623, and 624 for extracting analytes from corresponding ones of the sector-shaped analyte extraction wells 611, 612, 613, and 614.

[0146] FIGURE 7 illustrates an example process for using the consumable 600 of FIGURES 6 and 6A through 6D in accordance with the present disclosure. During use, with the slider 609 at position 1, the user adds approximately 10 mL or other amount of whole blood into the upper sample chamber 601 through the fluid transfer port 602. The blood passes from the upper sample chamber 601 through ahole in the movable plate 608 into a first one (or more) of the sector-shaped well(s) 611, 612, 613, or 614 containing particles configured for extraction of gDNA. For example, the particles within the first sector-shaped well(s) 611, 612, 613, or 614 may be in the form of lyophilized microspheresincluding SDS, EDTA, Tween, and Proteinase K. The blood rehydrates the Proteinase K in the respective first sector-shaped analyte extraction well(s) 611, 612, 613, or 614, and whole blood cell digestion commences.

[0147] The user moves the slider 609 to position 2, which closes the first sector-shaped analyte extraction well(s) 611, 612, 613, or 614 and opens a second one (or more) of the sector-shaped analyte extraction well(s) 611, 612, 613, or 614 that contains particles configured for extraction of plasma. For example, the particles within the second sector-shaped analyte extraction well(s) 611, 612, 613, or 614 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, Pepsin, and HCL, together with lyophilized microspheres including a stabilizing gel, EDTA, and protease inhibitors. The user allows the consumable to remain at room temperature for about one hour or other time period, during which time the plasma entering the second sector-shaped analyte extraction well(s) 611, 612, 613, or 614 from the upper sample well 601 rehydrates the Pepsin and plasma stabilization microspheres.

[0148] After the time period has elapsed, the user moves the slider 609 to position 3, which closes all openings between the upper sample well 601 and the sector-shaped analyte extraction well(s) 611, 612, 613, and 614 and opens the air vent hole to initiate the exothermic reaction. The exothermic reaction heats the contents of at least some of the sector-shaped well(s) 611, 612, 613, or 614 to a temperature of approximately 70°C or other elevated temperature for about 5 minutes or other time period. The heat from the activated heat source material in the intercalating region 605 melts or dissolves the encapsulation on particles within at least some of the sector-shaped analyte extraction well(s) 611, 612, 613, and / or 614 that are in the form of encapsulated lyophilized microspheres. For example, those encapsulated lyophilized microspheres may include alpha cyclodextrin in one of the sector-shaped analyte extraction well(s) 611, 612, 613, or 614; alpha cyclodextrin, MOPS, and a first type of capture beads in another of the sector-shaped analyte extraction well(s) 611, 612, 613, or 614; and alpha cyclodextrin, MOPS, and second type of capture beads in a third of the sector-shaped analyte extraction well(s) 611, 612, 613, or 614. The release of the alpha cyclodextrin facilitates SDS capture of nucleic acids, and release of the buffer components neutralizes the Pepsin. After the time period has elapsed, the foil over the analyte extraction openings 621, 622, 623, and 624 is pierced, and the analytes are pipetted out or otherwise removed.

[0149] FIGURE 8 illustrates an example automation plate 800 for use with the consumable 600 of FIGURES 6 and 6A through 6D in accordance with the present disclosure. The automation plate 800 includes a plurality of receptacles each configured to hold one of the example consumables 600 in an inverted orientation. Each receptacle includes a recess accommodating a transfer port cap 801 and a hole receiving a registration projection 802 on the top of the respective consumable 600. In the inverted orientation shown in FIGURE 8, the analyte extraction openings are accessible. Theregistration projection 802 and the associated hole in the receptable facilitate robotic extraction of the different analytes for transfer, such as by a liquid handling robot or a magnetic rod.

[0150] FIGURES 9A, 9B and 9C collectively illustrate an alternative consumable 900 for multi-omics biological sample collection and preparation in accordance with the present disclosure. More specifically, FIGURE 9 A is a perspective view of the consumable 900 in a first orientation, FIGURE 9B is a sectional view of the consumable 900 taken along a vertical plane through FIGURE 9A, and FIGURE 9C is a perspective view of the consumable 900 in a second orientation inverted relative to the orientation in FIGURES 9A. In some implementations, the consumable 900 is used for multi-analyte sample extraction from blood, enabling workflow passivation and parallelization. For example, the consumable 900 may be configured to extract gDNA, RNA, cfDNA, and protein from blood.

[0151] As shown in FIGURE 9A, the consumable 900 includes a generally rectangular container including a fluid transfer port 902 and an external tab 909 that can be pulled. As shown in FIGURE 9B, the upper half of the container includes an upper sample well (or chamber) 901 into which a blood sample passes from the exterior through the fluid transfer port 902. The upper sample well 901 is separated by a filter 906, a pump mechanism 907, and a movable plate 908 from analyte extraction wells 911, 912, 913, and 914. In some implementations, the filter 906 may include a plasma separation membrane, and the pump mechanism 907 may include a micro-pillar array for pumping blood plasma through the filter 906 as described above. The movable plate 908 is connected to the external tab 909 and includes an opening aligned with an analyte extraction well 911 when the movable plate 908 and the external tab 909 are in a first position and openings aligned with the analyte extraction wells 912, 913, and 914 when the movable plate 908 and the external tab 909 are in a second position.

[0152] A heat source 905 is disposed between at least some of the analyte extraction wells 911, 912, 913, and 914 (such as between well 911 and the exterior, between wells 911 and 912, and between wells 912 and 913 in the example depicted). In some implementations, the heat source 905 may include a combination of cellulose, iron, activated carbon, and vermiculite for an exothermic reaction upon exposure to air. Each of the analyte extraction wells 911, 912, 913, and 914 has a corresponding foil-covered analyte extraction opening 921, 922, 923, and 924.

[0153] The consumable 900 includes particles within the analyte extraction wells 912, 913, and 914. In some implementations, the particles may be in the form of lyophilized microspheres, encapsulated lyophilized microspheres, or both. Based on the composition of the particles therein, different analytes can be extracted from blood in each of the analyte extraction wells 911, 912, 913, and 914. For example, the particles within the analyte extraction well 911 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, and Proteinase K and encapsulated lyophilized microspheres including alpha cyclodextrin. The particles within the analyte extraction well912 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, Pepsin, and HO and encapsulated lyophilized microspheres including alpha cyclodextrin, MOPS, and a first type of capture beads. The particles within the analyte extraction well 913 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, Pepsin, and HC1 and encapsulated lyophilized microspheres including alpha cyclodextrin, MOPS, and a second type of capture beads. The particles within the analyte extraction well 914 may be in the form of lyophilized microspheres including stabilizing gel, EDTA, and protease inhibitors. In FIGURE 9C. the foil covering the analyte extraction openings 921, 922, 923, and 924 and the foil covering the opening 908 to the heat source 905 are visible.

[0154] FIGURES 10A, 10B, 10C, 10D, and 10E collectively illustrate an example spongebased consumable 1000 for multi-omics biological sample collection and preparation in accordance with the present disclosure. More specifically, FIGURE 10A is a perspective view of the consumable 1000, FIGURE 10B is a cutaway view of the consumable 1000 taken along a first vertical plane through FIGURE 10A, FIGURES 10C and 10D are cutaway views of the consumable 1000 taken along a second vertical plane through FIGURE 10 A, and FIGURE 10E is a cutaway view of the consumable1000 taken along the second vertical plane through FIGURE 10A before use.

[0155] As shown in FIGURE 10A, the consumable 1000 includes a generally rectangular container including a fluid transfer port 1002, an external slider 1009, and analyte extraction openings 1021, 1022, 1023, and 1024. As shown in FIGURE 10B, the upper half of the container includes an upper sample well (or chamber) 1001 into which a blood sample passes from the exterior through the fluid transfer port 1002. The upper sample well 1001 is separated by a filter 1006 from a pump mechanism 1007. In some implementations, the filter 1006 may include a plasma separation membrane as described above. Also, in some implementations, the pump mechanism 1007 may be a compressible sponge. In the example of FIGURES 10 A through 10E, plasma from blood within the upper sample well 1001 filters based on gravity through the plasma separate membrane and is collected by the sponge. The slider 1009 moves the sponge from one side of the container below the upper sample well1001 to the opposite side. As visible in FIGURE 10B, the container also includes an external tab 1010 projecting from a plate beneath the sponge that moves with the sponge. The external tab 1010 may be pushed to compress the sponge. The consumable 1000 also includes an analyte extraction well 1011 that extends across a width of the container, is adjacent to the region within which the sponge moves, and is disposed below the fluid transfer port 1002 in the example shown. A one-way valve separates the upper sample well 1001 from the analyte extraction well 1011.

[0156] As shown in FIGURE 10C, the analyte extraction well 1011 is disposed below a corresponding analyte extraction opening 1021. The consumable 1000 also includes analyte extraction wells 1012, 1013, and 1014 below corresponding analyte extraction openings 1022, 1023, and 1024.The analyte extraction wells 1012, 1013, and 1014 are physically separated from the upper sample well 1001 and are separated from the pump mechanism 1007 by the filter 1006.

[0157] FIGURES 10B, 10C, and 10D illustrate operation of the consumable 1000. During use, the user loads blood into the consumable 1000 via the fluid transfer port 1002 and waits approximately 30 minutes or other time period while plasma diffuses across the plasma separation membrane into the sponge below the upper sample well 1001 when the slider 1009 is in a first position. Concurrently, blood passes through the one-way valve between the upper sample well 1001 and the analyte extraction well 1011. After the time period has elapsed, the user moves the slider 1009 from the first position to a second position at which the sponge is located below the analyte extraction wells 1012, 1013, and 1014. The user pushes on the external tab 1010 to compress the sponge and pump plasma from the sponge into the analyte extraction wells 1012, 1013, and 1014.

[0158] As shown in FIGURE 10E, the consumable 1000 includes particles within the analyte extraction wells 1011, 1012, 1013, and 1014. In some implementations, the particles may be in the form of lyophilized microspheres, encapsulated lyophilized microspheres, or both. Based on the composition of the particles therein, different analytes are extracted from blood in each of the analyte extraction wells 1011, 1012, 1013, and 1014. For example, the consumable 1000 may be configured to extract gDNA, RNA, cfDNA, and protein from blood. In these implementations, the particles within the analyte extraction well 1011 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, and Proteinase K and encapsulated lyophilized microspheres including alpha cyclodextrin and the Proteinase K inhibitor TCK. The particles within the analyte extraction well 1012 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, and Trypsin and encapsulated lyophilized microspheres including alpha cyclodextrin, a trypsin inhibitor, and a first type of capture beads. The particles within the analyte extraction well 1013 may be in the form of lyophilized microspheres including SDS, EDTA, Tween, trypsin, and deoxyribonuclease (DNAse) and encapsulated lyophilized microspheres including alpha cyclodextrin, a trypsin inhibitor, and a second type of capture beads. The particles within the analyte extraction well 1014 may be in the form of lyophilized microspheres including either EDTA and protease inhibitors or a stabilizing gel.

[0159] The addition of blood or blood plasma promptly dissolves the (unencapsulated) lyophilized microspheres, forming a mixture and performing a first biological sample preparation step (e.g., whole blood cell digestion). Each of the encapsulated lyophilized microspheres may be configured to be triggered by time (e.g., about 30 minutes or other time period) within a liquid medium. Release of the components within the encapsulated lyophilized microspheres into the mixture may perform a second biological sample preparation step (e.g., enzyme neutralization). The analyte extraction well 1011 may be sized and contain particles configured to extract approximately 25 pL or other amount of analyte. The analyte extraction well 1012 may be sized and contain particlesconfigured to extract approximately 3 mL or other amount of analyte. The analyte extraction well 1013 may be sized and contain particles configured to extract approximately 1.5 mL or other amount of analyte. The analyte extraction well 1014 may be sized and contain particles configured to extract approximately 50 pL or other amount of analyte.

[0160] FIGURE 11 conceptually illustrates an example cfDNA extraction from blood using any of the consumables 100, 300, 600, 900, or 1000 in accordance with the present disclosure. In many cases, cfDNA is the key to valuable clinical diagnoses such as noninvasive prenatal testing (NIPT) or oncology testing. However, an analyte of interest may be sparse in blood / plasma and the extraction process may be complicated, requiring large instrumentation for automation and / or many touch points. In the present disclosure, a hydrophilic pillar array 1107 between a capture receptacle and a well is used to drive plasma from approximately 10 mL or other amount of blood across an asymmetric polysulfone membrane 1106, yielding plasma through a passive process absent of any external instrumentation. Plasma filtration from whole blood using the pillar array 1107 and the membrane 1106 may occur over approximately 15-30 minutes or other time period at room temperature or other temperature.

[0161] The passive workflow may utilize two types of lyophilized microspheres within the analyte extraction well below the pillar array 1107 and the membrane 1106. The first type of lyophilized microspheres may contain an enzyme (pepsin) to strip off histones from cfDNA and render an analyte accessible for capture. Histone stripping may occur over approximately 15-30 minutes or other time period at room temperature or other temperature. The second type of lyophilized microspheres may contain MOPS to neutralize the solution and inactivate an enzyme, as well as alpha cyclodextrin to sequester SDS, GITC, and PEG. pushing the cfDNA onto magnetic beads. To render the workflow passive, the second type of lyophilized microsphere may be encapsulated and released upon exposure to heat (e.g., >70-72°C or other temperature for approximately 15 minutes or other time period), which maximizes cfDNA capture. Existing approaches to cfDNA extraction are labor- intensive (e.g., requiring washes) and may result in one or more of contaminates, loss of small fragments and the associated data, and / or enzyme activity within the sample.

[0162] In other implementations, a one millimeter (mm) thick or other sponge may be used to collect plasma filtered by membrane 1106 and may release the collected plasma via compression. This approach might risk push-back of the plasma into the blood since the membrane 1106 is not unidirectional. To mitigate that risk, designs leveraging pushing or turning of the consumable to separate the sponge prior to dispensing plasma into the analyte extraction wells may be employed. In still other implementations, use of a hydrophilic pillar array or other pumping mechanism may be more controlled.

[0163] Integration of a porous membrane 1 106 into biological sample collection consumables as disclosed herein improves the user experience and enables analyte extraction during transit. Among other things, this may help to reduce sample degradation that can occur during lengthy shipment.

[0164] While certain materials (like blood or blood plasma) are described as being processed using specific reagents with specific temperatures and times using the described consumables, these details are for illustration and explanation only and do not limit the scope of the disclosure. The consumables may be used to process any suitable materials, the consumables may include any suitable reagents, and the consumables or the reagents may be used at any suitable temperatures for any suitable times depending on the implementation. The described consumables are not limited to processing the specific materials using the specific reagents with the specific temperatures and times described above.

[0165] 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

1. WHAT IS CLAIMED IS:

1. An apparatus comprising: a well within a container configured for holding a biological sample, the well containing first particles and including a portion containing a heat source and second particles; and a seal over an opening into the portion of the well containing the heat source, wherein removal or rupture of the seal initiates an exothermic reaction by the heat source; wherein the first particles are triggered by addition of the biological sample into the well; and wherein the second particles are triggered by the exothermic reaction by the heat source.

2. The apparatus of claim 1, wherein a first biological sample preparation processing step corresponding to the first particles occurs during a first period of time following the addition of the biological sample into the well.

3. The apparatus of claim 2, wherein a first biological sample preparation processing step comprises histone stripping.

4. The apparatus of claim 2, wherein the first particles comprise an enzyme.

5. The apparatus of claim 4, wherein: the enzyme comprises pepsin; and the first particles further comprise sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), polysorbate, and hydrochloric acid (HC1).

6. The apparatus of claim 2, wherein a second biological sample preparation processing step corresponding to the second particles occurs during a second period of time following the initiation of the exothermic reaction by the heat source.

7. The apparatus of claim 6, wherein a second biological sample preparation processing step comprises nucleic acid capture.

8. The apparatus of claim 6, wherein the second particles comprise beads.

9. The apparatus of claim 8, wherein the second particles further comprise a-cyclodextrin, 3-(N-morpholino)propanesulfonic (MOPS) acid, guanidine thiocyanate (GTC), and polyethylene glycol (PEG).

10. The apparatus of claim 1 , further comprising: a pump mechanism and a filter membrane over the portion of the well containing the second particles, the pump mechanism configured to drive a portion of plasma in the biological sample through the filter membrane, the filter membrane configured to pass the portion of the plasma from the biological sample into the portion of the well containing the second particles.

11. The apparatus of claim 10, wherein the pump mechanism comprises a pillar- array.

12. The apparatus of claim 10, wherein the container is a cubic container with the pump mechanism and the filter membrane disposed diagonally therein.

13. The apparatus of claim 12, wherein: a first surface of the container comprises a guide for mounting the container on an automation plate; and a second surface of the container comprises the opening into the portion of the well containing the heat source and an aspiration hole covered by foil.

14. The apparatus of claim 10, wherein: the first particles comprise a first type of the first particles and a second type of the first particles; and the container comprises: a cylindrical well disposed over the pump mechanism and the filter membrane, the cylindrical well containing the first type of the first particles; a movable plate disposed below the cylindrical well, the movable plate comprising a plurality of openings therethrough; and a drum disposed below the pump mechanism and the filter membrane, the drum comprising a plurality of sector-shaped wells, the heat source disposed centrally to the plurality of sector-shaped wells, at least a first of the plurality of sector-shaped wells containing the second type of the first particles and at least a second of the plurality of wells containing the second particles.

15. The apparatus of claim 14, wherein the first particles comprise an enzyme.

16. The apparatus of claim 15, wherein the first type of the first particles compriseProteinase K.

17. The apparatus of claim 15, wherein the second type of the first particles comprise pepsin.

18. The apparatus of claim 17, wherein the second type of the first particles comprise a plasma stabilizer.

19. The apparatus of claim 14, wherein the second particles comprise one or more of «- cyclodextrin, sodium dodecyl sulfate (SDS), or a buffer.

20. The apparatus of claim 14, wherein the movable plate is in a first position when the biological sample is collected.

21. The apparatus of claim 20, wherein movement of the movable plate from the first position to a second position aligns a first of the plurality of openings through the movable plate with at least one of the plurality of sector-shaped wells, allowing passage of the biological sample from the cylindrical well into the at least one of the plurality of sector-shaped wells.

22. The apparatus of claim 21, wherein movement of the movable plate from the second position to a third position aligns a second of the plurality of openings with an air source, allowing passage of air into contact with the heat source.

23. The apparatus of claim 10, wherein: the first particles comprise a first type of the first particles and a second type of the first particles; the well comprises a plurality of biological sample processing wells; the heat source comprises a plurality of heat sources disposed between adjacent ones of the plurality of biological sample processing wells; the container comprises: a collection well disposed over the pump mechanism and the filter membrane; and a movable plate disposed below the collection well, the movable plate comprising a plurality of openings therethrough; and at least a first of the plurality of biological sample processing wells contains the second type of the first particles and at least a second of the plurality of biological sample processing wells contains the second particles.

24. The apparatus of claim 23, wherein: the first of the plurality of biological sample processing wells contains particles used for extraction of genomic deoxyribonucleic acid (gDNA) from the biological sample; the second of the plurality of biological sample processing wells contains particles used for extraction of one of cell-free deoxyribonucleic acid (cfDNA), circulating tumor DNA (ctDNA), cell- free fetal DNA (cffDNA), or environmental DNA (eDNA) from the biological sample; a third of the plurality of biological sample processing wells contains particles used for extraction of one of ribonucleic acid (RNA), cell-free RNA (cfRNA), or circulating tumor RNA (ctRNA) from the biological sample; and a fourth of the plurality of biological sample processing wells contains particles used for extraction of proteins from the biological sample.

25. The apparatus of claim 1, wherein: the well comprises a plurality of biological sample processing wells; the container comprises: a pump mechanism; a collection well disposed over the pump mechanism; and a filter membrane between the collection well and the pump mechanism and between the well and the pump mechanism; and the well is disposed above the filter membrane.

26. The apparatus of claim 25, wherein: the pump mechanism comprises a movable and compressible sponge below the filter membrane; in a first position, the sponge is below the collection well and absorbs a filtered portion of the biological sample; and in a second position, the sponge is below the well such that compression of the sponge pumps the filtered portion of the biological sample into the plurality of biological sample processing wells.

27. The apparatus of claim 1, wherein: the container comprises a capture receptacle over the well; and the apparatus further comprises a pump mechanism and a filter membrane between the capture receptacle and the well, the pump mechanism configured to drive a portion of plasma in the biological sample through the filter membrane, the filter membrane configured to pass the portion of the plasma from the biological sample into the well.

28. A method of using the apparatus of any one of claims 1 through 27, the method comprising: receiving the biological sample within the container; after a predetermined period, removing the seal over the opening to initiate the exothermic reaction; and removing fluid including an analyte from the well.

29. The method of claim 28, wherein: the well comprises a plurality of biological sample processing wells; removing fluid including an analyte from the well comprises removing fluid including a first analyte from a first of the plurality of biological sample processing wells; the method further comprises removing fluid including a second analyte from a second of the plurality of biological sample processing wells; and the first analyte is extracted from the biological sample concurrently with extraction of the second analyte from the biological sample.

30. An apparatus comprising: a well within a container configured for holding a biological sample; first particles and second particles within the container, the first particles and the second particles configured for performance of sequential biological sample preparation processing steps on the biological sample; and a pump mechanism and a filter membrane, the pump mechanism configured to drive a portion of plasma in the biological sample through the filter membrane, the filter membrane configured to pass the portion of the biological sample into the well.

31. The apparatus of claim 30, wherein: the first particles are disposed within a first portion of the container outside of the well; and the second particles are disposed within the well.

32. The apparatus of claim 30, wherein: a first of the sequential biological sample preparation processing steps is triggered by rehydration of the first particles by the biological sample; and a second of the sequential biological sample preparation processing steps is triggered by exposure of the second particles to an elevated temperature.

33. The apparatus of claim 32, wherein the container further comprises: a heat source proximate to the well; and one of a seal over an opening into the portion of the container containing the heat source, wherein removal or rupture of the seal initiates an exothermic reaction by the heat source, or a region for compression of a blister pack including the heat source, wherein the compression of the blister pack initiates the exothermic reaction by the heat source.

34. The apparatus of claim 30, wherein the container is a cubic container with the pump mechanism and the filter membrane disposed diagonally therein.

35. The apparatus of claim 30, wherein the container comprises: a cylindrical collection well disposed over the pump mechanism and the filter membrane, the cylindrical collection well containing the first particles; a movable plate disposed below the cylindrical collection well, the movable plate comprising a plurality of openings therethrough; and a drum disposed below the pump mechanism and the filter membrane, the drum comprising a plurality of sector-shaped wells, at least a first of the plurality of sector-shaped wells containing the second particles.

36. The apparatus of claim 35, wherein, when the movable plate is in a first position, access to the plurality of sector-shaped wells from the cylindrical collection well is blocked while the cylindrical collection well remains accessible for collection of the biological sample.

37. The apparatus of claim 36, wherein movement of the movable plate from the first position to a second position aligns a first of the plurality of openings through the movable plate with at least one of the plurality of sector-shaped wells, allowing passage of the portion of plasma in the biological sample into the at least one of the plurality of sector-shaped wells.

38. The apparatus of claim 37, wherein movement of the movable plate from the second position to a third position aligns a second of the plurality of openings with an air source, allowing passage of air into contact with a heat source in the container.

39. Tire apparatus of claim 30, wherein: the well comprises a plurality of biological sample processing wells;the container comprises: a collection well disposed over the pump mechanism and the filter membrane; and a movable plate disposed below the collection well, the movable plate comprising a plurality of openings therethrough; and at least a first of the plurality of biological sample processing wells contains the second particles.

40. The apparatus of claim 30, wherein: the well comprises a plurality of biological sample processing wells; the container comprises a collection well disposed over the pump mechanism and the filter membrane; the filter membrane covers openings into the plurality of biological sample processing wells; the pump mechanism comprises a movable and compressible sponge below the filter membrane; in a first position, the sponge is below the collection well and absorbs a filtered portion of the biological sample; in a second position, the sponge is below the well such that compression of the sponge pumps the filtered portion of the biological sample into the plurality of biological sample processing wells; and at least a first of the plurality of biological sample processing wells contains the second particles.

41. A method of using the apparatus of any one of claims 30 through 40, the method comprising: receiving the biological sample within the container; and removing fluid including an analyte from the well.

42. The method of claim 41, wherein: the well comprises a plurality of biological sample processing wells; removing fluid including an analyte from the well comprises removing fluid including a first analyte from a first of the plurality of biological sample processing wells; the method further comprises removing fluid including a second analyte from a second of the plurality of biological sample processing wells; and the first analyte is extracted from the biological sample concurrently with extraction of the second analyte from the biological sample.

43. An apparatus comprising: a container configured for holding a biological sample, the container including a collection well containing first particles configured for performance of a first biological sample preparation processing step on a biological sample received in the collection well; and a well including a plurality of biological sample processing wells in fluid communication with the collection well, at least a first of the plurality of biological sample processing wells containing second particles configured for performance of a second biological sample preparation processing step on a portion of the biological sample received from the collection well.

44. The apparatus of claim 43, wherein: the second particles comprise second particles of a first type and second particles of a second type; the second particles of the first type are configured for biological sample preparation processing targeting a first nucleic acid molecule; the second particles of the second type are configured for biological sample preparation processing targeting a second nucleic acid molecule; the first of the plurality of biological sample processing wells contains the second particles of the first type; and a second of the plurality of biological sample processing wells contains the second particles of the second type.

45. The apparatus of claim 44, wherein: the plurality of biological sample processing wells comprises: the first biological sample processing well containing the second particles of the first type; the second biological sample processing well containing the second particles of the second type; a third biological sample processing well containing the second particles of a third type; and a fourth biological sample processing well containing the second particles of a fourth type: the second particles of the first type are configured for biological sample preparation processing targeting genomic deoxyribonucleic acid (gDNA); the second particles of the second type are configured for biological sample preparation processing targeting cell-free deoxyribonucleic acid (cfDNA);the second particles of the third type are configured for biological sample preparation processing targeting ribonucleic acid (RNA); and the second particles of the fourth type are configured for biological sample preparation processing targeting proteins.

46. The apparatus of claim 43. wherein the container comprises a pump mechanism and a filter membrane between the collection well and the well.

47. The apparatus of claim 46, wherein: the pump mechanism and the filter membrane are disposed above the collection well; the collection well is cylindrical; the well is a drum; and the plurality of biological sample processing wells are sector-shaped.

48. The apparatus of claim 47, wherein the pump mechanism comprises a pillar array.

49. The apparatus of claim 47, further comprising: a heat source disposed centrally to the plurality of biological sample processing wells.

50. The apparatus of claim 47, further comprising: a movable plate disposed below the collection well, the movable plate comprising a plurality of openings therethrough; wherein, when the movable plate is in a first position, access to the plurality of biological sample processing wells from the collection well is blocked while the collection well remains accessible for collection of the biological sample; and wherein movement of the movable plate from the first position to a second position aligns a first of the plurality of openings through the movable plate with the plurality of biological sample processing wells, allowing passage of a portion of the biological sample into the plurality of biological sample processing wells.

51. The apparatus of claim 46, wherein: the filter membrane is disposed below the collection well and the plurality of biological sample processing wells; the pump mechanism comprises a movable and compressible sponge below the filter membrane;in a first position, the sponge is below the collection well and absorbs a filtered portion of the biological sample; and in a second position, the sponge is below the plurality of biological sample processing wells such that compression of the sponge pumps the filtered portion of the biological sample into the plurality of biological sample processing wells.

52. A method of using the apparatus of any one of claims 43 through 51, the method comprising: receiving the biological sample within the container; and removing fluid including an analyte from the well.

53. The method of claim 52, wherein: the well comprises a plurality of biological sample processing wells; removing fluid including an analyte from the well comprises removing fluid including a first analyte from a first of the plurality of biological sample processing wells; the method further comprises removing fluid including a second analyte from a second of the plurality of biological sample processing wells; and the first analyte is extracted from the biological sample concurrently with extraction of the second analyte from the biological sample.

54. A method comprising: collecting a first biological sample in a collection well of a biological sample container, where the biological sample container includes: the collection well; a first biological sample processing well; and a second biological sample processing well; preparing the biological sample; extracting a portion of a first solution in the first biological sample processing well using a first fluid transfer vessel; and extracting a portion of a second solution in the second biological sample processing well using a second fluid transfer vessel.

55. The method of claim 54, wherein: preparing the biological sample is performed using first particles provided in the collection well prior to collection of the biological sample;extracting a portion of a first solution in the first biological sample processing well is performed subsequent to the initial biological sample preparation processing step using second particles of a first type provided in the first biological sample processing well prior to the collection of the biological sample; and extracting a portion of a second solution in the second biological sample processing well is performed subsequent to the initial biological sample preparation processing step using second particles of a second type provided in the second biological sample processing well prior to the collection of the biological sample.

56. The method of claim 54, wherein: the portion of the first solution is extracted from the biological sample container into a first analyzer for analysis of characteristics of the first nucleic acid molecule; and the portion of the second solution is extracted from the biological sample container into a second analyzer for analysis of characteristics of the second nucleic acid molecule.

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