Method and system for sample processing for sequencing, including host cell depletion

Abstract

Methods and systems are provided for automated preparation of biological samples for nucleic acid analysis and sequencing, including selective depletion of host cells and host nucleic acids to enrich pathogenic material. The methods are applicable to various types of samples and are compatible with sequencing-based workflows. Host cell depletion may be achieved using one or more filtration, mechanical disruption, chemical lysis, or nuclease treatment steps. Host cells are removed using a multi-stage filtration process comprising sequential filtration through a large-pore filter to remove host cells and debris, followed by filtration through a smaller-pore filter to further reduce host material while allowing pathogens to pass, thereby enabling fully automated processing without centrifugation. Sub-micron filtration may be used to remove bacterial and fungal cells and selectively enrich viral particles. Nucleic acids are purified, optionally amplified or enzymatically processed, and prepared for sequencing.

Description

METHOD AND SYSTEM FOR SAMPLE PROCESSING FOR SEQUENCING, INCLUDING HOST CELL DEPLETIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 749,571, filed on January 25, 2025, and entitled “METHOD AND SYSTEM FOR SAMPLE PROCESSING FOR SEQUENCING, INCLUDING HOST CELL DEPLETION.” The entire contents of said application are incorporated herein by reference.TECHNOLOGICAL FIELD

[0002] The following disclosure is directed to methods to selectively remove host or other large cells from a sample to enrich a sample for nucleic acids from pathogens. In one embodiment of the disclosure, the sample is pre-centrifuged at low speed to remove a portion of the host or larger cells and then is filtered to further reduce the host or large cell component. In an alternative embodiment, a sonication / bead-beating approach is used to remove host or larger cells from a sample selectively. These methods can be combined with a system using a cartridge to automate host cell depletion, isolation of nucleic acids from pathogens, and library preparation for sequencing to provide a system to automate sample preparation for pathogen sequencing library preparation.BACKGROUND OF THE INVENTION

[0003] Biological and chemical testing processes often are complex and require laboratory facilities and trained personnel. Many process steps increase the likelihood of human error and, thus, incorrect results. Complex processes have been automated with larger automated systems. However, there exists a need to automate processes in a deployable format that is easy and low-cost to use. In particular, analytical testing and biological analysis can benefit from a platform that can be readily reconfigured for different processes.

[0004] In general, chemical and biological testing use a variety of standard techniques: heating, mixing, disruption of samples, filtration, chromatography, separation, etc. Many disposable testing cartridges have been developed that carry out a limited number of process 133156225.1steps. There is a need for a simple system with a disposable cartridge that, can be configured to carry out any of these common process steps in more complex testing protocols.

[0005] In the field of genomics, rapid and convenient nucleic acid-based testing can provide actionable results at the point of care. The need to send samples to core laboratories can result in treatment delays that can adversely impact patients. In remote and under¬ developed areas, health care is not readily available to residents in a timely and accessible manner. Inadequate access to healthcare facilities such as hospitals and clinics, or even health product / service retailers (e.g., drug stores), seriously hinders any effort to achieve timely diagnosis and treatment of patients, especially those suffering from an infectious disease, making it difficult to properly diagnose and treat those individuals. Despite many advances in technology over the past decades, there still exists a pressing need for new and improved diagnostic tools that are highly mobile and capable of performing complex molecular testing to generate rapid, reliable, and accurate diagnostic results, regardless of location.

[0006] Over the past decade, the field of genomics has seen drastic improvements in sequencing. Next-generation sequencing (NGS) is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, the clinical use of NGS has been mostly focused on the human genome for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples where NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, the ability to detect fastidious or non-culturable organisms, and the ability to detect co-infections.

[0007] One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore, for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. In particular, NGS can be overwhelmed by a large amount of host cell nucleic acids. Patient samples may have a large ratio of host cells to pathogens. In addition, host cells have genomes thousands of times larger than bacteria and hundreds of thousands of times larger than most viruses. In order to have high sensitivity for low-concentration pathogens, host nucleic acids may be depleted by several orders of magnitude.33156225.1 2

[0008] A saponin enrichment process has been demonstrated to deplete host cells from respiratory samples. This process works well for the enrichment of bacterial nucleic acids. However, it does not perform well in the enrichment of viral nucleic acids, particularly from enveloped viruses. Saponin selectively disrupts eukaryotic membranes while having little effect on bacteria. Unfortunately, saponin disrupts many viruses limiting this approach to bacterial enrichment.

[0009] Furthermore, multiple nucleic acid extraction systems are often necessary to monitor several targets originating from distinct groups of organisms such as SARS-CoV-2 and AMR bacteria in wastewater. Most established methods are specific to either DNA or RNA, or to either bacteria or viruses. The goal is to develop a process that enriches whole viruses and bacteria, including their nucleic acids, while preferentially destroying eukaryotic cells.

[0010] A major feature of this disclosure is to provide a process that can be automated in a low-cost disposable to enable point-of-care testing. The process needs to be capable of being carried out by components compatible with disposable cartridges.BRIEF SUMMARY OF THE INVENTION

[0011] The current invention provides a method for depletion of larger eukaryotic cells from samples, leaving pathogens, including bacteria, viruses and fungi for analysis. In particular, the method is to isolate pathogens from host cells prior to preparation of a sequencing library.

[0012] Aspects of the following disclosure are directed to embodiments of a method for preparing a sequencing library from a biological sample in a disposable cartridge. In some embodiments, the method includes introducing the biological sample into the disposable cartridge and depleting host cells or host-derived material from the sample while retaining pathogenic material comprising one or more of bacteria, fungi, or viruses. In some embodiments, the method further includes purifying nucleic acids from the retained pathogenic material within the cartridge. In some embodiments, the method further includes preparing a sequencing library from the purified nucleic acids within the cartridge, wherein the host cell depletion, nucleic acid purification, and library preparation are performed without removal of the sample from the disposable cartridge.33156225.1 3

[0013] In some embodiments of the method, the depleting of the host cells comprises selectively removing intact eukaryotic cells while allowing pathogenic organisms or viral particles to remain in the sample. In some embodiments of the method, the depleting of the host cells is performed using one or more of filtration, centrifugation, mechanical disruption, chemical lysis, enzymatic treatment, or combinations thereof. In some embodiments of the method, the depleting of the host cells comprises sequential filtration of the sample through a plurality of filters integrated into the disposable cartridge. In some embodiments of the method, the sample is first passed through a primary filter having a pore size of about 15 to 30 microns to remove host cells and large debris. In some embodiments of the method, filtrate from the primary filter is subsequently passed through a secondary filter having a pore size of about 2 to 10 microns to further remove host cells while allowing pathogens to pass. In some embodiments of the method, the sequential filtration prevents clogging of downstream filters and enables automated fluid flow through the cartridge without centrifugation. In some embodiments of the method, the depleting of the host cells comprises centrifuging the biological sample to pellet host cells and debris, followed by filtration of a supernatant. In some embodiments of the method, the filtration is performed within the disposable cartridge following introduction of the supernatant. In some embodiments of the method, the centrifugation is performed at a speed sufficient to pellet host cells while retaining pathogenic organisms or viral particles in the supernatant. In some embodiments, the method further comprises digesting cell-free nucleic acids after the depletion of the host cells.

[0014] In some embodiments of the method, the digesting comprises contacting the sample with a nuclease that degrades free DNA or RNA not protected within intact pathogens. The method of claim 11, further comprising inactivating the nuclease prior to nucleic acid purification. In some embodiments of the method, nuclease inactivation is performed using a chaotropic agent. In some embodiments, the method is configured to selectively enrich viral particles. In some embodiments of the method, enriching viral particles comprises filtering the sample through a sub-micron filter to remove bacterial and fungal cells. In some embodiments of the method, the sub-micron filter has a pore size of about 0.1 to 0.5 microns. In some embodiments of the method, the sub-micron filter has a pore size of about 0.22 microns. In some embodiments of the method, viral enrichment is performed after the depletion of the host cells using filtration or centrifugation. In some embodiments of the method, preparing the sequencing library comprises one or more of reverse transcription, fragmentation, tagmentation, amplification, adapter ligation, or combinations thereof. In some embodiments of the method, the sequencing library is compatible with long-read sequencing.433156225.1

[0015] Aspects of the following disclosure are directed to embodiments of a disposable cartridge for preparing a sequencing library from a biological sample. In some embodiments, the disposable cartridge includes a sample input region configured to receive the biological sample and one or more host cell depletion components configured to selectively remove host cells or host-derived material while retaining pathogenic material comprising one or more of bacteria, fungi, or viruses. In some embodiments, the disposable cartridge includes one or more nucleic acid purification regions configured to isolate nucleic acids from the retained pathogenic material and one or more reaction regions configured to prepare a sequencing library from the isolated nucleic acids. In some embodiments, the disposable cartridge is configured to perform host cell depletion, nucleic acid purification, and sequencing library preparation without removal of the sample from the cartridge.

[0016] In some embodiments of the disposable cartridge, the host cell depletion components include a plurality of filters arranged for sequential filtration of the sample. In some embodiments, aspects of the following disclosure are directed to a system for preparing a sequencing library from a biological sample. In some embodiments, the system includes a sample input region configured to receive the biological sample, one or more host cell depletion components configured to selectively remove host cells or host-derived material while retaining pathogenic material comprising one or more of bacteria, fungi, or viruses, one or more nucleic acid purification regions configured to isolate nucleic acids from the retained pathogenic material, and one or more reaction regions configured to prepare a sequencing library from the isolated nucleic acids. In some embodiments, the disposable cartridge is configured to perform host cell depletion, nucleic acid purification, and sequencing library preparation without removal of the sample from the cartridge. In some embodiments, the system further includes a reader configured to interface with the disposable cartridge and to actuate fluid movement. In some embodiments, the system is configured to perform host cell depletion, nucleic acid purification, and sequencing library preparation within the disposable cartridge.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows a perspective view of an embodiment of a shuttle (2) sitting in a rail of a housing (1). The shuttle is shown with multiple chambers for reagents. The shuttle is shown with two syringe barrels (3). The syringes make fluidic contact with ports in the lower portion of the side walls of the shuttle to form a fluidic connection with the33156225.1 5chambers in the shuttle when the syringe and port are aligned. A chamber is also shown on the back side (opposite the syringe plungers).

[0018] Figure 2 shows a perspective view of an embodiment of a shuttle showing reagent chambers (4) and fluid access ports (5) on the side wall. A flexible over-mold is shown covering the side walls and base of the shuttle to form a better seal between the shuttle and the housing. A second row of ports are shown, which align with ports in the rail to form a vent to avoid pressure changes in the reagent chamber when moving fluids.

[0019] Figure 3 shows a perspective top view of an embodiment of a housing (1) showing the channel for the shuttle. Openings (7) in the base of the shuttle allow access to reaction chambers in the base of the shuttle by the magnets and heaters. Openings for heaters would be larger and would allow heaters to make contact with the reaction chambers in the base of the shuttle. Reaction chambers (8) are also shown in the base of the housing, which can be used for heating, including thermo cycling, and optical measurements. These reaction chambers have thin film walls on two sides for optical measurements or dual-sided thermocycling heating. An overflow chamber (6) is shown which is fluidically connected to the reaction chambers in the housing to allow for pressure relief during filling.

[0020] Figure 4 shows a perspective bottom view of an embodiment of a housing (1) showing chambers (8) and flow channels (9). An opening for access to the base of the shuttle is also shown (7).

[0021] Figure 5 schematically shows an embodiment of a layout of reagent chambers for sample and library preparation. This is a top view and does not show the reaction chambers in the base of the shuttle. This figure shows that the sample preparation reagent chambers are larger to handle about one milliliter of sample. The library preparation chambers are smaller to minimize the amount of enzymes needed. This version shows reagent chambers in both the shuttle and the housing. On the left, larger chambers are used for sample preparation. The first chambers (LI and L2) contain the lysis buffers and magnetic particles. The next chamber (S) is a sample chamber. The sample chamber is large enough to contain sufficient sample material for testing. Preferably, the chamber is between 0.5 and 3 milliliters in volume. The sample chamber may have sufficient volume to hold the sample to be tested and the lysis buffer with the magnetic particles. Alternatively, the two chambers may be merged, and the lysis buffer is preloaded into the sample chamber. After the sample and lysis buffer are combined, they are mixed and then if needed sonicated to disrupt the sample. After disruption, the disrupted sample and magnetic beads are pushed through a chamber at the bottom of the shuttle which is located directly above a magnet. Magnetic particles are collected33156225.1 6in a chamber in the base of the shuttle. The shuttle is moved to allow the syringe to pull in the magnetic particle wash buffer (MW). After washing, including disruption with sonication, the nucleic acids are eluted from the pellet using the elution buffer (ME). In this iteration of the cartridge shows the dry reagent chambers are located on the housing (D). Enzyme resuspension buffer (EB) and resuspension buffer (R) are located in the shuttle.

[0022] Figure 6 schematically illustrates a bottom view of an embodiment of the shuttle and the wings of the housing with a layout of reaction chambers. Multiple reaction chambers are shown in the shuttle base (10 and 11). Magnetic separation and heated reaction chambers are shown. In this iteration, the magnetic (11) and heated chambers (10) are linearly arranged in two offset lines. A dual-sided optical detection chamber is also shown in the housing base (12). This is only one option. All chambers may be in one line or alternatively shuffled. In a preferred embodiment the sonicator is in line with the magnetic separation chambers to allow for the chambers to be moved over both the magnet and the sonicator.

[0023] Figure 7 shows a perspective view of an embodiment of a lid structured to cover the reagent chambers in the shuttle. Unstable reagents, such as enzyme mixes, are held on or in pins (13) that extend into chambers in the shuttle. The lid is stored separately from the housing, thus keeping unstable reagents separated for long-term storage. These reagents are directed to the appropriate chambers via their position on the lid. Multiple pins are incorporated in the lid to hold the necessary enzyme pellets for multiple steps.

[0024] Figure 8 shows a sectional view taken along A-A of the embodiment of Figure 7 showing a dry reagent pellet (14) inserted into the end of a pin (13) on the lid.

[0025] Figure 9 shows a cross-sectional view of an embodiment of the housing (1), shuttle (2), and lid (19).

[0026] Figure 10 shows a close-up view of the embodiment of Figure 9.

[0027] Figure 11 shows a perspective view of an embodiment of the shuttle with multiple chambers on the upper surface. Ports are found on the outer face of the shuttle to allow a fluidic connection to the syringe barrels on the housing.

[0028] Figure 12 shows a perspective bottom view of an embodiment of the housing with a fluidic chamber and channel connections. In addition, an opening is shown that resides under the shuttle to allow physical contact of the reader to the shuttle. Possible needs for contact include magnets, heaters, and sonicators.

[0029] Figure 13 shows a perspective top view of an embodiment of a cartridge with the housing and shuttle. The shuttle is located partially out of the rail in the housing to give a better view of the shuttle.33156225.1 7

[0030] Figure 14 shows a top view of an embodiment of a cartridge, including the shuttle and housing. A lid is also shown.

[0031] Figure 15 shows a perspective top view of an embodiment of the shuttle.

[0032] Figure 16 shows a bottom view of an embodiment of the shuttle.

[0033] Figure 17 shows a perspective top view of an embodiment of the housing.

[0034] Figure 18 shows a perspective bottom view of an embodiment of the housing with portions of the housing being transparent so that elements with the housing can be observed.

[0035] Figure 19 shows an exploded view of an embodiment of the cartridge and its associated packaging.

[0036] Figure 20 shows an embodiment of a cartridge shuttle configured for automation of an agnostic diagnostic cartridge to prepare pathogen nucleic acids from patient samples for sequencing.

[0037] Figure 21 shows a top view of the embodiment of the shuttle of Figure 20.

[0038] Figure 22 shows the base of the embodiment of the cartridge shuttle of Figure 20. Multiple reaction chambers (10) have been included for multiple magnetic bead clean-up and thermal incubation steps.

[0039] Figure 23 shows a perspective view of the embodiment of the shuttle of Figure 21 in the housing of Figure 20.

[0040] Figure 24 shows a perspective view of the embodiment of the shuttle of Figure 21 in the housing of Figure 20 with a sample cap (25) and reagent storage lid (19).DETAILED DESCRIPTION OF THE INVENTION

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The term 'comprising' is intended to be open-ended and does not exclude additional elements or steps. Singular forms include plural referents unless the context clearly dictates otherwise.

[0042] In preferred embodiments, host cell depletion is performed using sizebased separation techniques, including filtration with or without centrifugation. Mechanical disruption techniques, including bead beating or sonication, represent alternative embodiments33156225.1 8that may be used alone or in combination with size-based separation depending on sample type and application.

[0043] The following disclosure is directed to embodiments of a system and methods for the selective depletion of host cells and their nucleic acids, RNA or DNA, from a sample to enrich nucleic acids from pathogens: bacterial, viral or fungal.

[0044] In a preferred embodiment of the disclosure, a multi-step process is provided to deplete nucleic acids from host cells to facilitate the sequencing of pathogenic material. First, a biological sample is centrifuged at low speed to remove larger non-pathogen cells and debris. Second, the supernatant is removed and filtered through a filter with pore size of 1-20 microns. Although larger cells will remain in the supernatant, the amount of larger cells is depleted so that the remaining material can pass through a filter without blocking fluid flow. The filtration step then removes most of the remaining larger cells, minimizing host nucleic acids that could otherwise overwhelm the sequencing. A third step of a nuclease treatment can remove any free nucleic acids in the sample further removing host nucleic acids. Digestion of free nucleic acids is optional, since free nucleic acids may include a significant fraction of pathogenic nucleic acids.

[0045] Prior to centrifugation and / or filtration, the sample may be mixed with reagents to minimize sticking of the pathogens to host cells or other pathogens. Aggregates may form that would cause pathogenic material to spin down or be caught by the filter along with larger cells. Aggregation can be inhibited by the addition of salts, adjusting pH, or addition of polymers to block pathogen interactions with other cells or viruses.

[0046] This process may be used to remove host cells from patient samples. But it may be used more broadly to remove larger cells from any sample, including environmental, veterinary, and food samples.

[0047] Small, portable, and low-cost centrifuges are available which can be deployed into the field for use. Some units are also battery-powered. Pelleting of larger cells can be carried out at speeds under 1,000 rpms. Preferentially, centrifugation is carried out at 500 rpms or less. More preferred is centrifugation at 300 rpm or less. The goal is to pellet most host cells while maintaining pathogens, including viruses, bacteria, and fungi in the supernatant. Centrifugation removes the bulk of large cells and debris from the sample before introducing the sample into the preparation cartridge. A portion of the larger particles may remain in the supernatant, with the priority being to retain most of the pathogens in the supernatant.933156225.1

[0048] After centrifugation, the sample is transferred to a sample preparation cartridge that automates the remaining steps.

[0049] The sample is first introduced into a sample chamber. In some embodiments, the sample chamber has an opening in the bottom of the chamber with a filter welded above the opening. A syringe pulls the sample down through the filter. The filter has a pore size large enough to allow pathogenic cells and viruses through while retaining larger cells. Prior to filtration, in some embodiments, reagents may be added to disrupt interactions between cells to prevent the pathogens from clumping or sticking to larger cells. Reagents may include salts, buffers to raise pH, or other materials such as polymers, which can inhibit interactions between pathogens or pathogens and larger cells or debris.

[0050] With low-speed centrifugation, the pellet of cells will be loose and some cells will be removed with the supernatant. This problem could be more significant when individuals with less experience in sample handling run the process. In some embodiments, to reduce the number of host or contaminating cells and debris, the sample is filtered using a membrane with pores large enough to allow bacteria and fungi through but retain eukaryotic and host cells. The filter pores are preferably larger than one micron to allow bacteria to pass. Preferably, the pore size is two or three microns. Pore size may be up to 10-20 microns.

[0051] The combination of pre-centrifugation and filtration is advantageous for this disclosure. For many samples, the amount of large cells and debris is such that a filter will be blocked, leading to loss of sample and decreased assay sensitivity. To improve performance, filtration removes most of the remaining host cells, eukaryotic cells, and / or debris. Including the filtration step in the cartridge minimizes handling steps, simplifies training and lab equipment needed, decreases variability in sample handling, and minimizes labor costs.

[0052] If needed, a nuclease treatment step can be included in order to deplete cell-free nucleic acids. A nuclease inactivation step is then required to inactivate the nucleases before the purification of nucleic acids from the pathogens. Inactivation of nucleases and disruption of pathogens can be carried out in a single step if the treatment step rapidly inactivates the nucleases, such as a guanidine treatment step.

[0053] Prior to library preparation, the nucleic acids may be purified to remove any potential inhibitors of library preparation or sequencing. In an alternative and preferred embodiment, host cell depletion is carried out using a multi-stage filtration process that eliminates the need for a centrifugation step and enables full automation within a disposable cartridge. In this embodiment, the sample is first passed through a primary filtration33156225.1 10membrane having a pore size sufficiently large to retain host or other large eukaryotic cells while allowing pathogens and smaller particles to pass. Preferably, the primary filter has a pore size in the range of 15 to 30 microns, more preferably 20 to 25 microns.

[0054] The primary filtration step removes the majority of host cells and large debris, thereby reducing the particulate load presented to downstream filtration steps. By removing these larger components first, the likelihood of clogging or flow restriction in subsequent filters is substantially reduced.

[0055] After passage through the primary filter, the filtrate is directed to a secondary filtration membrane having a smaller pore size selected to retain remaining host cells while allowing bacteria, fungi, and viruses to pass. Preferably, the secondary filter has a pore size in the range of 2 to 10 microns, more preferably 2 to 5 microns.

[0056] The combination of the primary and secondary filtration steps enables efficient host cell depletion while maintaining fluidic reliability, thereby allowing the process to be fully automated within a cartridge using syringe-driven or pressure-driven flow, without the need for centrifugation or external preprocessing equipment.

[0057] In a further embodiment, the filtration process is configured to selectively remove bacterial and fungal cells while retaining viral particles. In this embodiment, the sample, optionally following host cell depletion using one or more larger-pore filters, is passed through a sub-micron filtration membrane having a pore size in the range of 0.1 to 0.5 microns, preferably approximately 0.22 microns.

[0058] This sub-micron filtration step removes intact bacterial and fungal cells while allowing viral particles and viral nucleic acids to pass, thereby enabling selective enrichment of viruses for downstream nucleic acid purification, library preparation, and sequencing.

[0059] The filters used in the multi-stage filtration process may be membrane filters, track-etched filters, depth filters, or composite filters, and may be permanently integrated into the cartridge or mounted over fluidic ports within the shuttle or housing. In preferred embodiments, the filters are welded, bonded, or heat-sealed into the cartridge to ensure leak-free operation and compatibility with automated fluid handling.

[0060] In an alternative embodiment of the disclosure, the depletion of host cells or contaminating eukaryotic cells may be carried out using a bead beating approach. This approach does not have the efficiency of removing host cells that the precentrifugation / filtering process has, but it has the advantage of allowing automation of all steps in a cartridge.33156225.1 11

[0061] Sonication and bead beating show a preference for disruption efficiency based on the particles' size relative to the target's size. It is not based on the genus or other specific factors. Sonication has been used to provide mechanical force for disruption. Disruption can be driven using sonication. Ultrasonic energy can be introduced into a sample chamber. Ultrasonics results in cavitation which creates pulses of energy that can break apart the sample. If needed, beads may be used in the sample. During sonication, the beads are agitated and move through the sample to crush and grind the material.

[0062] Bead beating has been used to isolate live virus from tissue samples. It has also been using to enhance the isolation of bacterial and viral DNA / RNA from wastewater. In each case, there was improved isolation as determined by PCR amplification. In these processes, bead beating was used to disrupt the host tissue to release bacteria and viruses. PCR amplification selectively copied the pathogenic targets for detection. Bead beating has been shown to be an effective sample lysis process for a wide variety of sample types. Compared to chemical lysis approaches, bead beating using standard reagents has wide applicability and can be done faster, in a matter of minutes, for most sample types. Bead beating efficiency varies depending on bead characteristics and the energy driving the beads.

[0063] The major variables for bead function are size, composition and shape. The smaller the particles used in the grinding media, the smaller the average particle size and the smaller the lowest-limiting particle size produced during pulverization. Matrix particle size should be selected based on the size of the particles you wish to obtain in your lysate. The shape of the grinding media is also a major determining factor in how cells are disrupted. Dull media, such as spherical beads, utilize cascade impaction (hammering) as the main force for cell lysis. Sharp and angular-shaped media will primarily generate mechanical shear forces (chopping and cutting), which can quickly open difficult cell walls, grind fibrous or elastic animal tissue, or crack spores or oocytes. Shear forces are preferable when isolating stable molecules such as DNA, stable proteins, structural polysaccharides, and small molecules or metabolites. RNA and specific easily denatured proteins can be quickly degraded by shear forces, so care needs to be taken when using angular media. For the isolation of these molecules, smooth impactor grinding media can be much more forgiving. In a preferred embodiment of the disclosure, the beads are moved using ultrasonic energy. Ultrasonics does have the problem of generating shear forces which can break down DNA and RNA. However, if the pathogens remain intact, the bacterial membrane or viral coat should protect the nucleic acids from shearing.33156225.1 12

[0064] As bacteria tend to be very small, 1-3 μm in length or diameter, smaller beads normally work better for cell disruption. This is a very significant factor in cracking bacteria. Generally, the mass of the beads greatly exceeds the mass of the bacteria, thus both glass and zirconium work well. However, glass beads tend to clump more than denser zirconium beads which settle more effectively in lysates after processing. Other organisms of comparable size, such as cyanobacteria, can also be processed in a similar manner.

[0065] On the other hand, human cells are much larger. A single bacterial cell is about 1 pm in diameter. A typical human (eukaryotic) cell is 25 pm in diameter, so an average cell can hold more than 10,000 bacteria. Accordingly, particles of 1 or more millimeters are used to disrupt human cells. Larger particles will be less likely to disrupt bacteria and far less likely to hit viruses. Irregularly shaped particles can also provide spaces for bacteria or virus to fit without being crushed by the bead-beating process.

[0066] There are several options for the material composition of the beads. For example, glass beads have a density of 2.5 g / cc and is the most common bead media used for 'Beadbeating'. Zirconia / Silica beads have a density of 3.7g / cc (50% denser than glass - good for spores and most tissues). Silicon Carbide sharp particles (not a bead) have a density of 3.2 g / cc. These particles may work faster on tough tissue samples because the particles have sharp cutting edges. Their superiority over less expensive Zirconia / Silica beads is under investigation. See Brein's comments below. Garnet (an iron-aluminum silicate, sharp particle) has a density of 4.1 g / cc. Like dense, sharp-edged SiC particles, it accelerates the lysis of tough tissue. Unlike SiC sharp particles, garnet particles fragment during beadbeating. This property can be useful when homogenizing soft tissue or fecal and soil samples containing bacteria. No mix of bead sizes will be needed. Starting with microvials half filled with 2 mm Garnet particles, the sample is rapidly dispersed. At the same time, the beadbeating has produced, in situ, much smaller Garnet fragments which can efficiently disrupt the microorganisms. Zirconia has a density of 5.5g / cc (100% denser than glass) - This ceramic bead, used for tough tissue, is chemically inert and resistant to fragmentation. Chrome- Steel and Stainless Steel have a density of 7.9g / cc. These heavy beads are most often used for dry-grinding leaves and seeds. Depending on the size of the bead, 1 to 5 steel beads are used in reinforced 2ml polypropelyne vials. Ordinary screw-cap polypropylene micro-vials can crack or leak when used with steel beads. Chrome-steel beads often are a good substitute for stainless steel beads. While stainless steel beads are often selected because of their corrosion resistance, chrome-steel beads are inexpensive...indeed, they are cheap enough to be used as "disposable",33156225.1 13thus eliminating cleaning and cross-contamination concerns. Eventually, chrome-steel beads will rust in contact with aqueous media, so they should be stored dry.

[0067] In addition to bead characteristics, disruption is also affected by energy input, time, and frequency of the energy input. By varying settings in a system, the power, time and frequency can be adjusted to give good host cell disruption while minimizing bacterial and viral disruption.

[0068] Frequency is an essential variable in managing sonication. If the system inputs energy at the resonant frequency, energy transfer to the beads will be efficient and decrease heating, which can degrade nucleic acids. US Pat. No. 6,819,027 to Saraf discloses one ultrasonic control system that can be used with the system of the present disclosure. Saraf and cited patents within that patent describe control circuits that continually adjust the system's frequency to maintain resonance.

[0069] Optimally, the power, time, and frequency would be determined to give a high level of host cell or eukaryotic cell disruption with minimal disruption of bacteria or viruses. Bacterial and viral nucleic acids should be protected by the intact organism against shearing during this process, but care should be taken to keep the target nucleic acids intact for efficient sequencing.

[0070] The one objective is to enrich pathogenic nucleic acids to a level in which sequencing can identify the pathogens with a high degree of sensitivity. This process can be coupled with other approaches for pathogen sequence enrichment to obtain high levels of sensitivity. For example, magnetic beads that can bind methylated DNA from eukaryotes can be used to remove eukaryotic DNA from a sample selectively. This can be done after the nucleic acids are purified from the pathogens and host depletion has been carried out. If sequencing is done using the Oxford Nanopore sequencers, adaptive sequencing can be used to push host nucleic acids out of the pores to allow for more sequencing of pathogenic material. In this way, multiple enrichment approaches can be utilized to reach the intended enrichment levels.

[0071] The present disclosure provides processes that can be automated. In a preferred embodiment of the disclosure, the process is automated in a linear slide cartridge and system designed to automate complex chemical and biological analysis. The preferred cartridge system is described in PCT Application PCT / US23 / 81407, incorporated herein.

[0072] In some embodiments, the disposable cartridge utilizes a novel design with a shuttle 2 on a rail in a housing 1 (Fig. 1). The outer side walls 2a, 2b of the shuttle contact the inner walls of the rail to form a seal. Shuttle ports 5 defined in the walls of the33156225.1 14shuttle 2 and rail ports defined in the rail of the housing 1 allow for the valving of multiple fluidic channels by moving the shuttle 2 along the rail 1. Locating the shuttle 2 in various positions to align the shuttle ports 5 with the rail ports creates fluid paths that allow for the movement of fluids or air. Either the walls of the housing lb, lb or the walls of the shuttle 2a, 2b which are in contact can be over-molded with a low durometer material to improve sealing. The linear design enables the device to move from step to step in a linear fashion, minimizing cross-contamination of reagents. The device is manufactured as two separate components.

[0073] The housing 1 also has two or more syringe barrels 3. Each syringe barrel 3 has a moveable plunger to pull in and expel fluids or air. A syringe motor drives the movement of a plunger in the syringe barrel 3. The motor is preferably a linear drive motor. The syringe barrel 3 can be capped with a stripper cap that has a central opening for the plunger rod of the syringe motor to enter the syringe barrel and move the plunger. The stripper cap holds the plunger in the barrel 3 when the syringe motor withdraws the plunger rod, restraining the plunger in the barrel and keeping the liquids sealed within the cartridge.

[0074] The syringe barrel 3 is connected to a port in the rail wall la. The port, which opens on the inside surface of the rail wall la can be aligned with the ports 5 on the shuttle to allow for the syringe shuttle reservoirs and reaction chambers 4 to be fluidically connected. As the shuttle’s two parallel exterior surfaces 2a, 2b slide along the side walls of the housing rail la, lb, the port for a syringe 3 can align with the plurality of chambers fluidly connected by a plurality of ports 5 along the exterior surfaces at different positions, wherein sliding the shuttle aligns one of the shuttle ports 5 with a fixed port on the housing such that fluid may be moved into and out of the chambers 4 in response to displacement of the moveable plunger.

[0075] In a preferred embodiment of the disclosure, the disposable cartridge housing 1 has two or more syringe barrels 3a, 3b having at least two different volumes. The larger volume syringe barrel 3a is particularly useful for sample processing where larger volumes are preferred. Whereas the smaller syringe barrel 3b is preferred for enzymatic reactions where it is desirable to limit the fluid volume to lower costs.

[0076] Referring to FIG. 1 there is shown a shuttle 2 set in the housing 1 of the instant invention. In some embodiments, the shuttle 2 is held within the housing 1. In some embodiments, the shuttle 1 sits in a rail formed by two side walls la, lb and a floor 1c (FIG.3) in the housing 1. One or more syringe barrels 3 are attached to the first side wall la of the housing 1. Each syringe barrel 3 houses a plunger, which can be moved by a plunger rod from the reader. A port in the side wall 1 a opens into the syringe barrel and to the internal surface33156225.1 15of the side wall 1 a in the rail. When aligned with a port 5 in the side of the shuttle 2, it allows for a fluidic connection between the syringe and a reservoir or chamber 4 within the shuttle. The shuttle 2 is a component containing a plurality of reservoirs 4 opening on the top surface of the shuttle, which are capable of storing reagents, being used as a sample chamber, receiving waste, or for other purposes. Additional reaction chambers 4 are in the base of the shuttle 2 but not visible here.

[0077] Referring to FIG. 2, there is shown a top view of the shuttle 2 of the instant invention. In some embodiments, the shuttle 2 has a plurality of chambers 4 capable of containing fluids or dried reagents. Chambers 4 can also be used to receive samples and to collect waste. In some embodiments, heat seal films, not shown cover the top 2d and bottom 2c surfaces of the shuttle 2 and seals the chambers 4 from the outside environment. Each chamber 4 has a port 5 in the lower portion of the wall 2a, which can be aligned to a port in the housing, which is in fluidic connection to a syringe barrel 3. This allows for fluids to be moved in or out of the chamber 4 using the syringe. A second port is located in the upper portion of the side wall 2a of a chamber that can be aligned with a port in the housing 1. This port, when aligned with the port in the housing wall 1 a, creates a vent to allow for air to move in and out of the chamber when the syringe is actuated to prevent back pressure or negative pressure within the chamber 4.

[0078] Referring to FIG. 3, a view of the housing or base 1 is shown. The housing consists of a rail with two side walls la, lb, a base 1 c, and one or more syringe barrels 3. The side walls la. lb may be slightly angled out from the channel, or in other words, may be positions at an angle relative to the base 1 c that is greater than 90°. A snap feature may also be included along the top of the side walls to hold the shuttle in place. In some embodiments, supporting ribs If (Figure 9) may also be located perpendicular to the side walls outside of the channel to support the side walls and to maintain pressure on the seal with the shuttle. Openings in the housing base 1 c inside the channel allow for various components from the system to directly contact the base 2c of the shuttle 2. Such components include but are not limited to magnets, sonicators, or heaters (including thermocyclers). In some embodiments, one or more chambers 6, 8 may be formed or defined in the base 1. In some embodiments, one or more of the chambers 6, 8 may comprise a heat seal forming the top and / or bottom of the one or more chambers 6, 8. In some embodiments, such a chamber may be used for heating or for optical access to carry out optical measurements. Figure 3 shows an apron le on the side opposite the syringe barrels 3. Aprons le may be located on either side of the rail. The apron le shown depicts two reaction / optical chambers 8 and a reagent chamber or overflow chamber33156225.1 166. The reaction or optical chambers 8 are sealed on the top and bottom with a plastic film to enclose the chamber. If optically transmissive films are used, the chamber 8 can be used for optical measurements of materials in the chamber 8. The chamber 8 may also be used for a variety of processes not limited to sonication, heating (including thermocycling), and magnetic separation.

[0079] Figure 4 shows a bottom view of a housing 1 with chambers 8 and flow channels 9. The chambers 8 and flow channels 9 are completed with a thin plastic seal covering the chambers 8 and / or channels 9. The chambers 8 are in a fluidic connection to the syringe barrels 3 via the channels 9. An opening 7 is also shown in the base 1c of the rail. A cartridge may have multiple openings 7 in the base of the housing 1. Openings in the base 1 c of the rail of the housing 1 can be used to allow access to the bottom side 2c of the shuttle 2. A variety of components can access the bottom 2c of the shuttle 2, including sonicators, heaters (including thermocyclers), optical sensors, and magnets.

[0080] Figure 5 shows an example of a layout of chambers for the cartridge. In this layout, there is a plurality of larger chambers for sample processing. These larger chambers preferably can hold more than 100 micro liters of solution and more preferably more than 500 ul of solution. The chambers may be of different volumes. One or more chambers may be used to receive the test sample. In this configuration, there are chambers to hold lysis buffer (LI and L2), magnetic particles (MP), a wash buffer (MW), and an elution buffer (ME) in addition to the sample chamber (S). A desalting column (C) is also included. Smaller chamber chambers are then used for library preparation. These chambers include the magnetic particle, wash buffer, and elution buffer. A resuspension buffer is also included to rehydrate dry reaction mixes and to dilute the final sample if needed. These smaller chambers are typically designed to hold less than 100 micro liters of solution. More preferably the volume of solution is less than 50 microliters. If needed, reagent chambers may also be located in the housing 1. Several small chambers are shown in this interaction of the design to hold dry reagents in the housing 1. The diamond-shaped chamber is an optical detection chamber to measure the final concentration of the nucleic acids prior to analysis. In this layout, a waste chamber is shown. Preferably, all waste is contained in the cartridge for easy disposal. More than one waste chamber may be included in the cartridge. This depiction shows how dried reagents may be located in the housing 1. In that case, wet reagents can be stored in the shuttle 2 and dry reagents in the housing 1 to increase the shelflife of the dried reagents. In some embodiments, the base or housing 1 and the shuttle 2 are stored separately and snapped together just before use.33156225.1 17

[0081] Figure 6 shows a schematic laying out one possible arrangement of chambers 4 on the bottom surface 2c of the shuttle 2. A series of chambers 4 for heating and magnetic separation are depicted. The chambers 4 will typically be located along a line running the length of the shuttle 2 so that the chambers will move over a stationary sonicator, heater, or manet as the shuttle slides along the rail. Preferably, a sonicator and magnet in the system will be located in line with one another so that particles can be pulled down with a magnet with the shuttle 2 in one position and then moved over a sonicator when the shuttle 2 is moved to a second position in order to resuspend the particles. In this figure, two lines of reaction chambers 10, 11 are shown. The top line 10 of diamond-shaped chambers 4 can be used for thermal reactions, with each chamber 4 being moved over a heater. The second line 11 of chambers 4 is able to align each chamber with a sonicator and a magnet. The chamber 4 can be moved over the magnet to attract and collect the magnetic particles into a pellet. The chamber 4 can then be moved over a magnet in order to disperse the magnetic particles for washing and elution. One or more lines of chambers 10, 11 may be included as needed. The number of chambers 4 in each line may also be varied as needed.

[0082] In one embodiment of the disclosure, the cartridge has one or more chambers 4 in or towards the base 2c of the cartridge shuttle 2, wherein the chamber or chambers 4 can be moved over a magnet and also over a sonicator or other source of disruptive energy.

[0083] Figure 7 shows a lid 19 with pins 13 holding dried or frozen reagents. When the lid 19 is placed on the shuttle 2, the pins 13 introduce the reagents to the chambers 4, where they can be rehydrated or melted prior to use. The pin 13 may be any structure that can restrain the solid reagent and hold it in position in the appropriate chamber 4.

[0084] Figure 8 shows a cutaway view of the pin 13 from Figure 7 along A- A. As shown, the pin 13 is structured to hold a dry reagent pellet 14. In this embodiment, the dry reagent pellet 14 is held within a recessed opening at the end of the pin 13. Any configuration that can restrain the dried reagent pellet 14 while making it accessible to the resuspension solution is acceptable.

[0085] Figure 9 shows a cutaway view of the housing 1 and shuttle 2 with the lid 19 attached. In this view, the small syringe barrel 3b is shown connecting to a chamber with a pin from the lid. Each of the pins 13 in the lid 19 goes to a separate chamber 4 in which the dried reagent 14 attached to that pin 13 can be dissolved and activated. The syringe can push a resuspension buffer into the chamber to dissolve the dry reagent and activate the solution.33156225.1 18

[0086] Figure 10 shows a close-up of the view in Figure 9.

[0087] Figure 11 shows a top view of a shuttle 2. In this shuttle 2, large chambers 4a are shown at one end, with smaller chambers 4b extending toward the other end. Ports 5 in the base of each chamber 4a, 4b or leading to reaction chambers in the base are shown along the base of the side wall 2a. Vent ports are not shown. A larger waste chamber 4c is shown running behind the smaller chambers 4b. Within the waste chamber 4c, a number of vent stacks 4d are shown, which extend from a channel that connects to the reaction chambers in the base, These vent stacks 4d allow for air to exit the chambers during filling to prevent back pressure.

[0088] Figure 12 shows a fluidic chamber 8 with channels in the base of the housing. An opening (7) for a component in the system to access the base of the shuttle is also shown. One or more openings 7 may be in the base 1 c of the housing 1 to allow access to the base of the shuttle.

[0089] Figure 13 shows a top view of a cartridge 100 with the shuttle 2 in the housing 1, with the shuttle slid out from the housing 1 to better show the shuttle 2.

[0090] Figure 14 shows a top view of the cartridge 100 with the shuttle 2 in the housing 1 and with the dry reagent lid 19 attached. No top film is shown.

[0091] Figure 15 shows another embodiment of the shuttle 2 with an alternative layout of reagent chambers 4 and ports 5. Additional chambers 4 and ports 5 have been added to increase the number of steps that can be automated in a single cartridge 100.

[0092] Figure 16 shows an alternative configuration of the shuttle 2 with additional reaction chambers 10 in the base 2c.

[0093] Figure 17 shows an alternative configuration of the cartridge housing 1.

[0094] Figure 18 shows an embodiment of a device / system 200 to run the process in the cartridge 100. In some embodiments, the cartridge 100 is located under a lid 18 in the top rear of the device 200. A touchscreen display 17 sits forward of the cartridge bay and angles downward. A motor 16 is shown engaging the end of the shuttle in the cartridge, which moves the shuttle 2 in the housing 1 of the cartridge 100. Two plunger motors 15 are shown which move the plungers in the syringe barrels 3 of the housing 1. A sonicator 20 is shown coming up against the housing 1 of the cartridge 100 to be able to sonicate samples in the cartridge 100.

[0095] Figure 19 shows the packaging for a cartridge 100 with a lid 19 holding dried reagents. In some embodiments, a plastic tray 21, preferably a vacuum- formed tray, has recessed areas to hold the cartridge 100, the lid 19 with dry reagents, and the sample cap. The33156225.1 19lid with dry reagents is packaged with a desiccant 25 to keep the dried reagents stable. The cartridge assembly 100 with the housing 1 and shuttle 2 (including heat seals) is packaged separately from the lid 19 with dried reagents. A film seal 22 is attached to the top of the tray.

[0096] Figure 20 shows an embodiment of a cartridge shuttle 2 configured for automation of an agnostic diagnostic cartridge to prepare pathogen nucleic acids from patient samples for sequencing. A round sample chamber 23 holds the patient sample. Additional reagent chambers 4 have been incorporated to enable pathogen enrichment and non-specific nucleic acid amplification to improve sensitivity. A larger waste chamber 24 has also been designed to handle the additional reagents needed.

[0097] Figure 21 is a top view of the shuttle 2 of Figure 20.

[0098] Figure 22 shows the base 2c of the shuttle 2 of Figure 20. Multiple reaction chambers 10 have been included for multiple magnetic bead clean-up and thermal incubation steps.

[0099] Figure 23 shows the shuttle 2 of Figure 21 in the housing 1 of Figure 20.[000100] Figure 24 shows the shuttle 2 of Figure 21 in the housing 1 of Figure 20 with a sample cap 25 and reagent storage lid 19.[000101] The shuttle 2 and housing 1 are preferentially made of an injection molded plastic. An over-molded compliant material may also be used on the sealing surfaces of the shuttle 2 and or housing 1. Ports are located on the sealing surface to allow the movement of fluids between the shuttle 2 and the housing 1. The upper side of the shuttle 2 has multiple chambers 4 for fluids. The bottom 2c of the shuttle 2 has channels 9 for moving fluids and reaction chambers 4 which can interface with the sonicator, magnets, and heaters of the reader. Some fluid chambers 4 may be open on the bottom 2c of the shuttle 2. Chambers 4 open on the base 2c of the shuttle 2 will also connect to a channel 9 in the base of the shuttle 2 to allow a fluidic connection to the ports 5. The top and bottom of the shuttle 2 are sealed with a plastic film. This will contain reagents in the shuttle 2. A film using aluminum or other material to prevent water vapor transfer can be used to improve the shelf life of the disposable and its reagents.[000102] The housing 1 comprises a rail with two planer surfaces la, lb that contact the two planer surfaces 2a, 2b on opposite sides of the shuttle 2. The planer surfaces la, lb contacting the shuttle 2 may be covered with a compliant coating to enhance sealing between the shuttle 2 and the rail.[000103] The housing also comprises one or more syringe barrels 3. A syringe comprises a cylindrical barrel and a plunger. An external motor 15 (Figure 18) engages the33156225.1 20plunger to move fluids with the barrel 3. The plunger pushes or pulls fluids into or out of chambers 4 or channels 9 in which it is fluidically connected. In a preferred embodiment of the disclosure, the syringe is driven by a linear shaft motor 15 (Figure 18). Each syringe barrel is connected to a port that opens out through one of the planar surfaces la, lb of the rail. When a port for the syringe barrel 3 is aligned with a port 5 from the shuttle 2, it forms a path for fluids or air to move from the syringe to the reaction chamber 4 or channel 9 in the shuttle 2. The syringe barrel 3 preferably has a lip or cap to retain the plunger in the barrel as the drive shaft is removed.[000104] In some embodiments, the housing 1 also comprises chambers for the storage of reagents. In particular, the housing contains the dried reagents in a preferred embodiment of the disclosure. The housing may also contain additional chambers to hold waste, desalting columns, and reaction or detection chambers.[000105] In a preferred embodiment of the disclosure, the housing 1 has at least two syringe barrels with different internal volumes. In many processes, different volumes of reagents are handled. A large syringe has a larger variability of volumes moved and results in significant inefficiency when small volumes are handled. For example, in sample processing and library preparation for DNA sequencing, larger volumes are needed for sample processing and smaller volumes for library preparation. In a preferred embodiment of the disclosure, the larger syringe has a volume greater than 100 micro liters. In a more preferred embodiment, the volume of the larger syringe is greater than half a milliliter. A volume of one milliliter is preferred. A larger volume allows for a larger sample and therefore, more target to be analyzed. However, as volume increases, the size of the cartridge 100 and costs will increase and may be considered.[000106] Ports 5 for channels 9 for chambers 4, such as reaction chambers, waste chambers, or venting other than liquid chambers, could be located on either face of the shuttle 2. The linear configuration allows for a step-wise progression of handling steps to occur as the shuttle 2 moves relative to the housing 1. Minimizing movement backward will prevent crosscontamination of reagents during the process being run in the cartridge 100. However, the shuttle 2 may be moved in either direction relative to the housing 1 during a procedure.[000107] For library preparation, multiple enzymatic steps may be required. Smaller volumes are desired due to the cost of the enzymes, so many enzymatic reactions are carried out in volumes of 10-50 microliters. Sample sizes need to be sufficiently large to ensure that a target will be present. In a preferred embodiment, the volume of the first syringe barrel33156225.1 213 a is 100 micro liters or greater, and the second syringe barrel 3b volume is 100 micro liters or less. Preferably, the volume of the smaller syringe barrel 3b is less than 30 microliters.[000108] In some embodiments, the cartridge 100 with the sample is inserted into the reader or system 200. The processing unit has a motor 16 that moves the shuttle 2 along the rail in the housing 1. Preferably, the motor 16 is a linear drive motor. The system 200 also has one or more linear actuators 15 to move the plungers in the syringe barrels 3. The system 200 may also have one or more magnets, heaters (including thermocyclers), and sonicators 20. The magnets, heaters, and sonicators are positioned to contact the cartridge 100. The magnets, heaters, or sonicators 20 may be actuated to come into contact with a portion of the cartridge 100 when needed. The system 200 also has electronics to control the motors, heaters, sonicators 20 and any active actuating mechanisms.[000109] The shuttle 2 is preferentially made of an injection molded plastic. An over-molded compliant material may also be used on the sealing surfaces 2a, 2b of the shuttle 2. Ports 5 are located on the sealing surface to allow the movement of fluids between the shuttle 2 and the housing 1. The upper side of the shuttle 2 has multiple chambers 4 for fluids. The bottom of the shuttle 2 has channels 9 for moving fluids and reaction chambers 4 which can interface with the sonicator 20, magnets, and heaters of the reader 200. Some fluid chambers 4 may be open on the bottom 2c of the shuttle 2. Chambers 4 open on the base 2c of the shuttle 2 will also connect to a channel 9 in the base 2c of the shuttle 2 to allow a fluidic connection to the ports 5. The top and bottom of the shuttle 2 are sealed with a plastic film, preferably a heat-activated sealing film. This will contain reagents in the shuttle 2. A film using aluminum or other material to prevent water vapor transfer can be used to improve the shelflife of the disposable and its reagents.[000110] The housing 1 comprises a rail with two planer surfaces la, lb that contact the two planer surfaces 2a, 2b on opposite sides of the shuttle 2. The planer surfaces la, lb contacting the shuttle 2 may be covered with a compliant coating to enhance sealing between the shuttle 2 and the rail 1.[000111] The housing 1 also comprises one or more syringe barrels 3. A syringe barrel 3 comprises a cylindrical barrel and a plunger. An external motor 15 engages the plunger to move fluids with the barrel 3. The plunger pushes or pulls fluids into or out of chambers 4 or channels9 in which it is fluidically connected. In a preferred embodiment of the disclosure, the syringe is driven by a linear shaft motor 15. Each syringe barrel 3 is connected to a port that opens out through one of the planar surfaces la, lb of the rail. When a port for the syringe barrel 3 is aligned with a port 5 from the shuttle 2, it forms a path for fluids or air to move from33156225.1 22the syringe barrel 3 to the reaction chamber 4 or channel 9 in the shuttle 2. The syringe barrel 3 preferably has a lip to retain the plunger in the barrel as the drive shaft is removed.[000112] The housing 1 also comprises chambers for the storage of reagents. In particular, in one embodiment, the housing 1 contains the dried reagents in a preferred embodiment of the disclosure. The housing may also contain additional chambers to hold waste, desalting columns, and reaction or detection chambers.[000113] For sample processing, the sample chamber can be in the shuttle 2 or the housing 1. In a preferred embodiment, the sample chamber is in the shuttle 2. The sample chamber is preferentially cylindrical with an internal curved bottom surface with a point of the chamber having the lowest surface and sloping up to the sides. A port is located at the low point and is fluidically connected to a syringe. This will allow the particulates to slide down to the center for better mixing when sonication is used. The sample chamber in the shuttle 2 should be positioned over the ultrasonic head in the reader to disrupt and or mix the sample with lysis buffer and magnetic particles.[000114] In one embodiment the system 200 has a disruptor. The disruptor is capable of mixing or breaking down the fluids contained in the reservoirs by applying an ultrasonic force. A preferred disruptor is a piezoelectric driven sonicating horn. In one embodiment of the disclosure, beads are in the disrupting chamber or reservoir to assist in mixing fluids or breaking down samples. The sonicator 20 applies ultrasonic energy, causing the beads to become excited and move through the fluid. In one embodiment a magnet is utilized to generate a magnetic field. The magnet can pull or push magnetic particles in a chamber.[000115] A basic sample lysis procedure would be to introduce the sample into the sample chamber. Add lysis buffer to the sample. Lysis buffers may contain detergents, salts, buffers, and chaotropic agents. A preferred chaotropic agent is guanidine hydrochloride or guanidine thiocyanate to disrupt proteins. Chaotropic agents are particularly useful in the disruption of viral protein coats. Magnetic particles are also added to the sample. Commercially available magnetic particles are available with a variety of binding properties. The particles are paramagnetic, and magnetic in the presence of a magnetic field. Nucleic acids bind to the particles under specific conditions and can be selectively pulled out of the sample mixture. The mixture of the sample, lysis buffer, and magnetic particles is designed to enable efficient binding of the target material to the magnetic particles. Once the particles are mixed with the sample and lysis buffer, the sample is passed over a magnet to collect the particles with the bound nucleic acids. The particles are washed to remove residual material.33156225.1 23[000116] Preferably, the pellet of magnetic particles is resuspended and drawn back down with the magnet. Resuspension can be carried out by moving the wash buffer back and forth with the syringe over the pellet or with the disruptor. After washing, air can be pushed over the pellet to remove the wash buffer. An extraction buffer is then put into the chamber, the pellet resuspended and again pulled down. The extraction buffer is then removed with the nucleic acids. Disruption of the magnetic beads is important to effectively remove inhibitory compounds that otherwise could be trapped within the pellet. In addition, disruption will improve the elution of the target-bound molecules from the pellet. Without disruption, some of the target material can remain caught within the pellet, lowering the overall efficiency of the process.[000117] In one embodiment, liquid reagents are stored in one component, preferably the shuttle 2 and dry reagents are stored in the other component, preferably the housing 1. In one embodiment of the disclosure, the shuttle 2 is filled with liquid all the required reagents and stored separately from the housing 1. For storage, the ports 5 on the shuttle 2 can be covered with a removable film to seal the shuttle 2 while it is outside the housing 1. The film should cover both the fluid transfer port and the vent port for the chambers 4. A snap mechanism can be used to lock the components together. Before use, the film can be removed and then the shuttle 2 inserted and snapped into the housing 1. This allows for liquid and dry reagents to be stored separately to maximize shelf life. Each component would be separately packaged. In a preferred embodiment, each component would be packaged in a plastic pouch with an aluminum foil layer or other water barrier incorporated to keep water transfer to a minimum during storage. By separating wet and dry reagents, it is possible to ensure the stability of sensitive reagents such as enzymes to remain active for one or more years without refrigeration.[000118] Preferentially, all ports 5 on the shuttle 2 connecting to a chamber 4 holding a liquid reagent would be located on the same face of the shuttle 4, including both access ports 5 for the fluid and vent ports for each chamber. This would allow for only one removable seal to contain all liquid reagents.[000119] During storage of the shuttle separately from the housing 1, a seal is applied to cover ports 5 including vent ports on one face of the shuttle 2. After removal of the seal, keep the ports on the upwardly facing side of the shuttle 2 and insert the shuttle 2 into the housing 1 keeping the ports 5 facing upwards until the shuttle 2 is snapped into the housing 2.[000120] The reader can provide additional pressure on the shuttle 2 to maintain an effective seal with the shuttle 2. In one embodiment, a post If with a taper to match the33156225.1 24taper on the outside of the rail 1 a protrudes through the base 1 of the cartridge and pushes against the outside wall la, lb of the rail. These posts prevent the rail walls from splaying outwards and maintain pressure on the shuttle. In addition, the reader can push down the top of the cartridge to maintain the pressure of the shuttle walls against the inside surfaces of the rail.[000121] The present disclosure also provides chambers for restraining freeze-dried pellets without the need for a restraining component. In the Cepheid cartridge, a plastic retaining bed is pushed in above the dried pellets. This keeps the pellet in the bottom of the chamber accessible by fluids that are moved into the chamber during operation. Without restraint, the pellet can bounce around the chamber during handling and can be broken apart into a powder that can stick to surfaces. Some of the powder may be above the fluid fill line, resulting in variability in reagent concentrations. To eliminate the need for a restraining component, a novel chamber design is provided. Chambers comprise side walls and a bottom or top wall. Due to molding constraints only, a bottom or top wall can be molded. The final wall of the chamber is formed using a film seal over the top or bottom of the insert or housing. For purposes of holding the dried pellet, the chamber is molded from the bottom with a top wall to hold the pellet against the film seal, which is the bottom wall, where the top wall is set high enough to hold the dried reagent against the bottom seal. A vent port is formed in the top wall to allow fluid to be pushed into the chamber. Alternatively, the top wall may be a grid or have multiple openings. The chamber is fluidically connected to the syringe via a channel in the base of the shuttle and ports up to and across to the syringe.[000122] The shuttle 2 is engaged by a drive motor 16 in the system 200. Once snapped into the cartridge 100 the motor 16 engages the shuttle 2 and then homes itself before beginning the protocol that is programmed. Preferably, the shuttle movement is minimized to prevent cross-contamination of reagents. In a preferred mode, the shuttle 2 moves in the same direction that it will during the protocol and homing to maintain the linear movement of the shuttle 2 from one end of the housing 1 rail to the other. The shuttle 2 can be moved back to an earlier position if needed and in some cases may be moved repeatedly in the back direction. Preferably, any backward motions will be for a limited distance. It is most desired to prevent reagents that could inhibit other steps from crossing by ports for those reagents used later in the process.[000123] As the shuttle 2 moves, some components in the reader 200 will preferably be actuated to contact the base of the shuttle 2 or housing 1 in certain positions. Those components include magnets, heaters, and ultrasonic heads. Actuating these33156225.1 25components minimize wear on the components in the reader and minimize the potential of tearing the film seal on the shuttle 2 as it moves. Each component can be actuated actively with a motor drive. A preferred method would be passive. In that embodiment, actuated components would be spring-loaded to push up against the base of the shuttle 2 or housing 1. Furthermore, spring-loaded components can make firmer contact with the base of the cartridge component, improving heat transfer, transfer of sonic energy, or magnetic pull.[000124] Features on the bottom 2c of the shuttle 2 can hold the spring-loaded components at a level such that they do not contact the base 2c of the shuttle 2 or housing 1 except when the shuttle 2 is in a position where a step will occur requiring that component. At that point, the feature would allow the spring to push the component up against the base of the shuttle 2 or housing 1. For example, a component is connected to a bar that rides along the base 2c of the shuttle 2. As the shuttle 2 moves the feature connected to the bar would have space for the bar to move upwards when it is in the position where contact is desired with the base 2c of the shuttle 2. Such components include, but are not limited to heaters, piezoelectric heating and cooling, magnets, and sonicators.[000125] The linear design of the shuttle 2 provides several advantages over prior art rotary cartridges, which have been discussed in the background section. In a rotor valve, ports are limited to a segment of the circumference of the rotor due to the need for the pins to be parallel to one another in the molding process. As the number of chambers is increased, the diameter of the rotor may also increase and may result in dead space in the interior of the rotor. A shuttle length can be modified without resulting in major changes to the reader if additional space is open at the ends of the disposable cartridge in the unit. The rotor designs of both the Cepheid and INT cartridges also have potential risks due to the interaction of incompatible reagent risks due to the rotor sweeping ports of chambers containing reagents past the syringe or other ports in the housing of the cartridge. Optimizing the cartridge layout can minimize cross-contamination, but for complicated processes, this is not always possible. A linear layout minimizes the reverse motion needed to access the chamber or ports.[000126] The shuttle 2 faces with ports 5 may seal against the faces of the rail la, lb such that the ports may align. In this position, the two components 1, 2 may make a seal to prevent liquid from leaking. When the shuttle 2 is snapped into the housing 1, the snap mechanism should hold the parts together with pressure on the sealing surface. In addition to the snap mechanism providing pressure on the seal, the lid of the device or other mechanical force from the device may be used to maintain pressure on the seal to prevent leakage.33156225.1 26[000127] For a system to be readily utilized in resource-limited environments, there is a need to optimize the shelf life of the disposable cartridge 100. For example, the Cepheid Xpert MTB / RIF cartridges are the most commonly utilized test for tuberculosis. Tuberculosis is primarily a disease occurring in resource-limited areas. However, the Cepheid test cartridge with reagents needs to be stored at 2–28 °C, following the manufacturer's recommendations. The manufacturer states that the cartridges are stable if kept at 2-45 °C for less than six weeks at 75% relative humidity. The cartridges are bulky when packed and require substantial storage space. An average household refrigerator can hold the supplies needed for two may pose challenges in relatively inaccessible areas that have complex customs clearance procedures. Planning is essential to prevent stock-outs and cartridges from expiring before they are used; orders should be based on the number of cartridges that have been used, the shelf-life of the cartridges, the lead time for delivery, and the expected time needed to clear customs. A need exists for a disposable cartridge that is stable without refrigeration for a longer period. Preferably, the cartridge and reagents would be stable for a minimum of three months without refrigeration. More preferred, the cartridge 100 and reagents would be stable for a minimum of six months or even twelve months without refrigeration.[000128] To ensure the stability of lyophilized reagents in a disposable, the current invention provides that the wet and dry reagents are stored separately in different components of a disposable where the components 1, 2 are snapped together before use. This can be done in several formats but the wet reagents should be stored separately from the dry reagents. Freeze-dried reagents are extremely hydroscopic and enzyme activity can be seriously degraded with small amounts of water.[000129] In a preferred embodiment of the disclosure, the housing 1 contains all dry reagents and is packaged separately from the shuttle 2. The shuttle 2 would contain all wet reagents. To restrain the dry reagents to prevent them from moving during handling of the cartridge 100, which can result in the disruption of the dried material, the chambers holding the dried reagents may have a perforated barrier molded into the chamber. The dried material, preferably a pellet, is placed in the chamber with the shuttle 2 inverted so that the pellet rests on the perforated barrier. Once all dry reagents are located in the appropriate chambers 4, the base 2c of the shuttle 2 is sealed with a film. The film may be attached by any compatible method, not limited to welding, heat sealing, or an adhesive. The dried material is then held between the film and the barrier to limit its movement during shipping and handling. A fluidic channel is formed between the molded shuttle 2 and the film to allow fluid to be introduced33156225.1 27into the chamber 4 to dissolve the dry reagent. A vent port 4d is located above the perforated barrier to allow air to move in and out of the chamber 4 as fluid is moved.[000130] In an alternative embodiment, the dry reagents are incorporated into a lid 19 that interfaces with the shuttle 2. The disposable is stored in two parts and combined just before use. This allows unstable reagents to be stored dry, refrigerated or frozen while the remainder of the cartridge with stable components can be stored under ambient conditions. In biological processes, some reagents, especially enzymes, are unstable at room temperature or higher temperatures and require freezing or refrigeration to provide a useful shelf life. This creates significant additional costs in shipping and storage.[000131] In a preferred embodiment, a lid which holds the unstable reagents. The lid 19 has a cap and one or more reagent- containing features 13. When inserted into a cartridge, the cap 19 seals the top of the reagent chambers 4 that receive the unstable reagents. When inserted, the lid 19 aligns the reagent features into corresponding reagent chambers 4. For freeze-dried reagents, the lid 19 can be stored in a separate packaging without any liquids. For liquid and frozen reagents, the lid 19 can be stored separately from the remainder of the cartridge, minimizing the size of the components that require cold-storage.[000132] In one embodiment, such as show in Figures 7 and 8, the reagentcontaining feature is a pin 13. A dried or frozen regent pellet 14 may be attached to the pin 13. Alternatively, the pin is hollow, and the dried, frozen or liquid reagent is contained within the pin. In another embodiment, the regent-containing feature is sealed with a film. The film may be a flat film over a chamber or maybe a film that forms a blister on a flat surface. When the feature is inserted into a reagent chamber, the film is ruptured to release the reagent or to make the dried reagent accessible to a buffer or water. Preferably the reagent-containing region is ruptured upon insertion into the reagent chamber. Alternatively, the reagent-containing feature has a removable film seal that is removed prior to insertion of the lid into the cartridge. In yet another embodiment, plungers or pins are seated in the top of the reagent- containing feature and are depressed to push the reagent out of the reagent-containing feature when or after it is inserted into the reagent chamber. In one variation, a freeze-dried reagent 14 is attached to or contained in the reagent- containing feature 13a. A buffer or water can be introduced into the reagent chamber to rehydrate the freeze-dried reagent contained in or on the reagent-containing feature of the lid.[000133] Dry reagents are located on features on the bottom side of the lid 19 which locate the dry reagents in the appropriate chambers when the lid 19 is placed on the shuttle 2. Preferentially, such a lid has features that ensure alignment with the appropriate33156225.1 28chambers 4. The lid 19 with dry reagents would be packaged separately from the remainder of the cartridge 100 in a watertight package. No fluids would be present in the lid package, providing a dry environment for long-term storage.[000134] Alternatively, the lid 19 can hold all of the wet enzyme solutions so that they can be stored frozen. The lid 19 would then introduce the reagents into the appropriate chambers of the cartridge 100 where they can be thawed before use. This approach requires cold storage, but a smaller lid 19 component can be stored frozen with the bulk of the cartridge 100 stored separately without the need for cold-storage for the larger cartridge.[000135] The invention provides a disposable cartridge 100 for chemical or biological analysis, comprising a first component 1 that holds lyophilized reagents and no liquid reagents, a second component 2 that holds all liquid reagents, and wherein when the two components are assembled into a single cartridge 100, the liquid, and dry reagents are connected fluidically. The disposable cartridge 100 can further comprise that the first and second components 1, 2 have multiple chambers for reagents, and channels or ports to move reagents within each component and between the two components.[000136] Many biochemical processes can be automated within the cartridge, such as biological sample processing, nucleic acid sequencing, and diagnostics. Although the discussion refers to biological processes in more detail, the system and cartridge can automate any chemical analysis procedures as well.[000137] Sample preparation is required for many biological analytical techniques. Target material, which can include nucleic acids, proteins, or other molecules, often may be separated and concentrated from the original sample before analysis.[000138] For many analytical techniques, isolation of the target material is required. For nucleic acid testing or sequencing, this entails isolation of the nucleic acids from the sample. The ability to incorporate sample processing in this cartridge is a key differentiator from other technologies. For nucleic acid diagnostics or sequencing, nucleic acids may be released from the cells, viruses, or carrier material. Disruption of the sample can be carried out using chemical, enzymatic or mechanical disruption techniques alone or in any combination. A preferred method of sample disruption utilizes a chaotropic salt. Preferably the chaotropic salt is a guanidine compound at high concentration. If needed, sonication and / or detergents can also be used to facilitate sample disruption. In the case of tissue samples, whole insects or other larger structures, bead beating may be used with sonication.[000139] A common chemical process for the disruption of cells and viruses is treatment with guanidine hydrochloride. Guanidine disrupts protein folding and is especially33156225.1 29useful in the disruption of viral protein coats. Detergents are often used to disrupt membranes. A combination of both can be used for complex samples. Ionic strength can also be used to break open cells.[000140] Many samples will also require mechanical force for disruption. Spores are small single cellular units that have a very durable coat. Larger samples such as tissue or even whole organism, such as mosquitoes can also be processed, but need mechanical disruption to break apart the sample. Disruption can be driven using sonication. Ultrasonic energy can be introduced into a sample chamber. Ultrasonics results in cavitation which creates pulses of energy that can break apart the sample. If needed, beads may be used in the sample. During sonication, the beads are agitated and move through the sample to crush and grind the material. Ultrasonication can disrupt cells, tissue, and other materials. Sonication can be done with or without the use of beads or other materials to beat or shear the sample. Sonication can also be used to heat samples or simply to mix reagents. US Pat. No. 6,819,027 to Saraf discloses an ultrasonic control system that can be used with the cartridge of the present disclosure. Patent ‘027 provides a method to maximize efficiency by dynamically detecting and maintaining peak operational resonance frequency.[000141] Sonication may also be used to heat or mix the sample. Input energy can be varied to control temperature. Mixing may be used, especially with magnetic beads that may not disperse after being collected. Disruption may facilitate washing the beads to remove inhibitory materials.[000142] Once a sample is disrupted, the target material may need to be cleaned and or concentrated before analysis. A readily available approach for cleaning and concentrating materials is the use of magnetic separation. Target analytes are bound to magnetic particles. The magnetic particles are pulled down over a magnet. The magnet can be either a permanent magnet, in which case the sample is moved over the magnet, or an electromagnet, where the magnet is turned off and on as needed. An advantage of purification methods involving the binding of nucleic acid to a solid surface is the ability to wash the bound material using solutions that retain the bound molecules on the solid surface while removing other non-related components, thus resulting in isolation and purification of the polynucleotides of interest from the sample solution. After the particles are collected, they may be washed to remove residual material. During the wash steps, the particles may be resuspended in the wash buffer. After washing, the analyte is eluted from the particles. This process provides the added capability to pull down target analytes from a larger sample and to elute in a small volume. Glass-coated magnetically responsive particles have also been33156225.1 30developed for nucleic acid isolation. Such particles bind directly or indirectly to nucleic acids. An example of a system that utilizes direct binding includes magnetically responsive porous glass beads (e.g., U. S. Pat. Nos. 4,233,169; 4,395,271; 4,297,337).[000143] A common purification technique uses filters to remove particles above a certain size. In addition, some filters can selectively bind an analyte. Filter material can be welded to the plastic surrounding the opening to a port. The sample can be passed through a filter under certain binding conditions, the filter washed and then the analyte eluted from the filter. Alternatively, analytes can be purified using column chromatography. Filters and / or columns may be incorporated into the fluidics of the disposable cartridge.Columns may be used to remove contaminants that may interfere with later steps. In particular, enzymatic steps are subject to inhibition by a variety of compounds. In one embodiment, a size exclusion column can be used to remove small molecule inhibitors. Small molecules are caught in pores in the matrix while the larger nucleic acids or proteins pass through the column. Such a column could remove 90-95% of the inhibitory compound from the sample. If needed, more than one column may be used to separate based on different techniques or multiple columns may be used in series to further decrease inhibitor concentrations.[000144] U. S. Patent 8,663,918 describes the incorporation of a column containing a matrix, such as a desalting matrix, in a disposable cartridge. In that design, a column matrix is held in a chamber with a fluid opening in the base of the chamber. The fluid is pushed up through the matrix and over a wall, separating it from an adjoining overflow chamber. The matrix is held in a tubular column with seals on the top and bottom. An additional cap is placed on the top of the column which directs the fluid over the side wall and into the adjoining chamber.[000145] Another embodiment of the current invention provides a design for incorporating a column matrix in a cartridge, without the need for additional plastic parts. A channel in the slide or housing having side walls and a roof or floor formed in that part, with two ports located at each end of the channel, the first part fluidically connected to the syringe and the second port fluidically connected to a chamber to collect the fluid after passage through the column. Filter membranes are placed over each port to restrain the column matrix material when it is rehydrated. The chamber has the open side sealed with a plastic seal, preferably a heat seal, to the top of the walls of the chamber. The column matrix can be dried, set in the channel, and then sealed into the cartridge.33156225.1 31[000146] In the case of next-generation sequencing using nanopores, the quality of the nucleic acids is important. DNA should be double-stranded. The nucleic acids should contain minimal insoluble material and not be colored or cloudy. Proteins should be removed, potentially with the generous use of proteinase K. It should be protected from DNA-damaging conditions such as intercalating fluorescent dyes or UV radiation. It should not contain chelating agents (e.g., EDTA), divalent metal cations (like Mg 2+), denaturants (like guanidinium salts, phenol), or detergents (like SDS, Triton-X100). It should not contain carryover contamination from the starting organism / tissue (e.g., heme, humic acid, chitin, polysaccharides, polyphenols, etc.)[000147] Embodiments of the cartridge 100 of the present disclosure may also incorporate a detection mechanism. Detection and / or quantitation may be carried out using optical or electrical methods. Optical methods are often used with dyes or fluorescent markers incorporated into the target material or in a probe that is hybridized to the target material. Optical testing can be used to verify the quantity and quality of the nucleic acids. Optical detection processes can measure absorbance at one or more wavelengths or fluorescence.[000148] In a preferred embodiment, a flow cell has an optically clear wall to allow for optical sensing of the product. In an alternative embodiment, the flow cell has an electrical sensor as one wall of the cell, where electrical sensors have a connection external to the disposable to make contact electrically with the reader. Electrical detection may be carried out on sensors as in US Patent no. 7,851,149 to Braun et al..[000149] In some applications, such as agnostic diagnostics for infectious disease, a two-step lysis protocol can be employed. To deplete host cell nucleic acids, lysing conditions that will disrupt host cells but not pathogenic viruses, bacteria and / or parasites are used. One such approach that has been used widely is to treat the sample with a low-strength detergent such as saponin. The nucleic acids from the host cells are then digested with nucleases. The nucleases are inactivated and the nucleic acids are then isolated from the pathogens.[000150] Inactivation of the nucleases is necessary to prevent degradation of the pathogen NA in the next steps. Although many approaches have been used to inactivate nucleases, guanidine treatment is a preferred method and it is compatible with the plastics used in the cartridge. Guanidine is also very effective at breaking open bacteria and viruses in the same treatment step, further streamlining the process (Oberacker et al. PLoS Biol 17(1): e3000107 (2019). Competing systems struggle with eukaryotic pathogens and encapsulated viruses; the guanidine lysis can overcome these issues.33156225.1 32[000151] Guanidine is an efficient disrupter of protein folding and is effective at disrupting cells, bacteria, viruses and parasites. After the inactivation of the nucleases, the guanidine may be removed so it does not interfere with later enzymatic steps. The primary cleanup step that can be incorporated in the current cartridge is magnetic bead capture of the target material with washing. The system will bind the treated NA to magnetic particles, which are then collected and washed. Dispersing the pellet during washing using mixing or low-energy sonication will enable effective removal of the denaturant. In the process, nucleic acids are bound to the magnetic particles. The particles are then pulled down over a magnet and a wash buffer is flowed over the magnetic particles. The chamber 4 is then moved over a sonicator and a low level of energy is used to disrupt the pellet of magnetic particles and nucleic acids. The chamber 4 is then moved back over the magnet, the particles pelleted and then washed again. If needed, this process can be repeated. Disrupting and repelleting the magnetic particles allows for contaminants caught in the pellet to be released and washed away. The nucleic acids are then eluted from the magnetic particles using an elution buffer. Further reduction of small molecule inhibitors can be carried out by passing the eluted material through a column matrix, such as a desalting or nucleic acid binding matrix.[000152] The preferred differential lysis approach in an automated cartridge 100 is to use a mild detergent and mixing or low-energy sonication. These disruption approaches provide a simpler process than typical column and centrifuge-based protocols and were demonstrated by the team previously for automated PCR (unpublished data). Mild detergents can lyse host cells without inactivating introduced nucleases, such as DNase and / or RNase; these nucleases can thus be added directly to the lysed sample mixture without the need for an interim clean-up step. (Wu, et al. Respir Res 22, 310 (2021)). Non-denaturing detergents such as Saponin, Triton, Tween, and NP40 at concentrations that disrupt host cells while leaving pathogens largely intact (Quadt, et al. Parasitol Res 119, 4297-4302 (2020)). For more difficult samples, such as solid tissue or mycobacterium samples, we will evaluate sonication alone or in combination with detergents and more durable nucleases. Alternate pathogen enrichment methods are also available, including separation of methylated nucleic acids and CRISPER-based enrichment methods. (Thoendel, et al. Journal of Microbiological Methods, 127: 141-145 (2016)). Preferably, the conditions are optimized for the recovery of long NA (greater than 400 base pairs [bp]) for workflows with nanopore sequencers. In a more preferred embodiment, the fragments are more than 2,000 base pairs in length.[000153] Often, to analyze RNA sequences, the RNA is first converted to DNA via reverse transcription. This involves mixing the purified nucleic acids with a reverse33156225.1 33transcription mix, including a reverse transcriptase, and incubating the sample and enzyme for a period of time to produce DNA copies of the RNA. However, more recently, Oxford Nanopore has demonstrated the ability to directly sequence RNA using its nanopore technology. A key requirement for handling RNA is to prevent RNA from degradation by RNases. This cartridge design facilitates the protection of RNA by allowing for the use of procedures or chemicals that can inactivate nucleases and then keeping the RNA sealed in the cartridge and protected from external RNase contamination during the remainder of the protocol.[000154] Many protocols use an amplification step, either PCR, reverse transcriptase PCR, or isothermal amplification to improve sensitivity. Applications of nucleic acid amplification methods include the detection of rare cells, pathogens, altered gene expression in malignancy, and the like. Nucleic acid amplification is potentially useful for both qualitative analyses, such as the detection of nucleic acids present in low levels, as well as the quantification of expressed genes. The latter is particularly useful for the assessment of pathogenic sequences as well as for the determination of gene multiplication or deletion associated with malignant cell transformation. A number of methods for the amplification of nucleic acids have been described, e.g., exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification. An example of exponential nucleic acid amplification method is polymerase chain reaction (PCR) which has been disclosed in numerous publications, (see Mullis et al. Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, Methods in Molecular Biology, White, B. A., ed., vol. 67 (1998); Mullis EP 201,184; Mullis et al., U. S. Pat. Nos. 4,582,788 and 4,683,195; Erlich et al., EP 50,424, EP 84,796, EP 258,017, EP 237,362; and Saiki R. et al., U. S. Pat. No. 4,683,194). Linked linear amplification is disclosed by Wallace et al. in U. S. Pat. No. 6,027,923. Examples of ligation-based amplification are the ligation amplification reaction (LAR), disclosed by Wu et al. in Genomics 4:560 (1989) and the ligase chain reaction, disclosed in EP Application No. 0320308 B 1. Hampson et al. (Nucl. Acids Res. 24(23):4832-4835, 1996) describe a directional random oligonucleotide primed (DROP) method for use as part of global PCR amplification.[000155] In a typical PCR reaction, template DNA sequences lying between the ends of two defined oligonucleotide primers can be amplified in 1 to 2 hours. Three sequential steps are normally employed: (i) double-stranded DNA is denatured (D) to a single-stranded form at a high temperature (90° C. to 95° C.), (ii) the resulting single-stranded DN A strands are annealed (A) to oligonucleotide primers at ~40° C. to 60° C., and (iii) primer-template33156225.1 34complexes are elongated (E) using a thermostable DNA polymerase such as Thermus aquaticus (Taq) Polymerase at ~72° C.[000156] One cycle of these three steps (denaturation / annealing / elongation) results in a two-fold amplification of a DNA fragment whose 5' and 3' ends are defined by sequence-specific annealing of the oligonucleotide primers to the DNA template. Therefore, 30 PCR cycles result in a 230-fold (~106-fold) amplification of a particular DNA sequence.[000157] Isothermal target amplification methods include transcription-based amplification methods, in which an RNA polymerase promoter sequence is incorporated into primer extension products at an early stage of the amplification (WO 89 / 01050), and a target sequence or its complement is amplified by transcription and digestion of the RNA strand in a DNA / RNA hybrid intermediate. (See, for example, U. S. Pat. Nos. 5,169,766 and 4,786,600). These methods include transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), Nucleic Acid Sequence Based Amplification (NASBA), and variations thereof. (See Guatelli et al. Proc. Natl. Acad. Sci. U. S. A. 87:1874-1878 (1990); U. S. Pat. Nos.5,766,849 (TMA); and 5,654,142 (NASBA)).[000158] Other approaches exist which can also be incorporated into a disposable cartridge. For example, MICROBEnrich employs hybridization capture technology to remove human, mouse, and rat RNA (both mRNA and rRNA) from complex host-bacterial RNA populations, leaving behind enriched microbial total RNA. In the first step of the MICROBEnrich procedure, host-bacterial total RNA is incubated with an optimized mixture of capture oligonucleotides that bind to the mammalian 18S and 28S rRNAs and polyadenylated RNAs. Next, the rRNA / oligo nucleotide hybrids and all polyadenylated mRNAs are removed from the mixture with oligonucleotide-derivatized magnetic beads. After magnetic separation, the bacterial RNA remains in the supernatant and can be precipitated with ethanol.[000159] DNA sequencing technology is rapidly evolving. Next Generation Sequencing (NGS) technologies have greatly improved DNA sequencing capacity, speed and lowered costs dramatically. The current invention can automate the multiple steps of sample handling and library preparation needed before nucleic acid sequencing. Illumina is the leading sequencing technology company utilizing a sequencing by synthesis approach. However, many different technologies are in the market or in development. The current invention can run a wide variety of sample and library preparation protocols tailored for any of these technologies.[000160] The preferred sequencing technology to combine with the current invention is nanopore sequencing. Nanopore sequencers are smaller, easier to use, and lower33156225.1 35cost than the alternatives. Combining nanopore sequencing with the current invention provides an end-to-end solution that is fully automated, low-cost, and easy to use. In a preferred embodiment of the disclosure, a nanopore sequencing flow cell is built into the cartridge to provide a complete sequencing solution.[000161] After amplification, the nucleic acid library may be prepared for sequencing. This entails several steps, including end preparation and nick repair, ligation of adapters, and a determination of the concentration of the product. Numerous processes and kits are available for library preparation, they can be optimized for output to increase sensitivity, speed for rapid processing, increasing the yield of long nucleic acids for ultra-long reads, processing samples with ultra-low inputs and targeted sequencing. Nanopore sequencing also requires the attachment of a protein to facilitate feeding the strand through the pore. Embodiments of the current cartridge 100 can automate many of the library preparation protocols. The number of reagent reservoirs, reaction chambers, and clean-up steps needed will define the layout of the cartridge 100.[000162] Embodiments of the disclosed cartridge 100 can also incorporate a chamber for optical measurements. The optical chamber can be in the shuttle or in the housing. Preferentially, the optical chamber would be in the housing 1. Optical measurements can be made of many analytes, including nucleic acids, proteins, cells, viruses, or chemical compounds. Optical sensing can be colorimetric, including the use of dyes, a measurement of transmission or absorption, detection of particles in solution or analytes bound to a surface, or other optical measuring techniques. As an example, before sequencing, it may be desired to quantitate the nucleic product to load a known amount of material into the sequencing system. Based on the concentration, a buffer may be added to the sample to achieve the target concentration. Alternatively, an optical signal is quantitated during qPCR. In that case, the optical chamber may also be a chamber for one or more steps of thermocycling. The resulting signal is quantitated and used to calculate the nucleic acid concentration.[000163] The cartridge 100 design is adaptable so that workflows will produce optimized NA products for both short- and long-read sequencing. The cartridge 100 can incorporate unique reagents and steps such as adenylation or linker attachment, or targeted amplification of classes of NA. It can also include barcoding reagents for sequencing multiple samples thus decreasing costs and increasing throughput.[000164] A key concern with nanopore sequencing is accuracy. Barcoding technology can be used to increase accuracy. (Karst, S. M., Ziels, R. M., Kirkegaard, R. H. et al. High-accuracy long-read amplicon sequences using unique molecular identifiers with33156225.1 36Nanopore or PacBio sequencing. Nat Methods 18, 165–169 (2021). https: / / doi.org / 10.1038 / s41592-020-01041). This approach could be automated into thedisposable of the current invention, greatly improving the accuracy of a nanopore sequencer with barcoding.[000165] Barcoding can also be used to lower sequencing costs. If a barcoding step is carried out in a cartridge 100 and multiple cartridges have unique barcodes incorporated in the reaction mixtures, products from multiple cartridges can be pooled and run on a single sequencing reaction. The sequences can be sorted via the barcodes. This allows multiple sequences to be run on the same sequencing run, lowering sequencing costs.[000166] Agnostic diagnostic (one that detects any pathogen) capability can be a powerful tool to help real-time public health response. Next-generation sequencing (NGS) technologies can identify any pathogen present in a sample, including novel pathogens. This invention provides a process for nucleic acid preparation for nanopore sequencing, including pathogen enrichment and sample preparation. The process has been developed to be able to run on an automated system. In one approach, the process can be run on a robotic micro-titer plate system for high to moderate throughput. Alternatively, the process can be integrated into a single disposable cartridge which automates and standardizes all steps of the process. It is also able to be combined with library preparation in high or single-throughput automated systems.[000167] The key advantages of this approach are the following capabilities: • Lowering the sample -to-result time to under 24 hours• Reducing interference from host RNA and DNA• Minimizing the need for ancillary equipment and training[000168] The resulting material from this process can be used for diagnostics or nucleic acid sequencing. With variations of the library preparation process, the resulting nucleic acids can be run on a wide variety of sequencing systems, including traditional processes such as Maxim-Gilbert and high-throughput sequencing, which includes nextgeneration "short-read" and third-generation "long-read" sequencing methods. Sequencing applications include exome sequencing, genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChlP-sequencing), and epigenome characterization. Nanopore sequencing provides a unique advantage for point-of- care sequencing since it can be carried out in a low-cost portable unit, such as the Oxford Nanopore Minion.33156225.1 37[000169] As a first step, pathogen nucleic acids can be enriched relative to the host genetic material. Otherwise, the sequencing system will be overwhelmed with nucleic acids from the host preventing the collection of pathogen sequence information. Multiple processes are utilized in the lab to enrich pathogen materials. Several of these are capable of being incorporated into the current cartridge solution.[000170] One method to enrich pathogen nucleic acids is to selectively lyse host cells, degrade the host nucleic acids, inactivate the nucleases, and then lyse the pathogens and purify their nucleic acids. Such methods can increase the relative concentration of pathogen nucleic acids by 100-1,000 fold.[000171] Lysis of cells / viruses / other pathogens can be carried out via a variety of methods or combinations thereof, including heating, enzymatic treatment, chemical treatment, or mechanical disruption. Host cells, eukaryotic cells, are often easier to break open than bacteria, viruses, spores, and other pathogens. Preferably, the conditions used to lyse host cells or other sample cells will differentially break open those cells while leaving most pathogens intact. Previous studies have shown the effectiveness of differential lysis of host cells.[000172] A preferred approach for differential lysis in an automated cartridge would use a mild detergent and limited physical disruption via mixing or low-energy sonication. Such an approach provides for a simpler overall process flow. Nucleases can be added directly to the lysed sample mixture without the need for an interim clean-up step. Under mild detergent conditions, host cells can be lysed, but introduced nucleases will remain active. Some nucleases are also more resistant to inhibition by detergents and may be preferred. Saponin-based differential lysis procedures have been used to selectively reduce host nucleic acid concentrations. Wu, N., Ranjan, P., Tao, C. et al. Rapid identification of pathogens associated with ventilator-associated pneumonia by Nanopore sequencing. Respir Res 22, 310 (2021). Other detergents can be used at concentrations to disrupt host cells while leaving pathogens largely intact, such as Triton, Tween, and NP40. Zwitterionic detergents also are preferred. Denaturing detergents may also be used at lower concentrations. At higher concentrations, denaturing detergents can disrupt pathogens, so the concentration of denaturing detergents should be kept low.[000173] Once host cells are lysed, added nucleases, DNAase, and / or RNAase can be added to preferentially digest the available nucleic acids from the lysed host cells. Pathogenic nucleic acids will be protected since the pathogens largely remain intact. If the lysis treatment for the host cells results in a product that inhibits or inactivates the nucleases, then the sample will need to be cleaned before nuclease treatment.33156225.1 38[000174] After lysing the released nucleic acids, the sample needs to be treated to inactivate the nucleases to prevent degradation of the pathogen nucleic acids in the next steps. Multiple approaches have been used to inactive nucleases, including chemical treatment, heating, and enzymatic treatment. The preferred approach is to use a concentrated Guanidine solution. Guanidine treatment has long been a preferred method for nuclease inactivation. Other chemical treatments include the use of chloroform and / or phenol. This approach is very efficient but puts additional requirements on the materials used to assemble the cartridge. Guanidine also is very effective at breaking open bacteria and viruses in the same treatment step, further streamlining the process.[000175] An alternative approach to inactivate nucleases is to treat the mixture with EDTA.[000176] After chemical inactivation of the nucleases, it is essential to efficiently remove the chemical agent or it will interfere with later enzymatic steps in library preparation and sequencing. To remove guanidine or another inactivating agent, the treated nucleic acids can be bound to magnetic particles, collected, and washed. Dispersing the pellet during washing, either using mixing or low-energy sonication will enable the most effective removal of the chemical denaturant. After magnetic clean up, the sample can be passed over a desalting column to further reduce the level of denaturant and other small molecule enzyme inhibitors from the sample. Magnetic clean-up has the advantage over precipitation and centrifugation since it is more easily incorporated into an automated workflow.[000177] The current invention provides a method for selectively removing nonpathogen cells from a sample, wherein the sample is treated with one or more cell disruption techniques at a level sufficient to disrupt the non-pathogen cells in the sample while leaving most of the pathogens intact, introducing one or more nucleases into the treated sample and incubating the mixture to allow the nucleases to degrade nucleic acids released from the disrupted cells, adding a chaotropic salt at a final concentration greater than 0.5 molar, adding magnetic particles to the sample and binding nucleic acids from the pathogens to the magnetic particles, collecting the magnetic particles and bound nucleic acids with a magnet, washing the particles and bound nucleic acids with a wash buffer that maintains the binding of the magnetic particles and the nucleic acids and eluting the nucleic acids from the magnetic particles.[000178] The preferred disruption technique is to treat the sample with a low-strength detergent, in a preferred embodiment of the disclosure, the detergent is saponin. The sample may be treated with mild detergents and / or low-level to disrupt the non-pathogen cells.33156225.1 39During the process, the mild detergent is removed from the sample prior to adding the nucleases. Nucleases can be RNases, DNases, or a combination of RNase and DNase. In a preferred embodiment, the chaotropic salt is a Guanidine salt, preferably at a final concentration greater than one molar and more preferred at a final concentration greater than two molar. If needed, the method further comprises passing the eluted nucleic acids over a desalting column to remove any remaining inhibitors. In addition, the method may also further comprise non-specifically amplifying the nucleic acids eluted from the magnetic particles.[000179] Pathogens may be in low concentrations in a sample. After sample processing, the copy number of the target nucleic acids may be low. Amplification can be used to increase the sensitivity of the overall diagnostic process. Methods for amplification have been discussed above. “Sequence-independent single primer amplification” (SISPA) is a method based on nonspecific amplification using random primers and represents a universally applicable and already proven approach for NGS. This approach is preferred because it maintains an agnostic approach to pathogen detection, SISPA was first developed by Reyes and Kim (Reyes GR, Kim JP. Sequence-independent, single-primer amplification (SISPA) of complex DNA populations. Mol Cell Probes. 1991 Dec;5(6):473-81). It provides the capability to amplify nucleic acids non-specifically and thus maintain an agnostic diagnostic capability. This could improve sensitivity in agnostic diagnostics by increasing nucleic acid strands for sequencing without needing to target specific sequences. The first step is reverse transcription, where random hexamers labeled with a known specific sequence are directly incorporated into the cDNA. After denaturation, annealing, and double-strain synthesis (Klenow reaction), the yielded dsDNA is amplified using the second SISPA primer consisting of the corresponding known specific sequence without the random hexamers. Hence, it allows the enrichment of the viral genome without the need for virus-specific primers (Schulz A, Sadeghi B, Stoek F, King J, Fischer K, Pohlmann A, Eiden M, Groschup MH. Whole-Genome Sequencing of Six Neglected Arboviruses Circulating in Africa Using Sequence-Independent Single Primer Amplification (SISPA) and MinION Nanopore Technologies. Pathogens. 2022; 11(12): 1502.)[000180] The current invention provides embodiments of a method for agnostic diagnostic identification of pathogens in a sample comprising, disrupting the pathogens in a sample, isolating and concentrating the nucleic acids, preparing a sequencing library with the nucleic acids, sequencing the nucleic acid library, and analyzing the resulting sequence data for nucleic acid sequences indicating the presence of a pathogen, wherein all steps prior to sequencing are carried out in a single disposable cartridge. Additionally, the number of sequencing reads corresponding to a particular pathogen can be used to determine the amount33156225.1 40of pathogen in a sample. In a preferred embodiment of the disclosure, the sample would be treated initially to disrupt host cells in the sample, and the released nucleic acids would be degraded. In an alternative embodiment, the host cell nucleic acids would be preferentially removed from the sample after disruption of pathogens in the sample before preparing the sequencing library.[000181] The current invention also provide embodiments of a method for agnostic diagnostic identification of pathogens in a sample comprising preferentially disrupting host cells, digesting host cell nucleic acids, disrupting the pathogens in a sample, and inactivating the nucleases in the sample using a high concentration of a chaotropic salt, isolating and concentrating the nucleic acids, preparing a sequencing library with the nucleic acids, sequencing the nucleic acid library, and analyzing the resulting sequence data for nucleic acid sequences indicating the presence of a pathogen.[000182] In these methods, the isolated nucleic acids can be amplified prior to or during the library preparation. In a preferred approach, the amplification of the pathogen nucleic acids would be carried out using an agnostic amplification process.[000183] The agnostic diagnostic protocol can be modified to allow users to select optional processes on the controller, such as host cell depletion and amplification. The same cartridge can be used; the controller is programmed to skip certain steps.[000184] There are three magnetic clean-up steps. The first occurs in sample prep and should have separate reagent chambers. The last two are smaller volumes and are in the library preparation phase. Common chambers may be used for the reagents. Since magnetic separation chambers will have used particles when finished, each magnetic separation needs a separate chamber.[000185] The cartridge will need a waste chamber or chambers with a total volume of at least 3.0 mis. Waste can be placed in empty reagent chambers, as well as a dedicated waste chamber.[000186] The reverse transcription, Klenow, and PCR steps can use the same reaction chamber.[000187] In addition to nucleic acids, the cartridge of the current invention may also be configured to isolate, process, and prepare for analysis of a wide variety of analytes. A preferred analyte for analysis is proteins. Proteins may be extracted from complex biological samples. This may require disruption of tissue, cells, pathogens, etc., as well as isolation and purification of proteins. In a preferred embodiment, isolation of target proteins can be carried33156225.1 41out using antibodies specific to target proteins. Antibodies may be incorporated onto a filter, on beads (especially magnetic beads) or in a column matrix for use in isolation.[000188] During shipping and handling, the cartridge 100 movement may disrupt an unrestrained dry reagent pellet. If part of the dried material is in an area where the liquid cannot dissolve the material, the final concentration of reagents may not be sufficient to run the intended reaction efficiently. Therefore, a preferred embodiment contains the dried materials in a volume so that the dried material (1) does not move around and break apart and (2) is confined to the bottom of the chamber so that when water is introduced into the chamber the entire particle is dissolved.[000189] A method for restraining dried reagent pellets in a cartridge so that they do not move during shipment and handling has been disclosed previously, e.g. U. S. Patents 9,057,674, 8,758,701, and 8,187,557. Those patents specifically discloses placing a restraining component above the bead in a chamber. However, this approach introduces an additional component, i.e the restraining material which needs to be introduced into the chambers containing the dried materials. This increases the cost by increasing the number of components in the disposable and the handling steps in assembly.[000190] In one embodiment, the current invention provides a configuration that allows for a chamber built to provide the needed restraint without the introduction of a separate component. In a cartridge 100, an injection molded housing or shuttle can have chambers consisting of a floor or ceiling and side walls. The open side of the chamber can be sealed with a film that is heat-sealed, welded, or adhered to the cartridge component. The height of the chamber is such that the film restrains the pellet in the channel. An indentation, which is shaped to hold the pellet, can also be made in the ceiling or floor to hold the pellet more specifically. Fluid can be introduced into the cartridge 100 either via a port on a side wall or through a port on the floor or ceiling. The fluidic channel is in fluidic communication with a port on the other surface of the shuttle 2 or housing 1 that can be fluidically connected with the housing 1 or shuttle 2.[000191] In the current invention, the column matrix chamber preferentially would be in the base 2c of the shuttle 2 or the base 1c of the housing 1. The chamber would be molded with a top wall and side walls. The floor of the injection molded chamber would be open and the chamber would be sealed with a film. Two ports would be located in the top wall at each end of the chamber. A filter material can be welded to the port opening to hold the dried reagent within the channel. Two ports would allow the fluid used to rehydrate the particle to move into the chamber. The first port or opening would be fluidically connected to33156225.1 42a syringe via a pathway to allow for fluid to be pushed into the chamber. The second port or opening would allow for air to be vented out of the chamber to prevent back pressure while filling. The second port would be fluidically connected to a separate chamber to collect the solution after passing through the matrix. The solution could then be mixed with further chemistries in that chamber and / or removed by the syringe from that chamber to be used in follow-up steps. In a preferred embodiment, the component would be the shuttle 2. The ceiling of the chamber would restrain the dried reagent between the ceiling of the chamber and the seal on the bottom of the chamber. Filters can be welded to the ports to restrain the matrix material in the chamber after rehydration.[000192] In production, the shuttle 2 or housing 1 is placed with the chamber opening upwards and the dried materials are placed in each of the chambers. After filling the chambers, the housing would be sealed with a film over the bottom of the component. Filling and sealing of the bottom should be carried out in a dry environment to preserve the activity of the dried reagents.[000193] The preferred system 200 would be optionally powered by battery, small in size, and light in weight, thus permitting complete portable use at any location where patients may be, away from hospitals, laboratories, or even drug stores. The reader is capable of performing fully automated assays (optionally for detecting multiple analytes at the same time) and rapidly obtaining accurate results (typically within 1 or 2 hours and as fast, as 15-20 minutes). It is easy to operate, using one or more pre-manufactured test cartridges one can quickly obtain test results.[000194] This newly designed assay system 200 also includes components that provide secure cloud-based connectivity for conveying the diagnostic results from the portable testing device to a remote reporting system, which may be a centralized data collection or processing center or mobile devices such as hand-held devices used by a physician or a patient, to receive a diagnostic report. With such cloud-supported connectivity, data sharing can take place virtually instantaneously, not only allowing physicians to start treating patients without, any delay but also enabling monitoring and reporting.[000195] These important features circumvent the current limitations that tend to prevent or hinder early testing in poor, remote areas where laboratory facilities are few and testing capability is scarce. The combination of its deployment ability, its rapid and accurate diagnostic functionality, its technical sophistication yet ease of operation, and its cloud-based connectivity makes this new assay system the ultimate solution for emerging markets.33156225.1 43[000196] The reader may include a barcode reader or an RFID reader. Cartridges 100 may be labeled with a label with either a barcode or RFID tag to allow for tracking of the disposable through manufacturing and storage and for linking the cartridge and sample information. In addition, the system may have the capability to read either barcodes or RFID tags on samples that are analyzed in the system.[000197] A preferred use for the present disclosure is to automate diagnostics. This includes all forms of biological diagnostics, including testing for chemical analytes, protein chemistry, including immunochemistry, and genomic testing, including testing for the presence of specific nucleic acid sequences as well as nucleic acid sequencing. The present disclosure can automate a wide variety of techniques or procedures to enable complicated diagnostic protocols. Several key techniques are described below, but the current invention can automate many other techniques and is not limited to those described.[000198] One embodiment of the disclosure is a system 200 to automate polymerase chain reaction diagnostics, wherein the system isolates nucleic acids from a sample, amplifies target nucleic acids using PCR (if needed a reverse transcription step is also automated), and detects the amplified target or targets. All steps are automated within an embodiment of the cartridge 100. Sample preparation and amplification can be automated using some of the chambers needed for NGS preparation. Detection can be carried out optically in the optical flow cell. An array of capture probes can be patterned on the internal surface of the chamber to do an array base detection to multiplex detection and eliminate the need for colored indicators. Gold developer can be used to increase the optical density for improved sensitivity using the process disclosed in US Patent no. 7,851,149 to Braun et al. In Braun, a gold nanoparticle is attached to the PCR product which is bound to a capture probe on the surface of the array. A gold developer is used to grow gold metal around the nano-particle to give a stronger signal. Primers may also be used with binding regions in the primer such that the gold nan-particle targets a segment of the primer rather than the specific sequence in the target, thus allowing a common sequence to be used to target the PCR products.[000199] Another aspect of the current invention is a cartridge 100 and method for inactivating potentially pathogenic agents in the cartridge at the completion of the assay. Guanidine, or other denaturing solution, is used to disrupt the sample and will inactivate viruses, bacteria, and other pathogens. To render the cartridge 100 inert, the system 200 can be programmed to collect the guanidine waste left over from the disruption step, or alternatively, remaining unused guanidine or other disinfecting reagent stored in a separate chamber in the cartridge 100 and to introduce the denaturing or disinfecting reagent into all33156225.1 44channels and chambers that were exposed to the sample to inactivate pathogens from the sample in those channels or chambers. The denaturing reagent can be introduced into each chamber and mixed by moving the syringe back and forth. Additionally, the user can shake or invert the cartridge to ensure that the denaturing reagent coats the top surfaces of all chambers. This feature is beneficial for use in the field or in resource-limited settings where autoclaves may not be available for treating the waste cartridges.[000200] The invention provides a method for inactivating pathogens in the cartridge wherein denaturing solution is moved into each chamber or channel that has had contact with the sample before the sample was inactivated with the denaturing solution.[000201 ] A key feature of the present disclosure is the flexibility to run a wide variety of processes. When running a process, some of the reagent chambers, reaction chambers, and process capabilities may not be used. Preferentially, the cartridge 100 would be engineered with sufficient reagent chambers, reaction chambers, syringes and other components to allow for a wide variety of processes to be run in the cartridge. The order of steps may also be changed via the system’s programming. For a particular process which generates sufficient demand, specific cartridge components that would be made at high volume can be designed.[000202] While the invention has been described with reference to particular embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the scope of the disclosure.[000203] Therefore, it is intended that the invention is not limited to the particular embodiments disclosed as the best mode contemplated for carrying out this disclosure but that the invention will include all embodiments falling within the scope and spirit of the appended claims.[000204] The references cited herein provide additional details and are hereby incorporated by reference.ExamplesExample 1 - Host Cell Depletion and Bacterial Sequencing Using Staged Filtration.[000205] Contrived oral samples were prepared by spiking 106Escherichia coli cells per milliliter into human cheek swab samples. The samples were processed using a33156225.1 45host cell depletion workflow comprising filtration and enzymatic digestion of cell-free DNA, suitable for automation within a cartridge.[000206] Host cells were substantially depleted prior to nucleic acid extraction. Filtration removed the majority of intact human cells, and a nuclease treatment was used to digest residual cell-free human DNA. Purified nucleic acids were prepared for sequencing and libraries were generated using a rapid barcoding protocol compatible with a nanopore sequencer.[000207] Sequencing was performed on a nanopore flow cell for approximately 2 hours with six bar-coded samples. Approximately 2 gigabases of sequence data were generated. The sequencing results showed that 81% of reads (approximately 75,000 reads) mapped to E. coli. Approximately 6% of reads mapped to Streptococcus mitts, 5% of reads were of human origin, and the remaining reads mapped to other oral bacteria. The average read length was 3,859 bases, demonstrating that the host depletion and filtration workflow preserved long nucleic acid fragments suitable for long-read sequencing.Example 2 - Validation of Antimicrobial Resistance Detection After Filtration-Based Host Depletion.[000208] Sequencing data obtained from the E. coli samples processed according to Example 1 were analyzed using bioinformatic pipelines. The E. coli strain used contained a known mutation in the rpsL gene conferring resistance to streptomycin.[000209] Analysis of the sequencing data correctly identified the rpsL mutation and associated antimicrobial resistance, demonstrating that the filtration-based host depletion and nucleic acid preparation workflow preserved sequence fidelity and enabled downstream genomic analysis, including detection of resistance markers.Example 3 - Bacterial Sequencing of Mycobacterium tuberculosis After Host Cell Depletion.[000210] Contrived samples were prepared by spiking 107Mycobacterium tuberculosis cells per milliliter into cheek swab samples. Host cell depletion was performed using a combination of host removal steps followed by automated nucleic acid extraction.[000211] Sequencing libraries were prepared and six bar-coded samples were sequenced on a nanopore sequencer for approximately 3 hours. Sequencing results showed that approximately 45% of reads mapped to Mycobacterium tuberculosis, while only 0.2% of reads were of human origin. Genome coverage of M. tuberculosis was approximately 78%,33156225.1 46with an average depth of 22×, demonstrating effective host depletion and enrichment of bacterial nucleic acids suitable for whole-genome sequencing.[000212] Subsequent process optimization reduced the limit of detection for whole-genome sequencing of M. tuberculosis to approximately 105cells per milliliter or per swab, with further improvements projected using secondary depletion of non-target bacteria.Example 4 - Viral Enrichment and Sequencing Using Sub-Micron Filtration.[000213] Contrived viral samples were prepared by spiking adenovirus into cheek swab and saliva samples at concentrations of 20,000 copies per milliliter and 5,000 copies per milliliter. Samples were centrifuged to remove gross debris, and the supernatant was passed through a 0.22-micron filter to remove bacterial and remaining host cells, thereby enriching viral particles.[000214] Sequencing libraries were prepared and six to eight bar-coded samples were sequenced on a nanopore sequencer for approximately 3 hours. For samples containing 20,000 copies per milliliter, adenovirus reads represented approximately 20.5% of total reads, yielding 99% genome coverage with an average sequencing depth of approximately 604×. For samples containing 5,000 copies per milliliter, adenovirus reads represented approximately 1.5% of total reads, yielding 97% genome coverage with an average depth of approximately 133×.[000215 ] These results demonstrate that sub-micron filtration effectively removed host and bacterial cells while preserving viral particles suitable for sequencing.Example 5 - Detection of Viral Pathogens in Clinical Samples.[000216] Saliva, cheek swab, and tongue scraping samples were collected from a post-symptomatic patient with infectious mononucleosis. Samples were processed using a viral enrichment workflow including host depletion and filtration.[000217] Sequencing analysis detected Epstein-Barr virus (human gammaherpesvirus 4) in all sample types. Cheek swab samples yielded approximately 74% genome coverage with a median depth of 13×. Sequencing also detected the presence of Bohxovirus oralis in the same individual, demonstrating the capability of the workflow to identify multiple viral organisms in a single sample.[000218] In some embodiments, host cell depletion is performed solely by sequential filtration using a large-pore filter followed by a smaller-pore filter, without centrifugation, thereby simplifying automation and reducing equipment requirements.33156225.1 47[000219] hi some embodiments, host cell depletion is performed by centrifugation to pellet host cells, followed by filtration of the supernatant to remove residual debris.[000220] In some embodiments, after host cell depletion, cell-free nucleic acids are selectively digested using a nuclease prior to nucleic acid purification, improving pathogen signal-to-background ratios.[000221] In some embodiments, the method further comprises sub-micron filtration to selectively enrich viral particles by removing bacterial and fungal cells.[000222] The disclosed methods, cartridges, and systems share a single general inventive concept, namely automated host cell depletion integrated with nucleic acid purification and sequencing library preparation within a disposable cartridge. Alternative host, depletion techniques and enrichment strategies represent different, embodiments of the same inventive concept rather than separate inventions.[000223] The figures illustrate example cartridge and system architectures suitable for performing the disclosed methods. Other cartridge configurations, system layouts, and fluidic architectures may also be used to implement the methods described herein.[000224] The methods, systems, and cartridges described herein are applicable to clinical diagnostics, environmental monitoring, veterinary testing, food safety analysis, biodefense, and research sequencing applications, including use in decentralized or resourcelimited settings.33156225.1 48

Claims

1. ClaimsWhat is claimed:

1. A method for preparing a sequencing library from a biological sample in a disposable cartridge, the method comprising:introducing the biological sample into the disposable cartridge;depleting host cells or host-derived material from the sample while retaining pathogenic material comprising one or more of bacteria, fungi, or viruses; purifying nucleic acids from the retained pathogenic material within the disposable cartridge; andpreparing a sequencing library from the purified nucleic acids within the disposable cartridge,wherein the host cell depletion, nucleic acid purification, and library preparation are performed without removal of the sample from the disposable cartridge.

2. The method of claim 1, wherein depleting host cells comprises selectively removing intact eukaryotic cells while allowing pathogenic organisms or viral particles to remain in the sample.

3. The method of claim 1, wherein depleting host cells is performed using one or more of filtration, centrifugation, mechanical disruption, chemical lysis, enzymatic treatment, or combinations thereof.

4. The method of claim 1, wherein depleting host cells comprises sequential filtration of the sample through a plurality of filters integrated into the disposable cartridge.

5. The method of claim 4, wherein the sample is first passed through a primary filter having a pore size of about 15 to 30 microns to remove host cells and large debris.

6. The method of claim 5, wherein filtrate from the primary filter is subsequently passed through a secondary filter having a pore size of about 2 to 10 microns to further remove host cells while allowing pathogens to pass.33156225.1 497. The method of claim 4, wherein the sequential filtration prevents clogging of downstream filters and enables automated fluid flow through the cartridge without centrifugation.

8. The method of claim 1, wherein depleting host cells comprises centrifuging the biological sample to pellet host cells and debris, followed by filtration of a supernatant.

9. The method of claim 8, wherein the filtration is performed within the disposable cartridge following introduction of the supernatant.

10. The method of claim 8, wherein centrifugation is performed at a speed sufficient to pellet host cells while retaining pathogenic organisms or viral particles in the supernatant.

11. The method of claim 1, further comprising digesting cell-free nucleic acids after depletion of host cells.

12. The method of claim 11, wherein digesting comprises contacting the sample with a nuclease that degrades free DNA or RNA not protected within intact pathogens.

13. The method of claim 11, further comprising inactivating the nuclease prior to nucleic acid purification.

14. The method of claim 13, wherein nuclease inactivation is performed using a chaotropic agent.

15. The method of claim 1, wherein the method is configured to selectively enrich viral particles.

16. The method of claim 15, wherein enriching viral particles comprises filtering the sample through a sub-micron filter to remove bacterial and fungal cells.33156225.1 5017. The method of claim 16, wherein the sub-micron filter has a pore size of about 0.1 to 0.5 microns.

18. The method of claim 16, wherein the sub-micron filter has a pore size of about 0.22 microns.

19. The method of claim 15, wherein viral enrichment is performed after depletion of host cells using filtration or centrifugation.

20. The method of claim 1, wherein preparing the sequencing library comprises one or more of reverse transcription, fragmentation, tagmentation, amplification, adapter ligation, or combinations thereof.

21. The method of claim 1, wherein the sequencing library is compatible with long-read sequencing.

22. A disposable cartridge for preparing a sequencing library from a biological sample, comprising:a sample input region configured to receive the biological sample;one or more host cell depletion components configured to selectively remove host cells or host-derived material while retaining pathogenic material comprising one or more of bacteria, fungi, or viruses;one or more nucleic acid purification regions configured to isolate nucleic acids from the retained pathogenic material; andone or more reaction regions configured to prepare a sequencing library from the isolated nucleic acids,wherein the cartridge is configured to perform host cell depletion, nucleic acid purification, and sequencing library preparation without removal of the sample from the cartridge.

23. The disposable cartridge of claim 22, wherein the host cell depletion components comprise a plurality of filters arranged for sequential filtration of the sample.33156225.1 5124. A system for preparing a sequencing library from a biological sample, comprising: a disposable cartridge according to claim 22; anda reader configured to interface with the disposable cartridge and to actuate fluid movement,wherein the system is configured to perform host cell depletion, nucleic acid purification, and sequencing library preparation within the disposable cartridge.33156225.1 52

Images