System, apparatus and method for preparation, processing and analysis of nucleic acid samples

Abstract

An automated nucleic acid processing system includes a plurality of independent, asynchronously operating processing bays, each receiving a single biological sample. Each bay contains a robotic liquid handling module performing addition-only liquid transfers, a thermal cycling module executing programmable temperature profiles, a magnetic bead separation module, and a gripper mechanism transporting sample vessels between modules. A controller independently controls each processing bay, initiates sample processing without batch accumulation, and dynamically schedules workflow steps across all bays. Each processing bay receives a single-use closed consumable cartridge containing pre-measured reagents for nucleic acid extraction, library preparation, CRISPR-mediated depletion, and magnetic bead-based normalization. The system generates a sequencing-ready nucleic acid library from each biological sample without intermediate fluorometric quantification of nucleic acid concentration. An output interface provides a normalized sequencing library directly loadable onto a high-throughput sequencing instrument.

Description

SYSTEM, APPARATUS AND METHOD FOR PREPARATION, PROCESSING AND ANALYSIS OF NUCLEIC ACID SAMPLES RELATED APPLICATION

[0001] This application claims the priority of United States Provisional Patent application No.63 / 760,852 in the name of Keith Brown, which was filed on 20 February 2025, and titled “Automated Metagenomic Sequencing System for Pathogen Detection and Identification’’. The content of the provisional specification which was filed with the United States Patent and Trademarks Office on 20 February 2025 is expressly incorporated into the present complete specification by reference in its entirety and for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates to improvements in the preparation and processing of nucleic acid samples for analysis by next-generation sequencing. More particularly, the invention relates to an automated system, apparatus, and method, which may be used for extraction of nucleic acids, selective depletion of predetermined abundant nucleic acid sequences, library preparation, and workflow optimization to enable sensitive detection of low-abundance target nucleic acids in complex sample matrices. Byway of example, the invention has particular, but not exclusive, application to metagenomic sequencing techniques for infectious disease diagnostics, cancer genomics, rare disease detection, pharmacogenomics, prenatal testing, microbiome analysis, environmental surveillance, veterinary diagnostics, and agricultural applications. For convenience, the invention is described in the present specification in relation to an automated system, apparatus, and method for extraction of nucleic acids from biological samples, for selective depletion of predetermined abundant nucleic acid sequences using a CRISPR-based methodology, for library preparation including quantification-free adapter ligation and bead-based normalization, and workflow optimization through random-access asynchronous processing architecture, to enable sensitive detection of low-abundance target nucleic acids in complex sample matrices. It should be appreciated that the present invention is not limited solely to that use.BACKGROUND OF THE INVENTION

[0003] The discussion throughout this specification about the invention comes about due to the identification by the inventor of certain prior art problems in the field of the invention, and the desirability of identifying potential solutions to one or more of them. It is to be understood however that any discussion in this specification of documents, devices, acts, information or knowledge that existed on or before any priority date (“background information”) that the present invention claims is included solely to explain the context of the invention. It shouldnot be taken that any such discussion of background information forms a part of the prior art base in the field of the invention or part of the common general knowledge in the relevant art in the United States or elsewhere on or before the priority date of the disclosure and claims herein.

[0004] The detection of low-abundance target nucleic acids in complex biological samples presents significant technical challenges across multiple fields of molecular diagnostics and genomic analysis. In many applications, target nucleic acid sequences represent only a small fraction of total nucleic acid content extracted from samples. Abundant background sequences create a fundamental limitation in detection sensitivity — not because sequencing platforms lack capability, but because the sequencing depth required to achieve adequate coverage of rare target sequences becomes prohibitively expensive when those sequences must be recovered from beneath a vast excess of abundant background nucleic acids.

[0005] In the example of infectious disease diagnostics, host-derived ribosomal RNA and mitochondrial DNA frequently comprise 95-99% of extracted nucleic acids from clinical specimens, while pathogen sequences of diagnostic interest may represent less than 1% of the sample. In cancer genomics, circulating tumor DNA may comprise only 0.01-1% of total cell-free DNA. In prenatal applications, fetal cell-free DNA typically may comprise only 10-15% of total cell-free DNA in maternal plasma. And in microbiome analysis of host-associated specimens, host nucleic acids frequently exceed 80-90% of extracted material. In each of these applications, the fundamental challenge is identical: target sequences are obscured by abundant background nucleic acids.

[0006] Traditional approaches to addressing this challenge include ribosomal RNA removal using RNase H treatment with magnetic bead-based purification, achieving approximately 80-90% removal of ribosomal RNA but proving ineffective at removing host genomic DNA. RNase H-based methods are limited by hybridization of DNA probes to RNA templates where even partial complementarity (4-6 base pairs) can cause hydrolysis that lacks specificity, leading to off-target effects. Hybridization capture-based enrichment methods achieve high on-target rates but require prior knowledge of target sequences, introduce systematic bias, increase workflow complexity and cost, and cannot detect divergent or previously unknown target sequences. Computational filtering during bioinformatics analysis discards sequence data rather than enriching for targets, thereby reducing overall coverage depth and limiting sensitivity.

[0007] Existing approaches to overcoming these problems share common limitations: they either insufficiently deplete abundant background sequences, require prior knowledge of targets, introduce systematic biases, or discard potentially valuable sequence data. Recentadvances in CRISPR-Cas technologies have demonstrated potential applications beyond genome editing. The programmable specificity of CRISPR-guided nucleases enables selective targeting of specific nucleic acid sequences with high fidelity. However, comprehensive integration of CRISPR-based depletion with automated sample preparation, random-access processing architecture, quantification-free library preparation, and seamless integration with sequencing and bioinformatics systems has not been achieved.

[0008] The complexity of next-generation sequencing workflows has further limited widespread clinical adoption. Current metagenomic sequencing methods require multiple instruments and complex manual steps, thereby limiting their effectiveness in clinical settings. Manual sample preparation, library construction, and sophisticated bioinformatics analysis create barriers, particularly in time-sensitive environments such as hospital emergency departments and intensive care units. The need for fluorometric quantification of nucleic acid concentrations prior to library construction introduces additional labor, error-prone manual calculations, and expensive optical instrumentation requirements.

[0009] Recent advances in CRISPR-Cas technologies have demonstrated potential applications beyond genome editing. The programmable specificity of CRISPR-guided nucleases enables selective targeting of specific nucleic acid sequences with high fidelity. However, comprehensive integration of CRISPR-based depletion with automated sample preparation, random-access processing architecture, quantification-free library preparation, and seamless integration with sequencing and bioinformatics systems has not previously been achieved.

[0010] There thus remains a need for an automated system that combines comprehensive detection capabilities with improved sensitivity, automated sample processing with minimal hands-on time, rapid turnaround, and user-friendly data interpretation — applicable across the diverse fields in which low-abundance target nucleic acid detection is required.

[0011] Further to the preceding comments, it has been recognized by the inventor that rapid and accurate detection of pathogens is crucial for diagnosing infectious diseases and implementing appropriate treatments. Traditional methods often require time-consuming culturing or are limited to detecting specific pathogens. Metagenomic sequencing has emerged as a powerful tool for comprehensive pathogen detection, but challenges remain in sample preparation, host Nucleic Acid depletion (RNA / DNA), and data analysis for clinical applications.

[0012] The field of infectious disease diagnostics has seen significant advancements in recent years, yet the global healthcare community continues to face substantial challenges in rapidly and accurately identifying pathogens, particularly in the context of emerging infectiousdiseases and potential epidemics and pandemics. Traditional diagnostic methods, while valuable, often fall short in providing comprehensive, timely, and useful information to enable suitably informed decisions to be made in the context of providing effective patient care and public health responses.

[0013] Conventional approaches to pathogen detection typically rely on culture-based methods, targeted molecular assays such as polymerase chain reaction (PCR), or serological tests. While these methods have their merits, they are often limited in scope, requiring prior knowledge of the suspected pathogen, and may lack the sensitivity or specificity needed for complex clinical scenarios. Furthermore, these traditional methods often struggle to detect novel or emerging (e.g., previously not known or poorly understood) pathogens, co-infections, or antibiotic resistance markers, which are critical factors in managing infectious diseases effectively.

[0014] The advent of next-generation sequencing (NGS) technologies has opened new avenues for pathogen detection and characterization. Metagenomic next-generation sequencing (mNGS) offers the potential for unbiased, comprehensive detection of pathogens directly from clinical samples. This approach allows for the simultaneous identification of a wide range of organisms, including bacteria, viruses, fungi, and parasites, without the need for specific primers or probes. However, the widespread adoption of mNGS in clinical settings has been hindered by several factors, including high costs, complex workflows, extended turnaround times, and challenges in data interpretation.

[0015] One of the primary obstacles in applying mNGS to clinical diagnostics is the overwhelming presence of host nucleic acids in patient samples. In many cases, pathogen nucleic acids represent only a tiny fraction of the total genetic material present, making their detection and accurate quantification challenging. This "needle in a haystack" problem often necessitates deep sequencing, which increases costs and analysis time.

[0016] Various methods have been developed to address this issue, including host depletion techniques and targeted enrichment strategies. However, these approaches often introduce biases, may require large sample inputs, or can be limited in their ability to detect novel or emergent pathogens. There remains a critical need for a method that can efficiently and selectively enrich for pathogen sequences while maintaining the unbiased nature of metagenomic sequencing.

[0017] Yet again, advances in CRISPR-Cas technologies have shown promise in genomic applications other than gene editing. The highly specific nature of CRISPR-guided nucleases offers potential solutions for selective depletion of unwanted sequences in complex mixtures of nucleic acids. However, the integration of CRISPR-based depletion methods into acomprehensive, automated diagnostic platform for infectious diseases has to date not been realized.

[0018] Furthermore, the complexity of mNGS workflows often requires specialized expertise and equipment, thereby limiting its accessibility and utility in many clinical settings. The need for manual sample preparation, library construction, and sophisticated bioinformatics analysis create barriers to widespread adoption of mNGS in clinical applications. An improved system would automate these processes, thus reducing hands-on time and the potential for human error, while also providing rapid and interpretable results.

[0019] The interpretation of mNGS data presents another significant challenge. The vast amount of sequence data generated requires sophisticated bioinformatics pipelines and databases to identify and classify detected organisms accurately. Moreover, distinguishing clinically relevant pathogens from commensal microbes or environmental contaminants requires careful analysis and often relies on the expertise of trained specialists. There is a pressing need for systems that can not only generate high-quality sequencing data but also provide clear, actionable reports that can be readily interpreted by clinicians.

[0020] In the context of public health and pandemic / epidemic / outbreak preparedness and response, the ability to detect and characterize novel or hard-to-detect pathogens rapidly is crucial. The COVID-19 pandemic has underscored the importance of having flexible, scalable diagnostic platforms that can adapt to emerging threats. Such systems must be capable not only of identifying known pathogens, but also of providing detailed genomic information that can inform strain typing, track mutations, and guide the development of targeted countermeasures in response to an actual infection and / or the threat of infection.

[0021] The integration of mNGS data with broader public health surveillance systems represents another area of unmet need. The potential for real-time, genomics-based surveillance of infectious diseases could revolutionize outbreak detection and response. However, realizing this potential requires overcoming challenges in data standardization, integration, and rapid analysis at scale.

[0022] The present invention therefore aims to address one or more of these limitations of current diagnostic approaches, or at the very least, to provide a useful alternative to the known approaches.SUMMARY OF THE INVENTION

[0023] In general, the present invention provides an automated nucleic acid processing system comprising:(a) a plurality of independent processing bays, each configured to receive a single biological sample and to operate asynchronously relative to the other processing bays;(b) within each processing bay:(i) a robotic liquid handling module configured to perform addition-only liquid transfers,(ii) a thermal cycling module configured to execute programmable temperature profiles,(Hi) a magnetic bead separation module, and(iv) a gripper mechanism configured to transport sample vessels between the modules;(c) a controller comprising at least one processor and memory storing executable instructions configured to:(i) control each processing bay independently,(ii) initiate processing of a sample in any processing bay without requiring batch accumulation of samples, and(iii) dynamically schedule workflow steps across the plurality of processing bays;(d) a single-use closed consumable cartridge receivable within each processing bay, the cartridge containing pre-measured reagents for nucleic acid extraction, library preparation, CRISPR-mediated depletion, and magnetic bead-based normalization;(e) wherein the system is configured to generate a sequencing-ready nucleic acid library from each biological sample without requiring intermediate fluorometric quantification of nucleic acid concentration; and(f) an output interface configured to provide a normalized sequencing library directly loadable onto a high-throughput sequencing instrument.The plurality of processing bays preferably comprises four independent processing bays.

[0024] The CRISPR-mediated depletion is preferably performed after initial library amplification such that abundant nucleic acid sequences function as carrier molecules prior to depletion.

[0025] CRISPR cleavage preferably removes sequencing adapters from targeted fragments, preventing amplification of cleaved fragments.

[0026] The library preparation preferably comprises adapter ligation chemistry capable of accommodating nucleic acid input quantities from about 10 pg to about 1 pg without adjustment based on measured concentration.

[0027] The magnetic bead-based normalization preferably comprises contacting the amplified library with beads having a finite binding capacity sufficient to produce standardized output concentration without optical quantification.

[0028] The cartridge preferably comprises lyophilized CRISPR ribonucleoprotein complexes stable at ambient temperature for at least six months.

[0029] The cartridge preferably comprises machine-readable lot information and the controller is configured to prevent processing upon detection of an expired or incompatible cartridge.

[0030] The controller preferably comprises a dynamic scheduling engine configured to assign priority to selected samples based on stored priority parameters.

[0031] The priority parameters may comprise one or a combination of:clinical urgency,time since sample receipt,pathogen risk classification, anduser-defined priority.

[0032] Generally, the present invention also provides a method for automated preparation of a sequencing-ready nucleic acid library from a biological sample, the method performed within an integrated multi-bay automated instrument, comprising:(a) extracting nucleic acids from the biological sample using magnetic bead-based purification;(b) performing quantification-independent adapter ligation to generate a sequencing library without measuring nucleic acid concentration;(c) performing limited-cycle amplification of the library;(d) contacting the amplified library with CRISPR-associated ribonucleoprotein complexes configured to cleave predetermined abundant nucleic acid sequences;(e) removing cleaved fragments using magnetic bead purification;(f) performing post-depletion amplification to selectively enrich uncleaved library molecules; and(g) normalizing the amplified library using magnetic beads having a finite binding capacity sufficient to produce a standardized sequencing-ready output without fluorometric quantification,wherein the method is performed on individual samples asynchronously without batch processing.

[0033] In the above method the the CRISPR-associated ribonucleoprotein complexes preferably comprise guide RNAs targeting ribosomal RNAand mitochondrial DNA sequences.

[0034] In the above method the CRISPR-mediated depletion preferably increases representation of low-abundance target sequences relative to host-derived sequences.

[0035] Preferably the method produces a normalized library directly loadable onto a nextgeneration sequencing flow cell without manual dilution calculations.

[0036] In the above method each sample may be initiated upon arrival into an available processing bay without waiting for additional samples.

[0037] Generally, the present invention also provides a single-use cartridge for automated nucleic acid library preparation, the cartridge comprising:(a) a plurality of sealed reagent chambers containing reagents for nucleic acid extraction, adapter ligation, amplification, CRISPR-mediated depletion, and magnetic beadbased normalization;(b) one or more lyophilized CRISPR ribonucleoprotein complexes targeting predetermined abundant nucleic acid sequences;(c) magnetic beads configured for both purification and saturating normalization; (d) an integrated sample input vessel;(e) an integrated library output vessel; and(f) a machine-readable identifier encoding lot-specific and protocol-specific information,wherein the cartridge is configurable for insertion into a processing bay of an automated multi-bay instrument and enables completion of library preparation without external reagent addition or intermediate nucleic acid quantification.

[0038] The reagent chambers preferably comprise foil seals having differential peel strengths configured for sequential robotic access.

[0039] The incompatible enzymatic reagents may be co-stored in lyophilized form under differential stabilization conditions.

[0040] In preferred embodiments of the system, the controller is configured to reprioritize workflow steps in real time in response to arrival of an urgent sample, without interrupting processing of previously initiated samples.

[0041] In preferred embodiments of the system reprioritization reduces time-to-sequencing initiation for the urgent sample relative to non-prioritized samples.A. system, method, and apparatus for preparing a nucleic acid library from a biological sample for next-generation sequencing analysis

[0042] In a first broad aspect, the invention generally provides a system for preparing a nucleic acid library from a biological sample collected from a host organism for next-generation sequencing analysis, the system comprising:(a) means for extracting nucleic acids from the biological sample;(b) means for selectively depleting from the extracted nucleic acids, at least one predetermined abundant nucleic acid sequence using a CRISPR-based method, thereby enriching for target nucleic acid sequences of interest;(c) means for completing generation of a sequencing library from the enriched nucleic acid sequences;(d) a sequencing means adapted to perform high-throughput sequencing on the sequencing library; and(e) a bioinformatics analysis means adapted to:(i) process data derived from the high-throughput sequencing performed on the sequencing library; and(ii) detect the presence of the target nucleic acid sequences of interest based on analysis of sequence data from the sequencing library.

[0043] In embodiments of the system, the host organism is either a:(a) human; or(b) non-human animal species.

[0044] In embodiments of the system, the target nucleic acid sequences comprise one or more pathogen-derived nucleic acids, and the abundant nucleic acid sequences comprise one or more host organism-associated nucleic acids.

[0045] In embodiments of the system, the pathogen comprises one or more of:(a) a bacterial species;(b) a viral species;(c) a fungal species, or(d) a plant species (or material derived from a plant species); or(e) any two or more species drawn from (a) to (d); or(f) a parasitic species.

[0046] Preferably, in the system, the target nucleic acid sequences comprise one or more tumour-derived nucleic acids, and the abundant nucleic acid sequences comprise one or more wild-type genomic sequences.

[0047] In embodiments of the system, the tumour-derived nucleic acids comprise one or more circulating tumour DNA (ctDNA) present in a liquid biopsy specimen.

[0048] In embodiments of the system, the target nucleic acid sequences comprise one or more rare genetic variants associated with disease, and the abundant nucleic acid sequences comprise one or more common polymorphic alleles.

[0049] In embodiments of the system, the target nucleic acid sequences comprise fetal cell-free DNA, and the abundant nucleic acid sequences comprise maternal cell-free DNA.

[0050] In embodiments of the system, the abundant nucleic acid sequences comprise human ribosomal RNA (rRNA).

[0051] In embodiments of the system, the abundant nucleic acid sequences comprise mitochondrial DNA (mtDNA) or mitochondrial derived RNA.

[0052] In embodiments of the system, the abundant nucleic acid sequences comprise bacterial 16S or 23S ribosomal RNA.

[0053] In embodiments of the system, the abundant nucleic acid sequences comprise repetitive elements such as LINE, SINE, Alu, or other highly abundant long non-coding RNA, microRNA, PIWI RNA.

[0054] In embodiments of the system, the abundant nucleic acid sequences comprise RNA derived from abundant housekeeping genes, or structural mRNA genes.

[0055] In a further broad aspect, the present invention generally provides a method for detecting the presence of at least one pathogen species in a biological sample collected from a host organism, the method comprising the steps of:(a) generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whetherone or more pathogen species is or are present in the biological sample, where the steps for generating the sequence library comprise:(1) extracting one or more predetermined nucleic acids from the biological sample; (2) depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(3) completing the generation of the sequence library;(b) performing a high-throughput sequencing on the sequence library; and(c) performing a bioinformatics analysis in the sequence library in order to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library.

[0056] Embodiments of the method aspect of the invention include those described above in relation to the system aspect of the invention (mutatis mutandis).

[0057] In a further broad aspect, the invention generally provides an apparatus for detecting the presence of at least one pathogen species in a biological sample collected from a host organism, the apparatus comprising:(a) means for generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whether one or more pathogen species is or are present in the biological sample, where the means comprise:(1) means for extracting nucleic acids from the biological sample;(2) means for depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(3) means for completing the generation of the sequence library;(b) a sequencing means adapted to perform high-throughput sequencing on the sequence library; and(c) a bioinformatics analysis means adapted to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library.

[0058] Embodiments of the method aspect of the invention include those described above in relation to the system aspect of the invention (mutatis mutandis).

[0059] In a yet further broad aspect, the invention generally provides a method of diagnosing the presence of an infection in a host organism, the method comprising the steps of:(a) collecting a biological sample from the host organism;(b) generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whether one or more pathogen species is or are present in the biological sample, where the steps for generating the sequence library comprise:(4) extracting one or more predetermined nucleic acids from the biological sample; (5) depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(6) completing the generation of the sequence library;(b) performing a high-throughput sequencing on the sequence library; and(c) performing a bioinformatics analysis in the sequence library in order to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library.

[0060] Embodiments of the method of diagnosis aspect of the invention include those described above in relation to the system aspect of the invention (mutatis mutandis).A system and method for automated preparation of a nucleic acid library from a biological sample for next-generation sequencing analysis

[0061] In a yet further broad aspect, the invention generally provides a system for automated preparation of a nucleic acid library from a biological sample for next-generation sequencing analysis, the system comprising:(a) an automated sample preparation means configured to:(1) extract nucleic acids from the biological sample;(2) selectively deplete at least one predetermined abundant nucleic acid sequence from the biological sample using a CRISPR-based method comprising ribonucleoprotein complexes of guide RNA molecules and a Cas nuclease; and (3) complete preparation of a sequencing library without requiring intermediate fluorometric quantification of nucleic acid concentration;(b) a sequencing means adapted to perform high-throughput sequencing on the prepared library; and(c) a bioinformatics analysis means adapted to process sequencing data and detect the presence of one or more target nucleic acid sequences in the sample.(d) wherein the target nucleic acid sequences comprise sequences associated with one or more pathogenic organisms, and the predetermined abundant nucleic acid sequences comprise host-derived ribosomal RNA, mitochondrial DNA, and / or bacterial ribosomal RNA.

[0062] In embodiments of this aspect of the system, target nucleic acid sequences comprise sequences associated with one or more pathogenic organisms, and the predetermined abundant nucleic acid sequences comprise host-derived ribosomal RNA, mitochondrial DNA, and / or bacterial ribosomal RNA.

[0063] In embodiments, the target nucleic acid sequences comprise tumor-derived nucleic acids including somatic mutations, gene fusions, or structural variants.

[0064] In embodiments, the target nucleic acid sequences comprise rare genetic variants associated with genetic disease.

[0065] And in embodiments, the target nucleic acid sequences comprise fetal cell-free nucleic acids, and the predetermined abundant nucleic acid sequences comprise maternal cell-free DNA or RNA.

[0066] And in embodiments, the target nucleic acid sequences comprise microbial community nucleic acids, and the predetermined abundant nucleic acid sequences comprise host-derived nucleic acids.

[0067] And in embodiments, the automated sample preparation means comprises a plurality of independent processing bays, each capable of processing a single biological sample independently without requiring sample batching, enabling random-access asynchronous sample processing.

[0068] In some preferred embodiments of the system, the plurality of independent processing bays comprises four processing bays.

[0069] And in some embodiments, the automated sample preparation means further comprises a single-use consumable support pack for each biological sample, the support pack housing in separate chambers all reagents, pipette tips, sample tubes, output tubes, and waste compartments required for complete sample preparation.

[0070] And in embodiments, the consumable support pack comprises lyophilized reagents providing ambient temperature stability, including lyophilized CRISPR-Cas ribonucleoprotein complexes.

[0071] And in embodiments, the consumable support pack comprises foil-sealed reagent wells with calibrated differential peel strengths.

[0072] And in embodiments, the consumable support pack comprises a barcode encoding lotspecific information, and the system is configured to halt processing upon detection of expired reagents or protocol mismatch.

[0073] And in embodiments of the system, completing preparation of the sequencing library comprises:(a) adapter ligation using a terminal transferase and truncated T4 RNA ligase- mediated mechanism accommodating nucleic acid inputs from 10 pg to 1 pg without quantification-based adjustment; and(b) bead-based normalization using magnetic beads with finite binding capacity to produce standardized library output concentrations.

[0074] And in embodiments, the CRISPR-based depletion is performed after initial library amplification such that abundant nucleic acids act as carriers during early processing steps, and wherein double-strand breaks remove sequencing adapters from cleaved fragments.

[0075] And in embodiments, the guide RNA molecules comprise a modular set that is substitutable to adapt the system to different target applications without modification of the instrument hardware.

[0076] In embodiments, the automated sample preparation means comprise:(a) a robotic liquid handler providing three-axis motion control;(b) a gripper mechanism for automated transport of sample and library tubes between processing stations;(c) a thermal cycler with heated lid control; and(d) a magnetic bead separation station.

[0077] And in embodiments of the system, the automated sample preparation means performs all liquid handling operations using addition-only transfers without requiring optical quantification instrumentation.

[0078] In a yet further broad aspect, the invention generally provides a method for automated preparation of a nucleic acid library from a biological sample for next-generation sequencing analysis, the method comprising the steps of:(a) extracting nucleic acids from the biological sample;(b) selectively depleting at least one predetermined abundant nucleic acid sequence using a CRISPR-based method comprising ribonucleoprotein complexes of guide RNA molecules and a Cas nuclease;(c) completing preparation of a sequencing library without requiring intermediate fluorometric quantification;(d) performing high-throughput sequencing on the prepared library; and(e) performing bioinformatics analysis to detect the presence of one or more target nucleic acid sequences.

[0079] In embodiments of the method, the step of completing preparation comprises:(a) pre-depletion PCR amplification;(b) CRISPR-based depletion with magnetic bead cleanup to remove cleaved fragments;(c) post-depletion PCR amplification with index primers; and(d) bead-based normalization producing standardized library output.

[0080] In embodiments, the method is performed using a plurality of independent processing bays enabling random-access asynchronous processing of individual samples without batch accumulation.

[0081] In preferred embodiments of the method, the biological sample comprises a clinical specimen and the target nucleic acid sequences comprise pathogen-associated sequences.

[0082] In preferred embodiments of the method, the biological sample comprises tumor tissue, circulating tumor cells, or cell-free plasma, and the target nucleic acid sequences comprise tumor-derived sequences.

[0083] And in embodiments, the biological sample comprises a microbiome-associated specimen and the predetermined abundant nucleic acid sequences comprise host-derived nucleic acids.

[0084] In a yet further broad aspect, the invention generally provides an apparatus for automated preparation of a nucleic acid library from a biological sample for next-generation sequencing analysis, the apparatus comprising:(a) a plurality of independent processing bays, each configured to receive a single-use consumable support pack;(b) a robotic liquid handler providing three-axis motion control for executing addition- only liquid transfers;(c) a gripper mechanism for automated manipulation and transport of sample and library tubes between processing stations;(d) a thermal cycler providing temperature control for enzymatic reactions;(e) a magnetic bead separation station; and(f) an electronic control system comprising a processor, touchscreen display, and software configured to manage independent asynchronous operation of each processing bay.

[0085] Embodiments of this apparatus aspect of the invention include those described above in relation to the system and method method for automated preparation of a nucleic acid library aspects of the invention (mutatis mutandis).Consumable support pack

[0086] And in a yet further broad aspect, the invention generally provides a single-use consumable support pack for use with an automated nucleic acid library preparation system, the support pack comprising:(a) a plurality of foil-sealed reagent chambers containing wet and / or lyophilized reagents for nucleic acid extraction, library preparation, and CRISPR-based selective depletion;(a) one or more integrated pipette tips;(b) a sample input tube;(c) a library output tube;(d) waste compartments; and(e) barcode identification encoding lot-specific information including reagent expiration and guide RNA panel identity.

[0087] In some embodiments of the consumable support pack, the foil-sealed reagent chambers comprise differential peel strengths calibrated for sequential robotic access.

[0088] And in embodiments of the consumable support pack, the lyophilized reagents comprise CRISPR-Cas ribonucleoprotein complexes stable at ambient temperature for at least six months.

[0089] And in embodiments of the consumable support pack, co-storage of enzymatically incompatible reagents is achieved through differential lyophilization at different temperatures. Detection of pathogens in biological samples

[0090] In a further broad aspect, the present invention generally provides a system for detecting the presence of at least one pathogen species in a biological sample collected from a host organism, the system comprising:(a) means for generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whether one or more pathogen species is or are present in the biological sample, where the means comprise:(1) means for extracting nucleic acids from the biological sample;(2) means for depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(3) means for completing the generation of the sequence library;(b) a sequencing means adapted to perform high-throughput sequencing on the sequence library; and(c) a bioinformatics analysis means adapted to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library. The system may thus be used in order to detect a single infection in the collected biological sample, or more than one. Where the latter applies, the pathogens may be either organisms from the sametaxonomic classification (for example, different species of bacteria) or from different taxonomic classifications (for example, bacteria as opposed to viruses).

[0091] For clarity, wherever used throughout this specification, the invention:(A) is interchangeably called the “system”; and(B) includes and also refers to the apparatus and method and all other aspects of the invention disclosed in this specification, such that - unless the context specifically requires otherwise - references in this specification to the “apparatus” or the “method” (or to any other aspect of the invention disclosed in this specification) refer to (and include) any and all the disclosed alternative aspects of one and the same invention as the “system”.

[0092] In some embodiments, the system not only detects the presence of a pathogen, but additionally identifies the species of the pathogen (or any two or more pathogen species) detected in the biological sample.

[0093] In some embodiments of this and any other aspect of the invention, the host organism is a human being. The invention is thus directly applicable to infection diagnosis and control in human medicine.

[0094] In yet other embodiments of this and any other aspect of the invention, the host organism may be a non-human animal species.

[0095] The invention is therefore also directly useful in - amongst other applications - the diagnosis and control of infections in veterinary medical applications.

[0096] For the purposes of this and any other aspects of the invention, the at least one pathogen species comprises:(a) a bacterial species;(b) a viral species;(c) a fungal species;(d) a plant species (or material derived from a plant species);(e) a zoonotic pathogen; or(f) any two or more species drawn from (a) to (e).

[0097] In this and any other aspects of the invention, the biological sample may include any suitable material, such as one or more of the following:(a) whole blood, blood serum or blood plasma;(b) urine;(c) cerebrospinal fluid;(d) saliva;(e) a nasopharyngeal secretion;(f) milk;(g) semen;(h) tears (lacrimal fluid);(i) synovial fluid (joint aspirate);(j) bronchoalveolar lavage (BAL);(k) sputum (induced or expectorated);(l) vaginal discharge;(m) urethral discharge;(n) wound exudate; and / or(o) ascitic fluid (peritoneal fluid)(p) pleural fluid(q) pericardial fluid(r) amniotic fluid; and / or(s) stool specimens (after appropriate processing).

[0098] Persons of skill in the field of the invention will appreciate that this list is not exhaustive.

[0099] Persons of skill in the field of the invention would appreciate that prior to using any of the samples (a) to (s), a given sample may require preliminary processing. So, for example, as mentioned above, stool samples may require appropriate processing before being presented to the system for pathogen detection in accordance with the techniques of the invention. A system configured in accordance with the invention could, however, additionally comprise means for preliminarily processing a sample so that the sample may undergo processing and analysis in accordance with the invention. Such internal means could include, for example, means for lysing one or more cells included in the sample so as to expose their intracellular contents, means for extracting one or more targeted molecular species contained within either such cells or in any liquid supernatant comprised within the sample, and optionally, means for removing any unwanted materials from the sample prior to undergoing analysis.

[0100] In this and any other aspects of the invention, the system could alternatively be configured to analyse biological samples that are not substantially in liquid form when collected from the host organism. So for example, the system could be configured such that the sample to undergo analysis is drawn from a biological material that is solid, semi-solid or quasi-solid,or which alternatively, contains material which is not in liquid form. By way of example, materials in this category include any one or more of the following:(1) tissue biopsy materials, including fresh, frozen, or formalin-fixed paraffin- embedded (FFPE) tissue following the completion of any appropriate preliminary processing required in relation to a given tissue biopsy sample;(2) cellular materials, including cultured cells and cell lines, isolated cell populations and sorted cells derived from the use of flow cytometry procedures;(3) swab specimens, such as Nasopharyngeal, oropharyngeal, wound, or environmental swabs suspended (as appropriate) in transport medium; (4) stool samples: after the performance of appropriate preliminary processing steps, such as homogenization and clarification; and(5) dried blood spots: after reconstitution in or with an appropriate buffer.

[0101] Persons of skill in the field of the invention will appreciate that this list is not exhaustive.

[0102] In the invention, the means for extracting nucleic acids from the biological sample generally comprise the removal of DNA associated with the host species from the biological sample.

[0103] In some embodiments, the means for extracting nucleic acids from the biological sample comprise the removal of RNA associated with the host species from the biological sample.

[0104] Irrespective of whether extraction of DNA or RNA associated with the host is desired, the means for extraction typically will include means for lysing cellular material contained within the sample, as well as means specifically for extracting the nucleic acid type(s) to be removed. In preferred embodiments, the means for lysing cellular materials and / or for extracting the predetermined nucleic acid(s) are chosen by reference to the nature of at least one of the nucleic acids to be extracted and / or with predetermined extraction performance characteristics, and not by reference to the character of other materials contained in the sample.

[0105] In preferred embodiments, the means for lysing cellular material contained within the sample comprise a lysis buffer. Preferably, the lysis buffer is capable of disrupting (ie, lysing) one or more of the following:(a) bacterial cell walls;(b) mammalian cellular membranes(c) viral envelopes; and(d) fungal cell wallscontained in the sample.

[0106] In some preferred embodiments, the lysis buffer contains:• 400mM Tris-HCI (pH 8.0);• 60mM EDTA;• 5% SDS (sodium dodecyl sulfate); and• 10mM DTT (dithiothreitol).

[0107] Persons of skill in the field of the invention will be aware that commercially available buffers exist which have this composition. However, such persons will appreciate that buffers having a different composition would alternatively also be suitable for use in the invention.

[0108] In alternative embodiments, the means for lysing cellular material contained within the sample are adapted to lyse plant cells. In this context, it is known that there are plants which contain cells that can adversely affect human and / or non-human animal health. (Pollens released from plant species and algal infections are examples of potential pathogens in this category, although algae are arguably not considered to be “true” plants). In those embodiments in which the use of the invention is intended to analyse plant material, it will be appreciated that suitable lysis means may need to be able to lyse plant cell wall components (such as cellulose, hemicellulose and / or lignin, for example).

[0109] In some embodiments, the performance of one or more preliminary processing step(s) on the sample may be required, before the sample is suitable for presentation to (and for processing by) a system in accordance with the invention. So for example, in some embodiments, one or more preliminary preparatory step(s) may need to be performed by means or processes extrinsic to the system, before the sample is ready to be presented to it. This may be required where, for example (and as mentioned earlier), the sample comprises stool materials derived from a host organism, and which need to be homogenized and clarified before being suitable for processing by the system. And by way of further example, where the sample contains plant material, it may be necessary for plant cells contained in the sample to undergo treatment in order to lyse the cells concerned, before presenting the pre-processed sample to the system. Such pre-processing steps may take any appropriate form in order to render the sample suitable for processing via the system. Such measures would include, by way of example, physical steps (such as physically cutting, grinding, crushing, agitating, stirring, or shaking or irradiating the sample), as well as chemical and / or biological pretreatments. Pre-treatment for this purpose would also include purification, refinement orclarification processes or treatments, the nature of which would readily be appreciated by ordinary persons of skill in the field of the invention.

[0110] All such pre-treatments are apprehended for use in association with the invention.

[0111] In preferred embodiments, the means for lysing cellular material contained within the sample form an integral part of the system. In even more preferred embodiments, such means are either automated or automatable, such that the invention reduces the necessity for human involvement in performing the detection of pathogens.

[0112] In embodiments of the invention, the means for depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, comprise the use of:(a) one or more ribonucleoprotein complexes that target human rRNA, mitochondrial DNA or RNA, and / or bacterial 16S rRNA, together with(b) guide RNAs and(c) high-fidelity SpyCas9.

[0113] In some embodiments, the guide RNAs comprise single-guide RNAs (sgRNAs).

[0114] In some embodiments, the means for completing the generation of the sequence library comprise the use of:(a) a sequence library amplification means; and(b) a quantification means.

[0115] In some embodiments, the means for completing the generation of the sequence library comprise the use of:(a) a Polymerase Chain Reaction (PCR) method before the depletion from the biological sample, of at least one predetermined nucleic acid associated with the host organism; and(b) optionally, a post-depletion PCR method involving the use of at least one index primer.

[0116] In some embodiments, after performance of sequence amplification on the sequence library, optionally, further refinement of the sequence library may be carried out by:(a) depleting from the sequence library, any residual nucleic acid sequences associated with the host organism, via the use of one or more post-amplification CRISPR techniques;(b) digestion of primers in the sequence library, so as to prevent adapter primer formation; and / or(c) the use of modular probe sets that allow adaptation to specific host sequences, in order to attain more accurate pathogen detection.

[0117] In some embodiments of the invention, the sequence library undergoes:(a) quantification by a nucleic acid quantification means;(b) fragmentation analysis; and(c) normalization of the sequence librarybefore the sequence library undergoes high-throughput sequencing by the sequencing means.

[0118] In some embodiments, after the performance of steps (a), (b) and (c) listed in the preceding paragraph, the sequence library then undergoes high-throughput sequencing by the sequencing means.

[0119] In some embodiments, after the sequence library has undergone high-throughput sequencing by the sequencing means, the sequence library then undergoes analysis by the bioinformatics analysis means.

[0120] In some embodiments, the sequencing means comprises means for cooperating with the bioinformatics analysis means, in order to facilitate the formatting of sequencing data derived via the performance of the high-throughput sequencing, for presentation or transfer to and analysis by the bioinformatics analysis means.

[0121] In some embodiments, the bioinformatics analysis means comprise either:(a) physically integrated with the rest of the system; or(b) an online means.

[0103] In some embodiments, after the bioinformatics analysis is performed, the bioinformatics analysis means generates a report of the findings of its analysis on the sequence library.

[0104] In embodiments, the report is either:(a) a hard copy report; or(b) an electronic report, being one that is capable of being transferred from one piece of hardware to another by either a wired or a wireless communications connection (such as, for example, via a text message or an e-mail message, or via direct computer to computer communication).

[0105] In some embodiments, the system additionally entails the use of a container that comprises two or more internal chambers, where the individual chambers store, and, in use of the system, supply the reagents required in order to perform sequentially, the steps of:(a) extraction of the nucleic acid sequences from the biological sample(b) generation of the sequence library from the biological sample collected from the host organism;(c) Depletion of abundant background sequences from the sequence library;(d) Amplification of the non-background sequences from the library; and(e) Normalization of the amount of sequences from the resulting sequence library;

[0106] In some embodiments, the container takes the form of a single use cartridge, which is disposed after the performance of steps (a) to (e) (listed in the preceding paragraph) on a given biological sample, and replaced by an unused like cartridge in order to perform those steps on any other biological sample. And in some preferred embodiments, after the biological sample is collected from the host organism, the performance of any of those steps (a) to (g) is automated. And in some particularly preferred embodiments of the system, the biological sample is collected across two or more collection vessels, such that the performance of steps (a) to (g) on a given biological sample is replicated across all such collection vessels. In some embodiments of the invention, it is particularly preferred that the performance of steps (a) to (e) is performed simultaneously across two or more collection vessels, which will expedite the time taken to analyse the biological sample and generate a report about whether the system has detected that it contains one or more pathogens (and where applicable, to identify that the pathogen or pathogens concerned).

[0107] In another broad aspect, the invention also generally provides a method for detecting the presence of at least one pathogen species in a biological sample collected from a host organism, the method comprising the steps of:(a) generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whether one or more pathogen species is or are present in the biological sample, where the steps for generating the sequence library comprise:(1) extracting one or more predetermined nucleic acids from the biological sample;(2) depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(3) completing the generation of the sequence library;(b) performing a high-throughput sequencing on the sequence library; and(c) performing a bioinformatics analysis in the sequence library in order to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library.

[0108] Embodiments of the method aspect of the invention include those described above in relation to the system aspect of the invention (mutatis mutandis).

[0109] In yet another broad aspect, the invention also generally provides a method of diagnosing the presence of an infection, or infections, in a host organism, the method comprising the steps of:(a) collecting a biological sample from the host organism;(b) generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whether one or more pathogen species is or are present in the biological sample, where the steps for generating the sequence library comprise:(1) extracting one or more predetermined nucleic acids from the biological sample;(2) depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(3) completing the generation of the sequence library;(c) performing a high-throughput sequencing on the sequence library; and(d) performing a bioinformatics analysis in the sequence library in order to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library.

[0110] Embodiments of the method aspect of the invention include those described above in relation to the system aspect of the invention (mutatis mutandis).

[0111] In a further broad aspect, the invention also generally provides an apparatus for detecting the presence of at least one pathogen species in a biological sample collected from a host organism, the apparatus comprising:(a) means for generating a sequence library from the biological sample, to undergo a metagenomic sequencing and bioinformatics analysis in order to detect whether one or more pathogen species is or are present in the biological sample, where the means comprise:(1) means for extracting nucleic acids from the biological sample;(2) means for depleting from the biological sample, at least one predetermined nucleic acid associated with the host organism, where the depletion is achieved by the use of a CRISPR-based method; and(3) means for completing the generation of the sequence library;(b) a sequencing means adapted to perform high-throughput sequencing on the sequence library; and(c) a bioinformatics analysis means adapted to:(1) process data derived from the high-throughput sequencing performed on the sequence library; and(2) detect the presence of the at least one pathogen in the sample based on the detection of a nucleic acid sequence associated with the pathogen in the sequence library.

[0112] Embodiments of the apparatus aspect of the invention include those described above in relation to the system aspect of the invention (mutatis mutandis).

[0113] As explained in the general disclosure of the preceding aspects, the present invention therefore relates broadly to a system (and an associated apparatus and method), embodiments of which are specifically designed for metagenomic next-generation sequencing (mNGS) in clinical diagnostic settings, aimed at detecting and, where applicable, identifying pathogens. And as previously explained, the system (and its associated apparatus and method aspects) thus broadly relate to a system that is automated in order to detect and identify pathogens. The system addresses some of the key limitations of existing workflows, such as the need for manual processing, batch-based delays, and sensitivity issues resulting from the presence of high host nucleic acid content within a biological sample. Current metagenomic sequencing methods require multiple instruments and complex manual steps, thus limiting their effectiveness, especially in detecting low biomass pathogens that arecontained amidst abundant host genetic material contained in a sample. The known methods also entail inefficiencies, including adapter dimer formation and contamination risks, which delay results, potentially generate inaccurate results, increase costs, and hinder the ability to respond to both individual patient needs and the needs of the broader public, for example, in public health emergencies.

[0114] The present invention integrates several key components to streamline the process of RNA / DNA extraction, library preparation, and host sequence depletion. The system includes an Integrated Processing Module, which automates critical steps such as RNA / DNA extraction, adapter ligation, and PCR amplification. A CRISPR-based depletion system is employed to remove host sequences, significantly improving the sensitivity for detecting pathogens. Additionally, the system features a random-access architecture with multiple independent processing stations, thereby enabling parallel sample processing and eliminating batch delays. This allows urgent cases to be prioritized and processed immediately, without waiting for other samples in the batch. A single-use consumable cartridge containing lyophilized reagents simplifies storage and shipping, while ensuring a closed system that minimizes contamination risks.

[0115] Furthermore, the system includes a sequencer integration interface, automating data formatting for platforms such as the Illumina® MiniSeq (Illumina, Inc - www jHumina cem) and linking to cloud-based bioinformatics for seamless analysis. The system enhances pathogen signal-to-noise ratios by removing host sequences before (and where appropriate, after amplification), ensuring more accurate pathogen detection. Key innovations of the system include primer digestion that prevents adapter dimer formation, post-amplification CRISPR depletion for improved specificity, and modular probe sets that allow adaptation to different host sequences. Clinical validation studies (Seehave shown promising results, including a 98.7% efficiency in host sequence depletion and 100% sensitivity and 100% specificity when compared with FDA-approved PCR panels for respiratory pathogens at Ct values up to 35 (Chan et al, 2023), with end-to-end processing time of between 6-12.5 hours.

[0116] In the various embodiments disclosed above, advantageously, the invention allows both hospital laboratories and medical and veterinary clinics to deploy mNGS for syndromic infectious disease testing, outbreak surveillance, antimicrobial resistance profiling, and rapid response to novel pathogens during public health emergencies. The combination of automated host depletion, random-access mNGS processing, and integrated CRISPR / bioinformatics components advances clinical metagenomics, addressing existing technical and logistical challenges in pathogen detection.

[0117] As ordinary persons of skill in the field of the invention will appreciate, the fundamental improvements that the invention provides in the use of mNGS in biological sequencing extend beyond the aspects discussed previously. The invention therefore extends to, and embodies, all the following further general aspects.Rare Disease Genomics with Targeted Sequence Depletion

[0118] The inventor has realised that the invention may also usefully be deployed in genomic analysis in rare disease contexts.

[0119] Thus, in a further broad aspect, the invention also generally provides a method for detecting genomic variants associated with a disease, the method comprising the steps of:(a) extracting RNAfrom a biological sample collected from a host organism by the use of magnetic bead-based purification means under conditions optimized to preserve long nucleic acid fragments;(b) selectively depleting one or more predefined genomic regions of low diagnostic relevance using CRISPR-based cleavage mediated by one or more guide RNAs; (c) preparing a sequencing library from the remaining nucleic acids;(d) enriching genomic regions associated with one or more diseases by:(1) hybridizing the sequencing library with biotinylated oligonucleotide probes complementary to the genomic regions of interest at an elevated temperature;(2) capturing hybridized library molecules using streptavidin-coated magnetic beads; and(3) removing non-hybridized sequences through one or more washing steps; (e) amplifying the enriched library by PCR to generate sufficient material for sequencing;(f) sequencing the amplified library to a depth sufficient to achieve high coverage of the genomic regions of interest; and(g) analysing sequencing data derived from the sample in order to detect:(1) copy number variations through read depth analysis;(2) structural variants through paired-end read mapping; and(3) point mutations through comparison with a reference genome using disease-specific bioinformatic algorithms.

[0120] In the method, preferably, the magnetic bead-based purification means are configured to recover preferentially nucleic acid fragments greater than 5 kilobases in length (these are defined as being “long” nucleic acid fragments for the purposes of the invention).

[0121] In the method, preferably, the CRISPR-associated nuclease comprises Cas9 or a functional variant thereof.

[0122] In embodiments of the method, the predefined genomic regions of low diagnostic relevance comprise repetitive elements, highly conserved housekeeping genes, or genomic regions with low disease association.

[0123] In embodiments of the method, preferably, the enrichment means are configured to target a predefined disease gene panel comprising at least 10 genes associated with inherited or somatic disorders.

[0124] In the method, the sequencing module comprises a next-generation sequencing platform configured for paired-end sequencing.

[0125] In embodiments of the invention, the bioinformatics analysis means employ algorithms optimized fora specific disease selected from cancer, inherited genetic disorders, or infectious disease.

[0126] In embodiments of the method, it is preferred that CRISPR-based depletion is performed prior to hybridization capture enrichment.

[0127] And in embodiments of the method, it is preferred that the sequencing depth is at least 200* coverage across the enriched genomic region (or regions, if more than one). And in this and all other applicable embodiments of the invention, preferably, the sequencing coverage depth is at least 10 million read pairs per sample. In more preferred embodiments, the sequencing coverage depth is 20 to 40 million read pairs per sample.

[0128] And it is generally preferred that the method is automated or automatable, and that upon the conclusion of performing the method in any instance, the system or apparatus in which the method is performed generates a clinical report identifying detected genomic variants and correlating the variants with disease-associated annotations.

[0129] In this broad aspect, the invention further generally provides a system in which the method may be performed. The system comprises:(a) means for extracting RNAfrom a biological sample collected from a host organism by the use of magnetic bead-based purification means under conditions optimized to preserve long nucleic acid fragments;(b) means for selectively depleting one or more predefined genomic regions of low diagnostic relevance using CRISPR-based cleavage mediated by one or more guide RNAs;(c) means for preparing a sequencing library from the remaining nucleic acids;(d) means for enriching genomic regions associated with one or more diseases by:(1) hybridizing the sequencing library with biotinylated oligonucleotide probes complementary to the genomic regions of interest at an elevated temperature; (2) capturing hybridized library molecules using streptavidin-coated magnetic beads; and(3) removing non-hybridized sequences through one or more washing steps; (e) means for amplifying the enriched library by PCR to generate sufficient material for sequencing;(f) means for sequencing the amplified library to a depth sufficient to achieve high coverage of the genomic regions of interest; and(g) means analysing sequencing data derived from the sample in order to detect:(1) copy number variations through read depth analysis;(2) structural variants through paired-end read mapping; and(3) point mutations through comparison with a reference genome using disease-specific bioinformatic algorithms.

[0130] And in this broad aspect, the invention further generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Cancer Gene Fusion Detection

[0131] The inventor has divined that the invention may also usefully be deployed in the detection of recombination events in cancer specimens (in which gene fusions represent therapeutically actionable biomarkers) in a host organism.

[0132] Accordingly, in a further broad aspect, the invention also generally provides a method for detecting therapeutically actionable gene fusion events in a biological sample collected from a host organism, the method comprising the steps of:(a) extracting total RNAfrom the biological sample, where the sample comprises one or more of:(1) tumour tissue;(2) circulating tumour cells; or(3) circulating tumour RNAfrom the blood of the host organism, by the use of using magnetic bead-based purification means;(b) optionally depleting one or more abundant non-fusion-bearing transcripts from the extracted RNA;(c) reverse transcribing the RNA to generate cDNA;(d) ligating sequencing adapters to the cDNA and amplifying the cDNA so as to generate a sequencing library;(e) performing paired-end sequencing on the sequencing library; and(f) identifying, using a bioinformatics analysis means, sequence reads that map to two different gene loci to determine the presence of a gene fusion breakpoint.

[0133] Preferably, the optional depletion step comprises the step of contacting the RNA with one or more guide RNAs targeting ribosomal RNA.

[0134] It is further preferred that the optional depletion step comprises the step of targeting highly abundant housekeeping genes.

[0135] Preferably, the optional depletion step also comprises the step of targeting tissuespecific abundant transcripts.

[0136] It is further preferred that in the method, depletion increases the relative sequencing depth of rare fusion-containing transcripts.

[0137] It is yet further preferred that the method comprises the step of performing hybridization capture enrichment in order to target known fusion breakpoint sequences.

[0138] In embodiments of the method, preferably, the hybridization capture enrichment targets one or more entire gene regions known to participate in fusion events.

[0139] In yet other embodiments of the method, it is preferred that the paired-end sequencing enables identification of split reads spanning a fusion junction.

[0140] In yet other embodiments of the method, it is preferred that the method comprises mapping the fusion breakpoint with base-pair resolution.

[0141] In yet further embodiments of the method, it is preferred that the method comprises predicting a fusion protein sequence encoded by the detected fusion junction.

[0142] In yet other embodiments of the method, it is preferred that the method comprises that the selective transcript depletion means comprise programmable guide RNAs.

[0143] In yet other embodiments of the method, it is preferred that the bioinformatics module is configured to filter candidate fusion events based on read depth and breakpoint confidence metrics.

[0144] In yet other embodiments of the method, it is preferred that the biological sample comprises one or more circulating tumour RNA isolated from plasma.

[0145] Preferably further, in the method, detection of the gene fusion event identifies a therapeutically actionable biomarker for targeted cancer therapy selection.

[0146] In this aspect, the invention additionally provides a system in which the method may be performed. The system comprises:(a) an RNA extraction means configured to extract total RNA from a biological sample collected from a host organism, where the sample comprises one or more of:(1) tumour tissue;(2) circulating tumour cells; or(b) circulating tumour RNA from the blood of the host organism;(c) an optional selective transcript depletion means configured to reduce abundance of non-fusion-bearing transcripts;(d) a reverse transcription means configured to generate complementary DNA (cDNA) from the extracted RNA;(e) a library preparation means configured to ligate sequencing adapters and amplify the cDNAso as to form a sequencing library(f) a sequencing means configured to perform paired-end sequencing of the sequencing library; and(g) a bioinformatics analysis means configured to identify sequence reads mapping to two distinct gene loci and thereby detect a gene fusion breakpoint.

[0147] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system andthe apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Circulating Tumour DNA Detection

[0148] The inventor has also divined that the invention may also usefully be deployed in the ultra-sensitive detection of circulating tumour DNA in a host organism.

[0149] In essence, in this aspect, the invention provides a method of increasing the sensitivity of circulating tumour DNA detection in a plasma sample, comprising selectively depleting non-tumour-derived cell-free DNA prior to ultra-deep sequencing to enable detection of tumour-derived variants at or below 0.01% variant allele frequency.

[0150] Accordingly, in a yet further broad aspect, the invention also generally provides a method for ultra-sensitive detection of circulating tumour DNA (ctDNA) in a patient, the method comprising the steps of:(a) obtaining a biological sample comprising cell-free plasma derived from blood collected from the host organism following centrifugation so as to remove cellular material;(b) extracting cell-free DNA from the plasma using magnetic bead-based purification optimized for recovery of short DNA fragments characteristic of circulating cell-free DNA;(c) optionally selectively depleting non-tumour-derived cell-free DNA sequences from the extracted DNA;(d) optionally enriching the extracted DNA for one or more target somatic mutations; (e) sequencing the enriched DNA to ultra-high depth; and(f) identifying, using a bioinformatics analysis means which executes error-correction algorithms, one or more tumour-derived sequence variants present at a variant allele frequency of 0.01% or lower.

[0151] In embodiments, the method comprises a two-step centrifugation process configured to substantially remove cellular genomic DNA contamination.

[0152] In embodiments, the magnetic bead-based purification preferentially recovers DNA fragments between 100-200 base pairs.

[0153] In preferred embodiments, the selective depletion comprises the step of contacting the extracted DNA with guide RNAs targeting wild-type genomic sequences corresponding to genes in which tumour-associated mutations are sought.

[0154] In preferred embodiments, the selective depletion additionally comprises targeting common somatic mutations associated with clonal hematopoiesis.

[0155] In preferred embodiments, the selective depletion additionally reduces wild-type DNA background so as to increase the sensitivity of mutation detection.

[0156] In preferred embodiments, the enrichment step comprises targeted PCR amplification of previously identified tumour-specific mutations.

[0157] In preferred embodiments, the enrichment step comprises hybridization capture to target genomic loci corresponding to somatic mutations identified in a primary tumour from the host organism.

[0158] In preferred embodiments, the sequencing is performed to a depth of between 1 million and 10 million reads per sample. In particularly preferred embodiments, the sequencing is performed to a depth of between 20 million and 40 million reads per sample.

[0159] In yet further preferred embodiments, the method additionally comprises attaching unique molecular identifiers (UMIs) to individual DNA molecules prior to amplification.

[0160] In yet further preferred embodiments, the method additionally comprises collapsing reads sharing identical unique molecular identifiers in order to remove amplification and sequencing errors.

[0161] In yet further preferred embodiments, the step of identifying variants comprises applying statistical modelling in order to distinguish true variants from background sequencing errors.

[0162] In yet further preferred embodiments, the method additionally comprises the use of ctDNAto monitor minimal residual disease.

[0163] In yet further preferred embodiments, the method additionally comprises the detection of ctDNAto monitor responses by the host organism to treatment.

[0164] In yet further preferred embodiments, the method additionally comprises the detection of ctDNAto detect cancer recurrence prior to radiographic evidence.

[0165] In this aspect, the invention additionally provides a system in which the method may be performed. The system is one for detecting circulating tumour DNA in a plasma sample, the system comprising:(a) a sample preparation means configured to isolate cell-free plasma from blood; (b) a DNA extraction means configured to recover short cell-free DNA fragments using magnetic bead-based purification;(c) an optional selective depletion means configured to reduce abundance of non- tumour-derived DNA sequences;(d) an optional enrichment means configured to selectively amplify or capture one or more target somatic mutations;(e) a sequencing means configured to generate at least one million sequence reads per sample; and(e) a bioinformatics analysis means configured to perform molecular identifier collapsing and error correction to discriminate true low-frequency variants from sequencing artifacts.

[0166] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Environmental Surveillance

[0167] The inventor has also identified that the invention has applications beyond detection and identification of pathogens in living host organisms. The inventor has divined that the methods, systems, and apparatus of the invention may also usefully be deployed in the detection of pathogens in environmental samples, such as wastewater, where pathogen nucleic acids are vastly outnumbered by nucleic acids associated with environmental microorganisms.

[0168] Accordingly, in a yet further broad aspect, the invention also generally provides a method for detecting one or more pathogens in an environmental sample, the method comprising the steps of:(a) obtaining a biological sample comprising wastewater collected from an environmental source;(b) optionally concentrating the sample to increase the recovery of pathogen nucleic acids present in the sample;(c) extracting one or more predetermined nucleic acids from the sample;(d) selectively depleting one or more non-pathogenic nucleic acids from the extracted nucleic acids;(e) preparing a sequencing library from the depleted nucleic acids;(f) sequencing the library; and(g) identifying one or more pathogen-derived nucleic acid sequences present in the sample.

[0169] In preferred embodiments, the method comprises the additional step of estimating the relative abundance of the pathogen in the sample.

[0170] In yet further preferred embodiments, the method additionally comprises the step of identifying at least one pathogen detected in the biological sample.

[0171] In embodiments, the concentration step comprises filtration, centrifugation, precipitation, or ultrafiltration.

[0172] In embodiments of the invention, the extracted nucleic acids may comprise RNA, DNA, or both.

[0173] In preferred embodiments, the step of depletion comprises targeting human nucleic acids.

[0174] In preferred embodiments of the method, the targeted human nucleic acids comprise human ribosomal RNA.

[0175] In preferred embodiments of the method, the targeted human nucleic acids comprise human mitochondrial RNA.

[0176] In preferred embodiments of the method, the selective depletion step comprises targeting abundant environmental bacterial sequences.

[0177] In yet further preferred embodiments of the method, the selective depletion step comprises contacting the nucleic acids with guide RNAs configured to hybridize to non-pathogenic sequences.

[0178] In yet further preferred embodiments of the method, the selective depletion step increases the proportion of sequencing reads derived from pathogen nucleic acids.

[0179] In yet further preferred embodiments of the method, the sequencing is performed to a depth sufficient to detect pathogen sequences present at less than 0.1% of total nucleic acids.

[0180] In yet further preferred embodiments of the method, the step of identifying pathogen-derived sequences comprises aligning sequence reads to a pathogen reference database.

[0181] In yet further preferred embodiments of the method, the step of estimating relative abundance comprises calculating read counts normalized to total sequencing depth.

[0182] Preferably in the method, the pathogen comprises a virus, bacterium, fungus, or protozoan.

[0183] It is further preferred in the method that the pathogen is associated with a public health outbreak.

[0184] In this aspect, the invention additionally provides a system in which the method may be performed. The system is one for detecting pathogens in environmental wastewater, the system comprising:(a) a sample processing means configured to receive wastewater and optionally concentrate pathogen particles;(b) a nucleic acid extraction means configured to isolate total nucleic acids from the sample;(c) a selective depletion means configured to reduce abundance of non-pathogenic nucleic acids;(d) a library preparation means configured to generate a sequencing-ready nucleic acid library;(e) a sequencing means configured to generate sequence reads from the library; and (f) a bioinformatics analysis means configured to identify pathogen sequences and quantify their relative abundance.

[0185] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Vector-Borne Disease Surveillance

[0186] Arthropods, primarily blood-feeding insects and arachnids like mosquitoes, ticks, fleas, and flies, act as vectors by transmitting viruses, bacteria, and parasites to humans and animals, accounting for over 17% of all infectious diseases. They acquire pathogens by feeding on infected hosts and transmit them, often through saliva during subsequent bites or via faeces.

[0187] The inventor has also identified that the invention is useful in detecting the presence of pathogens present in arthropod species. For the purpose of this aspect of the invention, individual arthropods are captured for surveillance purposes, and biological samples are collected from them for analysis in accordance with the invention.

[0188] Thus, in this aspect, the invention generally provides a method for detecting one or more vector-borne pathogens in a biological collected from an arthropod species, the method comprising the steps of:(a) obtaining a biological sample from the arthropod species;(b) homogenizing and lysing the sample to release nucleic acids from arthropod tissues and any harbored pathogens;(c) extracting nucleic acids from the lysed sample;(d) selectively depleting non-pathogenic arthropod-derived nucleic acids from the extracted nucleic acids;(e) preparing a sequencing library from the depleted nucleic acids;(f) sequencing the library; and(g) detecting one or more vector-borne pathogens from the sequencing data.

[0189] In essence then, in this aspect, the method increases the sensitivity of pathogen detection in arthropod vectors, by selectively depleting arthropod ribosomal RNA, arthropod genomic DNA, and arthropod-associated microbiota nucleic acids prior to sequencing to increase representation of pathogen-derived sequences.

[0190] In preferred embodiments, the method additionally identifies at least one pathogen species present in the biological sample.

[0191] In the method, the arthropod is preferably selected from the group comprising:(a) a mosquito;(b) a tick;(c) a flea or(d) a fly.

[0192] However, in alternative embodiments, the arthropod may be from a different species to those mentioned in the preceding paragraph.

[0193] In embodiments, the selective depletion step comprises targeting arthropod ribosomal RNA.

[0194] In embodiments, the selective depletion step comprises targeting arthropod genomic DNA sequences.

[0195] In embodiments, the selective depletion step comprises targeting one or more nucleic acids derived from arthropod-associated microbiota.

[0196] In other embodiments, the targeted microbiota comprise one or more obligate symbionts.

[0197] In other embodiments, the selective depletion step comprises contacting nucleic acids with guide RNAs designed to hybridize to arthropod-derived sequences.

[0198] In other embodiments, the depletion step increases the proportion of sequencing reads corresponding to pathogen-derived nucleic acids.

[0199] In some embodiments, the pathogen comprises an arthropod-borne virus.

[0200] In some embodiments, the pathogen comprises a Rickettsia species.

[0201] In other embodiments, the pathogen comprises a spirochete.

[0202] In yet other embodiments, the pathogen comprises a parasitic organism.

[0203] And in some preferred embodiments, the method further comprises estimating the relative abundance of at least one pathogen species.

[0204] In this aspect, the invention also generally provides a system for detecting vector-borne pathogens in arthropod vectors, the system comprising:(a) a sample processing means configured to homogenize and lyse one or more arthropods;(b) a nucleic acid extraction means configured to isolate nucleic acids from arthropod and pathogen material;(c) a selective depletion means configured to reduce the abundance of arthropod- derived nucleic acids;(d) a library preparation means configured to generate a sequencing-ready library; (e) a sequencing means configured to generate sequence reads from the library; and (f) a bioinformatics means configured to identify one or more vector-borne pathogens.

[0205] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Veterinary Diagnostics

[0206] As indicated earlier, the invention is applicable in both human and veterinary medicine.

[0207] In this broad aspect, the invention generally provides a method for detecting one or more pathogens in a biological sample collected from a non-human animal, the method comprising the steps of:(a) obtaining a biological sample from a non-human animal;(b) extracting one or more nucleic acids from the biological sample;(c) optionally selectively depleting host-derived nucleic acids from the extracted nucleic acids, where the host-derived nucleic acids comprise species-specific ribosomal RNAand mitochondrial DNA;(d) preparing a sequencing library from the extracted or depleted nucleic acids; (e) sequencing the library; and(f) detecting one or more pathogens of veterinary significance from the sequencing data.

[0208] In preferred embodiments, the method additionally identifies the one or more pathogens present in the biological sample.

[0209] The method may be used with any non-human animal. Preferred non-human animal species for the purposes of the method include livestock (cattle, sheep, pigs and goats) and poultry.

[0210] A “biological sample” for the purpose of the method includes, without limitation, samples of any and all of the kinds of biological materials referred to in paragraphs

[0079] to

[0083] ,

[0211] In embodiments, the selective depletion step comprises contacting the extracted nucleic acids with guide RNAs designed to hybridize to species-specific ribosomal RNA sequences.

[0212] In embodiments, the selective depletion step comprises targeting host mitochondrial DNA sequences.

[0213] In embodiments, the guide RNA sets are selected based on the species from which the biological sample was obtained.

[0214] In embodiments, the selective depletion increases the proportion of sequencing reads corresponding to pathogen-derived nucleic acids.

[0215] For the purposes of this aspect of the invention, the pathogen may comprise one or more of:(a) a viral pathogen;(b) a bacterial pathogen;(c) a parasitic pathogen;(d) a fungal pathogen;(e) a zoonotic pathogen; and(f) a pathogen of plant origin.

[0216] In preferred embodiments, the invention allows the detection and identification of one or more economically significant pathogens affecting livestock or poultry.

[0217] In this aspect, the invention further generally provides a system for detecting one or more pathogens in a biological sample collected from an non-human animal, the system comprising:(a) a nucleic acid extraction means configured to extract nucleic acids from the biological sample;(b) a selective depletion means configured to reduce abundance of species-specific host ribosomal RNA and mitochondrial DNA;(c) a library preparation means configured to generate a sequencing-ready nucleic acid library;(d) a sequencing means configured to generate sequence reads; and(e) a bioinformatics analysis means configured to identify one or more pathogens of veterinary significance.

[0218] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Single-Cell RNA Sequencing with CRISPR-Based Depletion

[0219] The inventor has also identified that the invention is also useful for detecting lowly-expressed small RNA sequences derived from single cells.

[0220] Thus, in this broad aspect, the invention further generally provides a method for enhancing single-cell RNA sequencing, the method comprising the steps of:(a) isolating individual cells from a biological sample comprising tissue-derived cells or cultured cell populations, by using a single-cell capture methodology;(b) lysing the individual cells and reverse transcribing cellular RNA to generate complementary DNA(cDNA), where cell-specific barcode sequences are attached to transcripts originating from each individual cell;(c) pooling barcoded cDNAfrom multiple cells;(d) performing limited-cycle PCR amplification of the pooled barcoded cDNA under controlled thermal cycling conditions;(e) selectively depleting ribosomal RNA-derived sequences from the amplified barcoded cDNA using a CRISPR-based depletion system;(f) preparing a sequencing library from the depleted cDNA;(g) sequencing the library; and(h) generating a per-cell gene expression matrix from the sequencing data.

[0221] Preferably in the method, the single-cell capture methodology comprises microfluidic droplet-based capture.

[0222] In embodiments, the single-cell capture methodology comprises microwell-based capture.

[0223] In embodiments, the cell-specific barcode comprises a unique molecular identifier (UM / ).

[0224] In embodiments, the CRISPR-based depletion system comprises guide RNAs targeting 18S ribosomal RNA sequences.

[0225] In embodiments, the CRISPR-based depletion system comprises guide RNAs targeting 28S ribosomal RNA sequences.

[0226] In embodiments, the CRISPR-based depletion system further comprises guide RNAs targeting mitochondrial RNA species.

[0227] In embodiments, the depletion reduces the proportion of sequencing reads mapping to ribosomal RNA.

[0228] In embodiments, the depletion step increases the proportion of sequencing reads mapping to protein-coding transcripts.

[0229] In embodiments, the depletion step increases sensitivity for detection of lowly expressed genes.

[0230] And in embodiments, the step of library preparation comprises fragmentation and adapter ligation.

[0231] In this broad aspect, the invention further generally provides a system for enhancing single-cell RNA sequencing, the system comprising:(a) a single-cell capture means configured to isolate individual and optionally capture cells;(b) a reverse transcription means configured to generate barcoded cDNAfrom RNA of individual cells;(c) a thermal cycling means configured to perform limited-cycle PCR amplification of pooled barcoded cDNA;(d) a CRISPR-based depletion means configured to selectively remove ribosomal RNA-derived sequences from the amplified cDNA;(e) a library preparation means configured to prepare sequencing libraries;(f) a sequencing means; and(g) a bioinformatics analysis means configured to generate per-cell gene expression matrices.

[0232] In preferred embodiments, the system additionally provides means for generating reports from the performance of the method aspect of the invention.

[0233] And in this broad aspect, the invention further generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq)

[0234] The inventor has also identified that the invention is also useful in Assays for Transposase-Accessible Chromatin using sequencing.

[0235] Hence, in this aspect, the invention provides a method for improving assay for transposase-accessible chromatin (ATAC-Seq), the method comprising the steps of:(a) obtaining DNA fragments from cells or tissue that have been subjected to Tn5 transposase-mediated tagmentation, where sequencing adapters are inserted into accessible chromatin regions;(b) contacting the tagmented DNA fragments with a CRISPR-based depletion system comprising guide RNAs targeting mitochondrial DNA sequences;(c) cleaving mitochondrial DNA fragments while preserving nuclear chromatin-derived fragments;(d) removing cleaved mitochondrial DNA fragments through purification;(e) amplifying remaining intact adapter-containing DNA fragments; and(f) sequencing the amplified fragments to identify genome-wide open chromatin regions.

[0236] In embodiments, the Tn5 transposase is a hyperactive Tn5 transposase.

[0237] In embodiments, the guide RNAs are designed to target comprehensively, sequences distributed across the entire mitochondrial genome.

[0238] Preferably, in the method, cleavage of mitochondrial DNA fragments removes one or both sequencing adapters from the fragments.

[0239] Preferably further, in the method, cleaved mitochondrial DNA fragments lacking intact sequencing adapters are not amplified during subsequent PCR.

[0240] In embodiments, the purification step comprises bead-based size selection or magnetic bead-based cleanup.

[0241] In embodiments, the depletion step reduces the proportion of sequencing reads mapping to mitochondrial DNA.

[0242] Preferably, the depletion step increases the proportion of sequencing reads mapping to nuclear chromatin-derived fragments.

[0243] Preferably, the depletion improves sensitivity for detection of low-accessibility regulatory regions.

[0244] Preferably, the biological sample comprises mammalian cells.

[0245] It is further preferred that in the method, the biological sample comprises human cells.

[0246] Preferably, the sequencing methodology comprises paired-end sequencing.

[0247] In this aspect, the invention also provides a system for enhancing chromatin accessibility profiling, the system comprising:(a) an input means configured to receive Tn5-tagmented DNA fragments derived from cells or tissue;(b) a CRISPR depletion means comprising guide RNAs targeting mitochondrial DNA sequences across a mitochondrial genome;(c) a purification means configured to remove cleaved mitochondrial DNA fragments;(d) a PCR amplification means configured to selectively amplify intact adaptercontaining nuclear chromatin fragments; and(e) a sequencing and bioinformatics analysis means configured to identify open chromatin regions.

[0248] And in this broad aspect, the invention further generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Ribosome Profiling

[0249] The inventor has also divined that the invention is useful in a ribosome profiling methodology, in which ribosome-protected mRNA fragments are selectively recovered, indicating which mRNA molecules are actively translated.

[0250] In this aspect, the invention therefore provides a method for metagenomic characterization of a microbial community in a complex biological sample, the method comprising the steps of:(a) obtaining a biological sample comprising material from a microbiome source selected from gut contents, fecal material, skin swabs, oral samples, soil, water, or environmental material;(b) extracting the total nucleic acids from the biological sample, the total nucleic acids comprising microbial and non-microbial nucleic acids;(c) selectively depleting non-microbial nucleic acids from the extracted nucleic acids; (d) preparing a sequencing library from the remaining nucleic acids;(e) sequencing the library; and(f) performing bioinformatic analysis to generate taxonomic characterization of the microbial community.

[0251] In embodiments, the biological sample comprises a human microbiome.

[0252] In alternative embodiments, the biological sample comprises a non-human microbiome.

[0253] In the method, the selective depletion step preferably comprises contacting the nucleic acids with guide RNAs targeting human (or non-human animal) ribosomal DNA.

[0254] In embodiments, the selective depletion step comprises targeting human mitochondrial DNA. And in such embodiments, the selective depletion step preferably comprises targeting highly abundant human genomic sequences.

[0255] In embodiments, the depletion step preferably reduces host-derived sequences by between 80% and 95%.

[0256] In embodiments, preferably, the depletion step proportionally enriches microbial-derived nucleic acids.

[0257] And preferably, in the method, the sample comprises a high host-contamination sample.

[0258] In this broad aspect, the invention further generally provides a system for metagenomic characterization of microbial communities, the system comprising:(a) a nucleic acid extraction means configured to extract total nucleic acids from a microbiome-derived sample;(b) a selective depletion means configured to reduce abundance of non-microbial nucleic acids;(c) a library preparation means configured to generate a sequencing-ready library; (d) a sequencing module; and(e) a bioinformatics analysis means configured to perform taxonomic characterization of microbial sequences.

[0259] And in this broad aspect, the invention further generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Whole Genome Sequencing

[0260] The inventor has also divined that the invention is useful in whole genome sequencing.

[0261] This aspect is directed to comprehensive, unbiased genomic sequencing. The biological sample comprises genomic DNA extracted from diverse organism sources.

[0262] Thus, in this broad aspect the invention further generally provides a method for automated comprehensive genomic sequencing, the method comprising the steps of:(a) extracting genomic DNA from a biological sample using magnetic bead-based purification means optimized for recovery of high molecular weight DNA; (b) performing library preparation comprising on-bead transposome-mediated tagmentation, adapter ligation, and limited-cycle PCR amplification incorporating sample-specific index sequences;(c) automatically controlling a tagmentation reaction at approximately 55°C under precise thermal regulation;(d) performing automated bead-based library normalization; and(e) transferring normalized libraries for genome-wide sequencing, where the method is configured for random-access processing such that individual samples are processed upon receipt without batch accumulation.

[0263] In preferred embodiments of the method, the biological sample is derived from saliva.

[0264] In embodiments, the method further comprises the step of removing bacterial DNA contamination from the saliva-derived genomic DNA. In preferred embodiments, the step of bacterial contamination removal comprises selective depletion of bacterial nucleic acids.

[0265] In preferred embodiments, the magnetic bead-based purification preferentially recovers genomic DNA fragments greater than 20 kilobases.

[0266] Preferably, in the method, limited-cycle PCR amplification comprises fewer than 12 amplification cycles.

[0267] In preferred embodiments, the automated thermal regulation maintains temperature within ±0.5°C of 55°C during tagmentation.

[0268] It is generally preferred that in the performance of the method, random-access processing permits initiation of library preparation for a sample immediately upon loading into the system.

[0269] Preferably, the automated bead-based normalization produces sequencing-ready libraries without the need for manual quantification.

[0270] In embodiments of the method, genome-wide sequencing comprises whole-genome sequencing.

[0271] In alternative embodiments of the method, genome-wide sequencing comprises whole-exome sequencing.

[0272] In this broad aspect of the invention, the invention further generally provides an automated system for genome-wide sequencing, the system comprising:(a) a magnetic bead-based DNA extraction means configured to recover high molecular weight genomic DNA;(b) a library preparation means configured to perform on-bead transposome tagmentation, adapter ligation, and indexed PCR amplification;(c) a thermal control means configured to maintain tagmentation reactions at approximately 55°C with controlled temperature precision;(d) a normalization means configured to perform automated bead-based library normalization; and(e) a processing controller means configured to enable random-access processing of individual samples without requiring batch processing.

[0273] And in this broad aspect, the invention further generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Whole Exome Sequencing

[0274] In this further aspect, the invention generally provides a method for automated targeted sequencing of protein-coding exons, the method comprising the steps of:(a) extracting genomic DNA from a biological sample;(b) preparing a sequencing library from the genomic DNA;(c) incubating the sequencing library with biotinylated oligonucleotide probes complementary to target exonic sequences at approximately 65°C for about 90 minutes under automated temperature control;(d) capturing probe-target hybrids using streptavidin-coated magnetic beads;(e) performing automated stringent washing of the captured hybrids at elevated temperature and reduced salt concentration to remove off-target sequences; and (f) performing post-capture PCR amplification to generate enriched sequencing library material.

[0275] In some preferred embodiments, the genomic DNA is derived from a human sample.

[0276] Alternatively, in other embodiments, the genomic DNA is derived from a non-human animal sample.

[0277] Preferably, in the method, the hybridization temperature is maintained within ±0.5°C of 65°C.

[0278] Preferably, in the performance of the method, temperature control during hybridization is performed without manual intervention.

[0279] Preferably, in the method, stringent washing is performed at a temperature greater than 60°C.

[0280] In embodiments, stringent washing is performed at a salt concentration below 1 * SSC.

[0281] In embodiments, preferably, the hybridization probes collectively target substantially all protein-coding exons of a genome.

[0282] In alternative embodiments of the method however, the hybridization probes target a predefined subset of clinically relevant exons.

[0283] Preferably in the method, magnetic bead capture and washing are performed in an automated liquid handling system.

[0284] In embodiments, post-capture PCR amplification comprises limited-cycle amplification.

[0285] And in embodiments, the method comprises automated bead-based normalization following post-capture amplification.

[0286] In this aspect, the invention also generally provides an automated system for targeted exon sequencing, the system comprising:(a) a DNA extraction means;(b) a library preparation means configured to generate sequencing-ready libraries; (c) a hybridization means configured to incubate the library with biotinylated probes targeting protein-coding exons at approximately 65°C under controlled temperature conditions;(d) a magnetic bead capture means comprising streptavidin-coated beads;(e) a washing means configured to perform stringent washes at elevated temperature and low salt concentration; and(f) a PCR amplification means configured to amplify enriched on-target library fragments.

[0287] And in this broad aspect, the invention further generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of thesystem and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Targeted Gene Panel Sequencing

[0288] The inventor has divined that the invention is also useful in highly targeted sequencing of specific genes or genomic regions. The biological sample comprises genomic or complementary DNA.

[0289] Accordingly, in this further broad aspect, the invention generally provides method for highly targeted sequencing of selected genomic regions, the method comprising the steps of:(a) providing genomic DNA or complementary DNA from a biological sample;(b) performing automated multiplex polymerase chain reaction (PCR) amplification using a plurality of primer pairs simultaneously in a single reaction, in which each primer pair targets a specific exon or genomic region of interest;(c) automatically controlling thermal cycling to maintain a predefined annealing temperature during amplification;(d) removing unincorporated PCR primers;(e) ligating sequencing adapters to amplified products;(f) performing post-ligation PCR amplification incorporating sample-specific index sequences; and(g) sequencing the amplified products,where sequencing depth is preferentially weighted toward the targeted genomic regions.

[0290] In embodiments of the method, the plurality of primer pairs comprises at least 200 primer pairs.

[0291] In more specific embodiments, the plurality of primer pairs comprises at least 1,000 primer pairs.

[0292] In embodiments, the annealing temperature is maintained within ±0.5°C of a predefined setpoint.

[0293] Preferably, in the method, amplification comprises limited-cycle PCR.

[0294] Preferably, in the method, removal of unincorporated PCR primers comprises enzymatic digestion using exonuclease.

[0295] Preferably, in the method, enzymatic primer digestion reduces formation of primerdimer artifacts during adapter ligation.

[0296] In preferred embodiments of the method, the biological sample comprises human genomic DNA. In alternative preferred embodiments, the biological sample may comprise nonhuman genomic DNA.

[0297] Preferably, in the method, the targeted genomic regions comprise one or more clinically relevant genes.

[0298] Preferably, in the method, post-ligation amplification incorporates dual-index barcode sequences.

[0299] Preferably, in the method, up to 96 samples are processed simultaneously using unique barcode combinations.

[0300] Preferably, in the method, sequencing depth across targeted regions exceeds sequencing depth of non-targeted regions by at least 10-fold.

[0301] In this further broad aspect, the invention generally provides an automated system for targeted sequencing of selected genomic regions, the system comprising:(a) a reaction means configured to perform multiplex PCR amplification using hundreds to thousands of primer pairs in a single reaction;(b) a thermal cycling means configured to maintain precise annealing temperatures during amplification;(c) a primer digestion means configured to remove unincorporated PCR primers enzymatically;(d) an adapter ligation means;(e) a post-PCR indexing means configured to incorporate sample-specific barcode sequences; and(f) a processing controller means configured to enable high-throughput multiplex processing of multiple samples.

[0302] And in this further broad aspect, the invention generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).DNA Methylation Analysis

[0303] The inventor has divined that the invention is also useful in epigenetic analysis of DNA methylation patterns. The biological samples used in this aspect of the invention comprise genomic DNA.

[0304] Thus, in accordance with this further aspect, the invention generally provides a method for automated epigenetic analysis of DNA methylation patterns, the method comprising the steps of:(a) providing genomic DNA from a biological sample;(b) treating the genomic DNA with sodium bisulfite under automated temperature- controlled conditions for a duration of between 2 and 4 hours, thereby converting unmethylated cytosines to uracils while leaving methylated cytosines unchanged; (c) preparing a sequencing library from the bisulfite-converted DNA using a library preparation protocol adapted for bisulfite-treated DNA;(d) sequencing the library; and(e) determining methylated and unmethylated cytosines at single-base resolution.

[0305] In embodiments of the method, temperature during bisulfite treatment is maintained within ±0.5°C of a predefined setpoint.

[0306] In embodiments, in the method, bisulfite treatment comprises repeated thermal cycling steps to promote cytosine conversion.

[0307] In embodiments, the library preparation step comprises end repair and adapter ligation compatible with uracil-containing DNA.

[0308] In embodiments, the library preparation step comprises limited-cycle PCR amplification using uracil-tolerant polymerase.

[0309] In embodiments, the method further comprises the step of performing hybridization capture enrichment after bisulfite conversion.

[0310] In embodiments, the hybridization capture step targets predefined genomic regions.

[0311] In embodiments, the hybridization capture step targets CpG-rich regions.

[0312] In embodiments, the sequencing step comprises paired-end sequencing.

[0313] In embodiments, the step of determining methylation status comprises aligning sequencing reads to a reference genome and distinguishing cytosine-to-thymine conversions.

[0314] And in embodiments of the method, the methylation patterns are determined across substantially the entire genome.

[0315] In this further broad aspect, the invention generally provides an automated system for DNA methylation analysis, the system comprising:(a) a bisulfite conversion means configured to treat genomic DNAwith sodium bisulfite under controlled temperature conditions for 2-4 hours;(b) a thermal control means configured to maintain a predefined temperature profile during bisulfite treatment;(c) a library preparation means configured to generate sequencing libraries from bisulfite-converted DNA;(d) a sequencing means; and(e) a bioinformatics analysis means configured to identify methylated and unmethylated cytosines at single-base resolution.

[0316] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Chromatin Immunoprecipitation (ChIP)

[0317] The inventor has further divined that the invention is useful in genome-wide mapping of DNA-protein interactions. In this aspect of the invention, the biological sample comprises chromatin immunoprecipitated DNA representing sequences associated with specific DNA-binding proteins or histone modifications.

[0318] In this aspect, the invention provides a method for genome-wide mapping of DNA-protein interactions, the method comprising the steps of:(a) receiving chromatin immunoprecipitated (ChIP) DNA representing genomic sequences associated with a DNA-binding protein or histone modification;(b) performing low-input library preparation using magnetic bead-based purification configured to minimize dead volume and sample loss;(c) amplifying the library using thermal cycling under automated temperature control;and(d) sequencing the amplified library to identify genomic regions associated with the DNA-binding protein or histone modification.

[0319] In embodiments, the method, further comprises the step of processing a corresponding input DNA control sample in parallel.

[0320] In embodiments, both the ChIP DNA and the input control sample are subjected to identical library preparation conditions.

[0321] In embodiments, the method further comprises assessing sample quality prior to amplification.

[0322] In embodiments of the method, sample quality assessment comprises measuring DNA concentration.

[0323] And in embodiments of the method, sample quality assessment comprises measuring fragment size distribution.

[0324] In embodiments of the method, PCR cycle number is automatically increased or decreased based on measured sample quality.

[0325] In embodiments, the DNA-binding protein comprises a transcription factor.

[0326] And in embodiments, the histone modification comprises a methylation or acetylation mark.

[0327] In this further broad aspect, the invention also generally provides an automated system for preparing sequencing libraries from chromatin immunoprecipitated DNA, the system comprising:(a) a magnetic bead handling means configured for low-input DNA purification with reduced dead volume;(b) a thermal cycling means configured to maintain precise temperature control during library amplification;(c) a processing controller configured to process ChIP samples and corresponding input control samples in parallel; and(d) an adaptive amplification means configured to adjust PCR cycle number based on assessment of sample quality.

[0328] And in this further broad aspect, the invention generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).16S Ribosomal RNA Gene Amplicon Sequencing

[0329] The inventor has also divined that the invention is useful in microbial community profiling through targeted amplification of conserved ribosomal RNA genes. In this aspect of the invention, the biological sample comprises nucleic acids from complex microbial communities

[0330] In this further broad aspect, the invention generally provides a method for profiling microbial communities, the method comprising the steps of:(a) providing nucleic acids derived from a complex microbial community;(b) performing automated multiplex polymerase chain reaction (PCR) amplification of one or more conserved ribosomal RNA gene regions using primer pairs targeting conserved sequences flanking taxonomically variable regions;(c) automatically maintaining predefined thermal cycling conditions during amplification;(d) incorporating dual index barcode sequences into amplified products such that each sample receives a unique combination of two index sequences;(e) pooling a plurality of uniquely indexed samples;(f) sequencing the pooled samples; and(g) classifying bacterial and archaeal taxa present based on sequencing data.

[0331] In embodiments of the method, preferably, the conserved ribosomal RNA gene region comprises a 16S ribosomal RNA gene region. In more preferred embodiments, ribosomal RNA gene region comprises a 16S V3-V4 ribosomal RNA gene region. And in such embodiments, it is particularly preferred that the target gene length comprises approximately 1500 base pairs for the 16S V3-V4 region.

[0332] In embodiments of the method, the conserved ribosomal RNA gene region comprises an 18S ribosomal RNA gene region.

[0333] In embodiments of the method, the conserved ribosomal RNA gene region comprises a23S ribosomal RNA gene region.

[0334] In embodiments of the method, the primer pairs target conserved regions flanking one or more hypervariable regions.

[0335] In embodiments of the method, thermal cycling temperatures are maintained within ±0.5°C of predefined setpoints.

[0336] In embodiments of the method, the dual indexing step comprises incorporation of a first index on a forward adapter and a second index on a reverse adapter.

[0337] In embodiments of the method, up to 96 samples are processed simultaneously using unique dual index combinations.

[0338] In embodiments of the method, pooling of samples occurs prior to sequencing.

[0339] In embodiments of the method, classification comprises aligning sequencing reads to a ribosomal RNA reference database.

[0340] And in embodiments of the method, relative abundance of taxa is determined based on normalized read counts.

[0341] In this further broad aspect, the invention generally provides an automated system for microbial community profiling, the system comprising:(a) a reaction means configured to perform multiplex PCR amplification of conserved ribosomal RNA gene regions;(b) a thermal cycling means configured to maintain optimal amplification temperatures under automated control;(c) an indexing means configured to incorporate dual index barcode sequences into amplified products;(d) a processing controller configured to enable multiplex processing of up to 96 samples using unique index combinations; and(e) a bioinformatics analysis means configured to classify bacterial and archaeal taxa from sequencing reads.

[0342] And in this further broad aspect, the invention generally provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).Single-Cell RNA Sequencing Standard Workflow

[0343] The inventor has further divined that the invention is useful in single-cell RNA sequencing using droplet-based cell capture methodologies in which individual cells are captured with cell-specific barcodes, and mRNAis captured via poly(A) tail recognition. In this aspect of the invention, the biological sample comprises isolated individual cells or cell suspensions.

[0344] Accordingly, in this further broad aspect, the invention generally provides a method for single-cell RNA sequencing, the method comprising the steps of:(a) capturing individual cells from a biological sample comprising isolated cells or a cell suspension using a droplet-based capture methodology, in which each captured cell is associated with a cell-specific barcode;(b) capturing messenger RNAfrom the individual cells via poly(A) tail recognition and synthesizing complementary DNA (cDNA) incorporating the cell-specific barcode; (c) pooling barcoded cDNAfrom multiple cells;(d) performing automated limited-cycle PCR amplification of the pooled barcoded cDNA under controlled thermal cycling conditions;(e) performing automated library preparation comprising fragmentation, adapter ligation, and post-amplification indexing;(f) performing automated bead-based cleanup and normalization;(g) sequencing the prepared library; and(h) demultiplexing sequencing reads by cell-specific barcode and unique molecular identifier to generate per-cell gene expression matrices.

[0345] Preferably, the method also comprises means for generating and displaying or notifying to a person, the results derived from performance of the method.

[0346] In embodiments of the method, the droplet-based capture methodology comprises microfluidic droplet generation.

[0347] In embodiments of the method, the cell-specific barcode further comprises a unique molecular identifier (UMI).

[0348] In embodiments of the method, the limited-cycle PCR amplification comprises fewer than 15 cycles.

[0349] In embodiments of the method, the thermal cycling temperature is maintained within ±0.5°C of predefined annealing and extension temperatures.

[0350] In embodiments of the method, the fragmentation is enzymatic fragmentation.

[0351] In embodiments of the method, the adapter ligation incorporates sample-specific index sequences.

[0352] In embodiments of the method, the bead-based cleanup removes fragments below a predefined size threshold.

[0353] In embodiments of the method, the normalization produces sequencing-ready libraries without manual quantification.

[0354] In embodiments of the method, the sequencing comprises paired-end sequencing.

[0355] In embodiments of the method, the gene expression matrix enables identification of distinct cell populations.

[0356] And in embodiments of the method, the gene expression matrix enables characterization of cellular states.

[0357] In this further broad aspect, the invention generally provides an automated system for single-cell RNA sequencing, the system comprising:(a) a droplet-based cell capture means configured to associate individual cells with cell-specific barcodes;(b) a reverse transcription means configured to capture polyadenylated RNA and generate barcoded cDNA;(c) a thermal cycling means configured to perform limited-cycle amplification of pooled barcoded cDNA under predefined temperature conditions;(d) a library preparation means configured to perform fragmentation, adapter ligation, and index incorporation;(e) a bead-based purification and normalization means; and(f) a bioinformatics means configured to demultiplex sequencing reads and generate per-cell gene expression matrices.

[0358] And in this aspect, the invention further provides an apparatus in which the method may be performed. The features of the apparatus are essentially those defined above in relation to the system in this aspect of the invention. And preferred features of the system and the apparatus correspond with those described above in relation to the method of this aspect of the invention (mutatis mutandis).

[0359] In certain embodiments of the invention there is provided, a single-use cartridge adapted for automated nucleic acid library preparation comprising one or a combination of:lyophilized enzymatic reagents,CRISPR guide RNA sets,magnetic beads configured for saturation normalization,pipette tips,sample and output tubes,hazardous and non-hazardous waste compartments,wherein the cartridge is configured for insertion into an automated random-access nucleic acid processing instrument.

[0360] This single-use cartridge may be configured for insertion into an automated randomaccess nucleic acid processing instrument as disclosed herein.

[0361] In certain further embodiments the present invention provides a random-access automated nucleic acid processing system comprising:a plurality of independently operable bays, each comprising: (i) an independently controlled thermal module, (ii) a magnetic bead separation module, and (iii) a robotic liquid handling interface;a single-use consumable cartridge comprising: (i) lyophilized CRISPR-Cas ribonucleoprotein complexes targeting host ribosomal RNA, (ii) quantification-free adapter ligation reagents comprising terminal transferase and truncated T4 RNA ligase, (iii) magnetic beads having a predetermined DNA binding capacity configured to produce a standardized library mass upon saturation;a controller configured to: (i) extract nucleic acids, (ii) construct a sequencing library without fluorometric quantification, (iii) perform post-library CRISPR depletion after an initial amplification step, (iv) normalize the library by bead saturation without optical measurement, and (v) output a sequencer-ready library;wherein each processing station operates asynchronously relative to each other processing station.

[0362] In still further certain embodiments, the present invention provides a method of detecting pathogens in a biological sample comprising:(a) extracting nucleic acids;(b) constructing a sequencing library without measuring nucleic acid concentration; (c) amplifying the library;(d) contacting the amplified library with CRISPR-Cas ribonucleoprotein complexes targeting host ribosomal RNA;(e) selectively removing cleaved host-derived fragments;(f) re-amplifying uncleaved fragments;(g) normalizing the re-amplified library by contacting it with magnetic beads having finite binding capacity to produce a standardized output mass;(h) sequencing the normalized library;wherein the method increases pathogen read proportion relative to a non-depleted control.

[0363] In yet further certain embodiments, there is provided an automated nucleic acid sequencing preparation system comprising:an extraction module,a programmable nuclease depletion module,a library preparation module configured to accept variable input concentration without pre-measurement,a normalization module configured to produce standardized output concentration without optical quantification,a sequencing interface,wherein the modules operate in a closed system and are controlled to perform an asynchronous sample processing workflow.

[0364] Still further certain embodiments provide a method of enriching non-host nucleic acid sequences in a complex biological sample comprising:constructing an adapter-ligated library;selectively cleaving predetermined abundant sequences using programmable nucleases;removing cleaved fragments;amplifying remaining fragments;normalizing output concentration using finite-capacity solid-phase binding media.

[0365] Further certain embodiments provide a single-use cartridge containing one or a combination of:programmable nuclease complexes,adapter ligation reagents tolerant to variable input,normalization beads with defined binding capacity,waste containment matrix,encoded metadata identifying nuclease panel.

[0366] Further certain embodiments provide apparatus adapted to enrich non-host nucleic acid sequences in a complex biological sample, said apparatus including:processor means adapted to operate in accordance with a predetermined instruction set,said apparatus, in conjunction with said instruction set, being adapted to perform one or a combination of the method steps as disclosed herein.

[0367] Still further certain embodiments provide a computer program product including: a computer usable medium comprising a computer-readable recording medium storing instructions and having computer readable program code and computer readable system code embodied on said medium for enriching non-host nucleic acid sequences in a complex biological sample within a data processing system, said computer program product including:computer readable code within said computer usable medium for performing one or a combination of the method steps as disclosed herein.

[0368] Yet still further embodiments provide a computer-readable medium comprising machine-readable code, which, when executed by a processor, causes the processor to perform the method steps as disclosed herein.

[0369] In essence, embodiments of the present invention stem from the realization that the integration of post-library CRISPR-based host depletion with quantification-free adapter ligation chemistry, saturating bead-based library normalization, and random-access asynchronous processing architecture within a single closed, cartridge-based automated platform enables fully automated metagenomic next-generation sequencing workflows without requiring fluorometric nucleic acid measurement, manual batching of samples, or complex optical instrumentation. This integration overcomes the fundamental barriers that have prevented clinical adoption of metagenomic sequencing: (1) the overwhelming abundance of host nucleic acids obscuring low-abundance pathogen detection, (2) the labor-intensive requirement for precise nucleic acid quantification prior to library construction, (3) the operational delays imposed by batch-based processing architectures, and (4) the need for highly trained personnel to execute complex multi-step protocols. By performing CRISPR-mediated depletion after initial library construction (rather than before), the system exploits abundant host nucleic acids as carrier molecules during early workflow steps, preventing loss of low-abundance pathogen material while subsequently removing those same host sequences through targeted double-strand cleavage before final amplification. The quantification-free adapter ligation chemistry accommodates input concentrations ranging from 10 pg to 1 pg without manual adjustment, eliminating error-prone calculation steps. The saturating bead-based normalization produces standardized library concentrations through finite binding capacity rather than optical measurement. The random-access architecture enables samples to be processed individually and immediately upon arrival, with each of thefour independent processing bays operating asynchronously under dynamic scheduling that prioritizes urgent clinical samples. The single-use, foil-sealed consumable cartridge integrates all reagents (including lyophilized CRISPR RNP complexes, enzymatic mixes, and magnetic beads) in pre-measured aliquots, eliminating cold-chain dependencies and minimizing contamination risk. Collectively, these innovations transform metagenomic sequencing from a research technique requiring batch processing, extensive manual intervention, and specialized expertise into a walk-away automated diagnostic platform suitable for timesensitive clinical deployment in hospital laboratories, achieving sample-to-result turnaround times of 6-12 hours with 93% reduction in hands-on technician time compared to manual workflows.

[0370] At least one advantage provided by the present invention comprises the integration of the following into a single closed, automated, asynchronous, cartridge-based platform:• Post-library CRISPR depletion within an automated cartridge workflow• Quantification-free adapter ligation accepting 10 pg-1 g input• Saturating bead-based normalization eliminating fluorometric measurement• Independent random-access processing bays• Ambient-stable lyophilized reagent architecture• Full sample-to-sequencer closed workflow.BRIEF DESCRIPTION OF THE DRAWINGS

[0371] Fig. 1 depicts schematically, the workflow by which one or more pathogens in a biological sample would be analysed by using an exemplary apparatus (and the corresponding system and method) according to the present invention, in order to detect whether a biological sample (of the kind that would be typically presented for analysis in a clinical health care environment) contains a pathogen;

[0372] Fig. 2 is a first perspective external view of an instrument showing internal architecture in accordance with a preferred embodiment of the system according to the invention;

[0373] Fig. 3 is a second perspective external view of an instrument showing internal architecture in accordance with a preferred embodiment of the system according to the invention;

[0374] Fig. 4 is a perspective view of an exemplary container in accordance with preferred embodiments;

[0375] Fig. 5 is a schematic view of a location or position calibration mechanism in accordance with an embodiment of the preferred system;

[0376] Fig. 6 is a perspective view of support apparatus for containers in accordance with an embodiment of the preferred system;

[0377] Fig. 7 shows a loading and unloading workflow for the support apparatus of Fig 6 in accordance with an embodiment of the preferred system;

[0378] Fig. 8 is a perspective view showing the integration of modules in accordance with an embodiment of the preferred system;

[0379] Fig. 9 is an exploded view showing the integration of further modules in accordance with an embodiment of the preferred system;

[0380] Fig. 10 is a perspective view of a thermal module modules in accordance with an embodiment of the preferred system in an open configuration;

[0381] Fig. 11 shows an expanded view illustrating the integration of the thermal module of Fig. 10 in accordance with an embodiment of the preferred system;

[0382] Fig. 12 depicts a CRISPR-based host depletion mechanism of preferred embodiments;

[0383] Fig. 13 shows a timing chart for random sample arrivals across 8 positions for analysis in the preferred system;

[0384] Fig. 14 shows exemplary single use disposable cartridge layout with foil sealed reagent tubes, pipette tips, and disposable waste containers.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0385] In order that the invention may be further understood, one or more preferred embodiments will now be described by way of example only, with reference (where applicable) to the accompanying drawings.

[0386] Embodiments of the invention have particular, but not exclusive, application to metagenomic sequencing techniques for infectious disease diagnostics, cancer genomics, rare disease detection, pharmacogenomics, prenatal testing, microbiome analysis, environmental surveillance, veterinary diagnostics, and agricultural applications.Description of System Operation and Protocols - Overview of System Architecture

[0387] The system according to embodiments of the invention is a fully integrated, purpose-built sample preparation and analysis platform that automates a complete workflow from biological sample input through to normalized, sequencer-ready library output. The system architecture comprises six functional modules that work in concert to achieve walk-away automation: (1) bay drawer assemblies, (2) a liquid handling system, (3) a tube manipulation gripper, (4) a temperature-controlled thermal cycler, (5) a magnetic bead separation station, and (6) an electronic control infrastructure.Bay Drawer Assembly and Processing Architecture

[0388] Referring to Figs. 2 to 11 , an example of a preferred system according to the invention is shown, which, in the exemplary apparatus depicted, features four independent processing bays 206, each (i) with a dedicated thermal cycler, (ii) with a processing bay drawer, and (iii) capable of processing a single sample independently and through asynchronous operation, without requiring sample batching. Each bay 206 consists of a drawer assembly that securely houses a single-use consumable support pack as depicted in Fig. 4, containing all reagents, pipette tips, and sample / library tubes necessary for complete sample preparation of one specimen. Although the exemplary apparatus described here and in Figs. 2 to 11 comprises four independent processing bays, the present invention apprehends that the number of processing bays in a given apparatus / system is scalable. Hence, in alternative embodiments, an apparatus / system configured in accordance with the invention could, for example, comprise eight processing bays.

[0389] A preferred support pack or container assembly as shown may be sized according to final tip, tube, and reagent requirements. It may accommodate dry and wet reagent format for automated filling. A support pack barcode may be positioned for manual scan or for auto scan if deemed necessary. Additional barcodes on input and output tubes or reagents may also be provided.

[0390] Each drawer incorporates precision roller bearings for smooth actuation and an integrated hold-down mechanism 604 with end detents that maintain the drawer in both open and closed positions. Drawer closure is monitored through a dedicated closure sensor 612 that communicates with control software, ensuring the support pack is properly seated before automated processing begins. The drawer design includes an ergonomic carry handle 603 with integrated cable routing tie points. Support pack presence sensing is achieved through a dedicated sensor 611 that verifies the consumable is installed before permitting protocol execution, thus preventing operator error.

[0391] An important operational advantage of the four-bay architecture is true random-access sample processing. In this respect, samples need not arrive simultaneously or be batched; individual samples may be processed as they arrive. The control software manages each bay independently, with real-time status indicators displayed on the instrument's touchscreen interface 208. If, for example, a given sample, Sample A, completes library preparation at 10:00 AM but Sample B does not arrive until 11:30 AM, Sample A can be held at 4°C in its output tube while Sample B is simultaneously processed in an adjacent bay. This asynchronous processing capability is particularly valuable in clinical environments wheresample arrival times are unpredictable and where rapid access to results can inform immediate clinical decision-making.

[0392] Each bay has capability for processing one sample independently; operating on independent thermal and timing control; permitting immediate initiation without batch accumulation; and maintaining completed libraries in controlled temperature storage. Advantageously, this architecture eliminates batch dependency typical of 8-, 24-, or96-sample robotic platforms.Liquid Handling System

[0393] Referring to Fig.’s 2, 3 and 9 in particular, the system integrates a robotic liquid handler 207 providing three-axis (X, Y, Z) motion control with sub-microliter precision. The liquid handler 207 executes all liquid transfer, mixing, and dispensing operations throughout the sample preparation workflow, including: (1) precise delivery of buffer and reagent volumes ranging from 5 pL to 880 pL; (2) pipette mixing operations with adjustable velocity and frequency to ensure thorough homogenization; (3) sequential transfers from the support pack consumables to sample / library tubes; and (4) coordinated tip attachment and ejection for single-use tip utilization.

[0394] All workflow steps may be executed using addition-only liquid handling, obviating a requirement for subtraction or complex volume calculations. This eliminates the need for expensive fluorescence-based volume detection systems that would significantly increase instrument cost and maintenance complexity. The minimum validated pipetting volume is about 5 pL across all workflow steps.

[0395] Pipette tips may be incorporated as part of the single-use consumable support pack, and pre-loaded in organized strips as illustrated in Fig.’s 4 and 14. The liquid handler 207 rapidly picks up, uses, and discards tips into dedicated waste compartments within the support pack, eliminating the need for complex tip-washing or tip-dispensing systems on the instrument itself.Gripper System

[0396] The system incorporates a custom stainless-steel gripper mechanism 203, controlled via a linear actuator with a 25 mm stroke, to automate the manipulation and transport of sample tubes (1.5 ml_) and library tubes (0.2 mL PCR tubes) between processing stations including the liquid handling deck, magnetic bead collection station, and thermal cycler.

[0397] The gripper jaw geometry is engineered to securely manipulate both tube formats. Custom stainless-steel gripping surfaces minimize contamination risk and provide consistentengagement across repeated cycling. Position sensors on the Z-axis mount provide feedback to the control software regarding gripper position and tube capture confirmation.

[0398] The gripper 203 is integrated with the liquid handler gantry system 209, enabling coordinated motion where the gripper 203 and liquid handler 207 operate in sequence without collision. For example, after the liquid handler 207 completes addition of magnetic binding beads to a sample tube, the gripper 203 automatically retrieves the tube and transports it to the magnetic separation station. Following magnetic settling, the liquid handler 207 removes the supernatant, and the gripper 203 transports the tube back to the holding position for the next processing step.Thermal Cycler

[0399] The exemplary system also incorporates a thermal cycler 201 with a hybrid design combining a custom heat transfer block 621, 626 with a single dry well designed to accommodate a 0.2 mL PCR tube and a Peltier heating / cooling module 626. This hybrid design achieves rapid heating and cooling rates (target: 2°C / second ramp rate), heated lid control (temperatures up to 105°C to prevent condensation), and precision temperature stability of ±0.5°C across the thermal block.

[0400] The thermal cycler accommodates temperature profiles that may be required for the following enzymatic reactions within the library preparation workflow:1. RNA Fragmentation: 94°C for 2 minutes, followed by rapid cooling to 4°C.2. Reverse Transcription: 25°C for 15 minutes, 50°C for 50 minutes, 75°C for 10 minutes, 4°C hold.3. Exonuclease / rSAP Digestion: 37°C for 30 minutes, 95°C for 10 minutes, 22°C hold.4. Terminal Transferase Reaction: 37°C for 30 minutes.5. Adapter Ligation: 28°C for 30 minutes.6. PCR Amplification: 94°C for 2 minutes, followed by 20-25 cycles of 98°C / 20s, 55°C / 30s, 68°C / 45s, then 72°C for 2 minutes, 4°C hold.7. CRISPR-Cas9 Ribonucleoprotein Complex Formation: 42°C for 1 hour, 4°C hold.

[0401] Integrated position monitoring ensures tubes are properly seated and heating cycles complete successfully before the gripper 203 retrieves the tube for subsequent processing. Magnetic Bead Separation Station

[0402] The exemplary system incorporates a dedicated magnetic bead collection station employing a stationary magnet array positioned to accommodate two tube formatssimultaneously: one 1.5 mL sample tube (for RNA extraction) and one 0.2 mL PCR tube (for library purification). Magnet positions are adjustable to optimize bead pelleting efficiency.

[0403] The magnetic separation workflow operates as follows:1. Bead Capture: the gripper 203 transports the sample tube to the magnetic station following liquid handler 207 addition of magnetic beads and appropriate incubation.2. Settling: the stationary magnet field captures magnetic particles, causing beads to pellet to the tube wall (typically 3-5 minutes).3. Supernatant Removal: the liquid handler 207 carefully removes the supernatant without disturbing the bead pellet; tips are discarded to waste.4. Bead Resuspension: wash buffers are added while the tube remains at the magnet station for rapid re-pelleting, or the gripper 203 returns the tube to the deck for elution steps.5. Elution: elution buffer is added to the bead pellet, followed by brief incubation, repelleting, and recovery of purified nucleic acids in supernatant.

[0404] This magnetic separation process is performed repeatedly throughout the workflow during RNA extraction, library preparation SPRI cleanups, and final library normalization. Electronic Control Infrastructure and User Interface

[0405] The exemplary system incorporates electronic control architecture comprising an industrial-grade single-board computer (SBC), custom main board, network switch, touchscreen display, and integrated power distribution system, housed within a stainless-steel chassis.

[0406] The chassis comprises a precision aluminum extrusion frame, a rigid robot base plate with vibration isolation, a precision-machined drawer base plate for the four bay assemblies, a thermally insulated thermal cycler enclosure with fan-based active cooling, a segregated high-voltage power enclosure, a climate-controlled electronics enclosure, an organized cable management shroud, and vibration-isolating instrument feet.

[0407] A touchscreen user interface 208 is designed in accordance with IEC 62366-1 :2015 usability engineering principles and comprises:- Bay Status Indicators: large iconography displaying the status of each bay (Available, In Progress, Complete, Error) with circular progress indicators visible from at least 5 meters.- Workflow Wizard: a modal interface providing step-by-step protocol guidance.- Run Details Display: real-time display of Sample ID, Support Pack ID, and Protocol ID.- Bay-Specific Controls: state-dependent call-to-action buttons preventing operational errors.

[0408] The control software incorporates a hardware integration layer, a workflow scripting interface enabling rapid protocol development, and a programmatic control client supporting integration with laboratory information systems. A dynamic scheduling engine optimizes workflow across the four independent processing bays, enabling asynchronous operation and priority-based processing for urgent samples.

[0409] By way of an example of control flow, Fig. 7 illustrates a protocol for loading and unloading for receiving and ejecting a support pack.Sample Collection and Pathogen Inactivation

[0410] In the preferred aspect of the invention directed to infectious disease diagnostics, as described herein, clinical specimens are collected using standard clinical protocols and immediately immersed in a pathogen-neutralizing transport medium employing a chaotropic agent that rapidly denatures proteins, lyses cellular and viral membranes, and inactivates nucleases to prevent nucleic acid degradation. Complete inactivation (>4 log reduction) of high-consequence pathogens is achieved within 30 minutes at ambient temperature. Viral extraction occurs rapidly, within about 5 minutes.

[0411] This pathogen inactivation architecture supports two operational paradigms:

[0412] Higher Biosafety Level Laboratories (BSL-3 / BSL-4): A dual-aliquot approach wherein one aliquot is retained in non-inactivating viral transport medium for traditional microbiological workup, and the second aliquot is transferred to neutralizing medium for the automated sequencing workflow.

[0413] Lower Biosafety Level Laboratories (BSL-1 / BSL-2): Direct inactivation at the point of collection, rendering specimens non-infectious before transport and enabling safe processing at standard BSL-2 facilities or in BSL-1 locations according to local regulations.

[0414] Both paradigms converge on an identical downstream sample preparation workflow within the system.Automated RNA Extraction

[0415] Following sample loading, the system initiates automated nucleic acid extraction using magnetic bead-based purification. The extraction employs a method utilizing a chaotropic lysis reagent and magnetic binding beads, which may be selected for compatibility withautomated liquid handling at the system's minimum 5 pL pipetting volume. Automated pipette mixing (replacing manual plate-shaking) was optimized by increasing the quantity of magnetic beads to achieve approximately 1.8-fold higher RNA recovery. The method eliminates Proteinase-K digestion, thereby reducing extraction time by approximately 45 minutes.

[0416] The automated extraction workflow preferably comprises:1. Sample Preparation: the system adds lysis buffer and a lysis / binding enhancer to the sample tube.2. RNA Binding: magnetic binding beads are added and incubated to allow nucleic acid binding.3. Magnetic Separation: the sample tube is transferred to the magnet station; supernatant is removed.4. Sequential Washing: Wash Solution 1 and Wash Solution 2 are applied to remove contaminants.5. DNA Removal: DNase treatment degrades contaminating DNA.6. Rebinding and Purification: nucleic acids are rebound to beads with additional washes.7. Elution: preheated elution buffer releases purified nucleic acids.

[0417] Total extraction time is approximately 80 minutes.Library Preparation: Quantification-Free Workflow

[0418] A particularly advantageous feature of the system in preferred embodiments is quantification-free library preparation that eliminates the requirement for fluorometric or qRT-PCR quantification prior to library construction. Traditional NGS library preparation requires precise adjustment of adapter-to-template ratios to prevent adapter dimer formation, necessitating quantification steps that are labor-intensive, error-prone, and difficult to automate without expensive optical instrumentation.

[0419] The system employs a library preparation chemistry that accommodates varied nucleic acid inputs (10 pg to 1 pg) without manual adjustment, through a novel adapter ligation mechanism in which excess single-stranded adapter primers are digested by exonuclease prior to adapter addition.

[0420] The library preparation workflow consists of eight sequential addition-only steps:1. RNA Fragmentation: heat and Mg2+-based fragmentation (3-4 minutes) to generate optimal fragment lengths.2. First-Strand cDNA Synthesis: random primers with universal 5' adapter tails are used with reverse transcriptase.3. Excess Primer Removal: Exonuclease I and shrimp alkaline phosphatase (rSAP) remove excess primers and dNTPs.4. Second Adapter Ligation: terminal transferase adds 3' ribonucleotides to the 3' end of cDNA, followed by truncated T4 RNA ligase-mediated adapter ligation with 1.8* SPRI bead cleanup.5. First PCR Amplification: approximately 5 cycles to generate double-stranded library molecules using primers complementary to 3' and 5' adapters.6. CRISPR-Based Selective Depletion: double-stranded DNA libraries are incubated with CRISPR-Cas nuclease ribonucleoprotein complexes targeting predetermined abundant sequences, followed by 0.6* SPRI cleanup to remove cleaved fragments lacking adapters.7. Second PCR Amplification: approximately 15 cycles to enrich uncleaved target library molecules, with P5 / P7 index primers for sample multiplexing.8. Bead-Based Normalization: magnetic beads with finite binding capacity (1.2* bead:library ratio) produce standardized output of approximately 80 ng ± 5% coefficient of variation, enabling direct sequencer loading without quantification.

[0421] Total library preparation time is approximately 373 minutes.CRISPR-Based Selective Depletion

[0422] In preferred embodiments, the system employs programmable CRISPR-Cas nuclease-based selective depletion of predetermined abundant nucleic acid sequences after library construction but before final PCR amplification. Ribonucleoprotein (RNP) complexes comprising single-guide RNA molecules and high-fidelity Cas nuclease are incubated with the library at approximately 37°C for 30 minutes. Guide RNAs direct the Cas nuclease to complementary target sequences, resulting in site-specific double-strand breaks. Cleaved fragments lose their flanking sequencing adapters and cannot be amplified in subsequent PCR, effectively removing them from the final library while preserving low-abundance target sequences.

[0423] In a preferred embodiment for respiratory infectious disease diagnostics, the guide RNA panel comprises approximately 3,500 sgRNAs, including:- guides targeting human ribosomal RNA reference sequences;- guides targeting the top 1-3% most highly expressed human genes during respiratory viral infections;- guides targeting ribosomal RNAfrom 445 commensal organisms commonly found in nasal / oral microbiome samples.

[0424] All guide sets were computationally screened against 547 pathogenic strain genomes with no off-target hits detected.

[0425] Published validation demonstrates:- 89% depletion of human ribosomal RNAfrom nasal samples;- 99% reduction in human rRNA and 98% reduction in bacterial rRNA in clinical respiratory samples;- 3.6 to 6-fold enrichment of pathogenic sequences;- 100% sensitivity for SARS-CoV-2 detection (improved from 96.8% in non-depleted samples of Ct values up to 38);- Improved detection of antimicrobial resistance markers, with some resistance genes detected only in depleted libraries.References:1. Chan et al., 2023, Cell Reports Methods 3, 100463; and2. Ceron, S et al., Am J Clin Pathol February 2023;159:111-115

[0426] The CRISPR depletion approach is fundamentally target-agnostic: by designing appropriate guide RNA sets, the same methodology can selectively deplete any predetermined abundant nucleic acid sequence from any biological sample. Alternative guide RNA panels can target wild-type genomic sequences (for cancer applications), maternal cell-free DNA (for prenatal applications), plant chloroplast or mitochondrial DNA (for agricultural applications), arthropod ribosomal RNA (for vector-borne disease surveillance), or any other abundant background sequence, through simple substitution of the guide RNA cassette within the consumable support pack.

[0427] In some preferred embodiments, the CRISPR depletion system employs approximately 3500 single-guide RNAs (sgRNAs) targeting conserved regions of human ribosomal RNA (18S, 28S), human mitochondrial DNA, and bacterial ribosomal RNA (16S, 23 S). The Modular design of the consumable cartridge of the invention enables the sgRNA panel to be adapted, expanded or redesigned for different host organisms, sample types or clinical applications without modification to the instrument hardware or core workflow protocol.Single-Use Consumable Support Pack

[0428] Referring particularly to Fig. 4, the system employs a single-use consumable support pack that integrates all reagents, tips, tubes, and processing consumables required for complete preparation of a single sample. This design provides contamination prevention, complete traceability, and elimination of cold-chain logistics.

[0429] The support pack architecture comprises:Wet Reagent Strips (in foil-sealed wells of 1.2 ml_ and 0.2 mL formats):- Lysis reagent for nucleic acid extraction;- Ethanol solutions for various wash and preparation steps;- Magnetic binding beads forextraction, library purification, and normalization; - Wash buffers for sequential purification steps;- Elution buffer for nucleic acid recovery.Dry Reagent Strips (lyophilized, requiring reconstitution):- DNase master mix;- Reverse transcriptase and associated components;- Exonuclease and shrimp alkaline phosphatase;- Terminal transferase and ribonucleotides;- T4 RNA ligase and adapter sequences;- CRISPR-Cas ribonucleoprotein complexes specific to the intended application;- PCR master mixes;- P5 / P7 index primer mixes for sample multiplexing.Integrated Components:- Pipette tips in organized strips;- Sample input tube (1.5 mL) positioned for manual operator loading;- Output tube (0.2 mL PCR tube) for final normalized library;- Waste compartments for safe collection of discarded tips and hazardous supernatants;- Barcode identification at strategic locations for complete traceability.

[0430] The support pack incorporates a breakaway foil seal system with calibrated differential peel strengths: seals over enzyme compartments require stronger force to pierce (approximately 3.5 N), preventing accidental exposure, while seals over buffer compartments are calibrated for robotic piercing at approximately 1.2 N. A conical seal geometry directs piercing debris away from adjacent wells.

[0431] The lyophilization process employs differential lyophilization to enable co-storage of incompatible enzymes that would normally require separate storage conditions. Lyophilized reagents provide ambient temperature stability with shelf life exceeding six months at 25°C and 60% relative humidity, eliminating cold-chain requirements for global deployment.

[0432] Each support pack is encoded with a 2D DataMatrix barcode containing lot-specific reconstitution volumes, enzyme expiration dates, and the specific guide RNA panel identifier. The system reads this barcode before processing begins, and if expired reagents or a mismatch between the guide RNA panel and the selected protocol is detected, processing is automatically halted.Sequencer Integration

[0433] In the preferred embodiment, processed libraries are transferred to a next-generation sequencing instrument. The system provides sequencer-compatible normalized library output that can be loaded directly without additional quantification or manual dilution calculations.

[0434] The system normalizes output library to a fixed amount. This fixed amount is loaded onto the sequencer cartridge regardless of whether one, two, three, four, or more samples are being pooled. For example, if the loading amount is X, then one sample provides amount X, and four samples each contribute X / 4, totaling X for loading. The target utilization corresponds to 4-6 samples multiplexed per 25M-50M read flow cell.

[0435] In some embodiments, the sequencing instrument provides accelerated sequencing (approximately 2 hours for 100 bp single-end reads), real-time base calling with early-cycle data access enabling provisional target identification within approximately 45 minutes using as few as 20 bp of sequence data, and on-instrument filtering of host sequences before cloud transmission, protecting patient privacy and reducing bandwidth requirements.Bioinformatics Analysis

[0436] The bioinformatics analysis means comprise a cloud-based analysis platform that accepts sequencing data and executes species or variant classification analysis. In the preferred embodiment directed to infectious disease diagnostics, the bioinformatics pipeline employs:1. De novo assembly of reads into contiguous sequences with taxonomic assignment using average nucleotide identity.2. Read-based taxonomic profiling using k-mer classification for organisms not assembled into complete contigs.3. Comparison against curated reference databases.

[0437] The system is designed for agnostic detection, including novel organisms or variants that may not match existing database entries. Clinical reports are generated summarizing identified targets with associated confidence metrics, quantitative abundance estimates, and, where applicable, antimicrobial resistance markers or variant annotations. Reports are formatted for clinical interpretation and may be generated as hard copy or electronic reports transferable via wired or wireless communications.EXAMPLE 1 - clinical use for pathogen detection and / or identification

[0438] The present aspect of the invention relates generally to improvements in the ways by which pathogens that have infected an organism may be detected and identified, so that, as appropriate, the organism may subsequently be treated appropriately against the pathogen.

[0439] The aspect described here has particular application to detection / identification techniques that involve the collection and assay of a biological sample from the organism, and where the assay utilises nucleic acid sequencing techniques to detect and identify the pathogen.

[0440] The aspect described here also has particular application to the use of metagenomic sequencing techniques to detect and identify such pathogens.

[0441] The present embodiment is directed to detection and identification of pathogenic organisms present in clinical biological specimens. The biological sample comprises clinical material such as whole blood, plasma, serum, cerebrospinal fluid, nasopharyngeal secretion, respiratory specimens, urine, or other bodily fluids, cells, tissues or materials. The sample may alternatively comprise solid tissue material with appropriate lysis buffer treatment to disrupt cellular and viral membranes and expose intracellular nucleic acid content.

[0442] The system processes the biological sample through automated nucleic acid extraction using magnetic bead-based purification methodology. The extraction procedure comprises sequential steps of cell lysis through addition of lysis buffer containing detergents and chaotropic agents, binding of nucleic acids to magnetic bead surfaces, washing with ethanol and aqueous buffers to remove contaminants, optional nuclease treatment to remove specific contaminating nucleic acids, and final elution of purified nucleic acids.

[0443] Following nucleic acid extraction, the system may optionally perform limited-cycle polymerase chain reaction amplification to generate sufficient nucleic acid material for subsequent processing. This pre-amplification is performed using thermally controlled amplification across 8-12 PCR cycles depending on extracted nucleic acid quantity and quality.

[0444] The system then performs selective depletion of host-derived nucleic acids using CRISPR-based methodology. Ribonucleoprotein complexes are prepared comprising singleguide RNA molecules and high-fidelity SpyCas9 nuclease. Guide RNA molecules are designed to direct SpyCas9 nuclease to target sequences corresponding to human ribosomal RNA, human mitochondrial DNA, and bacterial 16S ribosomal RNA. Distinct guide RNA species are employed, each targeting different regions of the abundant background sequences, ensuring comprehensive coverage and high depletion efficiency.

[0445] In some embodiments, he CRISPR-based depletion reaction proceeds at approximately 37-'«C for 30 minutes in buffer solution providing optimal ionic strength and pH for SpyCas9 catalytic activity. However, the CRISPR-Cas9 ribonucleoprotein complex may be incubated with the double-stranded cDNA library at a temperature of 37°C to 42°C for 30 minutes to 60 minutes (and preferably at 42°C for 60 minutes) to achieve site-specific cleavage of target host sequences. During this incubation, guide RNAs direct SpyCas9 nuclease to complementary target sequences within the nucleic acid population, resulting in site-specific double-strand breaks in host nucleic acids. Cleaved host nucleic acid fragments lack sequencing adapters present on target pathogenic nucleic acids because adapter ligation occurs prior to CRISPR depletion, such that cleaved host fragments are physically separated from amplifiable sequences.

[0446] Following CRISPR cleavage, the system performs magnetic bead-based purification to remove cleaved host nucleic acid fragments. The purification selectively retains intact nucleic acid molecules bearing sequencing adapters while removing fragmented host NA lacking adapters. This separation is achieved through selective capture of full-length adapterbearing molecules on magnetic bead surfaces while allowing small fragmented molecules to be removed in wash steps.

[0447] The system then performs post-depletion amplification through limited-cycle PCR, amplifying remaining adapter-containing sequences. PCR reaction incorporates samplespecific index sequences in primer designs, enabling sample identification during multiplexed sequencing.

[0448] Enzymatic digestion is followed by magnetic bead-based cleanup to remove digestion products and excess primer.

[0449] The system then performs library normalization using bead-based methodology, eliminating the need for intermediate fluorometric quantification. Bead-based normalization adjusts library concentrations to standardized levels, enabling optimal flow cell loading for sequencing without the time and potential error associated with separate quantification steps.

[0450] The prepared library is transferred to sequencing instrument such as Illumina MiSeq , or comparable platform. The system automatically formats library data and sample metadata to be compatible with sequencing instrument input requirements, enabling seamless data transfer and sequencing run initiation.

[0451] Sequencing data are automatically transferred to bioinformatics analysis platforms. Sequencing reads are processed through taxonomic classification algorithms including de novo assembly of reads into contiguous sequences with average nucleotide identity-based taxonomic assignment, k-mer based read classification for organisms not assembled into complete contigs, and comparison against reference databases containing collections of known organisms. The system identifies sequences present in the sample and generates quantitative estimates of organism abundance based on read counts.

[0452] Clinical report generation summarizes identified organisms with associated confidence metrics, quantitative abundance estimates, and any detected antimicrobial resistance markers. The report is formatted for clinical interpretation, indicating whether specific organisms were detected or whether negative results reflect true absence or assay sensitivity limitations.

[0453] Validation studies (see in this regard, the references cited above) have demonstrated performance characteristics. System achieved 98.7% depletion efficiency for human ribosomal RNA, compared to approximately 85% efficiency with RNase H-based depletion methods. Off-target depletion rate was less than 0.01%. Sensitivity for detection in clinical samples reached 100%, with 100% specificity demonstrated through testing of negative control samples. Average recovery of reads mapping to pathogenic sequences reached 88,831 reads per million total reads with 10 ng RNA input.

[0454] A bullet point outline of the analytical pathogen detection / identification method performed by the use of the invention appears in the following paragraph. In the next paragraph, references to the “Instrument” refer to an apparatus configured in accordance with the present invention.

[0455] INPUT: Clinical specimen in pathogen-inactivating transport medium1STEP 1: AUTOMATED RNA EXTRACTION (Instrument Bay)• Sample lysis with chaotropic buffer• Magnetic bead-based nucleic acid binding• Automated washing (sequential buffer additions)• DNase treatment• Elution of purified RNA• Output: Purified total RNAI STEP 2: QUANTIFICATION-FREE LIBRARY PREPARATION (Instrument Bay) • RNA fragmentation (heat / Mg2+)• First-strand cDNA synthesis with universal adapters• Excess primer removal (exonuclease / rSAP)• Terminal transferase addition of 3' ribonucleotides• Adapter ligation (truncated T4 RNA ligase)• First PCR amplification (5 cycles with index primers)• Output: Indexed double-stranded cDNA libraryI STEP 3: POST-LIBRARY CRISPR-BASED HOST DEPLETION (Instrument Bay) • Incubation with pre-formed CRISPR-Cas9 RNP complexes targeting:o Human 18S / 28S rRNAo Human mitochondrial DNAo Bacterial 16S / 23S rRNA• Site-specific double-strand cleavage of abundant host sequences• Proteinase K inactivation• Magnetic bead purification (removes cleaved fragments <100 bp)• Output: Host-depleted library enriched for pathogen sequencesI STEP 4: POST-DEPLETION AMPLIFICATION (Instrument Bay)• Second PCR amplification (15 cycles)• Selective amplification of uncleaved (pathogen) library molecules• Output: Amplified pathogen-enriched libraryI STEP 5: BEAD-BASED NORMALIZATION (Cipher Bay)• Saturating magnetic bead binding (finite capacity normalization)• Ethanol wash• Elution at standardized concentration (no fluorometric measurement required) • Output: Normalized, sequencer-ready NGS library (20 L)I STEP 6: HIGH-THROUGHPUT SEQUENCING (Illumina MiSeq i100)• Automated loading from Cipher output tube• 100 bp paired-end or single-end sequencing• Real-time base calling with on-instrument de-hosting (optional)• Output: Raw sequencing data (FASTQ files)I STEP 7: BIOINFORMATICS ANALYSIS (BugSeq Cloud Platform)• Quality filtering and adapter trimming• Taxonomic classification against curated databaseso 8,400+ viral strainso 3.1 M AMR markerso Bacterial / fungal reference genomes• Pathogen identification and abundance quantification• Output: Clinician-ready diagnostic report.Automated Sample Preparation

[0456] This aspect incorporates the following key features:1. Automated nucleic acid extraction from various clinical sample types (e.g., blood, urine,cerebrospinal fluid)2. CRISPR-based host DNA depletion to enrich for pathogen nucleic acids3. Automated library preparation for sequencing

[0457] The system according to the invention is a fully automated, random-access sample processing system designed for clinical metagenomic sequencing, integrating multiple technologies into a single unified workflow. The system combines robotic liquid handling, thermal cycling, magnetic separation, and CRISPR-based depletion. Its architecture includes key subsystems such as a robotic core comprising a robot equipped with disposable pipette tips, four independent Peltier-based thermal cyclers with integrated magnetic separation offering ±0.1°C accuracy, and a reagent cartridge with active refrigeration to preserve temperature sensitive components at 4°C within each bay. The system also includes 2D barcode reader for sample and reagent traceability, as well as an HEPA-filtered positive pressure chamber for environmental control, ensuring clean and contaminant-free conditions (ISO Class 5 equivalent).

[0458] The processing of clinical specimens, such as nasopharyngeal swabs in viral transport medium, begins in a biosafety cabinet, or at the point of collection where a sample aliquot is transferred into a barcoded 1 ,5mL processing tube. A lysis buffer containing 400mM Tris-HCI, 60mM EDTA, 5% SDS, and 10mM DTT is added, along with an optional 5pL of MS2 phage RNA as a process control. The sample is then placed in the Cipher input drawer, where an overhead camera verifies the liquid level and barcode integrity before processing begins.

[0459] The nucleic acid extraction process follows an automated, seven-step procedure using magnetic bead-based purification. In the first phase, the system binds RNAto magnetic beads using a binding buffer. This is followed by two washing steps with ethanol and Tris buffers, a DNase treatment to remove any contaminating DNA, and an elution step with nuclease-free water containing Tween-20.

[0460] Subsequent RNA fragmentation is achieved by adding Mg++ to the eluate and heating to 94°C, generating RNA fragments between 150-500nt in size. Random hexamers tailed with a 5' universal adapter are then used for priming, followed by reverse transcription. After reverse transcription, the cDNA undergoes adapter ligation, which includes a terminal transferase reaction to create a 3-nt riboadenosine tail, followed by ligation using truncated T4 RNA ligase 2. The reaction products are purified using bead clean-up.

[0461] To reduce host contamination, the system incorporates CRISPR-mediated host depletion. This is achieved through pre-loaded ribonucleoprotein complexes targeting human rRNA, mitochondrial DNA, and bacterial 16S rRNA, using guide RNAs and high-fidelity SpyCas9. The depletion step occurs at 37°C -42C for 30-60 minutes, followed by bead cleanup to remove the cleaved fragments.

[0462] For library amplification, a dual-stage PCR strategy is employed. Pre-depletion PCR amplifies the target molecules, and post-depletion PCR is performed with index primers. In all applicable embodiments of the invention, it is preferred that:• Pre-depletion PCR involves 5 to 10 cycles (and 5 cycles is particularly preferred); and • Pre-depletion PCR involves between 10 to 20 cycles (and 15 cycles is particularly preferred).

[0463] The system’s innovations, including carrier effect utilization and the automated process, ensure high-quality results with minimal hands-on time. For instance, the system removes 98.7% of human rRNA compared to 85% using traditional RNase H methods and achieves an adapter dimer rate of less than 1%, with minimal cross-contamination between adjacent stations. This workflow reduces the hands-on time from 6 hours (manual) to just 15 minutes or less while ensuring high concordance with FDA-cleared PCR assays for respiratory pathogens.The system thus combines advanced technologies and automation, thereby optimizing clinical metagenomic sequencing with minimal user intervention and ensuring the efficient generation of highly reliable and reproducible results across multiple stages of processing.CRISPR-based Host Depletion

[0464] As explained, the present invention relates to an automated system for metagenomic sequencing, specifically integrating CRISPR-Cas9 technology to deplete host-associated nucleic acids from clinical samples. The system employs ribonucleoprotein (RNP) complexes that are sequence specific and designed to cleave abundant host sequences after librarypreparation. This enables the enrichment of low-biomass pathogen nucleic acids while retaining host material as a carrier during the initial processing steps. In clinical metagenomic sequencing, excessive host nucleic acids, such as human rRNAand mitochondrial DNA, often dominate sequencing libraries and obscure the detection of pathogen-related sequences. Traditional depletion methods, such as RNase H-based approaches, are limited by short hybridization requirements (4-6 bp) that lack specificity, leading to off-target effects. CRISPR-Cas9 systems, on the other hand, overcome these limitations by utilizing 20 bp guide RNAs (sgRNAs), which allow for precise targeting, enabling double-strand cleavage verification and minimizing non-specific interactions.

[0465] The system integrates the CRISPRclean® technology (Jumpcode Genomics https: / / www.jumpcodegenomics.com / ) into its automated workflow to perform host sequence depletion after the initial library amplification. This approach uses pre-loaded, lyophilized RNP complexes that target human rRNA (18S, 28S), mitochondrial DNA, and bacterial 16S rRNA (V3-V4 regions). The CRISPR depletion process is carried out after the first PCR reaction, allowing the host nucleic acids to act as carriers during library preparation. This step ensures that only the non-cleaved pathogen sequences are amplified in subsequent PCR cycles. Additionally, the system features a modular design that allows for the swapping of CRISPR probe cassettes to adapt to different host targets, such as bacterial rRNA in blood samples.

[0466] Improvements provided by the system include enhanced specificity due to the use of 20 bp sgRNAs (compared to the known RNase H method), which reduces off-target effects by requiring near-perfect complementarity for cleavage. (Traditional ribosomal RNA depletion methods employ RNase H-mediated hydrolysis based on short DNA oligonucleotide probes, as described in commercially available kits such as Illumina Ribo-Zero and NEB NEBNext rRNA Depletion. However, the short hybridization length of RNase H-based methods results in off-target cleavage and incomplete depletion efficiency (typically 80-90% removal).

[0467] The system also utilizes the carrier effect, which allows host nucleic acids to remain during the initial processing steps to prevent the loss of low-abundance pathogen material that may be lost by adherence to the walls of pipette tips or microtubes. Furthermore, the CRISPR-Cas9 system ensures complete inactivation of host sequences through the creation of doublestrand breaks, a more effective approach compared to single-strand nicking alternatives.

[0468] The CRISPR module within the system includes several components: independent Peltier-controlled thermal stations with ±0.1 °C accuracy, robotic liquid handling for dispensing premeasured RNPs (20 nM Cas9, 50 nM sgRNA) into library reactions, and magnetic separation using SPRI beads (0.6x ratio) to remove cleaved host DNA fragments after incubation. The RNP depletion process begins with the reconstitution of lyophilized RNPs innuclease-free water, followed by incubation of the library fragments with RNPs at 37°C-42C for 30-60 minutes. After incubation, magnetic beads bind intact DNA(>150 bp), while cleaved host fragments (<50 bp) are discarded in waste chambers. The final step involves the amplification of uncleaved pathogen libraries, which undergo index PCR to selectively amplify non-host sequences.

[0469] The design and manufacturing of sgRNAs are also integral to the system’s optimal performance. In the exemplary embodiments discussed in this specification, a total of approximately 3,500 sgRNAs are employed, targeting conserved regions of human rRNAand mitochondrial DNA. These sgRNAs are synthesized on microarrays or through standard chemical synthesis and undergo purification before being lyophilized for use. The quality of the sgRNAs is verified through gel electrophoresis, ensuring that at least 90% of the RNA is intact.

[0470] Performance characteristics of the system show a host depletion efficiency of 98.7% for human rRNA, compared to the 85% efficiency achieved by RNase H-based methods. The off-target rate is less than 0.01%, based on k-mer analysis. The system is compatible with a wide range of input RNA concentrations, from 10 pg to 1 pg.

[0471] Clinical validation (See in this regard, the references cited above) has demonstrated the system’s ability to achieve 100% concordance with FDA-approved respiratory pathogen panels across 412 clinical samples. This results in a 6.3x increase in viral read recovery compared to manual workflows, enabling the detection of pathogens at Ct (Cycle Threshold) values as high as 39. The CRISPR-based host depletion technology enables simultaneous detection of co-infections, strain typing for full-genome sequencing of pathogens, and antimicrobial resistance profiling, which are critical for clinical diagnostics and outbreak tracking.Random-Access Architecture of the Cipher System

[0472] The system incorporates a random-access architecture for clinical metagenomic sequencing, addressing significant limitations found in traditional batch-based sequencing systems. Specifically, the system allows for continuous, non-batched sample processing, offering substantial improvements in clinical diagnostics, especially in time sensitive environments such as emergency rooms or intensive care units (ICUs). Unlike traditional systems that require full instrument occupancy before processing can begin, the system allows for the immediate initiation of sequencing runs as soon as a sample is available. This advance in sample processing architecture enables the system to meet urgent diagnostic needs by reducing delays and improving efficiency.

[0473] The system achieves this through a tightly integrated design that includes three primary subsystems: modular processing stations, a dynamic scheduling engine, and a decoupled sequencing interface. Each subsystem plays a role in enabling the system's random-access capabilities.

[0474] In the exemplary embodiments described in this specification, the system comprises four independent modular processing bays, each capable of processing a single sample independently without requiring sample batching. Each such bay is equipped with a miniaturized Peltier thermal cycler. The thermal cyclers are capable of maintaining an exceptional temperature uniformity of ±0.1 °C, which is crucial for the accuracy of thermal amplification and other enzymatic reactions. Each station is further equipped with a multi-axis microfluidic manipulator capable of precise liquid handling, which includes capacitive liquid level sensing to prevent overfilling or underfilling during sample processing. Additionally, each station features a discrete magnetic separation station magnets, ensuring efficient separation of nucleic acids from other sample components. The stations operate asynchronously, allowing different workflow stages to be carried out simultaneously. For example, one station may be performing DNase treatment, while another station is executing CRISPR cleavage. This parallel processing maximizes throughput and minimizes the total processing time.

[0475] In practice, an important feature of the system’s functionality is its dynamic scheduling engine which ensures that processing is carried out efficiently and that clinical priorities are met. The dynamic scheduling engine comprises a software-based optimization algorithm, which executes on the single board computer (SBC) of an apparatus configured in accordance with the present invention. The algorithm uses a modified Hungarian algorithm to optimize multiple aspects of sample processing. Specifically, the engine minimizes overall processing time by scheduling tasks to run in parallel across the independent processing bays. It also incorporates a weighted priority scoring system that allows urgent samples — such as those from ICU (Intensive Care Unit) patients — to receive immediate attention, without disrupting existing or ongoing workflows. The priority score is determined based on several factors, including whether the sample is from an ICU patient, the pathogen risk index, and the time since the patient’s admission. Additionally, the engine uses predictive modelling to prevent resource contention, forecasting potential conflicts with reagent cartridge usage and triggering automatic replenishment alerts when needed.Decoupled Sequencing

[0476] The system additionally entails a decoupled sequencing capability, which, when utilised, decouples the sample processing workflow from the sequencing step. Processed libraries are queued in a temperature stabilized (4°C) carousel until they are ready forsequencing. The user of the system manually loads the sequencer cartridge (e.g., Illumina MiSeq i100) when one or more samples are ready for sequencing. This feature allows for sequencing without interrupting ongoing sample processing on the preparation device, ensuring that new samples can begin processing almost immediately after previous samples are loaded.Clinical Impact and Performance

[0477] The system’s random-access architecture is advantageous, particularly in clinical settings where rapid diagnosis is essential. One key benefit is the prioritization of urgent samples. This enables laboratories to keep pace with a steady stream of new samples, improving overall efficiency.

[0478] The system also enhances outbreak response capabilities. This ability to rapidly adapt to an evolving clinical situation is advantageous in terms of the preparedness of public and private health care facilities in relation to epidemics and pandemics and efficient outbreak management.Technical Differentiation from Batch Systems

[0479] When compared to traditional batch systems, the system’s random-access architecture offers several advantages:- Sample initiation in a system according to the invention is flexible and can occur at any time, whereas batch systems require a full batch of samples. For example, traditional benchtop NGS sample preparation systems require either the loading of 4, 8 or more samples simultaneously to be collected before processing can begin. Critically ill patients do not get sick in batches. Additionally, once a traditional system run starts, a new sample can not begin processing until the previous batch of samples has completed, causing significant delays in time to results for a critically ill patient sample.According to the invention, STAT samples (meaning those that require immediate, high-priority processing) can jump the queue and be processed immediately, whereas batch systems require manual intervention to re-prioritize urgent samples;- A system according to the invention offers continuous walkaway time (meaning that it allows laboratory workers greater ability than batch processing systems to work on other samples while the system is in operation), allowing for uninterrupted processing, while batchsystems often require significant waiting periods between runs;- A system according to the invention significantly reduces the risk of crosscontamination, as many of the traditional benchtop systems use open well strip tubes or plates to process multiple samples simultaneously for processing steps such as thermal cycling or magnetic bead isolation. The proximity between wells can cause cross contamination of nucleic acids between samples. The present invention provides individual sample processing separated by the individual bays, where all aspects including thermal cycling and magnetic bead isolation are distinct and separate between bays.

[0480] Apart from the other differences between the invention and hitherto known technologies in the field of the invention several aspects of the invention also set it apart from existing technologies:1. Asynchronous Thermal Control: Each modular processing station in the system operates with independent thermal control, allowing for simultaneous execution of different temperature protocols at each station. This eliminates thermal cross-talk between stations and optimizes processing efficiency.2. CRISPR Depletion: The system features a novel CRISPR depletion method, in which sgRNA complexes remain active across multiple stations. This allows for staggered addition of RNPs across different stations.

[0481] The system’s innovative random-access architecture offers practical advantages in clinical diagnostics. Hospitals using the Cipher system can process a highervolume of STAT samples during night and weekend shifts, resulting in improved diagnostic capabilities during off-hours. The system also helps reduce reagent waste as batch processing systems require loading of reagents for a full batch, whether or not a full batch of samples is processed. Additionally, its ability to maintain continuous throughput without dedicated “emergency” instruments makes it a potentially valuable tool for maintaining preparedness for emerging infectious diseases.

[0482] The system’s random-access architecture represents an important advance in the practice of clinical metagenomics, by transforming sequencing from a batch-based procedure to an on-demand diagnostic service. This advance is particularly critical for time-sensitive environments like hospitals, where urgent diagnostics can have life-or-death implications. The combination of modular stations, dynamic scheduling, and decoupled sequencing ensures that the system is well-suited to meet the growing demands of precision diagnostics and epidemic / pandemic / outbreak preparedness.Detailed Description of a Single-Use Consumable Cartridge for the System

[0483] With reference to FIG. 14 in a preferred embodiment, the present invention additionally provides a single-use, integrated consumable cartridge designed for automated metagenomic sequencing workflows. The cartridge houses a variety of thermolabile biological reagents — such as lyophilized enzymes, buffers, and CRISPR components — in a configuration that enables ambient temperature storage while maintaining full compatibility with robotic liquid handling systems. These advancements significantly enhance the operational efficiency of clinical metagenomics, especially in point-of-care diagnostics, by streamlining reagent handling and eliminating the need for cold-chain logistics.

[0484] The consumable cartridge is composed of several features that ensure both its functionality and efficiency in automated systems like those according to the present invention. Central to its design is the use of sequential workflow reagent tubes, which are arranged in foil-sealed tubes. The reagent tubes are pre-loaded with specific reagents, each designed to maintain stability in its lyophilized form at ambient temperatures. These reagents support various stages of the metagenomic sequencing process, including DNA lysis, binding, reverse transcription, CRISPR-mediated depletion, and PCR amplification. The reagent tubes, with precise positioning, are designed to match the pitch of robotic pipette systems in compliance with ISO 23783-2 standards, thus eliminating manual intervention during sequencing workflows. For instance, enzymes such as Proteinase K are stored in liquid-stable beads for up to 24 months, while CRISPR RNP complexes are lyophilized with a stabilizing BSA / trehalose matrix for up to 24 months of ambient-temperature stability. This advanced configuration ensures that the cartridge is able to deliver high-precision reagents at various points in the automated workflow with minimal risk of degradation overtime.

[0485] A particular aspect of the present invention is the integration of CRISPR RNP complexes directly into the consumable cartridge. These complexes, targeting specific genomic regions such as 18S / 28S rRNA and bacterial 16S rRNA, are pre-formed and lyophilized to maintain stability without the need for refrigeration. The cartridge integrates these RNPs into the metagenomic sequencing process according to the invention, without the requirement for cold-chain storage. This advance represents the first commercial integration of CRISPR RNPs into a reagent-containing cartridge for a diagnostic process that aims to detect the presence of a pathogen, thus making the overall diagnostic technology more accessible for clinical applications and reducing logistical challenges.

[0486] In the exemplary embodiments disclosed in this specification, the consumable cartridge incorporates a breakaway foil seal system, which is critical for preventing contamination and ensuring the proper sequential use of reagents.

[0487] The manufacturing process for the consumable cartridge also incorporates several innovative techniques that enhance its performance and reliability. One such advancement is differential lyophilization, which ensures the successful lyophilization of multiple enzymes with varying stability requirements. The process is optimized to ensure rapid reconstitution of these enzymes upon activation. This differential lyophilization enables the safe co-storage of incompatible enzymes, such as DNase and DNA polymerases, which would normally require separate storage conditions, thereby simplifying the cartridge's design.

[0488] The consumable cartridge further enhances its efficiency through the implementation of a barcode system. Each cartridge is encoded with a 2D DataMatrixit . co m / e r D ata at s x) ECC200 barcode, which includes crucial information such as the lotspecific enzyme expiration dates, and the unique CRISPR panel ID. This barcode is read by apparatus of the invention before the seals are pierced. If the system detects expired reagents or a mismatch between the cartridge’s CRISPR panel and the sample type (e.g., using respiratory rRNA RNPs on a blood sample), the process is automatically halted. This barcode-locked mechanism ensures that reagents are used correctly and safely, preventing costly errors in the diagnostic process.

[0489] Additionally, the cartridge features a waste containment system that addresses potential biohazardous waste handling. This system eliminates the need for external waste lines, allowing the cartridge to be used in clinical laboratory environments where contamination control and regulatory compliance are paramount.

[0490] A consumable cartridge for use in accordance with the present invention must be rigorously tested to meet high performance standards. Enzyme activity must be above 95% compared to fresh reagents, and cross-contamination levels must be minimized. Furthermore, the lyophilized reagents must show a low reconstitution volume coefficient of variation (CV), ensuring consistent performance across lots. A cartridge suitable for use in the invention must be designed for ambient storage with greater than or equal to six-month stability at 25°C and 60% relative humidity, making it suitable for global deployment without the need for refrigeration.

[0491] The cartridge’s design and functionality make it adaptable for a wide range of diagnostic applications, particularly in pandemic response scenarios. Additionally, the lyophilization process reduces cold chain costs, contributing to cost savings compared to traditional liquid reagents. The closed-system design ensures compliance with ISO 13485:2016 standards, making the cartridge suitable for use in regulated clinical environments.Sequencer interface

[0492] As previously explained, in preferred embodiments of the invention, the sequencing means comprise an integration interface. The integration interface for the sequencing means (sequencer) is an important component of the system,designed to automate aspects of next-generation sequencing (NGS) workflows, specifically library quantification, and cloud-based bioinformatics. In the embodiments of the invention disclosed in this specification, the system integrates two main subsystems: the quantification free Flow Cell Loading and the Cloud Integration, which together optimize sequencing efficiency, and scalability. The sequener employs a barcode-aware sequencing protocol to decode dual-index combinations, cross-referencing data from the Laboratory Information Management System (LIMS) to avoid index collisions. The flow cell loading volume is optimized based on the number of samples. This system enables the Miseq i 100 cycle count to export data in real-time (25- cycles) tor analysis. It should be noted that the sequencer can continue to run through the full cycles, but the data may be accessed after fewer cycles in real time.

[0493] In the embodiments disclosed in this specification, system connects the sequencer to BugSeq’s cloud-based bioinformatics pipeline (https: / / bugseq.com / ) via an API, facilitating secure, efficient data transfer and analysis. The system automatically uploads raw FASTQ files through BaseSpace Direct Upload (https: / / knowiedqe.iHumsna.conysoftwsre / clo d- software / software-cloud-software-referenci tagging them with critical metadata such as sample ID. Automated quality control checks are performed on the data, ensuring that over 50% of reads achieve Q30 quality scores and that the error rate remains below 0.01%, as determined by a Poisson distribution model.

[0494] This sequencer integration interface provides industrial and clinical advantages, particularly in flexible small-batch processing in regional hospitals, without requiring manual reconfiguration. It also ensures compliance with regulatory standards, automatically generating ISO 15189-compliant run reports, including operator IDs, timestamps, and QC metrics such as Q30 and cluster density. The system’s ability to process between 1 and 4 samples without reconfiguration, and its seamless integration with cloud bioinformatics platforms make it an important tool for next generation sequencing applications, offering significant improvements over existing workflows and eliminating manual intervention in clinical settings. Thus, the sequencer interface represents a significant innovation in the practical use of NGS in diagnostic applications, particularly in clinical diagnostics, where the need for automated, accurate, and scalable sequencing solutions is critical.

[0495] The sequencer interface for the system therefore offers an integrated and automated solution for NGS library preparation, easy quantification free flow cell loading, and bioinformatics integration. The system's advanced capabilities, alongside its compliance with regulatory standards, mean that it represents a significant advance in clinical diagnostics, especially in settings where bioinformatics expertise is limited.Bioinformatics Analysis

[0496] The bioinformatics pipeline consists of the following key steps:1. Quality control and preprocessing of sequencing reads2. Taxonomic classification3. Pathogen identification and abundance estimation4. Antimicrobial resistance gene detection5. Visualization and reporting of results, in the formats previously discussed.Example 2 - Rare Disease Genomics with Targeted Sequence Depletion

[0497] The biological sample comprises RNA extracted from blood, tissue, or other sources. The system extracts nucleic acids and performs optional selective depletion of common genomic regions providing minimal diagnostic information. Guide RNAs are designed to reduce sequencing depth devoted to uninformative regions while preserving complete coverage of genes relevant to disease. Optional hybridization capture enrichment for targeted disease gene panels can be performed. Bioinformatics analysis employs specialized algorithms for copy number variation detection through read depth analysis, structural variant detection through paired-end mapping, and point mutation detection through sequence comparison.Example 3: Cancer Gene Fusion Detection

[0498] The biological sample comprises tumor tissue, circulating tumor cells, or circulating tumor RNA extracted from patient blood. The system extracts total RNA and performs optional selective depletion of abundant non-fusion-bearing transcripts, including ribosomal RNA and highly abundant housekeeping genes, to increase sequencing depth devoted to rare fusioncontaining transcripts. Reverse transcription, adapter ligation, and library amplification are performed. Bioinformatics analysis identifies fusion junction sequences, maps breakpoints with base-pair precision, and predicts resulting fusion protein sequences.Example 4: Circulating Tumor DNA Detection

[0499] The biological sample comprises cell-free plasma. The system extracts cell-free DNA using purification optimized for short DNA fragments. Optional selective depletion targets normal cell-free DNA sequences, including known common somatic mutations in normal agingcells or highly abundant wild-type genomic sequences. Sequencing is performed to ultra-high depth (1-10 million reads per sample). Bioinformatics analysis employs unique molecular identifier collapsing for error correction, enabling detection of circulating tumor DNA at variant allele frequencies as low as 0.01%.Example 5: Environmental Surveillance

[0500] The biological sample comprises wastewater or other environmental samples. The system performs optional concentration steps, nucleic acid extraction, and selective depletion targeting human nucleic acids and abundant environmental bacterial sequences. Bioinformatics analysis identifies pathogens and provides quantitative estimates of relative abundance for surveillance purposes.Example 6: Vector-Borne Disease Surveillance

[0501] The biological sample comprises individual arthropod vectors including mosquitoes and ticks. The system processes samples through homogenization, lysis, and nucleic acid extraction. Selective depletion targets arthropod ribosomal RNA, arthropod genomic DNA, and abundant arthropod-associated microbiota. Bioinformatics analysis identifies vector-borne pathogens including arthropod-borne viruses, rickettsiae, spirochetes, and parasitic organisms.Example 7: Veterinary Diagnostics

[0502] The biological sample comprises blood, respiratory secretions, gastrointestinal samples, or tissue from non-human animals. The system performs nucleic acid extraction and selective depletion targeting species-specific host ribosomal RNA and mitochondrial DNA, with guide RNA sets designed appropriate to the specific animal species. Bioinformatics analysis identifies pathogens of veterinary significance.Example 8: Single-Cell RNA Sequencing with CRISPR-Based Depletion

[0503] Pooled barcoded cDNA from microfluidic single-cell capture is processed through the system. The system performs CRISPR-based depletion targeting ribosomal RNA and mitochondrial RNA sequences that dominate single-cell libraries. Removal of these sequences increases the proportion of reads mapping to protein-coding transcripts, improving sensitivity for detection of lowly expressed genes.Example 9: Small RNA Sequencing with Adapter Dimer Depletion

[0504] The biological sample comprises total RNA or size-selected small RNA. The system performs CRISPR-based depletion targeting adapter dimer sequences that form when unligated adapters ligate to each other rather than to RNA molecules, eliminating the need forgel purification. This is particularly beneficial for low-input samples where gel extraction results in substantial material loss.Example 10: ATAC-Seq with Mitochondrial DNA Depletion

[0505] Following Tn5 tagmentation performed prior to system processing, the system performs CRISPR-based depletion targeting mitochondrial DNA that accumulates in ATAC-Seq libraries due to open mitochondrial chromatin structure. Cleaved mitochondrial fragments lack sequencing adapters and are removed during purification, preserving nuclear chromatin-derived fragments for genome-wide open chromatin analysis.Example 11: Microbiome Sequencing with Host Depletion

[0506] The biological sample comprises material from gut, skin, oral, or environmental microbiome sources. The system performs selective depletion targeting non-microbial nucleic acids dominating the sample. For human microbiome samples, guide RNAs target human ribosomal DNA, mitochondrial DNA, and abundant genomic sequences. For plant-associated microbiome samples, guide RNAs target plant chloroplast DNA, plant mitochondrial DNA, and plant nuclear ribosomal DNA.Example 12: Ribosome Profiling

[0507] Ribosome-protected mRNA fragments are processed through the system. CRISPR-based depletion targets contaminating ribosomal RNA fragments and abundant transfer RNA species co-isolated with ribosomes. Bioinformatics analysis maps ribosome positions genome-wide, providing translational activity profiles.Example 13: Whole Genome Sequencing

[0508] The system performs automated genomic DNA extraction and standard library preparation employing tagmentation, adapter ligation, and limited-cycle PCR amplification. Random-access processing enables samples to be processed immediately upon receipt. In one embodiment, CRISPR-based depletion removes bacterial contamination from saliva-derived DNA.Example 14: Whole Exome Sequencing

[0509] Following library preparation, the system performs automated hybridization capture enrichment with biotinylated oligonucleotide probes at 65°C, streptavidin bead capture, stringent washing, and post-capture PCR amplification. The thermal cycler maintains precise temperature control during the 90-minute hybridization incubation without user intervention.Example 15: Targeted Gene Panel Sequencing

[0510] The system performs automated multiplex PCR amplification with hundreds to thousands of primer pairs simultaneously, followed by optional enzymatic primer digestion, adapter ligation, and indexing. High-throughput multiplexing enables simultaneous processing of up to 96 samples with unique barcode combinations.Example 16: DNA Methylation Analysis

[0511] The system performs automated bisulfite conversion with precise temperature control for 2-4 hours, followed by specialized library preparation and optional hybridization capture enrichment. Bioinformatics analysis identifies methylated and unmethylated cytosines at single-base resolution.Example 17: Chromatin Immunoprecipitation Sequencing (ChlP-Seq)

[0512] The system processes immunoprecipitated DNA, typically present in limiting quantities, through specialized low-input library preparation with minimized dead volumes. Automated adaptive PCR cycle adjustment based on sample quality assessment optimizes library complexity.Example 18: 16S Ribosomal RNA Gene Amplicon Sequencing

[0513] The system performs automated multiplex PCR amplification of conserved ribosomal RNA gene regions flanking variable regions, with automated dual-indexing and high-throughput multiplexing enabling simultaneous processing of up to 96 samples.Example 19: Standard Single-Cell RNA Sequencing

[0514] Pooled barcoded cDNA from droplet-based cell capture is processed through the system for automated amplification, library preparation, cleanup, normalization, and indexing. Bioinformatics analysis demultiplexes reads by cell barcode and unique molecular identifier to generate per-cell gene expression matrices.CLINICAL VALIDATION DATA

[0515] The following validation data were generated using the manual (non-automated) molecular workflow. These data demonstrate the performance of the underlying chemistry and are expected to be replicated or improved upon the automated system during instrument validation.

[0516] Site 1 (TGen, Phoenix, AZ): Clinical validation of CRISPR-enhanced NGS for SARS-CoV-2 detection. n=57 positive samples (Ct range 15.56-39.27), n=15 negative samples. Achieved 100% sensitivity and specificity for Ct <35.

[0517] Site 2 (PerkinElmer Clinical Lab, Valencia, CA): Independent validation demonstrating performance with 10-fold lower RNA input (1 ng vs. 10 ng) while achieving higher viral detection (192,582 vs. 88,831 reads per million).

[0518] Site 3 (UCLA): 412 clinical samples tested, achieving 100% concordance with FDA-approved respiratory pathogen panels and 6.3* increase in viral read recovery.

[0519] Host depletion efficiency: 98.7% for human rRNA, compared to approximately 85% for RNase H-based methods. Off-target rate: <0.01%. Adapter dimer rate: <0.01%. Crosscontamination rate: <0.001%.

[0520] Published reference: Cern S, Clemons NC, von Bredow B, Yang S. "Application of CRISPR-Based Human and Bacterial Ribosomal RNA Depletion for SARS-CoV-2 Shotgun Metagenomic Sequencing." Am J Clin Pathol. 2023; 159(2):111 -115.Industrial Applicability

[0521] The system has significant industrial applicability across multiple fields:

[0522] In clinical infectious disease diagnostics, the system enables hospital laboratories and clinics to deploy metagenomic NGS for syndromic testing, outbreak surveillance, antimicrobial resistance profiling, and rapid response to novel pathogens during public health emergencies.

[0523] In cancer diagnostics, the system enables detection of gene fusions, circulating tumor DNA, somatic mutations, and minimal residual disease with enhanced sensitivity through depletion of abundant background sequences.

[0524] In prenatal diagnostics, the system enables non-invasive prenatal testing with enhanced fetal DNA fraction through depletion of maternal background sequences.

[0525] In veterinary medicine, the system enables rapid pathogen detection in livestock, poultry, and companion animals.

[0526] In environmental monitoring, the system enables wastewater surveillance, food safety pathogen screening, and biodefense applications.

[0527] In research applications, the system enables single-cell RNA sequencing, microbiome analysis, chromatin accessibility profiling, and translational profiling with enhanced sensitivity through targeted depletion of abundant background sequences.INTERPRETATION OF THIS SPECIFICATION

[0528] It is to be understood that the invention could take many forms and be put to many different uses. All such forms and uses are embodied within the spirit and scope of the invention, which is to be understood as not being limited to the particular constructional or compositional or other details of the embodiments discussed or disclosed anywhere within this specification, but which extends to each novel feature and combination of features disclosed in or evident from this specification and the accompanying claims and drawings. All of these different combinations constitute various alternative aspects of the invention.

[0529] It will also be understood that the term “comprises” (or its grammatical variants), as used in this specification (including within the appended claims), is equivalent in meaning to the term “includes” and should not be taken as excluding the presence of other elements or features. Further, wherever used in this specification, the term “includes” is not a term of limitation, and is not to be taken as excluding the presence of other elements or features.

[0530] It is further to be understood that any reference to or discussion in this specification of background or prior art documents, devices, acts, information, knowledge or use (‘Background Information’) is included solely to explain the context of the invention. Any discussion of such Background Information is therefore not be taken as an admission in any jurisdiction that any such Background Information constitutes prior art, part of the prior art base or the common general knowledge in the field of the invention on or before the priority date of the appended claims or any amended claims later introduced into this specification.

[0531] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification(s). This application is intended to cover any variations uses or adaptations of the invention following in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

[0532] As the present invention may be embodied in several forms without departing from the spirit of the essential characteristics of the invention, it should be understood that the above described embodiments are not to limit the present invention unless otherwise specified, but rather should be construed broadly within the spirit and scope of the invention as defined in the appended claims. The described embodiments are to be considered in all respects as illustrative only and not restrictive.

[0533] Various modifications and equivalent arrangements are intended to be included within the spirit and scope of the invention and appended claims. Therefore, the specific embodiments are to be understood to be illustrative of the many ways in which the principlesof the present invention may be practiced. In the following claims, any means-plus-function clauses are intended to cover structures as performing the defined function and not only structural equivalents, but also equivalent structures. For example, although a nail and a screw may not be structural equivalents in that a nail employs a cylindrical surface to secure wooden parts together, whereas a screw employs a helical surface to secure wooden parts together, in the environment of fastening wooden parts, a nail and a screw are equivalent structures.The following sections I -VII provide further guidance to interpreting the present specification. I. Terms

[0534] The term “product” means any machine, manufacture, and / or composition of matter, unless expressly specified otherwise.

[0535] The term “process” means any process, algorithm, method, or the like, unless expressly specified otherwise.

[0536] Each process (whether called a method, algorithm or otherwise) inherently includes one or more steps, and therefore all references to a “step” or “steps” of a process have an inherent antecedent basis in the mere recitation of the term ‘process’ or a like term. Accordingly, any reference in a claim to a ‘step’ or ‘steps’ of a process has sufficient antecedent basis.

[0537] The term “invention” and the like mean “the one or more inventions disclosed in this specification”, unless expressly specified otherwise.

[0538] The terms “an embodiment”, “embodiment”, “embodiments”, “the embodiment”, “the embodiments”, “one or more embodiments”, “some embodiments”, “certain embodiments”, “one embodiment”, “another embodiment” and the like mean “one or more (but not all) embodiments of the disclosed invention(s)”, unless expressly specified otherwise.

[0539] The term “variation” of an invention means an embodiment of the invention, unless expressly specified otherwise.

[0540] A reference to “another embodiment” in describing an embodiment does not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise.

[0541] The terms “a”, “an” and “the” mean “one or more”, unless expressly specified otherwise.

[0542] The term “plurality” means “two or more”, unless expressly specified otherwise.

[0543] The term “herein” means “in the present specification, including anything which may be incorporated by reference”, unless expressly specified otherwise.

[0544] The phrase “at least one of’, when such phrase modifies a plurality of things (such as an enumerated list of things), means any combination of one or more of those things, unless expressly specified otherwise. For example, the phrase “at least one of a widget, a car and a wheel” means either (i) a widget, (ii) a car, (iii) a wheel, (iv) a widget and a car, (v) a widget and a wheel, (vi) a car and a wheel, or (vii) a widget, a car, and a wheel. The phrase “at least one of’, when such phrase modifies a plurality of things, does not mean “one of each of’ the plurality of things.

[0545] Numerical terms such as “one”, “two”, etc. when used as cardinal numbers to indicate quantity of something (e.g., one widget, two widgets), mean the quantity indicated by that numerical term, but do not mean at least the quantity indicated by that numerical term. For example, the phrase “one widget” does not mean “at least one widget”, and therefore the phrase “one widget” does not cover, e.g., two widgets.

[0546] The phrase “based on” does not mean “based only on”, unless expressly specified otherwise. In other words, the phrase “based on” describes both “based only on” and “based at least on”. The phrase “based at least on” is equivalent to the phrase “based at least in part on .

[0547] The term “represent” and like terms are not exclusive, unless expressly specified otherwise. For example, the term “represents” do not mean “represents only”, unless expressly specified otherwise. In other words, the phrase “the data represents a credit card number” describes both “the data represents only a credit card number” and “the data represents a credit card number and the data also represents something else”.

[0548] The term “whereby” is used herein only to precede a clause or other set of words that express only the intended result, objective, or consequence of something that is previously and explicitly recited. Thus, when the term “whereby” is used in a claim, the clause, or other words that the term “whereby” modifies do not establish specific further limitations of the claim or otherwise restricts the meaning or scope of the claim.

[0549] The term “e.g.” and like terms mean “for example”, and thus does not limit the term or phrase it explains. For example, in the sentence “the computer sends data (e.g., instructions, a data structure) over the Internet”, the term “e.g.” explains that “instructions” are an example of “data” that the computer may send over the Internet, and also explains that “a data structure” is an example of “data” that the computer may send over the Internet. However, both “instructions” and “a data structure” are merely examples of “data”, and other things besides “instructions” and “a data structure” can be “data”.

[0550] The term “i.e.” and like terms mean “that is”, and thus limits the term or phrase it explains. For example, in the sentence “the computer sends data (i.e., instructions) over the Internet”, the term “i.e.” explains that “instructions” are the “data” that the computer sends over the Internet.

[0551] Any given numerical range shall include whole and fractions of numbers within the range. For example, the range “1 to 10” shall be interpreted to specifically include whole numbers between 1 and 10 (e.g., 2, 3, 4, . . . 9) and non-whole numbers (e.g., 1.1, 1.2, . . . I.9).II. Determining

[0552] The term “determining” and grammatical variants thereof (e.g., to determine a price, determining a value, determine an object which meets a certain criterion) is used in an extremely broad sense. The term “determining” encompasses a wide variety of actions and therefore “determining” can include calculating, computing, processing, deriving, investigating, looking up (e.g., looking up in a table, a database, or another data structure), ascertaining and the like. Also, “determining” can include receiving (e.g., receiving information), accessing (e.g., accessing data in a memory) and the like. Also, “determining” can include resolving, selecting, choosing, establishing, and the like.

[0553] The term “determining” does not imply certainty or absolute precision, and therefore “determining” can include estimating, extrapolating, predicting, guessing and the like.

[0554] The term “determining” does not imply that mathematical processing must be performed, and does not imply that numerical methods must be used, and does not imply that an algorithm or process is used.

[0555] The term “determining” does not imply that any particular device must be used. For example, a computer need not necessarily perform the determining.III. Indication

[0556] The term “indication” is used in an extremely broad sense. The term “indication” may, among other things, encompass a sign, symptom, or token of something else.

[0557] The term “indication” may be used to refer to any indicia and / or other information indicative of or associated with a subject, item, entity, and / or other object and / or idea.

[0558] As used herein, the phrases “information indicative of’ and “indicia” may be used to refer to any information that represents, describes, and / or is otherwise associated with a related entity, subject, or object.

[0559] Indicia of information may include, for example, a symbol, a code, a reference, a link, a signal, an identifier, and / or any combination thereof and / or any other informative representation associated with the information.

[0560] In some embodiments, indicia of information (or indicative of the information) may be or include the information itself and / or any portion or component of the information. In some embodiments, an indication may include a request, a solicitation, a broadcast, and / or any other form of information gathering and / or dissemination.IV. Forms of Sentences

[0561] Where a limitation of a first claim would cover one of a feature as well as more than one of a feature (e.g., a limitation such as “at least one widget” covers one widget as well as more than one widget), and where in a second claim that depends on the first claim, the second claim uses a definite article “the” to refer to the limitation (e.g., “the widget”), this does not imply that the first claim covers only one of the feature, and this does not imply that the second claim covers only one of the feature (e.g., “the widget” can cover both one widget and more than one widget).

[0562] When an ordinal number (such as “first”, “second”, “third” and so on) is used as an adjective before a term, that ordinal number is used (unless expressly specified otherwise) merely to indicate a particular feature, such as to distinguish that particular feature from another feature that is described by the same term or by a similar term. For example, a “first widget” may be so named merely to distinguish it from, e.g., a “second widget”. Thus, the mere usage of the ordinal numbers “first” and “second” before the term “widget” does not indicate any other relationship between the two widgets, and likewise does not indicate any other characteristics of either or both widgets. For example, the mere usage of the ordinal numbers “first” and “second” before the term “widget” (1) does not indicate that either widget comes before or after any other in order or location; (2) does not indicate that either widget occurs or acts before or after any other in time; and (3) does not indicate that either widget ranks above or below any other, as in importance or quality. In addition, the mere usage of ordinal numbers does not define a numerical limit to the features identified with the ordinal numbers. For example, the mere usage of the ordinal numbers “first” and “second” before the term “widget” does not indicate that there must be no more than two widgets.

[0563] When a single device or article is described herein, more than one device / article (whether or not they cooperate) may alternatively be used in place of the single device / article that is described. Accordingly, the functionality that is described as being possessed by a device may alternatively be possessed by more than one device / article (whether or not they cooperate).

[0564] Similarly, where more than one device or article is described herein (whether or not they cooperate), a single device / article may alternatively be used in place of the more than one device or article that is described. For example, a plurality of computer-based devices may be substituted with a single computer-based device. Accordingly, the various functionality that is described as being possessed by more than one device or article may alternatively be possessed by a single device / article, or for that matter, a cartridge, a vial or other device or article such as a medical device or consumable product. In this respect, any reference in this specification to a support pack is to be treated as synonymous to a cartridge.

[0565] The functionality and / or the features of a single device that is described may be alternatively embodied by one or more other devices which are described but are not explicitly described as having such functionality / features. Thus, other embodiments need not include the described device itself, but rather can include the one or more other devices which would, in those other embodiments, have such functionality / features.V. Disclosed Examples and Terminology Are Not Limiting

[0566] Neither the Title nor the Abstract in this specification is intended to be taken as limiting in any way as the scope of the disclosed invention(s). The title and headings of sections provided in the specification are for convenience only, and are not to be taken as limiting the disclosure in any way.

[0567] Numerous embodiments are described in the present application, and are presented for illustrative purposes only. The described embodiments are not, and are not intended to be, limiting in any sense. The presently disclosed invention(s) are widely applicable to numerous embodiments, as is readily apparent from the disclosure. One of ordinary skill in the art will recognise that the disclosed invention(s) may be practised with various modifications and alterations, such as structural, logical, software, and electrical modifications. Although particular features of the disclosed invention(s) may be described with reference to one or more particular embodiments and / or drawings, it should be understood that such features are not limited to usage in the one or more particular embodiments or drawings with reference to which they are described, unless expressly specified otherwise.

[0568] The present disclosure is not a literal description of all embodiments of the invention(s). Also, the present disclosure is not a listing of features of the invention(s) which must be present in all embodiments.

[0569] Devices that are described as in communication with each other need not be in continuous communication with each other, unless expressly specified otherwise. On the contrary, such devices need only transmit to each other as necessary or desirable, and may actually refrain from exchanging data most of the time. For example, a machine incommunication with another machine via the Internet may not transmit data to the other machine for long period of time (e.g. weeks at a time). In addition, devices that are in communication with each other may communicate directly or indirectly through one or more intermediaries.

[0570] A description of an embodiment with several components or features does not imply that all or even any of such components / features are required. On the contrary, a variety of optional components are described to illustrate the wide variety of possible embodiments of the present invention(s). Unless otherwise specified explicitly, no component / feature is essential or required.

[0571] Although process steps, operations, algorithms, or the like may be described in a particular sequential order, such processes may be configured to work in different orders. In other words, any sequence or order of steps that may be explicitly described does not necessarily indicate a requirement that the steps be performed in that order. The steps of processes described herein may be performed in any order practical. Further, some steps may be performed simultaneously despite being described or implied as occurring non-simultaneously (e.g., because one step is described after the other step). Moreover, the illustration of a process by its depiction in a drawing does not imply that the illustrated process is exclusive of other variations and modifications thereto, does not imply that the illustrated process or any of its steps are necessary to the invention(s), and does not imply that the illustrated process is preferred.

[0572] Although a process may be described as including a plurality of steps, that does not imply that all or any of the steps are preferred, essential or required. Various other embodiments within the scope of the described invention(s) include other processes that omit some or all of the described steps. Unless otherwise specified explicitly, no step is essential or required.

[0573] Although a process may be described singly or without reference to other products or methods, in an embodiment the process may interact with other products or methods. For example, such interaction may include linking one business model to another business model. Such interaction may be provided to enhance the flexibility or desirability of the process.

[0574] Although a product may be described as including a plurality of components, aspects, qualities, characteristics, and / or features, that does not indicate that any or all of the plurality are preferred, essential or required. Various other embodiments within the scope of the described invention(s) include other products that omit some or all of the described plurality.

[0575] An enumerated list of items (which may or may not be numbered) does not imply that any or all of the items are mutually exclusive, unless expressly specified otherwise. Likewise,an enumerated list of items (which may or may not be numbered) does not imply that any or all of the items are comprehensive of any category, unless expressly specified otherwise. For example, the enumerated list “a computer, a laptop, a PDA” does not imply that any or all of the three items of that list are mutually exclusive and does not imply that any or all of the three items of that list are comprehensive of any category.

[0576] An enumerated list of items (which may or may not be numbered) does not imply that any or all of the items are equivalent to each other or readily substituted for each other.

[0577] All embodiments are illustrative, and do not imply that the invention or any embodiments were made or performed, as the case may be.VI. Computing

[0578] It will be readily apparent to one of ordinary skill in the art that the various processes described herein may be implemented by, e.g., appropriately programmed general purpose computers, special purpose computers, and computing devices. Typically, a processor (e.g., one or more microprocessors, one or more micro-controllers, one or more digital signal processors) will receive instructions (e.g., from a memory or like device), and execute those instructions, thereby performing one or more processes defined by those instructions.

[0579] A “processor” means one or more microprocessors, central processing units (CPUs), computing devices, micro-controllers, digital signal processors, or like devices or any combination thereof.

[0580] Thus, a description of a process is likewise a description of an apparatus for performing the process. The apparatus that performs the process can include, e.g, a processor and those input devices and output devices that are appropriate to perform the process.

[0581] Further, programs that implement such methods (as well as other types of data) may be stored and transmitted using a variety of media (e.g., computer readable media) in a number of manners. In some embodiments, hard-wired circuitry or custom hardware may be used in place of, or in combination with, some or all of the software instructions that can implement the processes of various embodiments. Thus, various combinations of hardware and software may be used instead of software only.

[0582] The term “computer-readable medium” refers to any medium, a plurality of the same, or a combination of different media, that participate in providing data (e.g., instructions, data structures) which may be read by a computer, a processor or a like device. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks and other persistent memory. Volatile media include dynamic random access memory (DRAM),which typically constitutes the main memory. Transmission media include coaxial cables, copper wire, and fibre optics, including the wires that comprise a system bus coupled to the processor. Transmission media may include or convey acoustic waves, light waves, and electromagnetic emissions, such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, an EPROM, a FLASH-EEPROM, any other memory chip or cartridge, a carrier wave as described hereinafter, or any other medium from which a computer can read.

[0583] Various forms of computer readable media may be involved in carrying data (e.g. sequences of instructions) to a processor. For example, data may be (i) delivered from RAM to a processor; (ii) carried over a wireless transmission medium; (iii) formatted and / or transmitted according to numerous formats, standards, or protocols, such as Ethernet (or IEEE 802.3), SAP, ATP, Bluetooth™, and TCP / IP, TDMA, CDMA, and 3G; and / or (iv) encrypted to ensure privacy or prevent fraud in any of a variety of ways well known in the art.

[0584] The term “computer-readable recording medium storing instructions” refers to any medium, a plurality of the same, or a combination of different media, that participate in providing data (e.g., instructions, data structures) which may be read by a computer, a processor or a like device. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. A computer-readable recording medium storing instructions disclosed, defined, or referred to in this disclosure does not encompass transitory media.

[0585] Thus, a description of a process is likewise a description of a computer-readable medium storing a program for performing the process. The computer-readable medium can store (in any appropriate format) those program elements which are appropriate to perform the method.

[0586] Just as the description of various steps in a process does not indicate that all the described steps are required, embodiments of an apparatus include a computer / computing device operable to perform some (but not necessarily all) of the described process.

[0587] Likewise, just as the description of various steps in a process does not indicate that all the described steps are required, embodiments of a computer-readable medium storing a program or data structure include a computer-readable medium storing a program that, when executed, can cause a processor to perform some (but not necessarily all) of the described process.

[0588] Where databases are described, it will be understood by one of ordinary skill in the art that (i) alternative database structures to those described may be readily employed, and (ii) other memory structures besides databases may be readily employed. Any illustrations or descriptions of any sample databases presented herein are illustrative arrangements for stored representations of information. Any number of other arrangements may be employed besides those suggested by, e.g., tables illustrated in drawings or elsewhere. Similarly, any illustrated entries of the databases represent exemplary information only; one of ordinary skill in the art will understand that the number and content of the entries can be different from those described herein. Further, despite any depiction of the databases as tables, other formats (including relational databases, object-based models, and / or distributed databases) could be used to store and manipulate the data types described herein. Likewise, object methods or behaviours of a database can be used to implement various processes, such as the described herein. In addition, the databases may, in a known manner, be stored locally or remotely from a device which accesses data in such a database.

[0589] Various embodiments can be configured to work in a network environment including a computer that is in communication (e.g., via a communications network) with one or more devices. The computer may communicate with the devices directly or indirectly, via any wired or wireless medium (e.g. the Internet, LAN, WAN or Ethernet, Token Ring, a telephone line, a cable line, a radio channel, an optical communications line, commercial on-line service providers, bulletin board systems, a satellite communications link, a combination of any of the above). Each of the devices may themselves comprise computers or other computing devices that are adapted to communicate with the computer. Any number and type of devices may be in communication with the computer.

[0590] In an embodiment, a server computer or centralised authority may not be necessary or desirable. For example, the present invention may, in an embodiment, be practised on one or more devices without a central authority. In such an embodiment, any functions described herein as performed by the server computer or data described as stored on the server computer may instead be performed by or stored on one or more such devices.

[0591] Where a process is described, in an embodiment the process may operate without any user intervention. In another embodiment, the process includes some human intervention (e.g., a step is performed by or with the assistance of a human).

[0592] It should be noted that where the terms “server”, “secure server” or similar terms are used herein, a communication device is described that may be used in a communication system, unless the context otherwise requires, and should not be construed to limit the present invention to any particular communication device type. Thus, a communication device mayinclude, without limitation, a bridge, router, bridge-router (router), switch, node, or other communication device, which may or may not be secure.

[0593] It should also be noted that where a flowchart or workflow is used herein to demonstrate various aspects of the invention, it should not be construed to limit the present invention to any particular logic flow or logic implementation. The described logic may be partitioned into different logic blocks (e.g., programs, modules, functions, or subroutines) without changing the overall results or otherwise departing from the true scope of the invention. Often, logic elements may be added, modified, omitted, performed in a different order, or implemented using different logic constructs (e.g., logic gates, looping primitives, conditional logic, and other logic constructs) without changing the overall results or otherwise departing from the true scope of the invention.

[0594] Various embodiments of the invention may be embodied in many different forms, including computer program logic for use with a processor (e.g., a microprocessor, microcontroller, digital signal processor, or general purpose computer and forthat matter, any commercial processor may be used to implement the embodiments of the invention either as a single processor, serial or parallel set of processors in the system and, as such, examples of commercial processors include, but are not limited to Merced™, Pentium™, Pentium II™, Xeon™, Celeron™, Pentium Pro™, Efficeon™, Athlon™, AMD™ and the like), programmable logic for use with a programmable logic device (e.g., a Field Programmable Gate Array (FPGA) or other PLD), discrete components, integrated circuitry (e.g., an Application Specific Integrated Circuit (ASIC)), or any other means including any combination thereof. In an exemplary embodiment of the present invention, predominantly all of the communication between users and the server is implemented as a set of computer program instructions that is converted into a computer executable form, stored as such in a computer readable medium, and executed by a microprocessor under the control of an operating system.

[0595] Computer program logic implementing all or part of the functionality where described herein may be embodied in various forms, including a source code form, a computer executable form, and various intermediate forms (e.g., forms generated by an assembler, compiler, linker, or locator). Source code may include a series of computer program instructions implemented in any of various programming languages (e.g., an object code, an assembly language, or a high-level language such as Fortran, C, C++, JAVA, or HTML. Moreover, there are hundreds of available computer languages that may be used to implement embodiments of the invention, among the more common being Ada; Algol; APL; awk; Basic; C; C++; Conol; Delphi; Eiffel; Euphoria; Forth; Fortran; HTML; Icon; Java; Javascript; Lisp; Logo; Mathematica; MatLab; Miranda; Modula-2; Oberon; Pascal; Perl; PL / I; Prolog; Python; Rexx; SAS; Scheme; sed; Simula; Smalltalk; Snobol; SQL; Visual Basic; Visual C++; Linuxand XML.) for use with various operating systems or operating environments. The source code may define and use various data structures and communication messages. The source code may be in a computer executable form (e.g., via an interpreter), or the source code may be converted (e.g., via a translator, assembler, or compiler) into a computer executable form.

[0596] The computer program may be fixed in any form (e.g., source code form, computer executable form, or an intermediate form) either permanently or transitorily in a tangible storage medium, such as a semiconductor memory device (e.g., a RAM, ROM, PROM, EEPROM, or Flash-Programmable RAM), a magnetic memory device (e.g., a diskette or fixed disk), an optical memory device (e.g., a CD-ROM or DVD-ROM), a PC card (e.g., PCMCIA card), or other memory device. The computer program may be fixed in any form in a signal that is transmittable to a computer using any of various communication technologies, including, but in no way limited to, analog technologies, digital technologies, optical technologies, wireless technologies (e.g., Bluetooth), networking technologies, and internetworking technologies. The computer program may be distributed in any form as a removable storage medium with accompanying printed or electronic documentation (e.g., shrinkwrapped software), preloaded with a computer system (e.g., on system ROM or fixed disk), or distributed from a server or electronic bulletin board over the communication system (e.g., the Internet or World Wide Web).

[0597] Hardware logic (including programmable logic for use with a programmable logic device) implementing all or part of the functionality where described herein may be designed using traditional manual methods, or may be designed, captured, simulated, or documented electronically using various tools, such as Computer Aided Design (CAD), a hardware description language (e.g., VHDLorAHDL), ora PLD programming language (e.g., PALASM, ABEL, or CUPL). Hardware logic may also be incorporated into display screens for implementing embodiments of the invention and which may be segmented display screens, analogue display screens, digital display screens, CRTs, LED screens, Plasma screens, liquid crystal diode screen, and the like.

[0598] Programmable logic may be fixed either permanently or transitorily in a tangible storage medium, such as a semiconductor memory device (e.g., a RAM, ROM, PROM, EEPROM, or Flash-Programmable RAM), a magnetic memory device (e.g., a diskette or fixed disk), an optical memory device (e.g., a CD-ROM or DVD-ROM), or other memory device. The programmable logic may be fixed in a signal that is transmittable to a computer using any of various communication technologies, including, but in no way limited to, analog technologies, digital technologies, optical technologies, wireless technologies (e.g., Bluetooth), networking technologies, and internetworking technologies. The programmable logic may be distributed as a removable storage medium with accompanying printed orelectronic documentation (e.g., shrink wrapped software), preloaded with a computer system (e.g., on system ROM or fixed disk), or distributed from a server or electronic bulletin board over the communication system (e.g., the Internet or World Wide Web).

Claims

CLAIMS1. An automated nucleic acid processing system comprising:(a) a plurality of independent processing bays, each configured to receive a single biological sample and to operate asynchronously relative to the other processing bays;(b) within each processing bay:(i) a robotic liquid handling module configured to perform addition-only liquid transfers,(ii) a thermal cycling module configured to execute programmable temperature profiles,(iii) a magnetic bead separation module, and(iv) a gripper mechanism configured to transport sample vessels between the modules;(c) a controller comprising at least one processor and memory storing executable instructions configured to:(i) control each processing bay independently,(ii) initiate processing of a sample in any processing bay without requiring batch accumulation of samples, and(iii) dynamically schedule workflow steps across the plurality of processing bays;(d) a single-use closed consumable cartridge receivable within each processing bay, the cartridge containing pre-measured reagents for nucleic acid extraction, library preparation, CRISPR-mediated depletion, and magnetic bead-based normalization;(e) wherein the system is configured to generate a sequencing-ready nucleic acid library from each biological sample without requiring intermediate fluorometric quantification of nucleic acid concentration; and(f) an output interface configured to provide a normalized sequencing library directly loadable onto a high-throughput sequencing instrument.

2. The system of claim 1 , wherein the plurality of processing bays comprises four independent processing bays.

3. The system of claim 1, wherein the CRISPR-mediated depletion is performed after initial library amplification such that abundant nucleic acid sequences function as carrier molecules prior to depletion.

4. The system of claim 3, wherein CRISPR cleavage removes sequencing adapters from targeted fragments, preventing amplification of cleaved fragments.

5. The system of claim 1, wherein library preparation comprises adapter ligation chemistry capable of accommodating nucleic acid input quantities from about 10 pg to about 1 pg without adjustment based on measured concentration.

6. The system of claim 1, wherein magnetic bead-based normalization comprises contacting the amplified library with beads having a finite binding capacity sufficient to produce standardized output concentration without optical quantification.

7. The system of claim 1, wherein the cartridge comprises lyophilized CRISPR ribonucleoprotein complexes stable at ambient temperature for at least six months.

8. The system of claim 1, wherein the cartridge comprises machine-readable lot information and the controller is configured to prevent processing upon detection of an expired or incompatible cartridge.

9. The system of claim 1 , wherein the controller comprises a dynamic scheduling engine configured to assign priority to selected samples based on stored priority parameters.

10. The system of claim 9, wherein the priority parameters comprise one or a combination of:clinical urgency,time since sample receipt,pathogen risk classification, anduser-defined priority.

11. A method for automated preparation of a sequencing-ready nucleic acid library from a biological sample, the method performed within an integrated multi-bay automated instrument, comprising:(a) extracting nucleic acids from the biological sample using magnetic bead-based purification;(b) performing quantification-independent adapter ligation to generate a sequencing library without measuring nucleic acid concentration;(c) performing limited-cycle amplification of the library;(d) contacting the amplified library with CRISPR-associated ribonucleoprotein complexes configured to cleave predetermined abundant nucleic acid sequences;(e) removing cleaved fragments using magnetic bead purification;(f) performing post-depletion amplification to selectively enrich uncleaved library molecules; and(g) normalizing the amplified library using magnetic beads having a finite binding capacity sufficient to produce a standardized sequencing-ready output without fluorometric quantification,wherein the method is performed on individual samples asynchronously without batch processing.

12. The method of claim 11, wherein the CRISPR-associated ribonucleoprotein complexes comprise guide RNAs targeting ribosomal RNAand mitochondrial DNA sequences.

13. The method of claim 11, wherein CRISPR-mediated depletion increases representation of low-abundance target sequences relative to host-derived sequences.

14. The method of claim 11, wherein the method produces a normalized library directly loadable onto a next-generation sequencing flow cell without manual dilution calculations.

15. The method of claim 11 , wherein each sample is initiated upon arrival into an available processing bay without waiting for additional samples.

16. A single-use cartridge for automated nucleic acid library preparation, the cartridge comprising:(a) a plurality of sealed reagent chambers containing reagents for nucleic acid extraction, adapter ligation, amplification, CRISPR-mediated depletion, and magnetic beadbased normalization;(b) one or more lyophilized CRISPR ribonucleoprotein complexes targeting predetermined abundant nucleic acid sequences;(c) magnetic beads configured for both purification and saturating normalization; (d) an integrated sample input vessel;(e) an integrated library output vessel; and(f) a machine-readable identifier encoding lot-specific and protocol-specific information,wherein the cartridge is configurable for insertion into a processing bay of an automated multi-bay instrument and enables completion of library preparation without external reagent addition or intermediate nucleic acid quantification.

17. The cartridge of claim 16, wherein the reagent chambers comprise foil seals having differential peel strengths configured for sequential robotic access.

18. The cartridge of claim 16, wherein incompatible enzymatic reagents are co-stored in lyophilized form under differential stabilization conditions.

19. The system of claim 1, wherein the controller is configured to reprioritize workflow steps in real time in response to arrival of an urgent sample, without interrupting processing of previously initiated samples.

20. The system of claim 19, wherein reprioritization reduces time-to-sequencing initiation for the urgent sample relative to non-prioritized samples.*****

Images