Method and system comprising a cartridge for sequencing a target polynucleotide

Abstract

A method for identifying target polynucleotide sequences from a sample using a cartridge-based system and for forming an array of amplified nucleic acid molecules on a solid support is described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 224,116, filed July 21, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to sample processing and nucleic acid sequencing. [Background technology]

[0003] Nucleic acid sequencing technology has advanced greatly in recent years. An important contribution to this progress was the advent of next-generation sequencing, or NGS (see, for example, The sequence of sequencers: The history of sequencing DNA (2016) Heather and Chain; Genomics 107:1-8). As an example, as part of the Human Genome Project, the first human genome was sequenced in about 13 years at a cost of at least hundreds of millions of US dollars. Today, under certain circumstances, it is possible to sequence a human genome in a few days at a cost of 1000 US dollars (see https: / / www.genome.gov / about-genomics / fact-sheets / Sequencing-Human-Genome-cost). Nucleic acid sequencing is now widely used in many fields and its value is well understood.

[0004] However, for many applications, the sequencing workflow (the processes required to prepare target polynucleotides contained in a sample for sequencing, perform the sequencing, and analyze the resulting data) is laborious, time-consuming, complex, and often expensive. Many of the steps are still performed manually and require highly skilled personnel. Even when certain processes within the workflow are automated, multiple devices and auxiliary components are required, and skilled human intervention is required at various points to perform the entire workflow. Also, the time from sample to result is several hours to days or more. Furthermore, the maximum allowable amount of sample input is low, which represents a further current limitation. Thus, the capacity and value of sequencing, including next-generation sequencing, may be significantly reduced in practical use. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] The sequence of sequencers:The history of sequencing DNA(2016)Heather and Chain;Genomics 107:1-8 [Non-Patent Document 2] https: / / www.genome.gov / about-genomics / fact-sheets / Sequencing-Human-Genome-cost Summary of the Invention

[0006] The systems and methods of the present invention recognize an existing need for a sequencing workflow that is fully automated (sample to report), requires no user intervention once a run is initiated, is rapid (sample to actionable results in hours), sensitive, accurate, cost-effective, and suitable for use at the point of need. As disclosed herein, the previously isolated steps for sample preparation, library preparation, and sequencing can be performed automatically on a single cartridge in a single benchtop device.

[0007] Disclosed are embodiments useful for rapid analysis of target polynucleotides, including determining their nucleotide sequence from a wide range of input sample types and quantities, using a fully automated system including cartridges, devices, and operation and analysis software that does not require human intervention after a run has been initiated (e.g., a sample reports). Further embodiments disclose the use of semiconductor chips for detection, and methods for generating nucleic acid clusters on surfaces, including surfaces of semiconductor chips. Embodiments are useful for a wide variety of applications, including clinical diagnostics, epidemiology and surveillance, oncology, genetic and genomic analysis including metagenomics, hospital infection control, basic and applied research, food testing, forensics, environmental testing, biothreat detection, animal health, agricultural testing, and the like.

[0008] In various embodiments, any of the reagents (e.g., primers, enzymes, and buffers) and / or systems described herein can be combined and provided in one or more kits. Thus, kits are contemplated that include any of the systems described herein, their components (e.g., cartridges or devices), and / or reagents and that are operable to perform any of the methods described herein.

[0009] Aspects of the invention can include a method for analyzing a target in a sample, including steps such as introducing the sample into a cartridge and introducing the cartridge into a device. The device can then be operable to manipulate the sample in the cartridge to automatically isolate target nucleic acids from the sample, amplify the isolated target nucleic acids, and sequence the amplified target nucleic acids using next generation sequencing. The sample can remain in the cartridge throughout the isolating, amplifying, and sequencing steps. Furthermore, the entire process may be performed within a single device without user intervention. In certain embodiments, the isolating, amplifying, and sequencing steps may be performed within 8 hours after introducing the sample into the cartridge.

[0010] The systems and methods of the invention can provide excellent sensitivity in isolating, amplified and sequenced target nucleic acids, including fungal nucleic acids present in the sample at levels as low as 3 copies, in certain embodiments. In certain embodiments, the target nucleic acids can include bacterial nucleic acids present in the sample at levels as low as 3 copies or viral nucleic acids present in the sample at levels as low as a single copy.

[0011] Another advantage of the systems and methods described herein is that the cartridges have a relatively small footprint, allowing for a smaller footprint for the analytical device device occupying less expensive laboratory space while providing for more manageable shipping, storage, and handling. As described herein, the external volume of a cartridge or device may refer to the total three-dimensional volume (e.g., the height x length x width of a rectangular prism-shaped cartridge) that the cartridge or device occupies. In certain embodiments, the cartridge may have an external volume of about 3 liters or less. The cartridge may have an external volume of about 2.5 liters or less. In some embodiments, the cartridge may have an external volume of about 2.1 liters or less. Similarly, in addition to a small relative external volume, the cartridges of the present invention may have a relatively small longest linear dimension (e.g., the longest height, length, or width of a rectangular prism-shaped cartridge) to allow for ease of handling and a compact device. In certain embodiments, the cartridge may have a longest linear dimension of about 200 mm or less. In some embodiments, the cartridge may have a longest linear dimension of about 160 mm or less.

[0012] The systems and methods of the present invention can accommodate a variety of assays and a variety of sample types and amounts, for example, through changes in reagents and / or the timing, sequence and duration of various steps in the workflow. In various embodiments, a single cartridge can perform multiple different assays and a single device can accept different cartridges as needed to perform different assays. The systems and methods of the present invention contemplate that different cartridge internals should be required to perform different assays (e.g., different sample preparation units for processing biological samples versus environmental samples) where the overall size and shape of the different cartridges are substantially the same to allow interoperability with a single device. In various embodiments, the sample can be selected from the group consisting of biological samples, clinical samples, environmental samples, and food samples. A particular advantage of the present invention is the ability to receive raw or minimally processed samples and automatically progress through the required workflow steps of the selected assay to provide sequencing results without user intervention and without removing the sample from a single cartridge or a single device. Thus, in certain embodiments, the sample can be an unprocessed biological sample obtained from a subject and prior to introduction into the cartridge.

[0013] Sample processing, which may include isolating the target nucleic acid in the sample, may include digesting proteins in the sample. Proteins may be digested using proteinase K. Isolating may include lysing organisms in the sample (e.g., bacteria, fungi, or host cells such as human cells) to release the target nucleic acid. Lysis may include mechanical lysis. In various embodiments, mechanical lysis may include flowing the sample through a lysis chamber in the cartridge and rotating a paddle in the lysis chamber. Mechanical lysis may further include adding zirconium beads to the lysis chamber prior to rotating the paddle in the lysis chamber. Isolating may include denaturing the target nucleic acid. Denaturation may include heat denaturation. Isolating may include capturing the target nucleic acid by annealing a target capture oligonucleotide to the target nucleic acid to form a complex, binding the complex on a solid support, and removing unbound material from the solid support. In certain embodiments, removing unbound material may include washing the solid support-bound complex with a wash reagent.

[0014] In some embodiments, an amplification step may be performed on the target nucleic acid bound to the solid support. The amplification step may be performed in the sample processing unit of the cartridge, instead of or in addition to an amplification step (e.g., incorporating any required barcodes, tags, or adapters) performed as part of library preparation in the library preparation unit of the cartridge. In certain embodiments, the method may include eluting the target nucleic acid from the washed solid support to prepare an isolated target nucleic acid. The amplification step for library preparation may be performed directly on the eluted target nucleic acid without any intervening steps.

[0015] In certain embodiments, the isolating step can automatically isolate the target nucleic acid from a sample having a volume of about 1 mL to about 25 mL. The isolating step may include only one purification step. The isolated nucleic acid can be amplified without quantification.

[0016] In some embodiments, the amplifying step may include performing a first amplification of the isolated target nucleic acid using a first primer set to produce a first amplification product, diluting and aliquoting the first amplification product into a plurality of aliquots, performing a second amplification of the target nucleic acid in the plurality of aliquots using a second primer set to produce a plurality of second amplification products, and pooling the second amplification products. In various embodiments, one or more primers in the first primer set may be identical to one or more primers in the second primer set. The amplifying step may further include purifying the pooled second amplification products to produce the amplified target nucleic acid. One or more of the first and second amplifications may include PCR amplification. The plurality of aliquots may include at least 10 separate aliquots. The first PCR amplification and the second PCR amplification may be performed without quantification. In some embodiments, the second primer set may be nested with respect to the first primer set. The amplifying step may include performing copy control on the amplified target nucleic acid prior to the sequencing step. The amplifying step may include only one purification step.

[0017] In certain embodiments, the amplified target nucleic acid may be sequenced without quantification. The step of sequencing may include immobilizing the amplified target nucleic acid on a semiconductor surface in a cartridge that includes an ion-sensitive field effect transistor (ISFET) sensor. All products of the step of amplifying may flow over the semiconductor surface without an intervening step. In some embodiments, the amplified target nucleic acid may be immobilized by a capture oligomer attached above the ISFET sensor, the capture oligomer hybridizing to a portion of the target nucleic acid. The surface may include an array of ISFET sensors having wells disposed thereon. At least one of the wells may be disposed above a plurality of ISFET sensors in the array of ISFET sensors. One or more of the wells may include a surface-bound forward primer that hybridizes to a portion of the target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, and the step of sequencing may include paired-end sequencing. In certain embodiments, one or more of the wells, or a gap between one or more of the wells, may include a plurality of binding-inactive oligomers that do not hybridize to the target nucleic acid. The amplified target nucleic acid may be immobilized by a universal capture oligomer attached above the ISFET sensor, which hybridizes to the universal binding site. In some embodiments, the amplifying step may include amplifying the isolated target nucleic acid using a primer that includes a universal binding site. In certain embodiments, the amplifying step may include ligating an adaptor to the isolated target nucleic acid, which adaptor includes a universal binding site. The sequencing step may include clonal amplification of the immobilized target nucleic acid, which may include recombinase polymerase amplification, rolling circle amplification, bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification.

[0018] In certain aspects, the system of the present invention may include a sample cartridge including a sample input, a sample preparation unit operable to receive a sample from the sample input and isolate a target nucleic acid from the sample, a library preparation unit operable to receive an isolated target nucleic acid from the sample preparation unit and amplify the isolated target nucleic acid, and a sequencing unit operable to receive an amplified target nucleic acid from the library preparation unit and sequence the amplified target nucleic acid. The system may further include an apparatus including a cartridge interface including physical and electronic connections operable to drive movement of the sample and reagents within the cartridge and communicate with the sequencing unit. One or more reagents required for isolating the target nucleic acid, amplifying the isolated target nucleic acid, and sequencing the amplified nucleic acid may be dry reagents, and the apparatus is operable to reconstitute the one or more reagents.

[0019] The system of the present invention may further include one or more reagent cartridges that contain one or more reagents necessary for isolating the target nucleic acid, amplifying the isolated target nucleic acid, and sequencing the amplified nucleic acid. The device may be operable to transfer reagents from one or more reagent cartridges to the sample cartridge. The use of reagent cartridges may avoid contamination and logistical issues from maintaining on-board reagent wells in the device. Additionally, the reagent cartridges may be sealed after manufacture, thereby avoiding the possibility of contamination or user error in manually refilling the reagent wells. Reagent cartridges may be assay specific and may contain reagents required for one complete assay or multiple assays. A user may insert the appropriate reagent cartridge along with the sample cartridge at the beginning of an assay run. In certain embodiments, the device may monitor the levels of reagents in the reagent cartridges, particularly when a single reagent cartridge contains reagent amounts for multiple assays, and notify the user when the reagent levels are low or insufficient to run a desired assay. The sample cartridge and / or one or more reagent cartridges may be equipped with a sealed pneumatic interface (SPI) port, and the device may be operable to transfer one or more reagents from one or more reagent cartridges to the sample cartridge via the SPI port using one or more pipettes.

[0020] In certain embodiments, the device may include a three degree of freedom pipette gantry operable to transfer one or more reagents. The system may be operable to isolate, amplify and sequence target fungal nucleic acids in a sample at levels as low as three copies, target bacterial nucleic acids present in a sample at levels as low as three copies, and / or target viral nucleic acids present in a sample at levels as low as a single copy.

[0021] The device of the present invention may have a total external volume of about 150 liters or less. In some embodiments, the external volume of the device may be about 135 liters or less. The device may have a longest linear dimension of about 700 mm or less. In certain embodiments, the device may have a longest linear dimension of about 650 mm or less. The sample cartridge may be operable to receive biological, clinical, environmental, and food samples, including unprocessed samples. Isolating the target nucleic acid may include, for example, the device digesting proteins in the sample by exposing the sample to proteinase K in the sample preparation unit. The sample preparation unit may be operable to lyse the organisms to release the target nucleic acid. To that end, the sample preparation unit may include a lysis chamber including a rotating paddle, and the device is operable to interface with the sample cartridge to flow the sample into the lysis chamber and rotate the rotating paddle to mechanically lyse the organisms in the sample. The lysis chamber may include zirconium beads to aid in lysis.

[0022] The device may be operable to provide thermal energy to the sample preparation unit to denature the nucleic acid therein. The device may further be operable to perform target capture by exposing a sample to the target capture oligonucleotide and the solid support in the sample preparation unit to anneal the target capture oligonucleotide to the target nucleic acid to form a complex and bind the complex to the solid support. The device may then introduce a wash buffer to the solid support-bound complex and separate the solid support-bound complex from the unbound sample. The device may then transfer the separated solid support-bound complex to a library preparation unit and amplify the solid support-bound target nucleic acid. The device of the present invention may be operable to introduce an elution buffer to the separated solid support-bound complex to elute the target nucleic acid from the solid support and transfer the eluted target nucleic acid to the library preparation unit for amplification. The device may further be operable to introduce an amplification reagent to the solid support-bound complex and amplify the target nucleic acid in the sample preparation unit.

[0023] In various embodiments, the cartridge and device may be operable to automatically accommodate a sample volume received by the sample input ranging from about 1 mL to about 25 mL. The device may be operable to interface with the library preparation unit of the sample cartridge to introduce necessary reagents and provide thermal energy to perform a first amplification of the isolated target nucleic acid using a first primer set to produce a first amplification product, dilute and aliquot the first amplification product into a plurality of aliquots, perform a second amplification of the target nucleic acid in the plurality of aliquots using a second primer set to produce a plurality of second amplification products, and pool the second amplification products. One or more primers in the first primer set may be identical to one or more primers in the second primer set. The device may be further operable to purify the pooled second amplification products to produce an amplified target nucleic acid.

[0024] The system of the present invention may further be operable to perform copy control on one or more of the isolated and amplified target nucleic acids to control the number of output copies transferred to the library preparation unit or the sequencing unit, respectively. The sequencing unit may include a semiconductor surface including an array of ion-sensitive field effect transistor (ISFET) sensors, each having a well disposed thereover, and the device is operable to immobilize the amplified target nucleic acids above the array of ISFET sensors, and the array of ISFET sensors electronically connects with the device via a cartridge interface electronic connection when a sample cartridge is disposed therein. The device may be operable to channel all output from the library preparation unit to the wells on the semiconductor surface.

[0025] The cartridge may include a capture oligomer attached above the array of ISFET sensors, the capture oligomer configured to hybridize to a portion of the target nucleic acid. At least one of the wells may be disposed above a plurality of ISFET sensors in the array of ISFET sensors. In some embodiments, one or more of the wells may include a surface-bound forward primer that hybridizes to a portion of the target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, the device being operable to perform paired-end sequencing. One or more of the wells and the gaps between the wells may include a plurality of binding-inactive oligomers that do not hybridize to the target nucleic acid. The cartridge may include a universal capture oligomer attached above the array of ISFET sensors, the universal capture oligomer configured to hybridize to a universal binding site.

[0026] The device may be operable to interface with a library preparation unit to amplify isolated target nucleic acids using primers that include universal binding sites. In some embodiments, the device may be operable to ligate adaptors to isolated target nucleic acids in the sample preparation unit or the library preparation unit, the adaptors including universal binding sites. The device may be operable to interface with a sequencing unit to perform clonal amplification of immobilized target nucleic acids, the clonal amplification may include recombinase polymerase amplification, rolling circle amplification, bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification. The physical and electrical connections between the device and the sample and / or reagent cartridges may include a pneumatic system for driving fluid movement between and / or within the cartridges. The cartridge interface may further include physical and electronic connections through which the device operatively communicates with one or more of the sample preparation unit and the library preparation unit. [Brief description of the drawings]

[0027] [Figure 1A]1A-1C are diagrams illustrating exemplary capture oligomers according to the present disclosure, including a capture sequence, a blocking moiety, a complement of the capture sequence (C'), an additional sequence (e.g., a third or fourth additional sequence), and a target hybridizing sequence, among other molecules. In FIG. 1A, the capture oligomer is annealed to a target polynucleotide (target), with the 3' end of the target polynucleotide annealing with the 5' end of the target hybridizing sequence. The capture sequence is annealed to C'. [Figure 1B] FIG. 1B illustrates an exemplary capture oligomer according to the present disclosure, which includes, among other molecules, a capture sequence, a blocking moiety, a complement of the capture sequence (C'), an additional sequence (e.g., a third or fourth additional sequence), and a target hybridizing sequence. In FIG. 1B, the 3' end of the target is extended to the blocking moiety, and the resulting target extension is annealed to the additional sequence and C', but the capture sequence is displaced and single stranded. The 3' end of the capture oligomer also extends along the target polynucleotide. [Figure 1C] FIG. 1C shows an exemplary capture oligomer according to the present disclosure, which includes a capture sequence, a blocking moiety, a complement of the capture sequence (C'), an additional sequence (e.g., a third or fourth additional sequence), and a target hybridizing sequence, along with other molecules. In FIG. 1C, the complex of FIG. 1B is annealed to a secondary capture reagent that includes the complement of the capture sequence and a binding partner or solid substrate. Meanwhile, excess capture oligomers, whose capture sequences remain annealed to the complement of the capture sequence, do not interact with the secondary capture reagent. [Figure 2A] FIG. 1C illustrates an embodiment of the present disclosure in which a complex such as that of FIG. 1B is annealed to a secondary capture reagent that contains the complement of the capture sequence and is associated with a solid substrate (in this case, a streptavidin-coated magnetic bead). The solid substrate may be part of the secondary capture reagent or may be associated with the secondary capture reagent through an interaction with a binding partner of the secondary capture reagent (e.g., biotin). [Figure 2B] FIG. 2B shows an embodiment of the present disclosure in which the extended capture oligomer-target complex from FIG. 2A has been eluted from the secondary capture reagent. [Diagram 3]FIG. 1 shows an embodiment of a capture oligomer according to the present disclosure comprising a stabilizing (clamp) sequence as a first additional sequence, a capture sequence, a linker, an internal extension blocker, a complement of the capture sequence, a stabilizing (clamp) sequence as a third additional sequence, a fourth additional sequence, and a target hybridizing sequence. [Figure 4A]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A target molecule is provided in which a first strand comprises a sequence Sf at its 5' end and Sr' at its 3' end, and a second strand comprises a sequence Sf' at its 3' end and Sr at its 5' end. Here and throughout, a sequence designation with ' indicates complementarity to a sequence having the designation without '. The target molecule can be, for example, an amplicon from a reaction previously performed using primers with sequences Sf and Sr. A first extension cycle (cycle 1) is performed in which a capture oligomer according to the present disclosure comprising a capture sequence C, an internal extension blocker (black circle), a complement of the capture sequence C', a fourth additional sequence A4, and a target hybridizing sequence THS complementary to Sr' anneals to the first target strand 1 (+). A deamplification oligomer comprising an additional sequence A2 and a target hybridizing sequence Sf* complementary to at least Sf' anneals to the second target strand 1 (-). Sf* may contain affinity enhancing modifications and / or additional complementary nucleotides for the second target strand to enhance its affinity for the target and, if present, promote binding competition with a primer having sequence Sf from a previous reaction. Extension of the capture oligomer and the reverse amplification oligomer produces products 2(-) and 2(+), respectively, with the first strand extending along the capture oligomer to produce product 1(+)e and the second strand extending along the reverse amplification oligomer to produce product 1(-)e. The capture sequence in the extended capture oligomer 2(-) is essentially replaced as described for FIG. 1B. A second reaction cycle (cycle 2) is performed, in which 2(-) anneals to the reverse amplification oligomer and results in extension to produce products 2(-)e and 3.1(+). Meanwhile, 1(+)e anneals to the capture oligomer and extension of the capture oligomer produces product 3.1(-). Further examples 1(-)e and 2(+) and 2(-)e and 3.1(+) are also generated from appropriate hybridization and extension events. This reaction scheme illustrates the inclusion of additional sequence at each end of the target while rendering the target captureable, for example, by incorporation of C in a form available for binding to a second capture reagent. [Figure 4B]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A target molecule is provided in which the first strand comprises a sequence Sf at its 5' end and Sr' and A4' at its 3' end, and the second strand comprises a sequence Sf' at its 3' end and Sr and A4' at its 5' end. The target molecule can be, for example, an amplicon from a reaction previously performed using a primer with sequences Sf and A4-Sr, where A4 is, for example, an additional sequence not originally present in the template. A first extension cycle (cycle 1) is performed in which a capture oligomer according to the present disclosure anneals to the first target strand (not shown), comprising a capture sequence C, an internal extension blocker (black circle), a complement of the capture sequence C', a fourth additional sequence A4, and a target hybridizing sequence THS complementary to A4'. A deamplification oligomer anneals to the second target strand (not shown), comprising an additional sequence A2 and a target hybridizing sequence Sf* complementary to at least Sf'. Sf* may contain affinity enhancing modifications and / or additional complementary nucleotides to the second target strand to increase its affinity to the target and promote competition for binding with a primer with sequence Sf from a previous reaction that exists. Extension of these complexes produces extended capture oligomer 2(-) and extended first target strand 1(+)e, as well as extended second target strand 1(-)e and extended de-amplification oligomer 2(+). The capture sequence in the extended capture oligomer 2(-) is essentially replaced as described for FIG. 1B. A second reaction cycle (cycle 2) is performed, in which 2(-) anneals to the de-amplification oligomer, resulting in extension to produce products 2(-)e and 3.1(+). Meanwhile, 1(+)e anneals to the capture oligomer, and extension of the capture oligomer produces product 3.1(-). Further examples of 1(-)e and 2(+) and 2(-)e and 3.1(+) are also generated from appropriate hybridization and extension events. This reaction scheme illustrates the inclusion of additional sequences at the end of the target distal from the capture oligomer binding site, as well as the use of a capture oligomer that can have a universal THS (i.e., binds (e.g., via amplification or ligation) an additional sequence A4' that can bind to the target in a previous step, thereby allowing the target to be captured by incorporating C (in a form available for binding). [Diagram 5] 1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. Among other things, a capture oligomer is provided that includes a 3' blocking moiety and a target hybridizing sequence (THS) that binds to sequence A1' in the target strand. A1' can be an additional sequence that was bound to the target in a previous step (e.g., via amplification or ligation). The capture oligomer further includes a sequence x that includes the complement of the capture sequence of the capture oligomer, and can also include a third or fourth additional sequence between the complement of the capture sequence and the THS. The target strand can be extended along the capture oligomer to displace the capture sequence from the complement of the capture sequence. The capture oligomer can be provided in a limiting amount (e.g., 1012 copies) relative to the target (e.g., 1014 copies). A primer is also provided in excess over the target (e.g., 1015 copies) that includes sequences A2 and Sf. This extension of the primer results in a strand that includes A2 at its 5' end and A1' at its 3' end. The target strand is also extended along the primer to include the sequence A2'. When a second extension cycle is performed (downward arrow), a mixture of products is formed including those discussed above and complexes of the target strand with the capture oligomer where the target strand includes A2 at its 5' end and A1' near its 3' end. This reaction scheme illustrates the production of single-stranded capturable products, including those where the target strand includes additional sequence (if a second extension cycle is performed). [Figure 6]This diagram shows (above the dashed line) how hybridization of a capture oligomer with an extendable 3' end to another capture oligomer can generate a dimer in which the capture sequence is displaced from C' upon extension. This dimer is now captureable and may interfere with downstream processes such as competing with capture of the desired target by occupying a secondary capture reagent (not shown), interfering with subsequent analysis (e.g., the dimer becomes part of the sequencing library, thereby reducing the output and quality of the subsequent sequencing run). Sx' is the complement of a portion of the target hybridizing sequence, with other elements as in the previous diagram. Below the dashed line is shown a capture oligomer with a blocking moiety at its 3' end (circled x), which prevents the formation of a dimeric extension product so that the dimer does not undergo displacement of C. [Figure 7A] FIG. 1 illustrates an embodiment in which a capture oligomer is used that includes a capture sequence, various intermediate elements (indicated with "..."), a reversible extension blocker (filled circle), and a target hybridizing sequence (THS). Prior to unblocking of the reversible extension blocker, the capture sequence and various intermediate elements (if present) are not templates for extension (e.g., target strand or amplification oligomer). This can promote more efficient and more specific extension or amplification by avoiding incorporation of additional sequences complementary to the capture sequence and various intermediate elements (if present) in the product (e.g., in any mispriming products that may be formed) throughout the extension or amplification process until the reversible extension blocker is unblocked, and following unblocking, the capture sequence and various intermediate elements (if present) can be incorporated. [Figure 7B]FIG. 1 shows an embodiment in which a first amplification oligomer is used that includes, from 3' to 5', a target hybridization sequence Sr, a reversible extension blocker (black square), an additional sequence A1, and any additional elements, such as an optional capture sequence (indicated by "..."). A second amplification oligomer is optionally used that includes, from 3' to 5', a target hybridization sequence Sf, a reversible extension blocker (open square; this may be the same or different from the reversible extension blocker in the first amplification oligomer), an additional sequence A2, and any additional elements (indicated by "..."; these may be the same or different from those of the first amplification oligomer) (as shown in the figure). Before unblocking the reversible extension blocker or the reversible extension blocker, the additional sequence and optional additional elements (if present) are not templates for extension (e.g., target strand or amplification oligomer). This can promote more efficient and more specific extension or amplification by avoiding the incorporation of additional sequences in the products (e.g., in any mispriming products that may be formed) and sequences complementary to various other elements (if present) throughout the initial extension or amplification process. One or more reversible extension blockers are not blocked (if two are present, unblocking can occur simultaneously or separately), and the additional sequences and any other elements present can be incorporated at later stages in the process, such as in later extension rounds. [Figure 8A]4A-4C show exemplary molecules and exemplary reaction schemes according to the present disclosure. Initial target strands 1(+) and 1(-) are as in FIG. 4A. Below the straight vertical arrow, a capture oligomer is provided that includes a target hybridizing sequence THS and additional elements A4, C', an internal extension blocker, and C as described for the oligomer in FIG. 4A, where THS binds to an internal site on the target strand and undergoes extension to produce product 2N(-). A displacer oligomer is provided that includes Sr, the extension of which displaces 2N(-) from 1(+) to produce 2(-). A de-amplification oligomer as in FIG. 4A is provided, the extension of which along 1(-) produces 2.1(+), and the extension of 1(-) along the de-amplification oligomer produces 1(-)e. As 2N(-) is displaced (left curved arrow), the deamplification oligomer anneals to 2N(-) and is extended, respectively, to produce products 2N(-)e and 2.2(+), which now contain A2' and C has been displaced from C'. This reaction scheme illustrates the use of a displacer oligomer to facilitate the generation of capturable products in only one cycle that contain additional sequences (e.g., adapters) on both ends of the target sequence. Additionally, this reaction scheme illustrates an embodiment in which the capture oligomer does not bind to a site that contains the 3' end of the target strand. [Figure 8B]8A and 8B show further exemplary molecules and further exemplary reaction schemes according to the present disclosure. The reaction scheme is substantially similar to that shown in FIG. 4A, except for 1) and 2) below. 1) The initial target strands 1(+) and 1(-) contain additional sequences including the target hybridization sequence THS, an optional spacer sequence S, and a displacer oligomer binding site D. These additional sequences are any user-defined sequences and can be incorporated into the target by an amplification reaction using, for example, an amplification oligomer containing Sr and a sequence tag containing THS, S, and D, and an amplification oligomer containing Sf. 2) The THS of the capture oligomer binds to the user-defined THS site. Otherwise, the reaction proceeds as shown in FIG. 8A, and the resulting product is shown in FIG. 8B. The optional spacer can be useful to improve the extension of the displacer oligomer and the subsequent displacement of the capture oligomer. Similar to the scheme shown in Figure 4A, this reaction scheme illustrates the use of a displacer oligomer to facilitate the generation (e.g., in only one cycle) of a capturable product that contains additional sequences (e.g., adapters) on both ends of the target sequence. Additionally, this scheme illustrates the use of an additional user-defined sequence that can serve as a binding site for both the capture and displacer oligomers. This design can generalize this approach, allowing for a simpler and much more cost-effective means of designing capture and displacement oligomers for use with different targets, including multiplex formats. [Figure 9]FIG. 1 illustrates the general principle of how a blocker oligomer can prevent hybridization between an additional sequence in an oligomer and its complement in the extension product. An amplification reaction is performed with a forward primer containing sequence f that hybridizes to target strand T(-) and a reverse primer containing sequence A (an additional sequence not present in the target) and sequence r that hybridizes to target strand T(+). Extension produces products 1(-) and 1(+). A blocker oligomer is provided that contains sequence A and a 3' blocking portion. In cycle 2, the forward primer extends along 1(-) to produce 2(+) and the reverse primer extends along 1(+) to produce 2(-). From cycle 3 onwards, the blocker oligomer anneals to 2(+), which means that hybridization of r to r' is required for the reverse primer to prime extension along 2(+). This can be beneficial if a mispriming event occurs that generates a small amount of by-product that has an incomplete complement of r but is extended to include A'. Without the blocker oligomer, the binding of the de-amplification oligomer to the misprimed by-product becomes more favorable due to the interaction between A and A' of the de-amplification oligomer, resulting in the amplification of more by-products than if a blocker oligomer was provided. (Meanwhile, the forward primer anneals to 2(-) and undergoes extension.) [Figure 10A]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. (i) A combination of a capture oligomer comprising a first and second portion of a capture sequence (C1 and C2), an internal extension blocker (black circle), a first and second portion of a spacer sequence (S1 and S2), and a target hybridizing sequence (THS) that binds to a site in a target strand including its 3' end, and (ii) a complementary oligomer comprising S1' and C2' is provided. Upon hybridization of the capture oligomer to the target and extension along the capture oligomer to the internal extension blocker of the target, incorporation of S' into the target strand displaces the complementary oligomer. The capture oligomer also extends along the target (note that in other embodiments described herein, the capture oligomer may be blocked and no such extension occurs). A secondary capture reagent is provided that comprises a binding partner or solid support (circled B) linked by a linker (zigzag line) to the complement C' of the capture sequence. The secondary capture reagent occupies a sufficient amount of the capture sequence, C2, to substantially prevent annealing of the secondary capture reagent to the capture oligomer, such that the secondary capture reagent captures oligomers bound to the extended target but anneals such that it does not capture oligomers bound to complementary oligomers. [Figure 10B]FIG. 10B illustrates an embodiment in which the oligomer combination is useful for capturing a target polynucleotide from a composition that optionally contains a fixed amount (eg, a limited amount or an amount not greater than a predetermined amount). This combination includes a capture oligomer comprising, from 5' to 3', a first portion of capture sequence C1, a second portion of capture sequence C2, an optional spacer sequence S, a second portion of target hybridizing sequence THS2, a first portion target hybridizing sequence THS1, and an optional blocking moiety (circled X); a separate complementary oligo comprising, from 5' to 3', THS2', S' (optional; may or may not be used if S is present in the capture oligomer), and C2' (where the complement of the element is indicated by "") and an optional blocking moiety (circled X) at the 3' end; and a secondary capture reagent comprising, from 5' to 3', C2', C1' (C1' or C2' may or may not be complementary to the entire length of C1 and C2), and the complement of the capture sequence including a binding partner (exemplified in this figure by a biotin molecule represented as a circled B). In the absence of target polynucleotide, the complementary oligomer binds to the capture oligomer, blocking access to the complete capture sequence to a sufficient extent to block binding of the complement of the capture sequence in the secondary capture reagent (see the complex of complementary oligomers to capture oligomer at the top of the figure). In the presence of target, the THS1 region of the capture oligomer binds to the target, followed by the energetically favorable THS2 region, which displaces the THS2' region of the separate complementary oligo from the capture oligomer. When this occurs, the C2' region of the separate complement is no longer stable enough to bind to the capture oligomer and therefore does not bind, thus leaving the complete capture sequence available for binding as shown under the first arrow. The complement of the capture sequence in the secondary capture reagent then binds to the capture sequence of the capture oligomer, as shown under the second arrow. This complex can then be isolated from the mixture, for example, by streptavidin-coated magnetic microspheres (described elsewhere in this disclosure) to capture and purify the target polynucleotide. Optionally, the capture oligomer may be present in combination in greater amount than the secondary capture reagent.Such oligomers and combinations are useful for capturing an amount (eg, a limited amount or an amount up to a predetermined amount) of a target polynucleotide from a composition. [Figure 11A] 11A-11C show exemplary molecules and exemplary reaction schemes according to the present disclosure. In FIG. 11A, a capture oligomer is provided that essentially contains the elements described for the capture oligomer of FIG. 4A, except that the THS binds to a site on the target strand that does not include the 3' end (which may be circular as shown or linear). A complementary oligomer is provided that contains (i) a target hybridizing sequence that anneals adjacent to the THS of the capture oligomer, and (ii) at least a partial complement of A4, which is insufficient to anneal to the capture oligomer in the absence of the target strand. [Figure 11B] 11A-11C show exemplary molecules and exemplary reaction schemes according to the present disclosure. In FIG. 11B, the complementary oligomer has undergone an extension, which displaces C and renders it available for capture using a secondary capture reagent (not shown). This scheme is useful for capturing circular molecules and / or represents an alternative approach for using a capture oligomer that does not bind to the 3' end of the target strand. [Figure 12]4A-4C show exemplary molecules and exemplary reaction schemes according to the present disclosure. A capture oligomer containing elements such as the capture oligomer of FIG. 4A with a second additional sequence A2, which may include a mixed nucleotide segment between C' and the internal extension blocker, anneals to a target strand at a site including its 3' end. The target strand also contains sequence A5 at its 5' end, which may be any sequence, a primer binding site used in a previous amplification reaction, or a sequence added during a previous step (e.g., amplification or ligation). Extension of the target strand along the capture oligomer adds sequences A4', C, and A2' to the 3' end of the target strand. The presence of A4 in the capture oligomer and A4' in the extended target strand is optional. The extended target strand can then be annealed to a splint oligomer containing sequences A5', A2, C', and A4, such that when the extended target strand is annealed to the splint oligomer, the target strand 5' and 3' ends are immediately adjacent. The extended target strand can then be circularized by ligation. The A2 and A2' sequences help ensure proper juxtaposition of the extended target strand 5' and 3' ends. This can be useful when C and C' are slippery repeat sequences (e.g., polyA and polyT or vice versa) that would otherwise inhibit the formation of a substrate for ligation. This scheme is useful for capturing and then circularizing target molecules, for example for use in rolling circle amplification procedures. [Figure 13]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A capture oligomer comprising a capture sequence C (comprising a first portion C1 and a second portion C2; not shown), an internal extension blocker (filled circle), a spacer sequence S (with a first portion S1 and a second portion S2; not shown), and a target hybridizing sequence THS is provided along with a de-amplification oligomer comprising sequence S2. THS and S2 serve to generate an amplified target (e.g., via PCR). A complementary oligomer comprising the complement of a first portion of spacer sequence S1' and the complement of a second portion of capture sequence C2' is added. C2' is insufficient to anneal to C of the amplified target if S' is annealed to S of the other strand of the amplified target. The complementary oligomer anneals to capture the oligomer not annealed to the amplified strand. A secondary capture reagent comprising C' and a binding partner or solid support (circled B) is added to capture the amplified target. The secondary capture reagent binds to the amplified target but not to capture oligomers that are not annealed to an amplified strand, and C is blocked to a sufficient extent by C2' of the complementary oligomer. [Figure 14] FIG. 13 shows the fold difference in output of methods using capture oligomers with and without clamp sequences. [Figure 15] Panel A - Solution-mediated surface-phase recombinase polymerase amplification (SM-RPA) The forward primer is in solution, while the reverse primer is immobilized on a surface. Upon initial hybridization of the template to the surface primer, the concerted action of the solution primer, recombinase, single-stranded DNA binding protein (ssDNA) and polymerase results in the synthesis of a complementary strand. Subsequent recombination and extension of the solution-phase primer results in the displacement of one strand into solution. These strands can then be locally recaptured. In Panel B - In comparison, in Bridge RPA or ExAmp (for example), both primers are co-immobilized on a surface, allowing only surface-phase amplification. [Figure 16]Figure 16 shows a model of cluster formation in bridge RPA (panel A) and SM-RPA on a dug-out chip (panels B and C). In bridge RPA (e.g., as in bridge PCR and ExAmp), the clusters are small (panel A). In SM-RPA, amplification using in-solution primers allows lateral growth of clusters, whose size is limited by spatial exclusion from neighboring clusters. Amplicons generated in solution are recaptured in close proximity to seeded template molecules, promoting the formation of clonal patches of amplicons larger than those generated in bridge amplification (panel B; clusters from three separate targets are indicated by *, ◆, and ‡). [Figure 17] FIG. 1 is a diagram of an exemplary surface (e.g., on-chip) template circularization method of the invention. First, during PCR2 in library preparation, a target template is fitted with partial first and second RCA primer binding sites at each end. During the copy control stage of the workflow, the second RCA primer binding site is extended to include a copy control adaptor and a second portion of the first RCA primer binding site. The template hybridizes to a surface-immobilized first RCA primer, which acts as a splint. This creates a structure with a gap between adjacent 5' and 3' ends. Addition of ligase fills the gap, creating a circle that can facilitate an RCA reaction in situ. [Figure 18]Figure 1 shows an embodiment of cluster formation using rolling circle amplification (RCA). (A) Hybridization of both ends of a linear RCA template to immobilized first primer molecules allows template circularization by ligase on a surface. (B) Once the gap is closed by ligase, an amplification mix containing a highly processive strand-displacing polymerase is added. (C) The polymerase extends the free 3' end of the first primer using the circle as a template. (D) The extended concatemeric first strand amplicon contains repeating units of second primer binding sites. These sites hybridize to immobilized second primer molecules and serve as templates for second strand synthesis. (E) As synthesis reaches the next unit, the extending second strands displace each other via the strand-displacing activity of the RCA polymerase. (F) The free second strands hybridize to the remaining free immobilized first primer molecules and promote further synthesis of the concatemeric first strands. The extending first strands then displace each other and the cycle continues. [Figure 19] FIG. 1 depicts a method for incorporating a primer-specific key sequence according to embodiment #1 herein. Truncation of the 3' end of Oligo 2 allows the remaining known sequence of Section 1A to be used as the key sequence. [Figure 20] FIG. 1 shows another variation of the method for incorporating a target specific key sequence according to embodiment #1 herein, in this case using both a 3' truncation and a 5' extension. Truncation of the 3' end of Oligo 4 allows the remaining known sequence of Section 3A to be used as the key sequence. [Figure 21]Depicts a method for incorporating a primer-specific key sequence according to embodiment #2 herein. In this example, a synthetic non-specific sequence at the 5' end (section 6B) of a surface-bound capture oligomer / primer (oligo 6) can be used as a known universal key sequence. Step (a): A sequencing template (oligo 5) provided in solution hybridizes to the complementary section 6A of oligo 6 (surface-bound). Step (b): After enzyme addition, polymerization can be performed from the 3' end of oligo 5 to section 6B of oligo 6. The resulting sequence can be used to set the signal base calling parameters. The 3' blocking moiety on oligo 6 prevents polymerization from this end. Step (c): The 3' end of oligo 6 is unblocked as described. Step (d): With the addition of additional polymerase (if necessary), the sequencing reaction can now proceed through the unknown region of oligo 5. [Figure 22A] FIG. 2 is a top view of a preferred embodiment of a sample preparation cartridge showing the reagent / assay configuration of chambers within the main cartridge body. [Figure 22B] FIG. 2 is a top view of a preferred embodiment of a sample preparation cartridge showing the reagent / assay configuration of chambers within the main cartridge body. [Figure 23A] 1 is a top view of a preferred embodiment of a sample preparation cartridge showing the main body and two additional functional fins. FIG. 2 shows an alternative microfluidic configuration of Mag Sep fins. "Mag Sep" stands for magnetic separation. [Figure 23B] 1 is a top view of a preferred embodiment of a sample preparation cartridge showing the main body and two additional functional fins. FIG. 2 shows an alternative microfluidic configuration of Mag Sep fins. "Mag Sep" stands for magnetic separation. [Figure 24A] 1 shows a 3D CAD rendering of one preferred embodiment of a sample preparation cartridge. [Figure 24B] 24B shows a photograph of an actual prototype cartridge constructed according to the design of FIG. 24A. [Figure 25A]1 is a top-down rendering of one preferred embodiment of a library prep cartridge showing the body and one additional functional fin. The reagent / assay configuration of the chambers within the main cartridge body is shown. "Mag Sep" stands for magnetic separation. [Figure 25B] 1 is a top-down rendering of one preferred embodiment of a library prep cartridge showing the body and one additional functional fin. The reagent / assay configuration of the chambers within the main cartridge body is shown. "Mag Sep" stands for magnetic separation. [Figure 26A] 1 shows a 3D CAD rendering of one preferred embodiment of a library preparation cartridge. [Figure 26B] 26B shows a photograph of an actual prototype cartridge constructed according to the design of FIG. 26A. [Figure 27A] FIG. 1 shows a top-down rendering of a preferred embodiment of a Cluster Generation / Sequencing (CA-Seq) cartridge, showing the reagent / assay configuration of chambers within the main cartridge body. [Figure 27B] FIG. 1 shows a top-down rendering of a preferred embodiment of a Cluster Generation / Sequencing (CA-Seq) cartridge, showing the reagent / assay configuration of chambers within the main cartridge body. [Figure 28A] 1 shows a 3D CAD rendering of one preferred embodiment of a Cluster Generation / Sequencing (CA-Seq) cartridge. [Figure 28B] 28B shows a photograph of an actual prototype cartridge constructed according to the design of FIG. 28A. [Figure 29] FIG. 1 shows an example of a flow cell assembly for use in the present invention. The valve (one possible configuration shown) can be bonded or welded to the fins. The film can be via solvent bonding, heat or laser welding, or pressure sensitive adhesive. The flow cell is heat staked through the PCB (printed circuit board). Not shown is the silicone gasket between the film and the chip. [Diagram 30]1 is a three-dimensional rendering of a sequencing reagent cartridge shown attached to a manifold for fluid control and other necessary functions, and a cluster generation / sequencing cartridge. One particular configuration of reagent bottles and their contents is shown, although other configurations are envisioned. [Diagram 31] FIG. 2 is a three-dimensional rendering of an exemplary manifold for sequencing reagent cartridges used for fluid control and other required functions. [Figure 32-1] FIG. 1 illustrates an example of a sequencing reagent delivery system. [Figure 32-2] FIG. 1 illustrates an example of a sequencing reagent delivery system. [Diagram 33] FIG. 1 shows two exemplary designs of an integrated assay cartridge. [Diagram 34] FIG. 1 is a diagram identifying the various components contained within the cartridge shown in the previous figure (not all components are identified or depicted in this figure). The functions identified (e.g., STC, PCR1&2, CC, etc.) are merely exemplary. The components can be utilized for various functions as required for a particular application. The numbered components are (in this example) 1) sample input port (2 in this configuration); 2) mechanical lysis unit; 3) specific target capture (STC) chamber; 4) PCR1 and PCR2 aliquotization chambers; 5) PCR1 and PCR2 reaction chambers; 6) copy control (CC) chamber; 7) condensation trap chamber (for STC and CC); 8) cluster generation and sequencing flow cell; 9) pneumatic connections for fluidic control; 10) SPI for accessing fluidic connections to the various chambers; 11) STC and CC fins (multiple thermal mixing chambers and bead capture regions); 12) amplification and dilution fins (double-sided thermal control). [Diagram 35]FIG. 1 illustrates the following steps of an exemplary mechanical lysis (ML) process for a given application (e.g., detection of pathogens in blood) performed within an integrated cartridge: 1) blood is collected from a vacutainer via a liquid handler; 2) blood is transferred to a specific target capture (STC) chamber, and then liquid reagents are added to the blood; 3) fluids are shuttle mixed between chambers while incubated via a heated device interface; 4) blood and reagent solutions are transferred to the mechanical lysis chamber, where the device's motor rotates paddles to achieve lysis. Note that not all parts of the cartridge are shown in this figure for ease of viewing the distinctive components. [Diagram 36] FIG. 1 illustrates the following steps of an exemplary specific target capture (STC) process for a given application (e.g., detection of pathogens in blood) performed in an integrated cartridge: 1) The lysed blood solution is returned from the ML chamber to the STC chamber. Capture beads are delivered to the chamber via port 1 using a liquid handler; 2) The beads are thoroughly mixed during a heated incubation step in the STC chamber; 3) A magnet is engaged to contact the serpentine channel as blood is drawn into the collection beads by the liquid handler; Wash and elution buffers are introduced via port (1). Note that not all parts of the cartridge are shown in this figure for ease of viewing the distinctive components. [Figure 37]The diagram illustrates the following steps of an exemplary target amplification (e.g., PCR1 and PCR2 for target enrichment and tag / adapter addition) process for a given application (e.g., detection of pathogens in blood) performed in an integrated cartridge: 1) The liquid handler extracts the target nucleic acid from the STC submodule; 2) The liquid handler loads the target solution into the PCR1 chamber and rehydrates the lyophilized reagents in the fluidic path; thermal cycling is performed on the chamber by the device; 3) After PCR1 is completed, the liquid handler extracts the PCR1 product and dilutions; 4) The liquid handler delivers the diluted PCR1 product to the PCR2 chamber and rehydrates the lyophilized reagents in the fluidic path; thermal cycling is performed on the chamber by the device. Please note that not all parts of the cartridge are shown in this figure to make it easier to see the characteristic components. [Figure 38] FIG. 1 illustrates the following steps of an exemplary copy control (CC) process for a given application (e.g., detection of pathogens in blood) performed within an integrated cartridge: 1) PCR2 products pooled through the liquid handler; 2) the liquid handler loads the PCR2 products into the copy control chamber; beads are prepared and delivered through the liquid handler; 3) fluid and beads are mixed and incubated through a heater interface; 4) a magnet engages the serpentine portion of the cartridge to collect beads as fluid is drawn into the liquid handler. Wash and elution buffers are introduced through the liquid handler to port (2). Note that not all parts of the cartridge are shown in this figure to make it easier to see the distinctive components. [Figure 39]FIG. 1 illustrates the following steps of an exemplary cluster generation and sequencing process for a given application (e.g., detection of pathogens in blood) performed in an integrated cartridge: 1) eluted templates are collected via a liquid handler; 2) templates and cluster generation reagents are introduced into the cluster generation / sequencing flow cell via the liquid handler; 3) the flow cell is incubated via a heated device interface; 4) sequencing reagents are delivered through the device fluidic manifold and sequencing is performed. Note that not all parts of the cartridge are shown in this figure to make it easier to see the distinctive components. [Diagram 40] FIG. 46 highlights exemplary features of the device shown in FIG. 45, shown here for a particular application or set of applications. The features are flexible to accommodate a wide range of applications. [Diagram 41] Cross-sectional schematic (left) and three-dimensional rendering of a sealed pneumatic interface (SPI) port. [Diagram 42] Figure 2 shows two exemplary designs of the reagent cartridge. Fluids can be accessed and moved in several ways, via the liquid handler (LH; e.g., pipette system) and the liquid manifold (film; e.g., on the device). Various valving methods are applicable, including sealing of the sealed pneumatic interface (SPI) port. In the middle diagram, corresponding to 1) foil-sealed lyophilized reagent storage (LH access); 2) foil-sealed liquid reagent storage (LH access); 3) waste volume for all sequencing waste (and possibly assay cartridge waste); 4) waste inlet port (manifold port and SPI); 5) nucleotide chamber (with SPI and manifold ports for fluid and CO2 scrubbing, respectively); 6) wash chamber (SPI and manifold ports); 7) soda lime chamber (for CO2 scrubbing); SPI and manifold ports are covered, for example, with either a removable seal or a puncturable foil. [Diagram 43]FIG. 1 illustrates another exemplary design of a reagent cartridge. Section A holds liquid reagents, section B holds dry reagents, section C is the interface with sequencing reagents and other bulk reagents as needed, and section W (negative space of the reagent cartridge) is for waste storage (IM is the connection to the device manifold). The liquid and dry reagent chambers / bottles are sealed with foil, which is punctured by the pipette tip of the liquid handler to reconstitute (dry reagents) and transfer the reagents. The design of the chambers and bottles allows for great flexibility in the reagents that can be stored on the cartridge, allowing for a large number of different assays / processes to be performed on the cartridge. Section B modules can be separated during manufacturing, filled, dried independently, and sequestered in a low humidity environment for storage. In some embodiments, section A can also be separated during manufacturing, filling, and storage. In a preferred embodiment, one or more chambers in the reagent cartridge contain a magnetic stir bar (see figure) that interfaces with a magnetic stir motor in the device when the cartridge is loaded into the device (e.g., useful for on-board preparation / mixing of reagents). [Diagram 44] FIG. 1 provides further details regarding the reagent cartridge and its function. [Diagram 45] 1 is a three-dimensional rendering of an exemplary device architecture for use in the system of the present invention. [Diagram 46] FIG. 2 highlights some of the cartridge loading mechanisms of the device shown in FIG. I-1. [Figure 47] Figure 47 highlights additional exemplary features (viewed from the opposite side) of the device shown in Figure I-1, shown here for a particular application or set of applications, the features being flexible to accommodate a wide range of applications. [Figure 48] FIG. 1-1 highlights additional exemplary features (focused on the electronics) of the device shown in FIG. I-1 (viewed from the rear), which are shown here for a particular application or set of applications, and are flexible to accommodate a wide range of applications. [Figure 49]FIG. I-1 highlights additional exemplary features (focused on cooling) of the device shown in FIG. I-1, here shown for a particular application or set of applications, which are flexible to accommodate a wide range of applications. [Figure 50] 1 is a graph of qPCR results demonstrating recovery of spiked antimicrobial resistance (AMR) targets direct from blood (DfB) using specific target capture (STC) oligomers. [Figure 51] Agarose gel electrophoresis confirming elution of ssDNA: Lane 1: DNA ladder, Lane 2: 5ng dsDNA P3 target and 100ng ssDNA (IDT ultramer), Lane 3: 5ng dsDNA P31 target and 100ng ssDNA (IDT ultramer), Lane 4: 5ng dsDNA P48 target and 100ng ssDNA (IDT ultramer), Lane 5: 68.1ng dsDNA from PCR2 reaction, Lane 6: 70.3ng of multiplexed (P3, P31 and P48 targets) fully captured ssDNA material, replicate 1, Lane 7: 71.3ng of multiplexed (P3, P31 and P48 targets) fully captured ssDNA material, replicate 2, Lane 8: 78.3ng of multiplexed (P3, P31 and P48 targets) fully captured ssDNA material, replicate 3. [Figure 52] 13 is an agarose gel electrophoresis image confirming elution of ssDNA from the NaOH eluate. Lane 1: DNA ladder, lane 2: ssDNA control, lanes 3 and 4: dsDNA control, lanes 5 and 6: NaOH ssDNA eluate. [Figure 53] Fluorescence microscopy images demonstrating in-well amplification of three synthetic DNA templates using SM-RPA. This is shown for three different starting copies of the synthetic DNA template, where amplified patches of target DNA were visible by the different intensities seen in the image of each target. [Figure 54]Fluorescence microscopy images confirming in-well clonal amplification products of target nucleic acid using RCA. Two different starting copies of the template were tested, and a separate chip was used for each template input. For a given template input, the same area was imaged (separate images shown for each fluorophore) showing distinct clusters of amplified target DNA products that are distinguishable in non-overlapping regions. [Figure 55] Figure 1 shows results from sequencing synthetic DNA templates directly immobilized on the chip surface. A scatter plot shows aligned read length on the x-axis and aligned read length-error on the y-axis, with corresponding histograms shown above and to the right, respectively. [Figure 56] Figure 1 shows the results of sequencing synthetic DNA templates using the direct hybridization method: A scatter plot shows aligned read length on the x-axis and aligned read length-error on the y-axis, with corresponding histograms shown above and to the right, respectively. [Figure 57-1] Figure 57-1 shows the results of automated sample-to-answer sequencing of pathogens spiked in whole blood. In Figure 57-1, a scatter plot shows aligned read length on the x-axis and aligned read length-error on the y-axis (labeled "effectiveReadLen"), with corresponding histograms shown above and to the right, respectively. [Figure 57-2] Figure 57-1 shows the results of automated sample-to-answer sequencing of pathogens spiked in whole blood. Figure 57-2 shows an analysis of the output indicating that the spiked pathogens were called correctly. [Figure 58A] FIG. 1 is a perspective view of an exemplary device. [Figure 58B] FIG. 58B is a front view of the device of FIG. 58A. [Figure 58C] FIG. 58B is a side view of the device of FIG. 58A. [Fig. 58D] FIG. 58B shows a cartridge interface assembly within the device of FIG. 58A. [Figure 58E] FIG. 58B illustrates an exemplary pneumatic pumping subunit within the device of FIG. 58A. [Fig. 58F]FIG. 58B illustrates an exemplary power subunit arrangement within the device of FIG. 58A. [Figure 58G] FIG. 58B illustrates an exemplary air handling and reagent cartridge air intake subsystem within the device of FIG. 58A. [Fig. 58H] FIG. 58B illustrates a liquid cooling subsystem within the device of FIG. 58A. [Fig. 58I] FIG. 58B illustrates the placement of an exemplary condensation management subsystem within the device of FIG. 58A. [Figure 59] FIG. 1 illustrates a pneumatic subsystem for use with the various devices described herein. [Figure 60] FIG. 1 illustrates an exemplary sample or assay cartridge, according to certain embodiments. [Figure 61] FIG. 1 illustrates an exemplary reagent cartridge, according to certain embodiments. [Figure 62] FIG. 1 illustrates an exemplary workflow for performing an assay using the devices and cartridges described herein. [Figure 63] FIG. 1 shows an exemplary sample or assay cartridge with a library preparation unit. [Figure 64] FIG. 1 shows an exemplary sample or assay cartridge with a sample input and mechanical lysis subunit. [Figure 65] FIG. 1 shows an exemplary sample or assay cartridge with specific target capture subunits. [Figure 66] FIG. 1 illustrates an exemplary flow cell and pipette reservoir in an exemplary assay cartridge. [Figure 67] FIG. 1 illustrates an exemplary SPI port configured for a 1 mL pipette tip. [Figure 68] FIG. 1 illustrates an exemplary SPI port configured for a 5 mL pipette tip. [Figure 69] FIG. 1 illustrates an exemplary specific target capture subunit according to certain embodiments. [Figure 70] FIG. 1 shows an exemplary library preparation unit or PCR fin. [Figure 71] FIG. 1 shows PCR results demonstrating successful mechanical lysis and observation of released target nucleic acid using an exemplary cartridge-compatible mechanical lysis subunit. [Figure 72] FIG. 1 shows PCR results demonstrating successful specific target capture using an exemplary cartridge compatibility subsystem. [Figure 73] FIG. 1 shows an electropherogram overlay of various PCR results from Example R. [Figure 74] FIG. 1 shows the sequencing results obtained from templates amplified using the direct hybridization method in Example S. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present disclosure provides oligomers, methods, compositions and kits useful for the rapid analysis of target polynucleotides, including the determination of their nucleotide sequence. Analysis can be performed from a wide range of input sample types and quantities using a fully automated system including cartridges, devices, and operating and analytical software without the need for human intervention after a run has been initiated (sample-to-report). Additionally, complete workflows are disclosed illustrating the chemical and mechanical aspects as well as other aspects of the invention.

[0029] Generally speaking, the workflow includes one or more of the following segments: 1) sample preparation, 2) library preparation, 3) copy control, 4) cluster generation, or 5) sequencing. Cartridge and device designs and concepts are disclosed that can perform all chemical workflow steps of each segment in an automated fashion. Embodiments are disclosed that include sequencing performed on the surface of a semiconductor chip. Software is described that controls all the functions of the device, as well as [software-controlled] algorithms for all stages of data analysis from raw data to the final report (i.e., what is the answer to the question that the test was performed on).

[0030] 1. Sample Type A wide range of sample types (an exemplary, non-exhaustive list of sample types is provided in the "Definitions" section) suitable for use with the disclosed invention are contemplated. In a preferred embodiment, the sample is in liquid form, and the entire sample or a portion thereof is introduced into the cartridge. In another preferred embodiment, the sample is in solid form, but is treated to bring the sample or a portion thereof into liquid form prior to introduction into the cartridge. Alternatively, the solid is processed into a suspension, slurry, emulsion, etc., and then introduced into the cartridge. In another embodiment, the solid sample is directly introduced into the cartridge and processed therein at or near the beginning of the workflow to bring the sample into liquid, suspension, emulsion, etc. form. Alternatively, the target polynucleotides can be extracted directly from the solid sample, either before or after introduction into the cartridge. In another embodiment, the sample is already in the form of a suspension, slurry, emulsion, etc., when obtained, and is either directly introduced into the cartridge or is treated prior to introduction into the cartridge in liquid, solid or gas form. In another embodiment, the sample is gaseous. Typically, in gas samples, the target polynucleotides are present in the sample in the form of aerosols or suspensions that may or may not be associated with cells. The gas sample may be directly introduced into the cartridge, and the polynucleotides may be collected, suspended, or otherwise collected from the gas sample and subsequently introduced into the cartridge. It is envisioned that the sample input port will accommodate this wide range of sample types. The sample input port may exist in a variety of forms for this to occur, including, but not limited to, 1) a universal sample port that directly accommodates all sample types, 2) a sample port designed for a specific sample type or group of sample types, where cartridges with different sample input ports are available for different assay types, and 3) a semi-universal sample port that accommodates the attachment of an adapter designed for a given sample type, such that the appropriate adapter is attached to the cartridge depending on the assay being performed.

[0031] Preferred sample types include, for example, whole blood as used in preferred embodiments of the disclosed invention, where the system is utilized for detection of bloodstream infections, antimicrobial resistance genes (see further details in ) and plasma, where used in preferred embodiments for detection of cell-free DNA (cfDNA), including circulating tumor DNA (ctDNA). Another preferred embodiment where the preferred sample type includes whole blood is the detection of targets in plasma, such as, but not limited to, cfDNA, ctDNA and various viral infections (e.g., HIV). In such an embodiment, the plasma can be separated from the whole blood, preferably in the assay cartridge, but can alternatively be performed in a sample collection tube or other device that ideally interfaces directly with the sample input port.

[0032] 2. Sample Preparation A wide variety of sample preparation methods and compositions (an exemplary, non-exhaustive list of sample preparation methods is provided in the "Definitions" section) are contemplated as suitable for use with the disclosed invention.

[0033] In a preferred embodiment, whole blood is the sample and the target polynucleotides are contained within various infectious agents (e.g., bacteria, fungi, viruses) present in whole blood (exemplary applications are bloodstream infections and detection of antimicrobial resistance genes). An exemplary sample preparation method of this embodiment includes the following steps: 1) mixing a whole blood sample with sample preparation reagents (e.g., including reagents useful for homogenizing the sample and lysing cells; in one aspect of this feature of this preferred embodiment, vigorous mixing serves to solubilize the sample); 2) further homogenizing the sample by incubation at high temperature and simultaneous digestion of proteins in the sample with proteinase K; 3) combining the homogenized and digested sample with bead-basing beads; 4) mixing the beads and sample at a relatively high speed (e.g., 8000 RPM), for example using a rotating impeller, thereby lysing the infectious agent and releasing the contained target polynucleotide into solution; and 5) heating the sample at high temperature (e.g., 95° C.) to denature the target polynucleotide in double-stranded form (this step also serves to remove proteins and other components and / or structures surrounding and / or associated with the target polynucleotide). 6) mixing the denatured target polynucleotide sample with a set of specific target capture (STC) oligonucleotides (STC oligos can be designed, for example, for a specific target, a set of specific targets, broad range targets, etc., as further discussed); 7) heating the STC oligo / target mixture (e.g., 60° C.) to promote annealing of the STC oligos with their specific target sequences; 8) combining the STC oligo / target mixture with streptavidin-derivatized paramagnetic particles; 9) mixing and then incubating the STC oligo / target mixture (e.g., 45° C.) to promote binding of the STC oligo / target mixture (the STC oligos include biotin molecules attached) to the beads; 10) immobilizing the STC oligo / target mixture / bead complex using a magnet; 11) washing the STC oligo / target mixture / bead complex; and 12) eluting the target polynucleotides, which are then ready for further processing in the workflow.

[0034] The above preferred embodiment has many advantages, including but not limited to: 1) the entire process is automated in a fully automated system within the cartridge; 2) once sample processing is completed, no user intervention is required to continue processing of the test / assay as the workflow continues uninterrupted within the closed cartridge; 3) large volumes of whole blood are accommodated within the cartridge improving overall test / assay performance including sensitivity; 4) the sample is homogenized, cells lysed and target polynucleotides are denatured and prepared for further processing in a fast and efficient automated protocol; 5) the specific target capture (STC) process results in a high degree of purification in a fast protocol; 6) the STC process gives a high degree of specificity regulated by the capture oligomer design and reaction conditions among other parameters (composition of the mixture, temperature, etc.); 7) the STC process gives a high degree of inclusiveness, again using capture oligomer design, reaction conditions, etc. all DNA sequences along the phylogenetic tree can be captured or excluded as required; 8) the eluted sample is ready for further processing without the need for analysis.

[0035] In other embodiments, various features of the preferred sample preparation embodiments described in paragraphs can be readily substituted with alternative features / methods in various combinations, including, for example: 1) Proteinase K can be replaced or augmented with one or more alternative enzymes, detergents, chaotropes (e.g., GuSCN), chemical agents including reducing agents, etc.; 2) Further sample homogenization after mixing with lysis reagent may not be necessary; 3) Cell lysis by bead bashing with zirconium beads can be replaced or augmented with alternative beads, balls, or other grinding media, sonication, detergents, chaotropes, reducing agents (DTT, β-mercaptoethanol) and / or other chemical agents; 4) Homogenization and cell lysis can be performed simultaneously; 5) The step of denaturing double-stranded target polynucleotides can be omitted if the target is otherwise available for further processing; 6) If target capture / separation / isolation is not required for a given application, the sample or a portion thereof can be directly utilized for further processing at this point. 7) STC [alone] includes, for example, a) non-specific target capture (e.g., Boom method; see other exemplary methods in the "Definitions" section), b) a combination of non-specific and specific target capture methods (e.g., Boom method followed by STC method), c) aptamer-based capture, d) filtration, e) isoelectric focusing, f) other methods listed in the "Definitions" section, g) combinations thereof.

[0036] In some embodiments, the target capture oligomer (TCO) may perform functions in addition to target capture alone. For example, the TCO may contain one or more tags useful in downstream processes (e.g., unique molecular identifiers (UMIs), universal amplification primer sites, promoters (e.g., T7 RNA polymerase promoters), adapters for sequencing, etc.). The TCO may also function as a primer for extension and amplification (which may act together with one or more tags to achieve a desired function). In some workflows, the annealing of the TCO and its extension by the polymerase may occur simultaneously or at different times (or overlapping times) in the same reaction mixture. In other embodiments, the annealing of the TCO may occur first, followed by extension, e.g., upon addition or combination with another reagent. In other methods, the annealing and subsequent separation of the TCO / target complex may occur first, followed by extension in a subsequent step, e.g., while the TCO / target complex is still immobilized on the solid support. In some of these last described workflows, the extension may occur, followed by a final wash of the immobilized TCO / target complex, followed by elution. In this case, for example, the target polynucleotide is now already purified, tagged, and ready to move on to the next part of the extended process, saving time, steps, etc. in the overall workflow.

[0037] In some embodiments, no sample preparation is required (ie, the target polynucleotides can be used "directly from the sample" in the first step of the process, e.g., amplification).

[0038] Due to the wide range of reaction chambers, storage chambers, fluid interconnectivities, valving configurations, reagent delivery options, and functionalities (including mixing, stirring, heating, transport, separation, magnetic, etc.) disclosed or contemplated in the cartridges of the present invention, each of these sample preparation workflow options can be accommodated within the framework of a rapid, fully automated workflow.

[0039] 3. Library Preparation A wide range of library preparation methods and compositions (an exemplary, non-exhaustive list of library preparation methods is provided in the "Definitions" section) are contemplated as suitable for use in the disclosed invention. Target polynucleotides may be provided as input for library preparation by a variety of routes, including directly from the sample or in the form of output from a wide variety of sample preparation methods. In one preferred embodiment, a portion or portions of the target polynucleotide are selectively enriched using amplification (which aids, for example, in targeted sequencing approaches). Other optional features of this preferred embodiment include incorporation of tags, optionally including adaptors, two or more separate rounds of amplification, optionally including the use of nested primers for the second and / or subsequent rounds if used, and copy control (see elsewhere herein; described as a separate feature, but which can also overlap with library preparation). Exemplary applications are bloodstream infections as well as detection of antimicrobial resistance genes. An exemplary library preparation method of this embodiment includes the following steps: 1) combining target polynucleotides with a first amplification reagent (as noted elsewhere herein, the target polynucleotides can come from a variety of sources. One preferred source is the output of the sample preparation method described above, which utilizes whole blood as the sample type); 2) performing a first amplification using PCR; 3) diluting the product of the first amplification; 4) mixing an aliquot of the diluted first amplification product with a second amplification reagent; and 5) performing a second amplification using PCR. The method includes the steps of: 6) performing an amplification, 7) combining and mixing the products of the second amplification with a capture reagent (including magnetic capture beads), 8) incubating the second amplification product / capture reagent mixture for 10 minutes at ambient temperature (about 20-26°C) with constant mixing, 9) immobilizing the second amplification product / bead complex using a magnet, 10) washing the second amplification product / bead complex, and 11) adding an elution reagent to the washed second amplification product / bead complex, mixing, and incubating for 1 minute at ambient temperature (about 20-26°C). The eluted library molecules are ready for further processing in the workflow.

[0040] Key features and advantages of the preferred embodiment of the library preparation workflow summarized in the above paragraphs include: 1) A very wide range of sample input types and quantities is acceptable. 2) The first amplification reaction (PCR1) is designed to have high sensitivity and fidelity. 3) The primers utilized in PCR1 are designed to target bacterial 16S and 23S, fungal 28S, and specific antimicrobial resistance (AMR) genes, and to amplify broadly across the spectrum of bacterial and fungal targets potentially contained within the sample. 4) No purification of the PCR1 amplicon is required prior to moving to the second amplification (PCR2), only a simple [automated] dilution (this differs from prior art protocols that require a purification step). 5) The primers used for PCR2 are designed to nest within the primer sites of PCR1, providing high specificity for one or more target polynucleotides of interest; 6) Aliquots from the same dilution of PCR1 product can be used to perform multiple different PCR2 reactions (e.g., one embodiment of the cartridge design (see "Assay Cartridge" section below) shows 10 separate chambers dedicated to separate PCR2 reactions), increasing the specificity of each reaction by reducing the complexity (i.e., the desired number of targets are covered in, for example, 10 different reactions instead of 1, thereby reducing the number of primers in any given PCR2 reaction) and / or increasing the multiplexing capacity of the system. 7) In one case of this preferred embodiment, at least one of the primers of the primer pair used in PCR2 is equipped with one or more biotin molecules, allowing for immobilization of its amplicon product using streptavidin-bound microspheres (or similar). 8) One or more tags, including adapters, can be added to PCR1, PCR2 or a combination thereof, thus including incorporation at both ends of the amplicon as desired. 9) The copy control process can be either as summarized above, or designed to overlap / be incorporated into the PCR2 step, or added earlier in the workflow, after the library preparation workflow steps (see details elsewhere in ).In summary, the method can use a wide variety of target polynucleotide input types and amounts, is highly sensitive and specific, can be used to easily incorporate adapter-containing tags, can be easily interfaced (or overlapped) with copy control processes, is simple (e.g., only one purification step), is rapid, and is easy to automate.

[0041] In other preferred embodiments, various features of the preferred library preparation embodiments described in paragraphs 1 and 2 may be combined, e.g., 1) different nested amplification configurations may be used, e.g., a) primers are nested only at one end of the target region, b) no nesting is used, c) the nesting pattern is different for different target polynucleotides, 2) PCR may be replaced by another amplification method, 3) one amplification reaction may be performed instead of two (where the required performance, e.g., sensitivity, specificity, multiplexing capacity, can be achieved and adapter-containing tags may still be incorporated if necessary), making the workflow even simpler and faster; 4) aspects of library preparation may overlap with sample preparation (discussed in more detail in the following paragraphs); 4) adapter-containing tags containing unique molecular identifiers (UMIs) may be attached to target regions using methods other than amplification, e.g., ligation. 5) Copy control methods can be added just after the end of the workflow cited in the above embodiment, or just before the purification step, or just before the PCR2 (or alternative amplification method) step, or in the second amplification step, or in the PCR1 (or alternative amplification method) step, or in the last one or a few cycles of one amplification step (if only one), or as early as sample preparation (aspects of copy control are discussed elsewhere), etc., and can be readily substituted in various combinations with alternative features / methods. Other library preparation methods that can be used in the methods of the present disclosure are listed in the "Definitions" section.

[0042] As mentioned above, aspects of library preparation can overlap with sample preparation. Below are some examples to illustrate this concept: 1) An oligomer that can function as a primer is annealed to the target polynucleotide during sample preparation. The primer can also serve other functions, such as that of a target capture oligomer. The oligo can anneal to the target polynucleotide with a desired level of specificity, adding to the overall specificity of the assay at this early stage (sample preparation) and thereby improving its performance. The oligo can further include a tag, including an adaptor containing a UMI, a universal primer site, etc., if desired. At some point, the oligomer is extended, for example simultaneously with annealing, after annealing in a second step (e.g., combining the annealing reaction mixture with the extension reaction mixture), after immobilizing the oligo / target complex (e.g., while still immobilized), after elution, etc., or essentially as part of the first phase of library preparation. This extension product can enter the remainder of the workflow already equipped with the tag and with the level of specificity already achieved, potentially saving steps and time in the overall assay. Also, in this manner, the first (or only) amplification step can utilize a specific opposing primer (with any desired level of specificity, potentially non-specific through the use of a randomizer) and, if desired, a universal primer that binds to [at least a portion of] the tag sequence of the oligomer. Furthermore, incorporating a tag containing an adaptor containing a UMI during sample preparation may allow the product [of the sample preparation] to bypass library preparation and proceed directly to other steps in the workflow, such as cluster generation and / or sequencing (in applications where sensitivity is sufficient and no additional tags, etc., are required). 2) Using a variation of method 1 immediately above, oligos can be annealed to each strand of a double-stranded target polynucleotide, each strand with a desired level of specificity, one or both containing a tag (including an adaptor containing a UMI). This may complete even more of the workflow at this early stage, saving steps and time and increasing overall specificity.Additionally, the first (potentially only) step of library preparation can be a universal amplification using primers that anneal to tag sequences on both ends of the target. These can be the same or different. Also, other elements required for other steps in the workflow can be incorporated into the target during the sample preparation step and / or in the universal amplification step (using universal primers with tags). 3) Tags (including adapters containing UMIs) can be ligated to target polynucleotides during sample preparation.

[0043] Due to the wide range of reaction chambers, storage chambers, fluid interconnectivities, valving configurations, reagent delivery options, and functionalities (including mixing, stirring, heating, transport, separation, magnetic, etc.) disclosed or contemplated in the cartridges of the present invention, each of these library preparation workflow options can be accommodated within the framework of a rapid, fully automated workflow.

[0044] 4.Copy Control "Copy Control" (CC) refers to compositions and methods in which the copy number of a molecule that is the output of a given process is controlled in a predetermined manner (e.g., limited amount, i.e., not more than a predetermined amount, or a specific, predetermined amount). Additionally, in certain workflows, it is desirable to incorporate additional sequences into the target polynucleotide (e.g., tag), such as the incorporation of adapters into a sequencing library. This can also be achieved in CC compositions and methods (see "Definitions" and the figures and their associated brief descriptions for further details). CC is included in many of the workflows disclosed herein. A wide variety of CC compositions and methods are disclosed in "Compositions, Kits and Methods for Isolating Target Polynucleotides," PCT / GB2021 / 050098, which is incorporated herein by reference in its entirety. All of the CC compositions and methods disclosed therein are applicable to the workflows of the present invention. Additionally, examples of CC methods are summarized in Figures 1-14, which are included herein (see accompanying description in "Brief Description of the Figures").

[0045] Preferred CC embodiments include a capture oligomer comprising, in a 5' to 3' direction: a capture sequence, an internal extension blocker, a complement of the capture sequence, and a target hybridizing sequence, where the complement of the capture sequence is configured to anneal to the capture sequence in the absence of the target hybridizing sequence and the extended target sequence annealed to the complement of the capture sequence. In some such preferred embodiments, the capture oligomer has the formula 5'-A1-CLB-A2-C'-A3-RB-A4-THS-X-3', where A1 is an optionally present first additional sequence. C is a capture sequence, L is an optional linker, B is an internal extension blocker, A2 is an optional second additional sequence, C' is the complement of the capture sequence, A3 is an optional third additional sequence, RB is an optional reversible extension blocker, A4 is an optional fourth additional sequence, THS is a target hybridizing sequence, and X is an optional blocking moiety. In some of these preferred embodiments, the capture sequence comprises a polyA or polyT sequence and the complement of the capture sequence comprises a polyT or polyA sequence.

[0046] Another preferred embodiment includes a combination comprising a capture oligomer and a complementary oligomer, where (a) the capture oligomer comprises, in a 5' to 3' direction, a capture sequence comprising a first and a second portion, an internal extension blocker, a spacer sequence comprising a first and a second portion, and a target hybridizing sequence, and (b) the complementary oligomer comprises, in a 3' to 5' direction: a complement of the second portion of the capture sequence, and a complement of at least a first portion of the spacer sequence, wherein the complement of the second portion of the capture sequence and the complement of at least a first portion of the spacer sequence are configured to simultaneously anneal to the capture oligomer in the absence of the complement of the spacer sequence. In some such preferred embodiments, the capture oligomer has the formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3', where A1 is an optional first additional sequence, C1 is a first portion of the capture sequence, C2 is a second portion of the capture sequence, B is an internal extension blocker, A2 is an optional second additional sequence, S1 is a first portion of the spacer sequence, S2 is a second portion of the spacer sequence, A3 is an optional third additional sequence, RB is an optional reversible extension blocker, A4 is an optional fourth additional sequence, THS is a target hybridizing sequence, and X is an optionally present blocking moiety. In some such preferred embodiments, the complementary oligomer has the formula: 5'-S1'-A2'-L-C2'-X-3', where S1' is the complement of at least a first portion of the spacer sequence, A2' is the optional complement of a second additional sequence that may be present in the capture oligomer, L is an optional linker, C2' is the complement of a second portion of the capture sequence, and X is an optional blocking moiety.

[0047] Yet another preferred embodiment includes a combination comprising a capture oligomer and a complementary oligomer, where (a) the capture oligomer comprises, in a 5' to 3' direction, a capture sequence comprising a first portion and a second portion, and a target hybridizing sequence comprising a second portion and a first portion; (b) the complementary oligomer comprises, in a 3' to 5' direction: a complement of the second portion of the capture sequence and a complement of the second portion of the target hybridizing sequence, configured such that the complement of the second portion of the capture sequence and the complement of the second portion of the target hybridizing sequence simultaneously anneal to the capture oligomer in the absence of the complement of the target hybridizing sequence. In some such preferred embodiments, the capture oligomer has the formula 5'-A1-C1-C2-A2-S-A3-THS2-THS1-X-3', where A1 is an optionally present first additional sequence, C1 is a first portion of the capture sequence, C2 is a second portion of the capture sequence, A2 is an optionally present second additional sequence, S is an optionally present spacer sequence, A3 is an optionally present third additional sequence, THS2 is a second portion of the target hybridizing sequence, THS1 is a first portion of the target hybridizing sequence, and X is an optionally present blocking moiety. In some such preferred embodiments, the complementary oligomer has the formula 5'-THS2'-A3'-S'-A2'-C2'-X-3', where THS2' is the complement of the second portion of the target hybridizing sequence and A3' is the optional complement of a third additional sequence that may be present in the capture oligomer. S' is the optional complement of a spacer that is optionally present in the capture oligomer, A2' is the optional complement of a second additional sequence that is optionally present in the capture oligomer, C2' is the complement of a second portion of the capture sequence, and X is an optional blocking moiety.

[0048] Another preferred embodiment is a method of capturing a target polynucleotide from a composition comprising contacting a target polynucleotide as described above and others disclosed in PCT / GB2021 / 050098, wherein a target hybridizing sequence of a capture oligomer anneals to the target polynucleotide at a site comprising the 3' end of the target polynucleotide, extending the 3' end of the target polynucleotide using a DNA polymerase having strand displacement activity, thereby forming a complement of the complement of the capture sequence annealed to the capture oligomer such that the capture sequence of the capture oligomer is available for binding, contacting the capture sequence of the capture oligomer with the complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the secondary capture reagent, and isolating the complex from the composition, thereby capturing the target polynucleotide.

[0049] Another preferred embodiment is a method of capturing a target polynucleotide from a composition, the method comprising contacting the composition with a combination capture oligomer and a complementary oligomer as described above and others disclosed in PCT / GB2021 / 050098 (claims 28-30 or 34-51); annealing a target hybridizing sequence of the capture oligomer to the target polynucleotide at a site that comprises the 3' end of the target polynucleotide; and extending the 3' end of the target polynucleotide using a DNA polymerase having strand displacement activity. contacting the capture sequence of the capture oligomer with the complement of the capture sequence and a secondary capture reagent comprising either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the secondary capture reagent; and isolating the complex from the composition, thereby capturing the target polynucleotide.

[0050] In some further preferred embodiments, combinations are provided that include a capture oligomer and a complementary oligomer, (a) The capture oligomer is arranged in a 5' to 3' direction as follows: a capture sequence comprising a first portion and a second portion; and a target hybridizing sequence comprising a second portion and a first portion; (b) A complementary oligomer having, in the 3' to 5' direction: the complement of the second portion of the capture sequence, and comprising the complement of a second portion of the target hybridizing sequence; The complement of the second portion of the capture sequence and the complement of the second portion of the target hybridizing sequence are configured to simultaneously anneal to the capture oligomer in the absence of the complement of the target hybridizing sequence. Figure 10B provides a diagram of an exemplary oligomer according to these embodiments. Optional additional elements may be present as described in further embodiments above and / or as shown in Figure 10B (e.g., any individual element of Figure 10B or any combination thereof).

[0051] This combination can be used to perform limited capture in that the complementary oligomer can be configured to bind to the free capture oligomer, not to the capture oligomer bound to the target polynucleotide. For example, the binding of the target hybridization sequence of the capture oligomer to the target polynucleotide can be more energetically favorable than the binding of the complement of the second part of the capture sequence to the second part of the target hybridization sequence. In the absence of the target polynucleotide, the complementary oligomer binds to the capture oligomer and blocks access to the capture sequence (C1+C2 in FIG. 10B) to a degree sufficient to block the binding of the capture sequence by the complement of the capture sequence in the secondary capture reagent, which can be any of the secondary capture reagents described elsewhere herein.

[0052] Accordingly, there is provided a method of capturing a target polynucleotide from a composition, comprising the steps of: contacting the composition with the above combination or any further embodiment thereof described herein, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide; contacting the capture oligomer with a complementary oligomer before or after the capture oligomer anneals to the target polynucleotide, wherein the complementary oligomer anneals to the free capture oligomer and partially occupies its capture sequence, and the complementary oligomer does not anneal to a complex containing the capture oligomer annealed to the target polynucleotide, and wherein annealing of the target hybridizing sequence to the target polynucleotide results in dissociation of the complementary oligomer from the capture oligomer if contacting the capture oligomer with the complementary oligomer occurs before the capture oligomer anneals to the target polynucleotide; contacting the capture sequence of the capture oligomer complexed with the target polynucleotide with a complement of the capture sequence and a secondary capture reagent comprising either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and isolating the complex from the composition, thereby capturing the target polynucleotide. Optional additional elements may be present as described in further embodiments above and / or as shown in Figure 10B (e.g., any individual element of Figure 10B or any combination thereof).

[0053] Another exemplary CC method in accordance with the present disclosure includes the steps of: 1) mixing the PCR2 amplicon (see other details in ) with CC reagents including the CC capture oligonucleotides described herein and PCT / GB2021 / 050098; 2) incubating the mixture at elevated temperature (e.g., about 95° C.) for about 3-5 minutes to denature any double-stranded target nucleic acid (amplicon) present; 3) incubating the mixture at moderate temperature (e.g., about 60° C.) for about 5-10 minutes to facilitate annealing of the CC capture oligomer to the target; 4) extending the 3′ end of the amplicon along the CC capture oligomer, thereby removing the target capture sequence (extension may occur during annealing if appropriate reagents are present in the reaction mixture). The process can be performed during the step or as a subsequent step if the reagents for extension are added separately), 5) mixing the extension reaction mixture with a secondary capture reagent comprising a complement of the capture sequence and (i) a binding partner or (ii) a solid support, present in a user-defined amount, 6) incubating at a moderate moderate temperature (e.g., about 45° C.) for about 3-10 minutes, thereby forming a complex comprising the target nucleic acid, the capture oligomer and the secondary capture reagent, 7) isolating the complex from the composition, thereby capturing the target polynucleotide, 8) washing the beads, 9) adding an elution reagent to the washed beads, mixing and incubating at ambient temperature (22-26° C.) for 1 minute. The eluted copy control molecule is ready for further processing in the workflow.

[0054] Alternatives to the exemplary CC workflow above include, but are not limited to, 1) performing the steps above after PCR1 (i.e., if PCR2 is not performed in a given workflow), 2) performing the steps above with target amplicons produced by methods other than PCR, 3) adding a universal sequence site as a tag during PCR1 or PCR2 (or alternative amplification methods), where the use of this universal tag acts as a universal binding site for designing a single CC capture oligomer for use with multiple targets (advantages include, but are not limited to, b) higher multiplexing capacity, c) consistent annealing properties across target amplicons, etc.), 4) adding tags to amplicons during PCR1 and / or PCR2 using a CC-based strategy (as disclosed in ), 5) adding all desired tags, including adapters, as part of the CC process. It is noted that the CC methods generally described above and elsewhere therein (including tag addition) are disclosed in further detail in GB 2021 / 050098.

[0055] 5. Cluster Generation "Cluster" refers to a grouping of molecules, e.g., nucleic acid molecules, bound to a solid support. "Cluster generation" refers to the process by which clusters are generated. Examples of cluster generation processes include amplification-based, e.g., clonal amplification, and non-amplification-based, e.g., hybridization of target molecules to oligonucleotides immobilized on a solid support at known specific regions (e.g., spots). Clusters can be monoclonal (typically the preferred configuration in this disclosure) or polyclonal.

[0056] One widely accepted method of cluster generation is clonal amplification by performing emulsion PCR of beads. In next-generation sequencing (NGS) applications, after clonal amplification, the beads are arrayed on the surface of a flow cell (see US Pat. No. 8,012,690). This technology has been incorporated into several NGS platforms, including Ion Torrent (Thermo) systems, ABI SOLiD, and Roche 454. Emulsion encapsulates the beads, amplification reagents, and individual template DNA molecules in isolated aqueous droplets (micelles), preventing cross-contamination. Although emulsion PCR is a proven technology, the workflow is complex, time-consuming (hours), and difficult to automate in cartridge format. Other widely accepted methods, such as those developed by Illumina (originally Solexa and Manteia), perform clonal amplification directly on the surface of a flow cell using bridge amplification. The first generation of methods for cluster generation using bridge amplification was based on a form of isothermal polymerase chain reaction driven by cycles of reagent flow (isothermal bridge PCR; see US Pat. Nos. 1,037,0652 and 7,972,820). In these methods, two PCR primers are co-immobilized on the surface of a flow cell and hybridized to a population of target DNA molecules containing matching adapters at both ends. Denaturing and extension reagents are then flowed in successive cycles, generating distinct clonal clusters, typically less than one micron in diameter. These small clusters contain relatively few copies of the target nucleic acid, making their use unacceptable in platforms that require larger amounts of target nucleic acid to achieve the required sequencing performance. Like emulsion PCR, isothermal bridge amplification is time consuming (typically more than 4 hours) and requires high reagent volumes. Second generation methods using bridge amplification are based on recombinase polymerase amplification (RPA) in the form of a method called exclusion amplification (ExAmp) (see US Pat. No. 9,169,513). RPA is an isothermal DNA amplification method that uses two primers of similar design to PCR (see US Pat. No. 7,270,981). In ExAmp, both primers are immobilized on the surface of an arrayed (patterned) flow cell.The regions between the amplification sites are primer-free and are used to prevent mixing between clonal populations. Instead of hybridizing template DNA molecules to a surface before the start of the amplification process, multiple targets are added along with the amplification mix. As a result, template hybridization and amplification occur simultaneously, and cluster clonality is achieved by amplification occurring at a faster rate than hybridization. This process is still relatively time-consuming (approximately 3 hours), but results in a higher density of clusters, which is beneficial for sequencing output. However, these are also small clusters containing relatively few copies of the target nucleic acid.

[0057] Apart from emulsion PCR and bridge amplification, another method to create clonal clusters is by rolling circle amplification (RCA). In this method, template DNA molecules are circularized before hybridizing with a single amplification primer and extending it. This generates long amplicon molecules containing concatemers of the target sequence, providing large amounts of DNA for downstream analysis. The sequencing platform developed by BGI and Complete Genomics uses a form of solution-phase RCA to create DNA nanoballs, which are then hybridized to patterned arrays (DOI:10.1126 / science.1181498). Creating and manipulating DNA nanoballs requires accurate and proper quality control, complicating their application to rapid cartridge-based formats.

[0058] Apart from the solution-phase approach, QIAGEN has invested in the development of exponential surface-phase RCA for the GeneReader platform (US2018 / 0105871, US2018 / 0112251, EP1916311, US9683255B2, US2018 / 0087099). This method is based on exponential RCA, where two (or more) amplification primers are used instead of one (US5854033, US6143495, 1101 / gr.180501, US6323009). In this approach, multiple targets are modified so that they contain adapter sequences at both ends, which are then used to ligate the template into a circle. The DNA circle is then hybridized to the surface of a flow cell, co-immobilizing the two amplification primers. The amplification primers are complementary to the adapters present in the circularized template, allowing for a "branched" exponential RCA reaction (DOI: 10.1093 / biomethods / bpx007). This protocol involves many steps and can be time-consuming. To effectively apply it to rapid cartridge-based systems, a simpler and faster workflow is needed.

[0059] A. Immobilization of oligonucleotides Cluster generation on a solid support typically requires immobilization of one or more oligonucleotides or polynucleotides to the solid support. For the purposes of this disclosure, any immobilization method that supports one or more steps related to the disclosed embodiments is acceptable. This includes, but is not limited to, covalent and non-covalent methods. Direct and indirect methods; attachment to specific areas of a surface (e.g., spotting, arrays, etc.) and global immobilization over the entire surface; attachment to flat surfaces, surfaces containing features (e.g., wells, pillars, pads), particles, etc.; and the like.

[0060] In some preferred embodiments disclosed herein, oligonucleotides were attached to the surface of wells fabricated on semiconductor chips. A common method used for such attachment includes click chemistry (see, e.g., Click Chemistry, a Powerful Tool for Pharmaceutical Sciences (2008) Hein, et al.; Pharm Res 25(10):2216-2230 and A Hitchhiker's Guide to Click-Chemistry with Nucleic Acids (2021) Fantoni et al., Chem Rev, 121:7122-7154). In certain embodiments, the oligonucleotides to be attached contained 5'-DBCO = 5'-terminal dibenzocyclooctyl (DBCO) moieties (see, e.g., Example N, "Sequencing of templates generated using in-well clonal amplification"). In such embodiments, one preferred method of conjugation involves activating the chip surface with an acrylamide-based polymer coating, followed by covalent attachment of 5'-modified oligonucleotides. An exemplary protocol includes the following general steps: 1) Clean the surface of the semiconductor chip surface by immersion in 2% Decon90 solution (Decon Laboratories LTD) for 5 minutes. 2) Rinse the surface with 18 MΩ water, followed by incubation in 0.1 M HCl for 5 minutes, then rinse again with 18 MΩ water and blow dry with nitrogen. 3) Clean the surface with 7% ammonium sulfate (NH 4 ) 2 SO 4 3) Incubate the chip surface for 30 min in a 0.6% wt / vol solution of MCP-click polymer with 5% azide content in 1000 mM NaCl; 4) Rinse with 18 MΩ water, blow dry with nitrogen, then bake at 80 °C for 15 min; 5) Cool the chip to room temperature; 6) Bind oligonucleotides to the surface: A) Microarray spotting - 150 mM sodium phosphate buffer pH 8.5 (Na 2 HPO 4 and NaH 2 PO 4) and sucrose monolaurate 0.01% wt / vol. In some embodiments, the spot volume is about 200-300 pL and the oligonucleotide concentration is about 40-200 μM; the spotted sample is incubated at 75% relative humidity for a minimum of about 16 hours. B) Flood-filling - The oligonucleotide solution (same buffer composition and oligo concentration as in Method A above) is flooded onto the surface of the chip in the flow cell. The input and output ports of the flow cell are sealed and the chip is incubated for a minimum of about 16 hours. 7) The chip (either Method A or B) is flood-filled with 0.1 M Tris buffer ((HOCH 2 ) 3 CNH 2 ) (pH 9) at 50° C. for 15 minutes, then rinsed in 18 MΩ water, blown dry with nitrogen and stored in the dark at 4% relative humidity until needed.

[0061] Other conjugation methods that have been successfully demonstrated for use with selected embodiments within the present disclosure include, but are not limited to, 1) polyacrylamide-based coatings containing succinimide groups (e.g., Code Link from Surmodics IVD, Inc.) for conjugation of primary amine-derivatized oligonucleotides, 2) biosorbent polymer coatings containing bromoacetamide (the reactive monomer N-(5-bromoacetamidylpentyl)acrylamide) for conjugation of thiophosphate containing oligonucleotides, and 3) conjugation approaches utilizing UV irradiation and poly(T)poly(C)5'-tagged oligonucleotides.

[0062] b. Cluster generation using solution-mediated recombinase polymerase amplification (SM-RPA) Disclosed herein is solution-mediated recombinase polymerase amplification (SM-RPA), which can be used to generate clonal nucleic acid clusters from multiple targets directly on a solid support, typically in 20-60 minutes, which is faster than current RPA-based (and other) methods in the art. Furthermore, the clusters generated in this manner produce "patches" of clonally amplified nucleic acid of larger size and greater number of molecules than the small clusters generated by other methods, which is beneficial for applications requiring larger amounts of immobilized material.

[0063] In a preferred embodiment of SM-RPA, one of the two primers (e.g., a reverse primer) is immobilized on a solid support and the other primer (e.g., a forward primer) is present in the solution phase. As a result, amplification occurs at the surface and a strand is released into the liquid phase, which can rehybridize locally. Figure 15 shows the primer configuration and mechanism of SM-RPA and compares it to bridge amplification. The resulting clonal population exists as a "patchwork" of immobilized DNA, as shown in Figure 16, where different patches from clonal amplification of three different target molecules are shown. The products "grow" laterally, expanding in all directions across the solid surface until they meet neighboring "patches". This preferred embodiment has been demonstrated from multiple targets on flat surfaces such as glass slides, and from drilled semiconductor chips. The resulting clusters were analyzed using a variety of methods, including postamplification hybridization with fluorescently labeled probes, as well as sequencing with ISFET signal detection. Experimental demonstration of SM-RPA is summarized in Example J, "In-well Amplification of Target Nucleic Acids Using Solution-Mediated Recombinase Polymerase Amplification (SM-RPA)."

[0064] c. Cluster generation using branched surface-phase rolling circle amplification (RCA) Disclosed herein is a method of rolling circle amplification and related compositions that can be used to generate clonal nucleic acid clusters directly from multiple targets on a solid support. In a preferred embodiment, the addition of adaptors required for ligation and amplification is completed transparently during other parts of the workflow. Furthermore, the method allows for the creation of circularized templates directly on the surface of a flow cell where a ligation splint oligomer acts as the first amplification primer. All these aspects simplify and make the workflow more efficient, while reducing the overall workflow duration, creating advantages over current ostensibly RCA methods.

[0065] FIG. 17 shows a preferred embodiment in which the creation of circularized templates for use in clonal amplification is performed transparently over three distinct process steps (see figure legend for explanation). FIG. 18 shows the actual branched surface-phase RCA clonal amplification process (see figure legend for explanation). This method produces separate clusters of clonally amplified DNA composed of concatemeric repeats of the target sequence, providing a high signal-to-noise ratio. More notably, both strands of the target molecule sequence are generated by extension of two amplification primers. A specific experiment demonstrating this embodiment is summarized in Example K, "In-Well Amplification of Target Nucleic Acids Using Rolling Circle Amplification (RCA)."

[0066] d. Cluster generation using hybridization As described elsewhere herein, clusters can also be generated using non-amplification-based methods, such as hybridization of target molecules to oligonucleotides immobilized on a solid support at known specific regions (e.g., spots). In a preferred embodiment of the present disclosure, an array of target-specific oligonucleotides is spotted (immobilized) in wells on the top of a semiconductor chip. These target-specific oligonucleotides act as both capture oligomers and sequencing primers. Target polynucleotide sequences are flowed into the wells, forming monoclonal clusters in which target molecules of a given sequence specifically hybridize with complementary immobilized oligonucleotides. The clusters are then sequenced using the immobilized oligonucleotides as sequencing primers. In a preferred embodiment of the sequencing step, the semiconductor chip contains an array of ISFET sensors that serve as a detection mode in the sequencing reaction. A specific experiment demonstrating this embodiment is summarized in Example M, "Sequencing synthetic templates using a direct hybridization method."

[0067] Where applicable (i.e., in situations where this approach achieves the analytical / assay / testing objective), cluster generation using hybridization offers several distinct advantages. For one, no clonal amplification step is required, thereby typically saving steps, reagents and time. Furthermore, in some applications, no copy control step is required, again typically saving steps, reagents and time. In still other situations, the library preparation step can be simplified, e.g., less amplification is required, fewer or no tag / adapter steps are required, and purification steps can potentially be omitted. In situations where the target polynucleotide is abundant and the sample type produces little or no inhibition, it may even be possible to proceed directly from the sample to cluster generation and then analysis (e.g., sequencing). Also, if sequencing is the analytical method of choice, the target denaturation and sequencing primer annealing steps that are typically performed after clonal amplification are not required, since the capture oligomer also serves as a sequencing primer.

[0068] 6. Sequencing A preferred embodiment of the present invention performs sequencing using a semiconductor chip that includes an array of ISFET sensors (see, e.g., U.S. Pat. No. 7,686,929). The chip also typically includes an array of wells that are attached to the surface of the chip and positioned above the ISFET sensors. A preferred method of sequencing generally includes the following steps (sequencing by synthesis): 1) immobilizing the nucleic acid template to be sequenced in one or more wells (methods of immobilization in wells include clonal amplification and direct hybridization; see elsewhere for details); 2) annealing the sequencing primer to the template (in some embodiments, annealing of the sequencing primer is part of the immobilization step); 3) binding a sequencing enzyme (polymerase) to the template / sequencing primer complex (disclosed below is also a new method of adding the sequencing primer and enzyme in the same step); 4) flooding the well with a given dNTP (in some cases, multiple dNTPs can be included together in a single flow); 5) incubating (in some cases, the flow of dNTPs is stopped, in other cases, it is not stopped). If a complementary nucleotide is present in the template, the introduced dNTP will be incorporated and a proton (one per nucleotide incorporated) will be released, which will be detected by the ISFET below the well; 6) Wash; repeat steps 4-6 with each of the other dNTPs; this completes one "cycle" (other cycle configurations can be used); 7) Repeat the cycle to obtain the sequence of the template.

[0069] a. Simultaneous annealing of sequencing primers and binding of sequencing enzyme As summarized above, annealing of the sequencing primer to the nucleic acid template and binding of the sequencing enzyme to the resulting complex are performed in separate steps. This is because the annealing step (which typically also involves denaturation of the template, which is often double-stranded at this point in the workflow) requires high temperatures (up to about 95°C), at which the sequencing enzyme is not stable. We disclose a method to combine the two steps, including the use of a thermostable sequencing polymerase (e.g., Tin(exo-)LF DNA polymerase from Optigene). Briefly, all components required for sequencing primers and enzymes are combined in one reaction mixture with the template. The temperature is raised (e.g., to about 95° C.) to denature the double-stranded template and then lowered (e.g., to about 60° C.) to support annealing of the sequencing primer and subsequent binding of the sequencing enzyme to the primer / template complex (binding of the sequencing enzyme can also occur, at least to some extent, later in the process if the temperature is lowered further, e.g., between about 20° C. and 45° C.).

[0070] b. Sequencing after cluster generation by clonal amplification In a preferred embodiment of a sequencing workflow involving a semiconductor chip as described above, sequencing is performed after cluster generation by clonal amplification (see III.B.5 above and elsewhere herein). Also, as described elsewhere herein, cluster generation is followed by primer hybridization and enzyme-bound sequencing, followed by sequencing. In some embodiments, the sequencing primers can be universal for all potential targets, utilizing universal primer binding sites that are incorporated into the template during a previous step of the process (e.g., library preparation). Alternatively, multiple sequencing primers can be used to initiate sequencing from a specific target. An experiment illustrating this mode is summarized in Example N, "Sequencing of templates generated using in-well clonal amplification."

[0071] c. Sequencing after cluster generation by direct hybridization In another preferred embodiment of the sequencing workflow including the semiconductor chip as described above, sequencing is performed after cluster generation by direct hybridization (see III.B.5 above and elsewhere herein). Also, as described elsewhere herein, in the direct hybridization method, the immobilized capture oligomers also serve as sequencing primers. Thus, the steps of the method after template immobilization (hybridization) include enzyme-linked sequencing, followed by sequencing. An experiment illustrating this mode is summarized in Example M, "Sequencing synthetic templates using the direct hybridization method."

[0072] d.Key layout i. Introduction In semiconductor sequencing, the use of known DNA sequences to calibrate base calling parameters such as set thresholds for null incorporation events (i.e., "0-mers"), single base incorporation events (i.e., "1-mers") and homopolymer incorporation (i.e., "2-mers", "3-mers", etc.) is generally achieved by the use of a universal key sequence (see, e.g., Genome sequencing in microfabricated high-density picolitre reactors (2005) Marguilies, et al., Nature, 437:376-380). This known sequence can be incorporated into the template at an early stage of the workflow (e.g., adding tags / adapters during library creation). While such methods are commonly implemented, they typically add reagent costs, complexity and time to the workflow. Disclosed herein are two alternative methods for introducing key sequences into the sequencing workflow.

[0073] ii. Embodiment #1. Target-specific key sequence In the first approach, a section of the target itself can be used as a calibration key sequence. For example, if the upstream amplification step employs the use of primers to enrich for a particular region (AKA targeted enrichment), the sequence of the primer itself is usually known. In that case, it is possible to use a portion of the known sequence of the primer as a key sequence to determine thresholds and other signal processing parameters. This can be achieved by cleavage of oligonucleotides used as sequencing primers, i.e., surface-immobilized capture oligomers / primers in cluster generation followed by direct hybridization methods of semiconductor sequencing.

[0074] When an array of target-specific surface-immobilized capture oligomers / primers is used in a direct hybridization approach, each capture oligomer / primer can be truncated by x bases at its 3' end (i.e., the end distal to the surface). The value of x can be specifically determined for each oligomer based on the sequence. The truncated bases form the key sequence of each specific primer, hereafter referred to as the target-specific key sequence. Ideally, the target-specific key sequence will generate at least 0-mer, 1-mer and 2-mer outputs (see the "Introduction" paragraph above). However, it is highly unlikely that all primers, or indeed any primers, in a given panel will have a sequence capable of generating such outputs. Thus, the exact target-specific key sequence must be determined on a case-by-case basis according to the properties deemed most desirable for a particular signal processing approach.

[0075] Figure 19 shows an example of a basic target-specific key sequence. For Oligo 1, sections 1A and 1C respectively describe the forward and reverse primer binding sites used for targeted upstream amplification. Thus, the specific sequences of these sections are known. Oligo 1, section 1B describes the region of interest, i.e., the unknown section of the template to be sequenced. Note that for the purposes of this example, the actual sequences of Oligo 1, sections 1B and 1C are not relevant and therefore will not be described. Oligo 2 is designed to be complementary to Oligo 1, section 1A, notably with the exception of the nucleotide truncation at the 3' end. In the embodiment described herein, Oligo 2 is immobilized on a solid support. Oligo 1 is provided in solution, with its section 1A specifically hybridizing to Oligo 2, with the exception of the critical sequence nucleotide at the 5' end.

[0076] Table E below provides exemplary sequences for use in this embodiment. Note that the last base of the known primer sequence, i.e., the last base at the 5' end of section 1A of Oligo 1, may not be used as part of the key sequence, since this base may be identical to the first base of the unknown section to be sequenced (Oligo 1, section 1B). Thus, although it is known that there is an incorporation of that base, it is not known whether this is a monomer or homopolymer incorporation event, and therefore the incorporation event is not useful for threshold setting and may introduce errors if used incorrectly by itself.

[0077] [Table 1]

[0078] After 3' cleavage, the surface phase primer may no longer have the same properties important for hybridization with the template as before cleavage, such as nucleotide length, melting temperature (Tm) and GC content (%GC). In particular, the melting temperature is now reduced due to the removal of bases. If a panel of primers is cleaved to various amounts to provide optimal target-specific key sequences, the template hybridization kinetics and thermodynamics may change significantly such that the overall efficiency of hybridization may be reduced. If multiple templates are designed to hybridize to multiple surface capture oligomers / primers, the change in hybridization properties may result in different hybridization efficiencies across the panel. In this case, it may be necessary to further modify the surface capture oligomer / primer to counteract the effects of its 3' cleavage. This can be accomplished in several ways, including but not limited to: 1) the addition of bases at the 5' end, i.e. proximal to the surface, to increase primer length and increase melting temperature; 2) the use of nucleic acid analogs such as locked nucleic acid (LNA) or peptide nucleic acid (PNA) within the surface-bound capture oligomer / primer.

[0079] Figure 20 provides another similar example of the target specific key sequence approach. Oligo 3 sections 3A and 3C respectively describe the forward and reverse primer binding sites used for targeted upstream amplification. In this example, a tail primer approach is used during upstream amplification to extend Oligo 3 with an additional synthetic section 3D. This section is not target specific, therefore section 3D can be either (a) universal or (b) target specific, as required. In this method, the additional section 3D is specifically designed to negate the effect of the 3' truncation required to generate the target specific key sequence. Oligo 3, section 3B describes the region of interest, i.e., the unknown section of the template to be sequenced.

[0080] Oligo 4 is designed to be complementary to Oligo 3 and contains complementarity to both the target specific portion of Oligo 3 (Oligo 3, section 3A) and to a further portion (Oligo 3, section 3D). Truncation at the 3' end allows for the generation of a target specific key sequence. In the embodiment described here, Oligo 4 is immobilized on a solid support. Oligo 3 is provided in solution, and its sections 3A and 3D specifically hybridize to Oligo 4, except for the critical sequence nucleotides at the 5' end.

[0081] Table F below provides exemplary sequences for use with aspects of this embodiment shown in FIG.

[0082] [Table 2]

[0083] iii. Embodiment #2. Fixed Universal Key Sequence In the second approach, an additional 5' portion of the surface-immobilized capture oligo / primer can be used to generate key sequences by temporarily preventing polymerization from the 3' end of the surface-immobilized oligo while allowing polymerization from the 3' end of the hybridized template.

[0084] In this embodiment, the 3' end of the surface-bound capture oligomer / primer is not cleaved but instead equipped with a reversible blocking chemical group to prevent polymerization. Examples of such blocking groups include, but are not limited to, 3'-O-(2-nitrobenzyl), 3'-hydroxyamine and 3'-O-azidomethyl. At the 5' end, the surface-bound capture oligomer / primer contains an additional known sequence associated for use as a key sequence (see above description of possible key sequence elements). Because this known sequence is additive, it has at most a limited effect on the specificity of template hybridization to the sequence-specific portion of the surface-bound capture oligomer / primer. It may therefore be common or universal to all surface-bound capture oligomers / primers in the panel. During template hybridization, a sequencing polymerase enzyme is added. Because the 3' end of the surface-immobilized capture oligomer / primer is blocked, sequencing will not begin from this end. However, the 3' end of the template is not modified, and therefore the sequencing reaction is initiated using the additional 5' portion of the immobilized capture oligomer / primer as a template. The sequencing output from this additional 5' section of the surface-bound capture oligomer / primer serves as a universal key sequence for setting the associated signal processing and base calling parameters.

[0085] Once sequencing of the 5' fragment is complete, the 3' blocking moiety is reversed / removed using an appropriate method. For example, the 3'-O-(2-nitrobenzyl) group can be photocleaved by exposure to 340 nm light, the 3' hydroxylamine can be deblocked with aqueous sodium nitrite, and the 3'-O-azidomethyl can be removed by reduction with tris(2-carboxyethyl)phosphine. After removal of the blocking moiety, additional polymerase is added to ensure that all primer-template complexes are bound by polymerase, and sequencing is resumed from the unblocked 3' end of the capture oligomer / primer bound to the surface. The sequencing data thus generated is analyzed using signal processing and base calling parameters that were set using data from the preceding sequencing of the universal 5' section of the capture oligomer / primer itself.

[0086] A schematic diagram of this second key sequence embodiment is shown in Figure 21. In step (a), the sequencing target oligo 5 specifically hybridizes to the perfectly complementary section 6A of oligo 6. Section 6A can be identical to the primer used upstream in the workflow to amplify the target. Oligo 6 is reversibly blocked at the 3' end, preventing extension. Upon addition of polymerase, the 3' end of oligo 5 can polymerize using section 6B of oligo 6 as a template. Section 6B can be designed to be used as a key sequence so that 0-mer and 1-mer as well as other nucleotide incorporation events are reported as needed as sequencing progresses through this section. Once sequencing of section 6B is complete, the blocking portion at the 3' end of oligo 6 is removed, as in step (c). Upon further addition of polymerase, if necessary, sequencing now proceeds from the 3' end of oligo 6 through the unknown target region of oligo 5.

[0087] 7. System Disclosed throughout are embodiments useful for rapid analysis of target polynucleotides, including determining their nucleotide sequence from a wide range of input sample types and quantities using an automated system. The system comprises an apparatus and at least one assay cartridge removably insertable into the apparatus. In preferred embodiments, the system further comprises at least one reagent cartridge removably insertable into the apparatus. In further preferred embodiments, the system comprises a semiconductor chip, and in some embodiments, the chip is embedded within the cartridge. In particularly preferred embodiments, the system further comprises a flow cell mounted on top of the chip (with or without wells) useful for delivering and removing fluids to and from the chip. In preferred embodiments, the system comprises software. In particularly preferred embodiments, the software comprises operational software (for controlling the system) and analytical software (for receiving, processing, and analyzing the output of the system).

[0088] A semiconductor chip The use of semiconductor chips containing field effect transistor (FET) arrays to detect chemical and / or biological reactions, including sequencing reactions, is well known in the art (e.g., U.S. Pat. Nos. 7,686,929; 8,685,228; 8,986,525; U.S. Patent Application Publication No. 2010 / 0137143). In the present disclosure, preferred embodiments include semiconductor chips containing ion-sensitive field effect transistor (ISFET) arrays useful as detection devices for various reactions, including nucleic acid sequencing reactions. In particularly preferred embodiments, the chip further comprises an array of wells disposed above and in fluidic contact with the ISFET array. In these embodiments, the sequencing reactions typically occur in the wells, and the release of ions is detected by the ISFET sensor. In particularly preferred embodiments, the chip further comprises a flow cell attached to the top of the chip (with or without wells) useful for delivery and removal of fluids to and from the chip / ISFET array. In a preferred workflow / system embodiment, the chip is integrated into a cartridge in which the entire workflow is carried out.

[0089] b. Assay cartridge As used herein, the term "assay cartridge" or "sample cartridge" refers to a device in which the steps of a particular test, assay, or segment thereof are performed. Typically, such devices comprise chambers that are fluidly connected to one another to various degrees. Exemplary designs of assay cartridges useful for performing the entire sequencing workflow from sample input to result are disclosed. A wide variety of sequencing-based assays as well as other complex assays can be performed within such disclosed cartridges. In one set of embodiments, different stages of the workflow are performed in "subsystem" cartridges, and in another set of embodiments, all stages of the workflow are performed in an integrated cartridge. In some cases, assays can be performed in conjunction with a reagent cartridge (in which the exemplary designs are also enclosed). Typically, tests are performed in such cartridges in conjunction with an instrument, but in some cases, tests and cartridge configurations that do not require or require minimal automation can be performed.

[0090] i. Subsystem Cartridge Exemplary designs are disclosed for sample preparation (see, e.g., Figures 22-24), library preparation (including copy control; see, e.g., Figures 25-26), and cluster generation / sequencing (see, e.g., Figures 27-28). In some cases, these processes (sample preparation, library preparation, cluster generation / sequencing) may be performed individually or in various combinations depending on the overall target and requirements of the test being performed. When two or three cartridges are used for a given test, the output of one cartridge can be manually transferred to the next cartridge, or the transfer can be performed automatically using an instrument.

[0091] Key features of the sample preparation cartridge design include, but are not limited to, 1) a flexible input sample system capable of accommodating relatively large sample volumes; 2) a uniquely designed chamber layout that allows for accommodation of relatively large sample and assay volumes within a relatively compact cartridge; 3) a fully integrated rotary valve that can draw and deliver fluids to multiple chambers and channels; 4) separate but connected appendages or "fins" that can perform complex operations while providing flexible cartridge designs across assay types; 5) serpentine channels for improved heating and magnetic separation characteristics; 6) chambers and associated features that allow for storage of both liquid and dry reagents on the cartridge; and 7) a large number of chambers, again within the context of a relatively compact cartridge, thus allowing numerous variations of sample preparation protocols to be performed.

[0092] Important features of the library preparation cartridge include, but are not limited to, 1) two built-in rotary valves to support complex assay flow with fluid delivery and recovery in multiple combinations of chambers and channels; 2) multiple amplification chambers / stations to allow multiple reactions to accommodate complex workflows, diluted samples, high levels of multiplexing, etc.; 3) chambers and related features to accommodate the addition of tags / adapters; 4) chambers and features to accommodate copy control; 5) separate but connected appendages or "fins" (one or more additional fins can be easily added) that can perform complex operations while providing a cartridge design that is flexible across assay types; 6) serpentine channels to improve heating and magnetic separation characteristics; 7) chambers and related features to allow for storage of both liquid and dry reagents on the cartridge; and 8) multiple chambers, again within the context of a relatively compact cartridge, and thus capable of implementing multiple variations of library preparation protocols.

[0093] Important features of the cluster generation / sequencing cartridge include, but are not limited to, 1) a fully integrated rotary valve that can pick up and deliver fluids to multiple chambers and channels; 2) in addition to the rotary valve, an on-board "selector" valve to provide even greater flexibility and options for fluid delivery; 3) multiple chambers in a relatively compact format that can support multiple forms of cluster generation and sequencing; 4) chambers and associated features that allow both liquid and dry reagents to be stored on the cartridge; 5) full fluidic connectivity to a flow cell (in a preferred embodiment, equipped with a semiconductor tip; see, e.g., FIG. 29) for delivery and retrieval of fluids from the solid support; and 6) full fluidic connectivity to a reagent cartridge (see elsewhere in) for delivery of large quantities of required fluids, such as sequencing reagents (e.g., dNTP solutions), if selected as an option (see, e.g., FIGS. 30, 31 and 32).

[0094] Details of one particular assay performed across the three subsystem cartridges described above, in this case detection of pathogens from blood, are provided in Example O, "Automated Sample-to-Answer Sequencing of Pathogens Spiked into Whole Blood."

[0095] ii. Built-in cartridge Also disclosed are exemplary designs of fully integrated cartridges capable of performing all steps of the workflow including sample preparation, library preparation, copy control, cluster generation and sequencing (see, e.g., Figures 33-39). When coupled with reagent cartridges and devices (see elsewhere in ), the entire next generation sequencing workflow can be performed from sample to result in a fully automated format without user intervention once a run has begun. This is done quickly in a relatively small size cartridge and device system making it useful in a wide variety of settings. This has not been achieved in the art thus far and therefore represents a new and novel system and associated workflow.

[0096] The integrated cartridge design includes several key features. Below is a brief description of some of these features. The three-dimensional design of the cartridge was chosen to accommodate many of the required features in a relatively small footprint / volume. Two form factor options for such a cartridge are shown in Figure 33. Both provide good functionality in a relatively small volume. The design shown on the left offers a narrower form factor that reduces the width of the device without increasing the depth of the device. Rotating the STC fins and associated thermal interface inward also reduces the width of the device. The ergonomics for cartridge installation into the device are ideal for both form factors.

[0097] In some preferred embodiments of the cartridge, fluid handling includes [direct] pneumatic and precision pipetting using a miniature 3 degree of freedom (DOF) gantry located on the associated device (see, e.g., FIG. 40). Pipette tip access to the chambers / channels in the cartridge is enabled via a sealed pneumatic interface (SPI) port (see FIG. 41). The SPI port can be configured with a cap 6701 as shown in FIGS. 67 and 68 to align the tip with the SPI port. The inner diameter of the opening in the cap 6701 can be fixed to fit the tip size specified for the fluid transfer process (e.g., 1 mL tip in FIG. 67 and 5 mL tip in FIG. 68). The SPI port can include a stepped pipette tip interface 6703 as shown in FIGS. 67 and 68 to accept and seal pipette tips of different sizes. As shown in the exemplary design in FIG. 33, the SPI valves are clustered closely together to minimize the range of movement required for the 3DOF gantry, thus reducing the engineering complexity, cost, and real estate usage of the device. Access to the SPI valve for the pipette tip is made possible through an opening in the cartridge body directly above the valve (see diagram). This opening is covered until use with a puncturable foil seal to prevent contaminants from entering the cartridge. An additional round hole is shown in the diagram, which is for staging of the pipette tip to be used in the assay. The sample input port (2 shown in Figure 33) is located to allow access to the top of the cartridge. Two cylinders that house vacutainer tubes (among other sample tubes) are shown, indicating that the system can accommodate large volumes of input sample. Furthermore, each sample port contains a separate SPI, allowing access to either a sample or a combination of samples at any point in the assay. Furthermore, the sample ports are located in close proximity to the lysis chamber, minimizing the sample migration path to the lysis chamber when housed within a chemical workflow.

[0098] FIG. 34 illustrates the cartridge features in further detail. As described elsewhere herein, the functionality associated with each of the features in this exemplary diagram is based on a particular assay or assay type, but the functionality is flexible and easily reused, reprogrammed, or otherwise adjusted to accommodate a wide variety of applications. The cartridge is designed with sub-module parts, each with a designated function or combination of functions. This is less complicated and more cost-effective to manufacture and load reagents. It also allows for easier modification of the cartridge for different applications (e.g., designing a new cartridge for an application with a separate sample preparation step involves redesigning and manufacturing only the sample preparation sub-module compared to the entire cartridge). The different sub-module sections are then easily assembled into a single, integrated cartridge. It should also be noted that additional benefits in using a pipettor for reagent delivery include the ability to achieve simple sample dilution in the pipette tip, effective mixing by pipetting reagents in and out of the chambers / channels (multiple times if necessary), delivery of pressure to the chambers / channels to drive fluid movement (pipette tip inserted into SPI and "dispensed" air), etc.

[0099] 35-39 show some of the basic steps of a workflow / assay (e.g., pathogen detection in blood) performed on the cartridge. The figure legend summarizes these basic steps. As noted in the legend, not all parts of the cartridge are shown in this figure to facilitate viewing of the featured components. Also, not all steps of the workflow are summarized. Note that assay chemistry / reactions can occur in the channels as well as in the chambers. Also note that the cartridge is designed to be highly flexible and can perform all process steps of many different workflow configurations. Additionally, as mentioned above, in some cases, one or more of the sub-module fins in the integrated cartridge can be interchanged to accommodate an even wider range of workflows and applications.

[0100] Another exemplary assay or sample cartridge is shown in FIG. 60 and FIG. 63-65, highlighting the major fluid handling subsystems, units, or "fins." FIG. 63 shows an exemplary library preparation unit 6300 or PCR fin with copy control functionality. FIG. 64 shows an exemplary sample input and mechanical lysis fin 6400, and FIG. 65 shows an exemplary specific target capture (STC) fin 6500. The STC fin 6500 can include thermal zones for heating as needed during the target capture and elution steps. In certain embodiments, thermal energy may be applied to the STC fin 6500 from one side (e.g., from the interior of the cartridge). The library preparation unit 6300 can include double-sided heating for PCR and copy control thermal steps. The STC fin 6500 of FIG. 65 is shown in more detail in FIG. 69. The STC chamber 6901 is used for sample preparation heating and mixing, and in the illustrated embodiment may be about 9.3 mL in volume and may be designed to hold a volume of up to about 6 mL. Additional fluidic functions of the STC fin 6500 are performed using an elution chamber 6903 for elution heating and an auxiliary chamber 6905 that can be used for PCR1 dilution, PCR2 pooling and copy control dilution if applicable. A lyophilized reagent pocket 6907 allows for loading after fin construction and can be arranged to seal with a film. The STC fin 6500 can include a condensation trap 6909 to contain any condensation that forms during the heating and mixing steps within the STC fin. The STC chamber inlet channel 6911 feeds the STC chamber 6901 and maintains the fluid within the heated region during the mixing and heating steps. The STC inlet air pressure line 6913 allows for distribution of air through the STC inlet, for example to push fluid back into the STC chamber 6901 without the use of a pipette tip. The STC and elution serpentine channel 6915 is included for the magnetic bead capture step.

[0101] The exemplary library preparation unit 6300 or PCR fin of FIG. 63 is shown in more detail in FIG. 70. Various thermal chambers 7001 are included as needed for PCR thermal cycling and direct hybridization elution steps. Thermal chambers 7001 are positioned to allow heating from both sides of the cartridge in the device for faster thermal gradients required for PCR amplification. Lyophilized reagent pockets 7005 can allow for loading of required reagents after fin construction followed by sealing with film. Direct hybridization chambers 7003 can be included for mixing and incubation (at room temperature if specified) for assays using direct hybridization methods. Optionally, a direct hybridization magnetic serpentine 7013 can also be included for magnetic bead capture. The library preparation unit 6300 can include a PCR2 bypass channel 7011 to allow for direct loading of the PCR2 channel from a single SPI valve. A PCR optical sensor 7007 and metering control 7009 can be included for closed loop control used for fluid positioning and metering.

[0102] The sample input may include an opening or docking interface 6403 for receiving a sample container, such as a vacutainer or vial, to allow the cartridge to take up a sample for the assay. If the assay uses mechanical lysis (e.g., via a rotating paddle), a mechanical interface 6405 may be included, allowing the device to drive the lysis unit, for example, via a motor and shaft that operably couples to the interface when the cartridge is inserted into the device. The sample input, mechanical lysis, and STC steps may generally be combined in a sample preparation step, and their functions may be part of a sample preparation unit in the sample cartridge. After processing in the library preparation unit (as illustrated in FIG. 63), the amplified nucleic acids may be directed to a sequencing unit, including, for example, a flow cell as described herein, for sequencing and analysis of the target nucleic acids isolated and amplified from the original sample. An exemplary sequencing unit / flow cell 6600 is shown disposed within the sample cartridge in FIG. 66. The sequencing unit / flow cell 6600 may be heated from below the cartridge as necessary for any sequencing steps. Additionally, FIG. 66 shows an exemplary pipette reservoir 6603 within the assay or sample cartridge. As described herein, various fluid transfer operations within the sample cartridge between a reagent cartridge or other external source and the sample cartridge may be performed automatically by the device, for example, using a pipetting gantry and SPI port as described below. Inclusion of the necessary pipette tips within the sealed cartridge that fit the required volumes and SPI ports used in the system increases ease of operation while reducing the risk of user error or contamination.

[0103] c. Reagent cartridge In a preferred embodiment, a separate reagent cartridge is utilized along with the assay cartridge to perform a given test / assay. Exemplary designs for the reagent cartridge are shown in Figures 42-44. Fluids can be accessed and moved in several ways, including via a liquid handler (LH; e.g., a pipette system) and a liquid manifold (LM). A variety of valving methods are applicable, including sealed pneumatic interface (SPI) ports. Reagents stored in the reagent cartridge may include liquid and dry reagents, assay-specific and general-purpose reagents, bulk reagents (reagents that are typically required in larger quantities), as well as other reagents and components as needed (e.g., CO2 of selected reagents). 2 Soda lime for use in scrubbing). Furthermore, the body of the reagent cartridge provides a relatively large volume that can be efficiently used for liquid waste generated during the performance of the test / assay. Furthermore, the number of reservoirs / units / wells / chambers can be varied as needed to meet the requirements of a given or set of tests / assays. Also, a given configuration of a reagent cartridge can be filled with a variety of reagents supporting several different tests / assays, although not all reagents are used for all tests.

[0104] There are several advantages to using separate reagent cartridges, including but not limited to the following: 1) Storing reagents in separate reagent cartridges significantly reduces the size and complexity of the assay cartridge (particularly evident when considering the volumes required for bulk reagents such as sequencing reagents), thus improving the efficiency of the assay cartridge and reducing manufacturing costs. 2) Assay and reagent cartridges can be manufactured, filled and stored separately, reducing complexity and costs and increasing efficiency. 3) Preparation and storage of dry reagents is much more efficient when allocated to the reagent cartridge compared to the assay cartridge, especially if section B of the reagent cartridge (used for dry reagents; see FIG. 43 and associated figure legends) is manufactured, filled, dried and stored in isolation. 4) Preparation and storage of liquid reagents is more efficient when allocated to the reagent cartridge compared to the assay cartridge, especially if section A of the reagent cartridge (used for liquid reagents; see FIG. 43 and associated figure legends) is manufactured, filled and stored separately. 5) In some embodiments, the same reagent cartridge can be used with different assay cartridges. 6) The reagent cartridge can be rapidly assembled with sub-component parts for a given test / assay specific application (e.g., but not limited to, sections A and B filled, dried (if applicable), stored separately, and bulk reagents filled separately (e.g., for sequencing) so that the sub-component parts can be most efficiently manufactured and stored, and can be manufactured in quantities to meet demand. 7) Dried reagents can be reconstituted directly in the reagent cartridge prior to transfer to the assay cartridge. 8) In preferred embodiments, the majority of the reagents in the reagent cartridge are in dry form, such that the majority of the liquid in the reagent cartridge, and in some embodiments all of the liquid, is water. 9) In preferred embodiments, one or more of the chambers in the reagent cartridge include a magnetic stir bar that interfaces with a magnetic stir motor in the device when the cartridge is loaded into the device (e.g., useful for on-board preparation / mixing of reagents). Another exemplary reagent cartridge is shown in FIG.

[0105] d.Device A wide variety of cartridge embodiments are disclosed in section (e) immediately above and elsewhere herein. In this section (and elsewhere herein) are disclosed device embodiments useful for automating the execution of tests / assays in the cartridge. Examples of device design and functionality are shown in Figure 40, Figures 45-49 and associated figure legends. The device is designed to have a relatively small footprint and is useful in a multitude of settings. Furthermore, all of the functions required to execute a complete complex workflow, including nucleic acid sequencing in the preferred embodiment, can be automated in cartridge format from sample entry to final report, all without user intervention once execution is initiated. In preferred embodiments, the device is equipped with a compact 3 degree of freedom (DOF) pipetting gantry that is utilized to execute a variety of functions including mixing, diluting, reconstituting (dried reagents) and moving fluids / reagents as well as delivering to the cartridge. As noted elsewhere in, in preferred embodiments, pipette tip access to the chambers and channels is via an SPI port. In a preferred embodiment of the cartridge, the SPI valves are clustered closely together to minimize the range of movement required for the 3DOF gantry, thus reducing the engineering complexity, cost, and real estate use of the device. The device design includes one or more of the following capabilities: heating, cooling (including for the CPU), magnetic separation, magnetic stirring, rotation of the lysis impeller (for mechanical lysis in the cartridge), generation and controlled use of pressurized gas (pneumatic system), sensing (e.g., temperature, pressure, flow rate, fluid level, etc.). It also includes full CPU / computer control of functions and features, including collection and analysis of output data such as sequencing signals from semiconductor chips with ISFET arrays. Other features are highlighted in the figures and associated legends.

[0106] Another exemplary device and components therein are shown in Figures 58A-59. Figure 58A shows a perspective view of an exemplary device with a display / user interface, barcode scanner, and analytical and reagent cartridge doors are shown. The dimensions of the exemplary device are shown in front and side views in Figures 58B and 58C. The layout of various internal subsystems of the exemplary device is shown in Figures 58D-58I. A cartridge interface assembly for receiving and interfacing a sample or assay cartridge and a reagent cartridge is shown in Figure 58D. The cartridge interface can include a door that can be opened or closed manually or automatically to allow insertion of the cartridge by the user but to allow a closed and controlled environment for analytical processing after insertion. In various embodiments, the cartridge interface can include fluidic and electronic connections to allow the device to control the movement of fluids within the cartridge and to communicate with the cartridge and various units therein (e.g., control of sequencing and receiving sequencing data for processing). In certain embodiments, the fluidic control can be pneumatic. Figure 58E shows an exemplary pneumatic pumping subunit located within the device for providing air pressure controlled by the analyzer or device to drive fluid movement within the cartridge. Such a pneumatic subsystem is further shown in FIG. 59 and can include syringes of various sizes (e.g., macro and micro) to allow for bulk fluid movement as well as precise control thereof. FIG. 58F shows an exemplary power subunit arrangement for providing power to the device. FIG. 58G shows an exemplary air handling and reagent cartridge air intake subsystem for controlling and handling any air entering the device and cartridge. FIG. 58H shows a liquid cooling subsystem for providing thermal management, for example, to cool a processor or other heat generating unit within the device. FIG. 58I shows an exemplary condensation management subsystem arrangement for further control of the environment within the system.

[0107] 8. How to use the system As described elsewhere herein, prior art sequencing workflows (the processes required to prepare target polynucleotides contained in a sample for sequencing, perform the sequencing, and analyze the resulting data) are laborious, time-consuming, complex, and often expensive. Many of the steps are still performed manually and require highly skilled personnel. Even when certain processes within the workflow are automated, multiple devices and ancillary components are required, and skilled human intervention is required at various points to perform the entire workflow. Also, the time from sample to result is several hours to days or more. Furthermore, the maximum allowable amount of sample input is low, which represents a further current limitation. Thus, the capacity and value of sequencing, including next generation sequencing, may be significantly reduced in practical use. Thus, there is a need for a sequencing workflow that is fully automated (sample to report), does not require user intervention once execution is initiated, is rapid (sample to actionable result in a few hours), is sensitive, accurate, cost-effective, and easy to use at the point of need.

[0108] Disclosed throughout are embodiments useful for just such rapid analysis of target polynucleotides, including determining their nucleotide sequence from a wide range of input sample types and quantities using an automated system. Also as disclosed elsewhere herein, the system comprises an apparatus and at least one assay cartridge removably insertable into the apparatus. In preferred embodiments, the system further comprises at least one reagent cartridge removably insertable into the apparatus. In further preferred embodiments, the system comprises a semiconductor chip, and in some embodiments, the chip is embedded within the cartridge. In particularly preferred embodiments, the system further comprises a flow cell mounted on top of the chip (with or without wells) useful for delivering and removing fluids to and from the chip. In preferred embodiments, the system comprises software. In particularly preferred embodiments, the software comprises operational software (for controlling the system) and analytical software (for receiving, processing, and analyzing the output of the system).

[0109] In this section, several methods of using the above-described system are disclosed. These are exemplary and are not intended to limit the scope of potential methods and related applications. Specific examples of the implementation of some of these methods of using the system are disclosed in the "Examples" section below.

[0110] a. Example general workflow overview As stated above and elsewhere therein, workflow refers to the processes required to prepare target polynucleotides contained in a sample for sequencing, perform the sequencing, and analyze the resulting data. More specifically, preferred embodiments may include one or more (in various combinations) of the following steps: sample processing; library preparation; copy control; cluster generation; sequencing; data acquisition; primary, secondary, and tertiary data analysis; assay calling (to perform tests / assays to answer questions answered); and report generation. The disclosed cartridge(s) and device(s) present a new and novel solution to a previously unsolved problem, namely, complete end-to-end automation of all steps of the workflow with short time-to-result and within the limits of a relatively small device (including cartridge) footprint.

[0111] As a first step, a sample containing a target polynucleotide is introduced into the cartridge. In preferred embodiments, this is accomplished via a sample input port on the cartridge. Additionally, the cartridge includes components incorporated into or attached to the cartridge to help facilitate sample transfer in a safe, efficient, and contamination-free manner. For example, in some embodiments, the cartridge includes a cylindrical structure into which a tube containing a sample (e.g., a standard vacutainer tube) is inserted. In preferred embodiments, placed at the bottom of the cylinder is a needle that is in fluid contact with at least one chamber in the cartridge via the input port. In these embodiments, the sample tube can be placed, for example, top-down into the cylinder and pushed onto the needle, whereby the needle penetrates the cap (forming a seal around the needle and maintaining a seal between the cap and the tube) and the contents of the tube, or a portion thereof, are transferred to the cartridge. Additional exemplary components that help facilitate sample transfer include, but are not limited to, a luer lock; an external chamber / container (fluidically connected to at least one chamber in the cartridge via a port) into which a liquid sample can be introduced (e.g., via pipetting) and then sealed (e.g., via a lid) such that the contents of the chamber / container are transferred to the cartridge when the cartridge is inserted into the device; a pierceable septum that allows a sample to be inserted into the cartridge via a syringe with a needle that pierces the septum; In some cases, an unprocessed sample is loaded into the cartridge. In some cases, the sample has undergone a user-selected pre-processing (e.g., processing performed in a sample collection tube or processing routinely performed for a particular sample type as standard practice prior to analysis). In some cases, the sample is the output of other user selected methods such as the output of cell culture, cloning and expression, amplification, nucleic acid extraction, expression of a swab in transport medium, liquefaction / homogenization of a solid sample, concentration of a sample (including a sample in a gas such as air) medium, cell lysis, separation (e.g., phase separation, sedimentation, centrifugation, fractionation (e.g., whole blood into plasma or serum, buffy coat and red blood cells), filtration, etc.Further off-board conventional processing methods include lysis of the organisms in a detergent-based liquid (e.g., as a transport medium in a collection tube), lysis of the organisms by vortexing the sample containing the organisms with or without the presence of beads, lysis by freeze / thawing in the collection tube, solubilization of the sample in detergents, chaotropes, organic solvents, denaturants, etc., with or without application of heat and / or stirring / vortexing, etc. These are just examples of the range of options, but the cartridges and devices are designed to perform tests / assays directly using the primary sample. The external components (if used) / sample inlet ports / cartridges can accept a wide range of sample input volumes, e.g., in some cases low microliters to 1 milliliter, in some cases 2-10 mL, in some cases 4-20 ml, and in some cases even larger sample input volumes. Similarly, the cartridges and devices are designed to accommodate this volume range and successfully perform the desired tests / assays. This is accomplished in several ways, including unique chamber designs (including combinations of large and small chambers, as well as uniquely shaped chambers), dynamic fluid control, processing performed "on the fly" as fluids pass / through / through processing elements (e.g., heaters, magnets, etc.), superior mixing capabilities, etc. This unique ability to accommodate such a wide range of sample volumes (including relatively high volumes) fills a need in the art and distinguishes the present disclosure from the prior art.

[0112] In some embodiments, once loaded into the cartridge, the sample undergoes various potential processing steps to prepare it for further downstream processing and / or analysis (AKA, sample preparation). In other embodiments, for example, if the sample matrix itself is relatively uncomplex and the target polynucleotides are already in a form suitable for further processing and / or detection, the sample can bypass the sample processing steps and move to a later step in the overall workflow, such as library preparation. In some tests / assays, the sample type is whole blood and the target polynucleotides are intracellular, e.g., within a pathogenic organism that infects the blood (as in sepsis, for example). In preferred embodiments, sample preparation includes the following general steps / processes: 1) Mix the blood with reagents that aid in homogenizing the sample and lysing the cells. 2) Heat the sample (with or without continuous movement and / or mixing). This helps to solubilize the sample (in further preferred embodiments, the method includes turbulent mixing. Features within the chambers / channels of the cartridge can optionally be included to improve turbulent mixing, such as three-dimensional features such as pillars, narrow junctions between mixing chambers, etc.). In some further preferred embodiments, the reagents include an enzyme such as proteinase-K that enzymatically degrades components of the sample (heating aids in activating the enzyme in the case of proteinase-K). 3) Lyse the cells. This can be accomplished using a number of methods (examples listed elsewhere in ). In one particularly preferred embodiment, lysis is accomplished using mechanical lysis, which involves mixing the sample at relatively high speed in the presence of beads. The cartridge is uniquely designed to incorporate a large volume mechanical lysis chamber with an impeller that engages with the motor of the instrument when the cartridge is loaded into the instrument. 4) Release and denature the nucleic acids. Depending on the cell type and the exact lysis method, the target polynucleotides may still be associated / bound / captured with features in the cell and / or the sample. Furthermore, the target nucleic acid may be in double-stranded form and must be rendered single-stranded (denatured) for the next step of the process to function properly. In a preferred embodiment, both release and denaturation are achieved by heating to a relatively high temperature (e.g., about 95°C).These processes can be further aided by including a reagent composition, such as a detergent, chaotrope or denaturant (or combination thereof), which / these can be included in the lysis reagent or can be added after lysis. Furthermore, heating to a relatively high temperature can be performed by heating the entire lysate as a whole or by heating portions of the lysate at a time, for example by running the lysate through a heated channel, for example a serpentine channel. 5) Isolate the target polynucleotide. A preferred embodiment is target capture one or more specific target capture oligomers (specific target capture, or STC). The denatured lysate is mixed with a hybridization reagent containing capture oligomers and the mixture is incubated at high temperature (e.g., at about 60° C.) during which the capture oligomers anneal to the target polynucleotides at a user-defined level of specificity (via the capture oligomer design). In some embodiments, the lysate is mixed with a hybridization reagent containing capture oligomers prior to release / thermal denaturation. The sample is then heated, for example to about 95°C for release / denaturation, and then the temperature is reduced, for example to about 60°C, for annealing of the capture oligomers. In a preferred embodiment, after annealing to capture the target polynucleotide / capture oligomer complexes onto the magnetic microspheres, the microspheres are retrieved from the mixture using a magnet and the remaining lysate is removed (sent to waste in the cartridge). Exemplary steps of how this is accomplished are included elsewhere herein. The beads are then optionally washed and the target polynucleotides are eluted. The target polynucleotides are now prepared for further downstream processing and / or analysis.

[0113] The sample preparation embodiments described above and discussed elsewhere herein have multiple advantages that distinguish them over the prior art, including, but not limited to: 1) the entire sample preparation workflow, including starting with a relatively large volume of a complex biological sample, such as blood, is performed in a fully automated fashion in a cartridge format, which is an integrated cartridge that is also used for the remainder of the test / assay (i.e., the prepared sample does not need to be transferred to a different cartridge, different device, etc. to continue the process); 2) complete mechanical lysis is achieved on the cartridge. 3) Complete release and denaturation of target polynucleotides even in large volumes is achieved on the cartridge in a short time frame by using an excellent heating technique to heat / incubate the sample as it passes through a cartridge-based system (channels in contact with an effective heating element, e.g., a serpentine channel). 4) The specific target capture configured in the disclosed cartridge composition confers many advantages in itself, including high purification efficiency, high volume reduction capacity, high specificity, and more broadly, high specificity control (specific capture along the entire phylogenetic tree, including at the subspecies, species, genus, family, etc. levels (by user-defined oligomer design; furthermore, oligomers can be designed to specifically exclude undesired polynucleotides in the sample, e.g., human DNA). 5) The STC method is highly extensible and supports high levels of multiplexing. Furthermore, the STC method is highly flexible in that new applications / assays can be easily and quickly developed by simply designing new oligo sets. 6) Only one purification step is involved in the entire process (sample preparation for next-generation sequencing typically involves multiple purification steps that add time, complexity and cost). 7) The isolated target polynucleotides of the entire eluted volume can proceed directly to the next step in the process (e.g., library preparation; in a typical next-generation sequencing workflow, the target polynucleotides must be quantified at this step, and only a portion of the prepared targets enter the library preparation process).

[0114] In some embodiments, after sample preparation (or, in some other embodiments, using the sample or a portion thereof directly, as described elsewhere herein), the sample undergoes various potential processing steps to prepare it for further downstream processing and / or analysis (commonly referred to as library preparation). In other embodiments, the sample can bypass the library preparation step and move to a later step in the overall workflow, such as, for example, cluster generation or sequencing. As mentioned above, in some tests / assays, the sample type is whole blood and the target polynucleotide is intracellular, e.g., within a pathogenic organism that infects the blood (as in the case of sepsis, for example). In these cases, the input to the library preparation is a target polynucleotide prepared from cells within a whole blood sample (examples of preferred sample preparation methods summarized above). In preferred embodiments, library preparation includes the following general steps / processes: 1) mixing the input sample with a first amplification reagent. In preferred embodiments, the first amplification reagent is stored on the cartridge (either in the assay or reagent cartridge) as a dry reagent and is reconstituted on the cartridge (using a liquid, e.g., water, stored in the cartridge (assay or reagent cartridge)). 2) A region of interest (ROI) in the target polynucleotide is amplified in a first amplification reaction. This increases the copy number of the ROI in the sample and increases the relative abundance of the ROI compared to other polynucleotides and / or polynucleotide regions in the mixture. In a preferred embodiment, the primers are designed to amplify a wide range of pathogenic organisms if present in the sample. For example, the primers are designed to amplify any bacteria or fungi in the sample, or a subset of each, depending on the requirements of the test / assay. This enriches these bacterial and / or fungal ROIs over human sequences or other potentially interfering sequences. In some embodiments, one or more tags / adapters are incorporated into the amplicon product. One exemplary amplification method is PCR. 3) The product of the first amplification reaction (AKA, PCR1 amplicon) is diluted. 4) An aliquot of the diluted PCR1 amplicon is amplified in a second amplification reaction. An exemplary amplification method is PCR (e.g., in this case, PCR2).In a preferred embodiment, at least one primer is nested compared to the corresponding primer in PCR1. This adds another level of specificity. In another preferred embodiment, multiple second amplification reactions are performed (e.g., PCR2.1, PCR2.2, PCR2.3; 2-10 or more second amplification reactions can be performed on the cartridge). Each of the second amplification reactions can use a different primer set than the first amplification reaction (to cover a broad range of target polynucleotides), or the same primer set as the first amplification reaction (e.g., to produce more end product), or any combination thereof across different second amplification reactions. Note that the first amplification reaction and / or one or more second amplification reactions can be configured to amplify more than one target (i.e., are multiplexed). In some embodiments, one or more tags / adapters are incorporated into the amplicon products of one or more second amplification reactions. 5) The amplicon products of all second amplification reactions are pooled together. 6) In some embodiments, the pooled products from the second amplification reaction are directly purified (while in other embodiments, a copy control process is performed first; see below). The output solution is mixed with a target capture reagent, the mixture is incubated, the desired amplicons are bound to paramagnetic beads, a magnet is used to immobilize the beads, the beads are washed, and the target amplicons are eluted. Details of such a procedure are given in Example O. In some embodiments, the amplicons are tagged with biotin (e.g., via a biotinylated primer). In such embodiments, the target amplicons are bound to streptavidin-coated magnetic beads in the incubation steps listed above. In some embodiments, the amplicons are provided with a common tag sequence, and a capture oligomer complementary to this common sequence is designed. In some embodiments, the capture oligomer is biotinylated. In such embodiments, the biotinylated capture oligomer binds to the amplicons containing the common tag sequence in the first incubation step.This reaction mixture is then combined with a second capture reagent comprising streptavidin-coated magnetic beads, and the resulting mixture is incubated in a second incubation reaction during which the amplicon / capture oligomer complexes are bound to the beads. In some embodiments, all components of the first and second capture reagents are in a single capture reagent, and the first and second incubations are combined into a single incubation.

[0115] As noted above, in some embodiments, a copy control (CC) protocol is performed on the pooled PCR2 amplicons. Acceptable CC protocols for use in the disclosed invention can utilize any one of the CC protocols disclosed in PCT / GB2021 / 050098 (various combinations of the disclosed features are also contemplated). Details of various CC compositions and methods are disclosed elsewhere herein. In a preferred embodiment, the pooled output from the PCR2 reaction is mixed with a CC reagent, including one or more CC oligomers. The mixture is then heated at about 92-95°C to denature the target amplicons. The mixture is then heated to promote annealing of the CC oligomer and complement (if present) and extension of the CC oligomer and target amplicon (the 3' end of the strand hybridized to the CC oligomer; examples of various heating schemes, as well as examples of compositions and reaction schemes are provided in the Examples section elsewhere herein and described in PCT / GB2021 / 050098). The resulting mixture is then mixed with capture oligomers and incubated to promote hybridization with the capture sequence incorporated in the target amplicon / complex.The resulting mixture is then mixed with magnetic beads and incubated, the bead / target complex is immobilized using a magnet, the beads are washed, and the target is eluted.In some embodiments, some, most, or all of the copy control process is performed simultaneously with one or more of the first amplification and / or second amplification reactions.

[0116] The library preparation embodiments (including copy control) described and discussed above and elsewhere herein have several advantages that distinguish them from the prior art, including, but not limited to, 1) in embodiments where the library preparation input sample is the output from the sample processing methods disclosed therein, the sample processing output material can be used directly without quantification or other characterization. In some preferred embodiments, the entire volume of the sample processing output is used as input, and in other embodiments, a portion of the output is used as input. In some embodiments, that portion of the primary sample is used as input material for library preparation. 2) The system can rapidly and effectively mix the initial input sample and the necessary reagents and other combinations of reactants throughout the process. 3) The method of target enrichment offers a wide range of test / assay applications (two sequential amplification reactions with nested priming of choice in the second amplification and seamless integration of the two reactions (including fully automated dilution, aliquoting, and distribution of aliquots into individual reaction chambers)) as well as flexibility (e.g., in some embodiments, only one amplification reaction is performed. In some embodiments, each of the separate second amplification reactions is configured (e.g., via primer design) to yield different selectivities, specificities, target sets, etc.). 4) High multiplexing capabilities. 5) High flexibility and ability to add tags / adapters. For example, tags / adapters can be added via tagging primers in any combination (e.g., in some embodiments, both PCR1 and multiple PCR2 reactions are performed. Each can be multiplexed, and each primer or primer set can contain the same or different tag as another primer or primer set). In other embodiments, tags / adapters can be added via ligation (in a fully automated system). In other embodiments, tags / adapters are added via a combination of ligation and the use of tagged primers. Furthermore, tagging can be combined to some extent with the copy control process to further simplify and streamline the overall process, thereby reducing workflow complexity and processing time.5) The system (cartridge + device) provides a novel method of diluting, aliquoting, and distributing a reaction mixture (e.g., an amplification reaction such as PCR1) into multiple new reaction mixtures (e.g., a second amplification reaction such as PCR2). 6) In a preferred embodiment, the entire process utilizes only a single purification step (e.g., "direct purification" for downstream use in the disclosed direct hybridization methods such as cluster generation, purification as part of the copy control process, etc.). Furthermore, no pre-analysis (e.g., quantification) of either the input sample (e.g., from the various disclosed sample preparation processes) or the first amplification reaction and its dilution is required. These features distinguish the disclosed method from the prior art, which requires multiple purification steps and [typically] pre-analysis steps. 7) The disclosed copy control process is novel and transparently and fully automated using the disclosed system. 8) The required reagents can be stored and eventually reconstituted (if necessary), mixed with other reactants in various combinations, and mixed with various methods within the assay and reagent cartridge, providing a unique and efficient method / pathway for reagent storage and use. 9) The entire process is fully integrated and automated by the system. 10) The process is quick.

[0117] In some embodiments, after library preparation (with or without copy control), the sample undergoes cluster generation on the surface of the solid support. In other embodiments, the sample can bypass the library preparation step and move directly to cluster generation. In a preferred embodiment, cluster generation is performed on the surface of a semiconductor chip. In a further embodiment, the semiconductor chip comprises an array of ISFET sensors. In yet a further embodiment, the surface of the semiconductor chip comprises wells. In some aspects, cluster generation involves direct hybridization of target polynucleotides to an array of specific target capture oligomers on the surface of the solid support. In some embodiments thereof, the capture oligomers can also function as sequencing primers if the analytical method is sequencing. An exemplary method for cluster generation using a direct hybridization method (and in this case followed by sequencing) is summarized in Example M. In other methods, cluster generation involves clonal amplification. An exemplary method for clonal amplification using recombinase polymerase amplification (RPA) is summarized in Example J. An exemplary method for clonal amplification using rolling circle amplification (RCA) is summarized in Example K.

[0118] The cluster generation embodiments (including copy control) described and discussed above and elsewhere herein have multiple advantages that distinguish them from the prior art, including but not limited to: 1) the output of the library creation step (with or without copy control) can be used directly in the cluster generation method. In some embodiments, the output material (solution) is transferred directly into a flow cell that covers the solid support. In this case, the volume of the flow cell defines how much of the output solution is utilized in the cluster generation step. The output solution does not require additional manipulations such as quantification, dilution, aliquoting, etc. 2) In a preferred embodiment, the cluster formation is performed directly on the surface of the semiconductor chip, on which the sequencing is also performed. This is unprecedented in the prior art. 3) As described and discussed elsewhere herein, the disclosed method of performing clonal amplification using RPA is novel and distinct from the art. 4) As described and discussed elsewhere herein, the disclosed method of performing circularized template formation (starting at the library preparation stage) and clonal amplification using RCA is novel and distinct from the art. 5) The entire process is fully integrated and automated by the system. 6) The process is rapid.

[0119] In some embodiments, after cluster generation, target nucleic acid is analyzed by sequencing. In a preferred embodiment, sequencing is performed on the surface of a semiconductor chip. In a further preferred embodiment, the semiconductor chip comprises an array of ISFET sensors. In a further preferred embodiment, the surface of the semiconductor chip comprises wells. Exemplary methods for sequencing using a semiconductor chip that comprises an ISFET sensor array and further comprises wells on its surface are summarized in Examples L (sequencing synthetic templates directly immobilized on the chip surface), M (sequencing synthetic templates using direct hybridization method), N (sequencing templates generated using in-well clonal amplification) and O (automated sample-to-answer sequencing of pathogens spiked into whole blood; the entire workflow starts with a whole blood sample and ends with sequencing and analysis of results).

[0120] Many advantages of semiconductor sequencing are well understood and documented in the prior art. These advantages included speed (faster than most sequencing-by-synthesis methods), no modified / labeled nucleotides are required, no optics are required for detection, fewer nucleotides and wash flows are required per given sequence length, etc. There are multiple additional advantages of the sequencing embodiments disclosed herein, including, but not limited to, 1) the entire process including cluster formation on the surface of the semiconductor tip, as well as all sample and library preparation steps prior to cluster generation (if required), are fully automated in a system including cartridges and devices. 2) All reagents required for sequencing are contained in a disposable reagent cartridge, and all waste fluids are contained within the reagent cartridge after a sequencing run. This allows for easy and efficient use and safe disposal of used components after a run. 3) The disclosed method of simultaneous sequencing primer annealing and sequencing enzyme binding offers simplicity and reduced run times. 4) The disclosed method of critical sequence introduction and use provides a novel method for calibrating a sequencing run. 5) The system can perform on-board pH titration of reagents used in sequencing, which is critical for ISFET-based sequencing.

[0121] Further embodiments of the invention include an on-board computer and auxiliary devices / electronics and software. Use of the computer and software includes control of the system and collection and analysis of signals / data generated by the system.

[0122] Exemplary user interface steps of a general workflow are shown in FIG. 62. First, a user can scan various information into the device using the barcode scanner on the front of the device. For example, an ID badge can be scanned to register the user or to allow access to certain assays / functions. User information can be stored along with any subsequent assay data to control access and / or for quality control or analytical purposes. A specimen or sample container can be scanned to read and record the sample data for an assay. For example, patient information tied to a blood sample may be encoded on a barcode on the vacutainer, and by scanning with the device prior to entering the sample or assay cartridge, the system can tie any subsequent analytical data to a particular sample or patient. The user can then scan a kit, which can provide the device with information regarding which assays should be run. This can eliminate user error by automatically generating an assay workflow for the desired assay, or can be used as a quality control check to ensure that the appropriate kit is being used for the user-selected assay entered into the user interface. The user can then remove one or more cartridges from their packaging and prepare them for insertion. In certain embodiments, the only user handling involved in the assay is opening the cartridge, removing the sterilization foil covering any ports, and loading the sample before inserting the cartridge into the device for automated processing of the assay. The user can insert a vacutainer into the sample cartridge, for example, via a sample input subunit that is specifically designed to accept a particular size / type of sample container. The cartridge(s) can then be loaded into the device, for example, through an automatically opened door. The device can then automatically run the desired assay and provide the results to a user interface (e.g., a display on the device) or to another computer via a network. The device can then eject the cartridge with all waste contained therein.Thus, the risk of user error and contamination is reduced, and because all fluids are contained within the cartridge, the device is ready for the next test without the need for cleaning or refilling with on-board reagents.

[0123] b. Options within a typical workflow The system is designed to have unparalleled throughput and flexibility for the execution of tests / assays, including in the field of molecular testing, including sequencing. A single integrated system contains one or more cartridges, many chambers of different sizes and functionality, advanced fluidic connectivity, large volumes, novel and various means of moving, transporting, combining, mixing, reconstituting and otherwise handling liquids (gases and solids, as in the reconstitution of dried, vitrified or other solid reagents or components), means of heating, magnetic separation, disruption, blending, sensing, titrating (e.g. pH titration), detection and analysis. All of this is controlled via an on-board computer, allowing a multitude of processing steps, in various orders, durations, conditions (e.g. temperature), etc. Within this context, a wide variety of different tests / assays can be performed, with a wide range of options for the different steps of each test / assay.

[0124] Many options for sample preparation are feasible within the scope of the claimed invention. For some samples, the primary sample loaded into the device may require an initial processing step, for example if the sample is too viscous, inhomogeneous, fibrous, gelatinous, etc. Within the scope of the claimed invention, examples of methods for initial processing of the sample include, but are not limited to, mixing with reagents including turbulent mixing, serpentine mixing, vortex mixing, etc., mixing with reagents such as detergents, denaturants, chaotropes, organic solvents, buffers, salts, etc., and various combinations thereof, and / or enzymatic digestion, e.g. with proteinase K (described in the general workflow above) and / or other proteases, nucleases, lipases, etc.; and / or chemical compounds, e.g. dithiothreitol, β-mercaptoethanol, other reducing agents, oxidizing agents, acids, bases, etc. During the initial processing steps, the sample can be mixed, heated, sonicated, etc. For some samples, no initial processing is required and the sample can go directly into the next step. For some whole blood samples, a separation system that is integrated into the cartridge and functions seamlessly within the system is used to separate the whole blood loaded into the cartridge into plasma and other components. All of the above is contemplated to be possible within the context of the disclosed system.

[0125] In some samples, the target polynucleotides are contained within cells, including within the nucleus and / or other structures. Within the scope of the claimed invention, examples of methods for cell lysis / liberation of the target polynucleotides include, but are not limited to, mechanical lysis with or without the presence of beads (e.g., bead bashing), sonication, heating, mixing (e.g., turbulent mixing), shearing (e.g., by passing through a small orifice), etc. Each of these processes may also include the action of a reagent that is mixed with the sample and participates in the lysis mechanism (detergent, denaturant, solvent, etc.; see above for more options). If the target polynucleotides are contained in the nucleus of the cells, a gentle lysis of the outer cell membrane may be performed first, separating the cell contents from the nucleus, followed by more stringent methods to lyse the nucleus. In some cases, the sample does not require lysis (e.g., the cells were lysed before the sample was loaded into the cartridge, the cells were lysed in an initial processing step, or the target nucleic acid was not present in the cells in the sample). In these cases, the sample can enter the next step of the process directly. All of the above is envisioned to be possible within the context of the disclosed system.

[0126] Other protocols that may be used when the target polynucleotide is contained within a cell, including within the nucleus, and / or other structures include capturing intact cells (e.g., via affinity capture using magnetic beads), washing away the remainder of the sample, and then lysing the cells (examples of lysis methods described above). All of the above are envisioned to be possible within the context of the disclosed system.

[0127] In some protocols, the target nucleic acid must be denatured (e.g., double-stranded to single-stranded) and / or released from association with other structures (e.g., DNA coiled around histones). Methods for doing so include heating, treatment with reagents (chemical and / or biological), mixing, sonication, etc. In some cases, this is not necessary and the sample can go directly to the next step in the process. All of these are contemplated in the context of the disclosed system.

[0128] In some cases, target polynucleotides are isolated. Within the scope of the claimed invention, examples of methods for isolating target polynucleotides include, but are not limited to, 1) specific target capture (STC) as described elsewhere herein. This includes limited target capture as described elsewhere herein and in PCT / GB2021 / 050098; 2) non-specific capture methods, such as solid-phase extraction (examples include, but are not limited to, silica-based methods, including solid-phase reversible immobilization (SPRI), solid-phase microextraction (SPME), boom and AMPure); 3) combinations of non-specific capture (e.g., solid-phase extraction) and specific target capture techniques (e.g., first boom, followed by STC, as described herein); 4) hybrid capture target enrichment strategies, such as the Agilent SureSelect method, which utilizes RNA capture probes or "baits" to pull down regions of interest. There are many different such hybridization-based methods, as well as other strategies for target enrichment, including but not limited to transposon-mediated fragmentation (tagmentation), molecular inversion probes (MIPs), and focused amplification procedures (e.g., as described elsewhere herein). Note that many of these procedures utilize fragmented nucleic acid targets. Note that many of these procedures can be combined with library preparation or used after library preparation, but are described herein in the context of methods that can be used in the sample preparation stage of the disclosed system (they are also referred to in the library preparation section). Note also that the first or initial stage of amplification or amplification can be performed on target polynucleotides captured on beads (sample preparation, library preparation, copy control, and cluster generation). It is envisioned that all of the above are possible within the context of the disclosed system.

[0129] In some cases, other processes can be performed during the sample processing segment of the workflow within the scope of the claimed invention, including, but not limited to, 1) fragmentation of the target polynucleotide; 2) addition of tags / adapters to the target polynucleotide, e.g., via tagged oligonucleotides that anneal to the target polynucleotide and are extended (during sample preparation and / or library preparation) or by ligation. 3) A unique molecular identifier can be incorporated into some or all of the target polynucleotide in the sample. In some cases, the target polynucleotide is already in a fragmented form, otherwise very short fragments in the sample, e.g., fragmented DNA in urine, circulating tumor DNA (ctDNA) in blood, cell-free DNA (various sample types), small RNA (various sample types), etc. Methods for processing these polynucleotide samples (including, but not limited to, tag / adapter addition, amplification, reassembly, capture, etc.) can be performed on the system. All of the above is envisioned to be possible within the context of the disclosed system.

[0130] Many options for library preparation are feasible within the scope of the claimed invention. In some embodiments, the input of the library preparation process is the output of a sample preparation method, and in some embodiments, it is the primary sample itself. Preferred embodiments of amplification-based methods of targeted enrichment are described elsewhere herein. Variations of these methods envisioned for use in the disclosed systems include, but are not limited to, tags / adapters incorporated in 1) one or three (or more) separate amplification reactions; 2) a wide range of scenarios (as part of a copy control process (as described elsewhere herein and in PCT / GB2021 / 050098), as a combination of processes in the sample preparation stage (see above) and the library preparation stage, in a combination of amplification reactions that are differentially incorporated between the ends of the same target polynucleotide and from target polynucleotide to target polynucleotide, in only one of the amplification reactions), etc. Amplification can be achieved using a wide variety of methods known in the art, including, but not limited to, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), nicking endonuclease amplification reaction (NEAR), transcription-mediated amplification (TMA); loop-mediated isothermal amplification (LAMP); helicase-dependent amplification (HDA); clustered regularly interspaced short palindromic repeats (CRISPR); strand displacement amplification (SDA); recombinase polymerase amplification (RPA), ligase chain reaction (LCR), and the like. There is a wide range of other library preparation methods known in the art that are contemplated for use with the disclosed systems. Some examples of a general workflow include, but are not limited to, 1) fragmentation, adapter ligation, amplification (typically including the addition of additional adapters).2) fragmentation, amplification (random, semi-random, specific; can include adapter addition), adapter addition; optional amplification (often including additional adapter addition); 3) amplification (which can include the examples listed above as well as whole genome amplification (e.g., Picoseq, DOPlify, REPLI-g (based on multiple displacement amplification, or MDA) and Ampli-1WGA), long-range PCR, amplification using semi-random and / or degenerate primers, etc.), fragmentation, adapter addition, optional amplification (often including additional adapter addition); 3) transposon-mediated fragmentation (tagmentation); 4) molecular inversion probe-based methods (MIP); etc. Some of the above techniques of library preparation can be followed with target enrichment (some of the techniques mentioned above, such as SureSelect). As mentioned above, these steps of the library preparation and sample preparation process can be performed in overlap across different units of the cartridge. Also, as mentioned above, UMIs can be added at various stages of the sample preparation and / or library preparation process (the process depends on the exact protocol). It is envisioned that all of the above is possible within the context of the disclosed system.

[0131] Copy control can be performed after library preparation, overlapping with the library preparation process, or not at all (depending on the application). In some cases, the tags / adapters utilized in the copy control process can also be incorporated during sample preparation. A wide variety of novel copy control methods have been described. Additionally, other copy control methods known in the art can also be implemented on the system.

[0132] Cluster generation methods are described herein. Other methods can be utilized, including the use of different surfaces and / or surface geometries, different surface immobilization chemistries, and different amplification methods. Other methods that the inventors have demonstrated to support cluster generation in the claimed invention include PCR, HDA, SLAM (a proprietary surface-phase amplification procedure; pending patent) and EM-Seq (a proprietary displacement-mediated amplification procedure; pending patent).

[0133] Sequencing on semiconductor chips can be performed on essentially any type of sequencing library, using any targeting strategy (see above for examples), i.e., not just sequencing amplicons from targeted enrichment, for example. Sequencing can also be performed after cluster generation using direct hybridization (above) with any number of targets and target sources (not just those used as examples). High-density arrays of capture oligomers with different specificities can be applied to the surface of the chip and utilized in this method.

[0134] A wide variety of data analysis methods have been developed, including analysis by triangulation (triangulation strategies, including STC, target enrichment and key sequence development, have also been utilized in oligonucleotide design).

[0135] definition It is understood that the disclosure is not limited to specific compositions or process steps and may vary as such. As used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" include plural references, and phrases such as "one or more items" include singular references unless the context clearly dictates otherwise. Thus, for example, a reference to "oligomer" includes a plurality of oligomers, etc. The conjunction "or" should be interpreted in an inclusive sense, i.e., equivalent to "and / or," unless an inclusive sense is unwarranted in the context. When "at least one" member of a class (e.g., oligomer) is present, a reference to "the" member (e.g., oligomer) refers to the member (if only one) present or at least one (if more than one) of the members (e.g., oligomer) present.

[0136] It will be understood that there is an implicit "about" before temperatures, concentrations, amounts, times, etc. discussed in this disclosure, such that slight and very small deviations are within the scope of the teachings herein. In general, the term "about" refers to slight variations in the amount of a component of a composition that do not significantly affect the activity or stability of the composition, e.g., within 10%, 5%, 2%, or 1%. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least by considering the number of reported significant digits and applying ordinary rounding techniques. All ranges should be construed to include the endpoints unless there is an express exclusion, such as "not including the endpoints". Thus, for example, "within 10 to 15" includes the values ​​10 and 15 and all intervening integer and (where appropriate) non-integer values. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not intended to be limiting of the teachings. Section headings are provided merely for the convenience of the reader and are not intended to limit the disclosure. To the extent that any material incorporated by reference is inconsistent with the express content of this disclosure, the express content shall control.

[0137] Unless otherwise indicated, embodiments herein that are described as "comprising" various components are also considered to "consist of" or "consisting essentially of" the described components. "Consisting essentially of" means that additional components, compositions, or method steps that do not substantially alter the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics may include, for example, the ability to hybridize to a target polynucleotide and undergo further binding and / or extension reactions as described herein.

[0138] "Sample" refers to materials that may contain target polynucleotides, including but not limited to biological samples, clinical samples, environmental samples, and food samples. Environmental samples include environmental materials such as surface materials, soil, water, sludge, air, and industrial samples, as well as samples obtained from food and dairy processing equipment, equipment, equipment, equipment, disposable and non-disposable items. "Biological" or "clinical" samples refer to tissues, liquids, or other materials derived from living or dead humans, animals, or other organisms that may contain target polynucleotides, including, for example, tissue samples, swabs, lavage fluids, aspirates, exudates, biopsy tissue, or fluids such as blood, spinal fluid, feces, semen, or urine. Samples can be treated to physically or mechanically disrupt tissue or cellular structures and release intracellular nucleic acids into solutions that may contain enzymes, buffers, salts, detergents, and the like to prepare the sample for analysis. Samples can also be aqueous or organic solvents, or combinations thereof, with or without other components (e.g., buffers, salts, detergents, emulsifiers, EDTA, and the like) that contain the target polynucleotides. These examples should not be construed as limiting the sample types applicable to this disclosure.

[0139] Sample preparation refers to a method or combination of methods that manipulate a sample containing a target polynucleotide to prepare the target polynucleotide for further downstream processing and / or analysis. Such methods include, but are not limited to, methods that release, make accessible, digest, remove binding components, concentrate, enrich, capture, separate, and / or isolate the target polynucleotide in the sample. Such methods also include methods that remove, neutralize, or otherwise potentially compete with, interfere with, obscure, or otherwise harm downstream analytes, components, contaminants, organisms (including dead or live and / or debris from such organisms) or other biological, organic, or inorganic materials from the sample containing the target polynucleotide. Such methods include, but are not limited to, 1) solubilizing, dissolving, homogenizing, digesting, or otherwise altering the physical or chemical properties of the sample to aid in the preparation of the target polynucleotide, including, but not limited to, heating, cooling, freezing, freeze-thawing, digestion (including using chemical or biological means including enzymatic means), sonication, dissolution using solvents, reagents and / or other chemical or biological means, agitation, shearing, mechanical agitation, etc.; 2) filtering the sample; and 3) concentrating the sample. 4) tagging, labeling, capturing, enriching, isolating or otherwise treating cells suspected of containing the target polynucleotide, including, but not limited to, tagging with cell-specific moieties including antibodies, lecithin, nucleic acids, proteins, peptides, aptamers, dendrimers, other cells, viruses, macrophages, other biological moieties, etc.; labeling cells with fluorescent dyes, radioactive labels, luminescent labels, mass labels, etc.; capturing, enriching or isolating cells using precipitation, centrifugation (including the use of density gradients), filtration, affinity capture (including via at least one of the cell-specific moieties listed in the tagging section above, including direct or indirect binding to a solid support, cell sorting, etc.).5) Methods that lyse, digest, rupture, partially lyse, shear or otherwise manipulate cells or other structures that contain or are otherwise associated with the target polynucleotide to make the target polynucleotide accessible or otherwise available for further processing or analysis, including, but not limited to, heating, cooling, freezing, freeze-thawing, boiling, exposing the cells to osmotic shock, treatment with solvents, chemicals or other reagents (including in combination with heating), sonication, sonication with simultaneous heating, beating / bashing, enzymatic treatment, agitation, shearing, (including mechanical shearing), etc. 6) Methods that solubilize, dissolve, homogenize, digest or otherwise modify the physical or chemical capabilities of the target polynucleotide to aid in further processing and / or analysis of the target polynucleotide, including, but not limited to, at least one of the methods listed in section (1) above in this paragraph.7) tagging, labeling, enrichment, concentration, enrichment, capture, separation and / or isolation of target polynucleotides, including but not limited to methods of tagging, binding, attaching, coupling, incorporating or otherwise attaching, whether directly or indirectly, covalently or non-covalently, to a nucleic acid, a nucleic acid segment, a plurality of nucleic acid segments, a protein, an enzyme, an aptamer, a lecithin, a dendrimer, an element, a molecule, or any other substance or moiety, including chemical, biological, organic and / or inorganic, further preparation, processing (including amplification) and / or analysis of the target polynucleotides, including all tagging methods commonly known in the art, labeling with fluorescent dyes, radioactive labels, luminescent labels, mass labels, etc., precipitation, GuSCN, CTAB, Chelex (including other resin types) and alkaline extraction, chromatography, column chromatography, filtration, use of density gradients, centrifugation including isoelectric focusing and other focusing techniques known in the art, including methods commonly known in the art Capture on solid supports including magnetic microspheres and other solid support materials, the use of non-specific target capture methods such as silica-based methods including solid phase extraction (SPE), ion exchange SPE, solid phase reversible immobilization (SPRI), solid phase microextraction (SPME), the Boom method, the AMPure method, directly and indirectly, as well as specific target capture methods including the use of random or semi-random target capture oligomers to capture various targets in a sample (including methods that utilize one or more oligonucleotides specific for a polynucleotide or group of polynucleotides of interest), where oligonucleotides are annealed to the target nucleic acid and this / these complexes are immobilized on a solid support, some methods of concentrating, capturing, separating, and / or isolating the target polynucleotide, or purifying the target nucleic acid in any other manner include one or more wash steps, some such methods include an elution step, and some methods use the target nucleic acid directly from the sample for further downstream processing (e.g., amplification) and / or analysis.8) Removal, neutralization or otherwise competing with, interfering with, obscuring or otherwise making less harmful downstream analytes, components, contaminants, organisms (including dead or live and / or debris from such organisms) or other biological, organic or inorganic material from the sample; depletion of non-target polynucleotides, including genomic DNA, including human genomic DNA; depletion of RNA, including rRNA; removal, digestion, or inactivation or other methods of proteins, enzymes, lipids, carbohydrates, biological material, organic material, inorganic material, cells, including whole cells and partially or completely lysed or otherwise degraded cells, and other components that may potentially interfere with downstream processing and / or analysis. Further methods of sample preparation are described in J. Dapprich, et al., The next generation of target capture technologies-large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity (BMC Genomics (2016), 17:486) and N Ali, et al., Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics (BioMed Research International (2017), Article ID 9306564, 13 pages), which are incorporated by reference in their entireties.

[0140] Specific target capture (STC) methods refer to methods or combinations thereof that are useful for tagging, separating, isolating, or otherwise differentiating specific target polynucleotides within a broader mixture of polynucleotides. This is in contrast to non-specific capture methods that generally operate on all polynucleotides in a mixture (although specific types can be differentiated, for example, based on length). STC methods typically differentiate polynucleotides based on nucleotide sequence (although other differentiation methods are acceptable, provided the desired level of specificity is achieved). A preferred method in this closure is the use of STC oligonucleotides (oligos) to differentiate targets based on sequence. An oligo or set of oligos is designed to anneal specifically to a given target, group of targets, set of targets, etc., without annealing at significant levels to non-target polynucleotides potentially present in the sample mixture. Essentially, any level of specificity can be achieved across the taxonomic spectrum by the STC oligo design in combination with the selected reaction conditions. For example, target polynucleotides can be differentiated at the subspecies / strain, species, genus, family, order, class, and / or phylum level, and even at the kingdom and domain level. In a preferred embodiment of the disclosed invention, in which the system is utilized for the detection of bloodstream infections as well as antimicrobial resistance genes, the STC oligos are designed to bind and selectively capture a broad range of bacterial and fungal targets as well as specific antimicrobial resistance (AMR) genes. Once the STC oligos anneal to their intended targets, the resulting complexes can be captured, immobilized, separated, isolated, etc. using several different methods, some examples of which are discussed elsewhere in this disclosure.

[0141] "Nucleic acid" and "polynucleotide" refer to polymeric compounds that include nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs linked together to form polynucleotides, including polymers that are conventional RNA, DNA, mixed RNA-DNA, and analogs thereof. The nucleic acid "backbone" can be composed of a variety of linkages, including sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA; WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, such as 2' methoxy or 2' halide substitutions. The nitrogenous bases may be any of the conventional bases (A, G, C, T, U), their analogs (e.g., inosine, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), derivatives of purines or pyrimidines (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having a substituent at the 5- or 6-position (e.g., 5-methylcytosine), purine bases having a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and WO 93 / 13121). Nucleic acids can contain one or more "abasic" residues, where the backbone does not contain nitrogenous bases relative to the position of the polymer (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases and linkages, or can contain both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), analogs containing one or more LNA nucleotide monomers with bicyclic furanose units locked to RNA that mimic the sugar conformation, enhancing hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Examples of oligomers that may affect the stability of the hybridization complex include PNA oligomers, oligomers containing 2'-methoxy or 2'-fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or steric association of the hybridization complex, including oligomers containing charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates). Methylated cytosine, such as 5-methylcytosine, may be used in combination with any of the above backbones / sugars / linkages, including RNA or DNA backbones (or mixtures thereof), unless otherwise noted. RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. Differences between RNA and DNA equivalents do not contribute to differences in homology, since the equivalents have the same degree of complementarity to a particular sequence. When referring to a range of lengths of oligonucleotides, amplicons or other nucleic acids, it is understood that the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).Unless otherwise indicated, T residues are understood to be interchangeable with U residues, and vice versa.The orientation of a nucleic acid polymer strand can be described as plus (+) sense (or positive sense, or simply the sense strand or coding strand) or negative (-) sense (or negative, or simply the antisense strand or non-coding strand).

[0142] "Target polynucleotide" refers to a polynucleotide that is to be prepared, separated, captured, isolated, enriched, amplified, detected, identified, and / or sequenced, etc., using the compositions or methods described herein. In some embodiments, the target polynucleotide comprises a sequence of DNA or RNA from an organism (e.g., any virus, prokaryote, eukaryote, protist, plant, fungus, insect, animal, mammal, or other biological entity, whether living or formerly living). Exemplary DNA includes genomic DNA, circulating tumor DNA, episomal or plasmid DNA, and mitochondrial DNA. Exemplary RNA includes messenger RNA, more generally transcribed RNA, ribosomal RNA, transfer RNA, small nuclear RNA, regulatory RNA, transfer-messenger RNA, small nucleolar RNA, guide RNA, interfering RNA, microRNA, other regulatory RNA, non-coding RNA, etc. (and where applicable, e.g., in the case of certain viruses, genomic RNA). The target polynucleotide may be of positive sense, negative sense, or both positive and negative sense (e.g., where both strands of the polynucleotide are targeted). Target polynucleotides also include one or more copies of the above nucleic acids to which additional sequences may be added, such as any of the additional sequences described herein. In some embodiments, target polynucleotides include non-naturally occurring sequences resulting, for example, from in vitro synthesis, ligation, site-directed mutagenesis, recombination, and the like.

[0143] "Oligomer" or "oligonucleotide" refers to a nucleic acid generally less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 2-5 nt and an upper limit of about 500-900 nt. Some particular embodiments are oligomers in a size range having a lower limit of about 5-35 nt and an upper limit of about 50-600 nt, and other particular embodiments are in a size range having a lower limit of about 5-20 nt and an upper limit of about 30-1500 nt. Oligomers can be purified from natural sources, but can be synthesized by using any well-known enzymatic or chemical method. Oligomers can be referred to by their functional name (e.g., capture oligomer, primer, promoter primer, or detection probe), but one of skill in the art will understand that such terms refer to oligomers. Oligomers can form secondary and tertiary structures by self-hybridizing or by hybridizing to other oligonucleotides or polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, triplexes, and quadruplexes. The oligomer may include modifications including those described elsewhere in this disclosure. In some cases, the oligomer may refer to non-nucleic acid-based polymers such as some aptamers and non-nucleotide-based binding partners (see, for example, Winnacker, M., & Kool, ET (2013). Artificial Genetic Sets Composed of Size-Expanded Base Pairs. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 52(48), 12498-508.). The oligomer may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. In some embodiments, the oligomer that forms the invasive cleavage structure is generated in a reaction (e.g., by extension of a primer in an enzymatic extension reaction).

[0144] "Arbitrary sequence" refers to any sequence that is selected, chosen, determined, designed, etc. by a user (with or without the aid of a computer program) typically to perform a desired function or purpose in a downstream process. In a preferred aspect, the arbitrary sequence is designed to be non-complementary or otherwise non-reactive to one or more target sequences under given conditions of the process. In some embodiments, the arbitrary sequence can be a randomly generated sequence or set of sequences, for example, for use as a unique molecular identifier or universal primer.

[0145] "Capture oligomer", "capture oligonucleotide", "capture probe", "target capture oligomer" and "capture probe oligomer" are used interchangeably to refer to a nucleic acid oligomer or derivative thereof that contains a target binding sequence (TBS) that can bind to one or more target sequences in a target nucleic acid. Binding can occur at different user-selected levels of specificity depending on the design of the system and the desired application and outcome. One binding mode involves hybridizing to the target nucleic acid at different levels of specificity, again selected by the user, ranging from high specificity to low specificity (including designing the capture oligomer to capture a desired target at any point in the taxonomic order; see discussion elsewhere in this disclosure). Target capture oligomers can also include segments (or entire oligos) of random or semi-random sequences. A capture oligomer may also include one or more of the following: (i) an extendable 3' end, (ii) a non-extendable (e.g., blocked) 3' end, (iii) a ligatable 5' end, (iv) a first ligand of a ligand pair (e.g., biotin of the ligand pair biotin / streptavidin), of which one or more copies may be present, (v) a tag sequence added to the 3' end, 5' end, or both, and / or inserted into the THS, (vi) a tag sequence or a combination of a sequence and one or more ligands. Exemplary tag sequences include capture sequences that can hybridize to a secondary oligomer immobilized, for example, on a solid support, or a non-immobilized secondary oligomer linked to a binding partner, to facilitate isolation of a complex comprising the capture oligomer, target, and secondary oligomer from other molecules in the composition. Other exemplary tag sequence options are described in the "Tag" definition section below. The nucleic acid component of the capture oligomer may include any of the forms or combinations thereof described in the "Nucleic Acid" definition section above. A capture oligomer can, in certain cases, function as something other than a capture agent, including, but not limited to, a primer, an amplification oligomer, a blocking agent, a portion of a site for cleavage / digestion, a displacer, a portion of a recognition site, and the like.The capture oligomer may also be any of the capture oligomers described in "Compositions, kits and methods for isolating target polynucleotides" (PCT / GB2021 / 050098), which is incorporated by reference in its entirety, and which describes in detail copy control related compositions, kits and methods (discussed elsewhere herein).

[0146] (Within the explicit context of nucleic acids; "amplification" can have different meanings in different contexts, e.g., the production of one or more copies of a non-nucleic acid molecule, an increase in a detectable signal such as fluorescence, an increase in an electrical signal, etc.) "Nucleic acid amplification" or "amplification" refers to the production of one or more copies of a target nucleic acid (or target polynucleotide, parent molecule, template, template molecule, etc.) sequence, or a complementary sequence thereof, or a portion thereof (i.e., an amplified sequence that contains less than the entire target nucleic acid). Examples of nucleic acid amplification procedures include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA) and others (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), These include transcription-related methods such as rolling circle amplification (RCA) (e.g., US Pat. Nos. 5,854,033 and 6,143,495), recombinase polymerase amplification (RPA) (e.g., US Pat. No. 7,666,598), ligase chain reaction (LCR) (e.g., European Patent Application Publication No. 0320308), loop-mediated amplification (e.g., Loop-Mediated Isothermal Amplification of DNA (2000) Nucleic Acids Res, 28(12):e63) and strand displacement amplification (SDA) (e.g., US Pat. No. 5,422,252). Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as QB replicase. PCR amplification uses a DNA polymerase, primers, and a cycling step (typically a thermal cycle, but other types of cycles such as chemical cycles can also be used) to synthesize multiple copies of two complementary strands of DNA or cDNA. Copies of the two complementary strands can be generated in a ratio other than 1:1, for example by asymmetric PCR (eg, Asymmetric PCR. In: Capinera JL (eds) Encyclopedia of Entomology, 2008, Springer, Dordrecht.).LCR amplification uses at least four separate oligonucleotides to amplify the target and its complementary strand by using multiple cycles of hybridization, ligation and denaturation. SDA uses primers that contain recognition sites for restriction endonucleases that nick one strand of a semi-modified DNA duplex containing the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Although certain embodiments use PCR, it will be apparent to one of skill in the art that the oligomers disclosed herein can readily be used as primers in other amplification methods, and that other amplification methods and primers can be used in general.

[0147] "Amplicon" or "amplification product" refers to a nucleic acid molecule generated in a nucleic acid amplification reaction and derived from a template nucleic acid. An amplicon or amplification product comprises an amplified nucleic acid sequence (e.g., a target polynucleotide / nucleic acid) that may be of the same or opposite sense as the template nucleic acid, comprises DNA or RNA, and comprises single-stranded or double-stranded products. In some embodiments, an amplicon has a length of about 100-30,000 nucleotides, about 100-10,000 nucleotides, about 100-5000 nucleotides, about 100-2000 nucleotides, about 100-1500 nucleotides, about 100-1000 nucleotides, about 100-800 nucleotides, about 100-700 nucleotides, about 100-600 nucleotides, or about 50-500 nucleotides.

[0148] "Amplification oligonucleotide" or "amplification oligomer" refers to an oligonucleotide that hybridizes to a target nucleic acid, or its complement, or tag sequence, etc., and participates in an extension or amplification reaction of a nucleic acid, serving, for example, as a primer and / or promoter primer, a displacer (with or without extension), a blocking agent (e.g., blocking binding or extension), facilitating cleavage or degradation, and helping to disrupt structures. Some capture oligomers can also function as amplification oligomers (see elsewhere herein), and some amplification oligomers can also function as capture oligomers. Amplification oligomers also encompass promoter donors that contain a promoter capable of initiating transcription, but not necessarily extendable by DNA polymerase, and that may include a 3' blocking portion. Certain amplification oligomers contain a target, complementary or tag hybridizing sequence of at least about 5 contiguous bases, and optionally at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous bases, that are complementary to a region of the target nucleic acid sequence or tag sequence, or its complementary strand. Other exemplary lengths or length ranges of target hybridizing or tag hybridizing sequences are described elsewhere herein and can be applied to amplification oligomers. The contiguous bases can be at least about 70%, at least about 80%, at least about 90%, or fully complementary to the target sequence to which the amplification oligomer binds. In some embodiments, the amplification oligomer comprises an intervening linker or non-complementary sequence between two segments of complementary sequence, e.g., the two complementary segments of the oligomer collectively comprise at least about 5 complementary bases, and optionally at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 complementary bases. In some embodiments, the amplification oligomer is about 10 to about 80 bases in length, and can optionally comprise modified nucleotides. The amplification oligomer can be optionally modified, e.g., by including a 5' region that is non-complementary to the target sequence.Such modifications may include functional additions such as primers, tags, or tags, promoters, or other sequences or moieties that are used or useful for manipulating, amplifying, capturing, immobilizing, or otherwise processing the target molecule.

[0149] "Primer" refers to an oligomer with a 3' end that hybridizes to a template nucleic acid and is extended by polymerization. A primer can be optionally modified, for example, by including a 5' region that is non-complementary to the target sequence. Such modifications can include functional additions such as tags, promoters, or other sequences, or other moieties, all of which can be used or are useful for manipulating, amplifying, capturing, separating, immobilizing, or otherwise processing the primer or target oligonucleotide or its complement. "Opposite primers" refers to at least one primer that is positive (+) sense and at least one primer that is negative (-) sense, each of which is complementary to one strand of a target polynucleotide or one copy of a strand of a target polynucleotide, and which participate in the production of an amplicon when used together in an amplification reaction (e.g., a primer pair in PCR).

[0150] Amplification oligomers or primers can in some cases function as other than amplification oligomers and primers, including, but not limited to, capture oligomers, blocking agents, parts of sites for cleavage, digestion, displacers, parts of recognition sites, and the like.

[0151] Unless otherwise specified, a first sequence is the "complement" of a second sequence (or, equivalently, is "complementary" to a second sequence), and the first sequence has sufficient length and content to anneal to the second sequence under reasonable binding conditions, which may be, but are not necessarily, the stringent hybridization conditions described herein, including, for example, annealing conditions used in standard PCR and other techniques involving primer or probe binding and extension.

[0152] "Tag" refers to any additional nucleic acid sequence other than the target hybridizing sequence that may be included in an oligomer or that may be added or inserted into a target polynucleotide. Any sequence present in addition to the target hybridizing sequence can function as a tag. In some uses, a tag can also refer to a moiety other than a nucleic acid that is attached to or otherwise included in an oligomer or target polynucleotide. A tag can be included, added, inserted, appended, etc. to a target polynucleotide or fragment thereof using any method known in the art, including, but not limited to, incorporation by extension or amplification, ligation by transposome chemistry, etc., utilizing an oligomer or fragment thereof that contains one or more tags and hybridizes specifically, semi-specifically, or non-specifically to the target polynucleotide or fragment thereof. Tags include, but are not limited to, adaptors (see below). Further examples of tags are stabilizing sequences, including promoters, mixed nucleotide elements described elsewhere herein, elements used for sample preparation and target capture, and clamps. Further examples of tags are described above in the definition of "sample preparation" and elsewhere in.

[0153] An "adapter" or "adapter" (these two terms are used interchangeably in this disclosure and have equivalent definitions) is a sequence that adapts the molecule to which it is added to provide one or more additional functions. For example, an adapter provides a binding site for another molecule, such as an amplification oligomer, a sequencing primer, or a capture oligomer. The binding site can be a universal binding site (e.g., for multiple capture oligomers all having the same binding site specificity, e.g., the same sequence, in a multiplex format, or a universal primer). Further examples of binding sites are binding sites for displacer oligomers, probes, or nucleic acid modifying enzymes (e.g., RNA polymerases, primases, ligases, RNAses (such as RNAse H), or restriction enzymes), or for attachment to a solid phase (including via a solid phase primer or capture oligomer), including for use in clonal amplification, or other functional element or elements useful for downstream applications, such as enrichment, library preparation, clonal amplification, or sequencing. Thus, sample barcodes or index sequences, key or calibrator sequences, molecular barcodes (containing unique molecular identifiers), sites for downstream cloning, and sites for circularization of target molecules are further examples of elements that can be included in adaptors.

[0154] "Library preparation" in its most general sense means the process of preparing a group of target polynucleotides for further downstream processing and / or analysis. Cluster generation, including by clonal amplification, is an example of a downstream processing step. Sequencing is an example of downstream analysis. Library preparation methods often include one or more steps to add adapter sequences or other tag sequences to some or all of the molecules in the library. However, this is not always the case, as some of the disclosed embodiments do not require the addition of adapters to the library molecules. Examples of this include some modes of direct hybridization sequencing methods described elsewhere in this disclosure. Library preparation also often includes one or more amplification steps, for example, to enrich one or more regions of the target polynucleotides, to add tags, including adapters, to the library molecules, and to increase the copy number of molecules that include the target regions and / or tags that include adapters. Adapters and tags can be added without amplification, for example, via ligation. Library preparation steps may overlap with sample preparation steps (e.g., tags including adaptors may be added during sample preparation and / or first extension products from target regions may be generated (see the "Sample Preparation" section herein)) and may be associated / overlapping with copy control (see elsewhere herein, including below).

[0155] "Copy control" refers to compositions and methods in which the copy number of a molecule that is the output of a given process is controlled in a predetermined manner. For example, in certain workflows, it is desirable to capture (or amplify and capture) or otherwise isolate a target polynucleotide (which may be, for example, naturally occurring DNA or RNA, or an amplicon) that is equal to or less than a predetermined amount (e.g., a maximum desired value for downstream applications such as sequencing library preparation; sometimes referred to as "limit capture" which still falls under the inclusive definition of copy control). Similarly, in certain workflows, it is desirable to capture (or amplify and capture) or otherwise isolate a predetermined, specific amount (e.g., a specific number of molecules or copies of a molecule) of a target polynucleotide (which may be, for example, naturally occurring DNA or RNA, an amplicon, or a sequencing library) for use in downstream applications (e.g., clonal amplification, including next generation sequencing workflows). Furthermore, in certain workflows, it is desirable to incorporate additional sequences into the target polynucleotide (tag), such as the incorporation of adaptors into a sequencing library. This can also be achieved in copy control compositions and methods. The present disclosure provides oligomers, compositions, and kits useful for isolating target polynucleotides and / or attaching tags such as adaptors. Isolation includes limited amount isolation (limited capture) and specific amount isolation (copy control).

[0156] "Clonal" or "monoclonal" refers to a population of identical units or copies of (at least one) precursor molecule, nucleic acid, polynucleotide, gene, genetic material, cell, etc. In this disclosure, it most often refers to a population of identical copies made from one or more copies of the same nucleic acid / polynucleotide template. In this disclosure, it can sometimes refer to a collection of identical molecules in a population that are, for example, clustered together (see below). In the most preferred embodiment, the monoclonality is about 100% (i.e., all or nearly all copies are identical). In other preferred embodiments, the monoclonality is about 90% or more, about 80% or more, or 70% or more. In some embodiments, the monoclonality is about 50-70%. "Polyclonal" refers to a population of units or copies that are not all identical, but are derived from at least two different precursor molecules, etc. Clonal amplification refers to nucleic acid amplification of typically a single precursor nucleic acid molecule (which may sometimes be two or more identical precursor molecules) to generate a set of identical copies. "Cluster" refers to a grouping of molecules, e.g., nucleic acid molecules, bound to a solid support. "Cluster generation" refers to the process by which clusters are generated. Examples of cluster generation processes include amplification-based, e.g., clonal amplification, and non-amplification-based, e.g., hybridization of target molecules to oligonucleotides immobilized on a solid support at known specific regions (e.g., spots). Clusters can be monoclonal (typically the preferred configuration in this disclosure) or polyclonal.

[0157] A "linker" is a sequence or non-sequence element, or combination thereof, that connects one part of an oligomer to another part. In some embodiments, a sequence linker comprises a sequence that does not hybridize to a target polynucleotide and / or other oligomers in the combination or composition. In some embodiments, a non-sequence linker comprises an alkyl, alkenyl, amide, or polyethylene glycol group [(-CH 2 CH 2 O-) n ] is included.

[0158] A "stabilizing sequence" is a clamp, mixed nucleotide region, or other sequence that functions to increase the stability of a duplex region and / or control the register of hybridization (e.g., when located adjacent to a sequence prone to slippage, such as a containing repetitive nucleotide, e.g., poly-dA or poly-dT sequence). An "alignment sequence" is a stabilizing sequence that controls the register of hybridization. In addition to the clamps and mixed nucleotide regions described elsewhere herein, stabilizing sequences include GC-rich sequences and sequences containing affinity-enhancing modifications.

[0159] An "internal extension blocker" is an element located within the sequence of a nucleic acid or attached to a nucleic acid that prevents extension of a complementary strand along the nucleic acid. Examples include non-nucleotidic linkers or one or more abasic sites, non-natural nucleotides, or chemically modified natural nucleotides, as well as reversible extension blockers, as described below.

[0160] A "reversible extension blocker" is an internal extension blocker whose blocking function can be reversed, i.e., allowing extension of the complementary strand. Exemplary reversible extension blockers are unnatural nucleotides that have a complementary nucleotide that is accepted by the polymerase and exhibits specificity compared to natural nucleotides (i.e., the polymerase does not add a natural base beyond the reversible extension blocker). Providing the complementary nucleotide reverses the blocking function. Examples of unnatural base pairs in which either member of the pair can function as a reversible extension blocker are Iso-dC or Iso-dG; xanthine or 5-(2,4 diaminopyrimidine); 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one; 4-methylbenzimidizole or 2,4-difluorotoluene; 7-azaindole or isocarbostyril; dMMO2 or d5SICS; or dF or dQ. Other examples of reversible extension blockers are one or more nucleotides that are chemically modified, where the modification is attached via a reversible bond, and the bond can be reversed by providing one or more of: a chemical, an enzyme, a change in temperature, a change in reagent composition, etc.; a reversible nucleic acid structural feature; or a molecule reversibly attached to the capture oligomer, which is optionally a protein, enzyme, lipid, carbohydrate, or chemical moiety.

[0161] "Hybridization" or "hybridize" refers to the ability of two fully or partially complementary nucleic acid strands to come together in a parallel or antiparallel orientation under specified hybridization assay conditions to form a stable structure with a double-stranded region. "Hybridization" and "hybridize" are synonymous with "annealing" and "annealing", respectively. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. These hydrogen bonds are most commonly formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid strand, although base pairing can also be formed between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, e.g., RLP Adams et al., The Biochemistry of the Nucleic Acids (11th ed. 1992).) Additionally, triple-stranded regions can be formed by hybridizing a third strand to a B-form DNA duplex via Hoosteen base pairing.

[0162] As used herein, the term "specifically hybridize" means that under given hybridization conditions, a probe, primer or other oligomer (e.g., capture oligomer) detectably hybridizes substantially only to the target sequence in a sample containing the target sequence (i.e., there is little or no detectable hybridization to non-target sequences). In particular, an oligomer can be configured to specifically hybridize to any one of a set of targets (e.g., sequences from a particular taxonomic group, such as a species, genus, or more organisms). In some embodiments, a probe, primer or other oligomer (e.g., capture oligomer) can hybridize to its target nucleic acid to form a stable oligomer:target hybrid, but in some cases cannot form a sufficient number of stable oligomer:non-target hybrids for amplification or capture. Amplification and capture oligomers that specifically hybridize to a target nucleic acid are useful for amplifying and capturing target nucleic acids, but are not useful for amplifying and capturing non-target nucleic acids, especially non-target nucleic acids of phylogenetically closely related organisms. Thus, the oligomer hybridizes to a target nucleic acid to a sufficiently greater extent than non-target nucleic acid, allowing the skilled artisan to accurately capture, amplify, and / or detect the presence (or absence) of nucleic acid from a specific target (e.g., a specific pathogen) as required. In general, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence reduces the degree or rate of hybridization of the oligonucleotide to its target region. However, the inclusion of one or more non-complementary nucleosides or nucleic acid bases can promote the ability of the oligonucleotide to distinguish non-target nucleic acid sequences.

[0163] "Stringent hybridization conditions" or "stringent conditions" refers to conditions that (1) allow an oligomer to hybridize preferentially to a target nucleic acid as opposed to a different nucleic acid (e.g., a nucleic acid having only a single nucleotide difference in identity with the target nucleic acid), or (2) allow only an oligomer having a higher affinity target hybridizing sequence to hybridize to a target (compared to an oligomer having a lower affinity target hybridizing sequence), e.g., the higher affinity target hybridizing sequence is longer than the lower affinity target hybridizing sequence and / or contains affinity-enhancing modifications that the lower affinity target hybridizing sequence does not contain. While the definition of stringent hybridization conditions does not change, the actual reaction environment that may be used for stringent hybridization may vary depending on factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and the sequences of target and non-target nucleic acids that may be present in the test sample. Hybridization conditions include temperature and composition of the hybridization reagent or solution. Exemplary stringent hybridization conditions using oligomers of the present disclosure correspond to temperatures of about 40° C. to 75° C., e.g., 40° C. to 50° C., 50° C. to 60° C., or 60° C. to 75° C., with monovalent cation concentrations in the range of about 0.4 to 1 M, divalent cation concentrations in the range of about 0 to 10 mM, and pH in the range of about 5 to 9. Further details of hybridization conditions are provided in the Examples section. Other acceptable stringent hybridization conditions can be readily ascertained by one of skill in the art.

[0164] "Label" or "detectable label" refers to a moiety or compound attached directly or indirectly to an oligomer that is detected or provides a detectable signal. Any detectable moiety can be used, such as radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles (e.g., latex or metal beads) that impart a detectable color, luminescent compounds (e.g., bioluminescent compounds, phosphorescent compounds, or chemiluminescent compounds), and fluorescent compounds (i.e., fluorophores). Embodiments of fluorophores include those that absorb light (e.g., have peak absorption wavelengths) in the range of about 495 nm to 690 nm and emit light (e.g., have peak emission wavelengths) in the range of about 520 nm to 710 nm, including those known as FAM™, TET™, HEX, CAL FLUOR™ (orange or red), CY, and QUASAR™ compounds. Fluorophores can be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore, reducing background fluorescence. Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHER™ (or BHQ™), Blackberry Quencher® (or BBQ-650®), Eclipse® or TAMRA™ compounds.

[0165] A "non-extendable" oligomer or an oligomer that includes a "blocking moiety at its 3' end" includes a blocking moiety (also referred to as the 3' terminus) close enough to its 3' end to prevent extension. Any blocking moiety close enough to the 3' end to block extension is considered to be "at" the 3' end for purposes of this disclosure, even if it is not attached to or instead absent from the 3' hydroxyl or oxygen. A blocking moiety near the 3' end is, in some embodiments, within 5 residues of the 3' end and large enough to restrict binding of a polymerase to the oligomer, while other embodiments include a blocking moiety covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, including alkyl groups, non-nucleotidic linkers, alkane-diol dideoxynucleotide residues (e.g., 3'-hexanediol residues), and cordycepin. Further examples of blocking moieties include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other labels that prevent extension; inverted nucleotides (e.g., linked via a 3' to 3' phosphodiester to the preceding nucleotide, optionally with an exposed 5'-OH or phosphate); or proteins or peptides linked to an oligonucleotide to prevent further extension of the nascent nucleic acid chain by a polymerase. Non-extendable oligonucleotides of the present disclosure can be at least 10 bases in length and can be up to 15, 20, 25, 30, 35, 40, 50, or more nucleotides in length. Non-extendable oligonucleotides containing detectable labels can be used as probes.

[0166] A "binding partner" is a member of a pair of moieties that can be used to form a non-covalent association. An exemplary set of binding partners is biotin and a biotin-binding agent. Further examples of binding partners include, but are not limited to, digoxigenin / anti-digoxigenin, and more generally, antibodies and their targets.

[0167] A "biotin binding agent" is an agent (e.g., a polypeptide) that can specifically bind to biotin. Streptavidin, avidin, and neutravidin are examples of biotin binding agents. Anti-biotin antibodies are also considered biotin binding agents.

[0168] The term "antibody" encompasses any polypeptide that comprises a functional antigen-binding region with complementarity determining regions and framework regions (e.g., VH and VL domains), including, but not limited to, scFv, Fab and full length antibodies (e.g., IgA, IgG, IgD, IgE, or IgM antibodies).

[0169] The terms "unique molecular identifier (UMI)", "unique identifier (UID)", "molecular barcode", "randomer", "random molecular tag", "random barcode", "primer ID", "molecular label", "single molecule barcode" and "single molecule identifier (SMI)" are used interchangeably to refer to polynucleotide sequences whose sequences may be random, non-random, partially degenerate or degenerate. UMIs can be used to barcode DNA molecules prior to PCR or sequencing methods so that individual DNA strands can be identified. Amplicons containing the same UMI are assumed to originate from the same DNA molecule. UMIs can discriminate between true errors and errors that arise due to PCR or sequencing methods. UMIs can be about 5-100 nucleotides long or longer as needed to facilitate differentiation between a larger number of DNA strands, and can be of variable or uniform length. In some embodiments, UMIs can be introduced in the first two PCR cycles or using methods such as ligation, transposition by polymerase, endonucleases, transposases or any other methods known in the art.

[0170] The term "triangulation" means combining two or more results or data from independent analyses to determine an answer with a high degree of confidence.

[0171] As used herein, a "combination" of oligomers refers to any multiple oligomers in close proximity to one another, e.g., in different containers or the same container in a kit, or in a composition or set of compositions juxtaposed to one another, e.g., in a plate, rack, or other container.

[0172] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Common definitions can be found in technical books related to the field of molecular biology, such as Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY). EXAMPLES

[0173] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0174] A. Recovery of spiked antimicrobial resistance (AMR) targets directly from blood (DfB) using specific target capture (STC) oligomers Oligomers (all provided by IDT; 3'invdT and 3'invdC are the inverted nucleotides at the 3' end)

[0175] Bacterial target organisms - Klebsiella pneumoniae, ATCC strain BAA-1898; contains the AMR carbapenemase gene (KPC); Staphylococcus aureus, ATCC strain BAA-2094; contains the AMR mecA gene (mecA)

[0176] Specific Target Capture (STC) Oligomer - KPC STC oligomers were designed to bind to the AMR KPC gene with the following sequence: KPC_STC_F (SEQ ID NO: 6): 5'-biotin / AAAAACACCGCGCTGACCAACCTC / 3'invdT KPC_STC_R (SEQ ID NO: 7): 5'-biotin / AAAACACAGCGGCAGCAAGAAAGC / 3'invdT The mecA STC oligomer was designed to bind to mecA with the following sequence: mecA_STC_F-INT (SEQ ID NO: 8): 5'-biotin / AAAAAAGGTACTGCTATCCACCCTCAAACAGGT / 3'invdT mecA_STC_R2 (SEQ ID NO: 9): 5'-biotin / AAAATTGAGTTGAACCTGGTGAAGTTGTAATCTGG / 3'invdT

[0177] STC oligomers were designed to capture targets with the following sequences: mecA:5'-biotin / sequence / 3'invdC

[0178] Quantitative PCR (qPCR) was performed using primers with the following sequences: PCR1 KPC_P28_PCR1_F (SEQ ID NO: 10): 5'-AACCATTCGCTAAACTCGAACAGG-3' KPC_P28_PCR1_R (SEQ ID NO: 11): 5'-CCTTGAATGAGCTGCACAGTGG-3' mecA_P40_PCR1_F (SEQ ID NO: 12): 5'-CATGAAAAATGATTATGGCTCAGGTAC-3' mecA_P40_PCR1_R (SEQ ID NO: 13): 5'-TGGAACTTGTTGAGCAGAGGTTC-3' PCR2 KPC_P28_PCR2_F (SEQ ID NO: 14): 5'-CTTTGGCGGCTCCATCGG-3' KPC_P28_PCR2_R (SEQ ID NO: 15): 5'-CTCCTCAGCGCGGTAACTTAC-3' mecA_P40_PCR2_F (SEQ ID NO: 16): 5'-GCTATCCACCCTCAAACAGGTGAAT-3' mecA_P40_PCR2_R (SEQ ID NO: 17): 5'-ATTCTTCGTTACTCATGCCATACATA-3'

[0179] Protocol / reaction conditions Reactions were performed in 15 mL Falcon conical polypropylene tubes (Corning 352097) by adding 50 μL, 25 μL or 15 μL of pathogen spike (10, 5 or 3 CFU / mL, respectively), 1 mg of proteinase K (20 mg / mL, Promega MC5008), 30 μL of STC oligo pool (20 pmol / 5 μL), 100 μL of antifoam Y-30 emulsion (Sigma-Aldrich A6457-100ML) and 1.667 mL of dissolved NS4X buffer formulation E (100 mM Tris pH 8.0, 16.675% SDS) to 5 mL of whole blood.

[0180] The reaction mixture was mixed by vortexing and inversion for approximately 15 seconds per sample and pulse spun to a relative centrifugal force (RCF) of 700 using a swinging bucket centrifuge (Centrifuge 5810, Eppendorf) for a total duration of 10 seconds.

[0181] Protein digestion and subsequent cell lysis-reaction mixtures were incubated for 15 min in a custom laboratory heat block (Custom, DNAe) preheated to 75°C, such that the samples reached an internal temperature of approximately 60°C after 15 min of incubation.

[0182] The mixture was then transferred to an 8 mL polypropylene screw-cap tube (Fischer Scientific, NC9691446) containing pre-measured 4 gr of 0.1 mm VHD ZrO mechanical lysis (ML) beads (GlenMills Grinding Media) and mechanical lysis (ML) was performed using an OMNI Bead Ruptor Elite (OMNI International) using the "Blood" program (3 cycles of 90 s "mix" on and 20 s "dwell" off at a speed of 6.6 m / s).

[0183] The mechanically lysed samples were allowed to cool at room temperature (approximately 20-26 °C) for 5 min. The ML tubes were then centrifuged at 700 RCF for 1 min and the liquid was transferred back to the original 15 mL tube, leaving behind the ML beads. The sample tubes were pulse-spun in the centrifuge at up to 700 RCF for a total of 10 s.

[0184] Proteinase K inactivation and DNA denaturation - The tubes were placed in a heat block preheated to 100°C and incubated for 30 minutes.

[0185] Capture of target DNA using biotinylated STC oligos - The tubes were then transferred to a heat block preheated to 60°C and incubated for 40 minutes.

[0186] Capture of target DNA-STC oligos with streptavidin beads - The tubes were removed from the 60 °C heat block, the contents were cooled slightly to ambient temperature (approximately 20-26 °C), and 1.2 mg of streptavidin beads (custom streptavidin beads, DNAe) were added. The tubes were then incubated at 45 °C for 10 minutes with constant mixing (1500 RPM) in a heat / cool shaking incubator (Benchmark Scientific, model HC5000-HC).

[0187] Bead separation - Samples were pulse spun in a centrifuge at 700 RCF for up to 10 seconds, sample tubes were placed on a magnetic rack (Invitrogen, DynaMag-15) for 5 minutes, and the supernatant was aspirated and discarded.

[0188] Washing beads with Wash-S buffer - The beads were washed by adding 1 mL of Wash-S buffer (50 mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl) and stirring using a magnet. The washed beads and buffer were then transferred from the 15 mL tube to a new 1.5 mL tube, then magnetized for 2 minutes, and the Wash-S buffer was removed and discarded. The beads were then washed again by adding 1 mL of Wash-S buffer, magnetized for 2 minutes, and then the Wash-S buffer was removed and discarded.

[0189] Washing of beads with Wash-T buffer (10 mM Tris pH 8.0, 0.01% Tween-20) - The sample was then washed with 1 mL of Wash-T buffer and magnetized for 2 minutes, then the buffer was removed and discarded. The bead washing with Wash-T buffer was then repeated once more.

[0190] Target elution from beads - The magnet was removed from 1.5 mL and 50 μL of added IDTE elution buffer (10 mM Tris pH 7.5, 0.1 mM EDTA; Integrated DNA Technologies). The sample was then mixed, pulse spun, and incubated at 75° C. for 3 minutes to elute the target DNA from the streptavidin beads. The tube was then magnetized for 2 minutes, and the eluate containing the target DNA was then transferred to a new 1.5 mL tube (DNA LoBind Tube, 022431021, Eppendorf).

[0191] Quantitation - Capture of mecA and KPC targets from whole blood was confirmed using two qPCR reactions using the primers described above.

[0192] Results and Conclusions Two organisms containing AMR targets, Klebsiella pneumoniae containing KPC, and Staphylococcus aureus containing mecA, were spiked into 5 mL of whole blood at 10, 5, or 3 CFU / mL, respectively, and the previously described protocol (steps 6-19 above) was performed. The qPCR data in Figure 50 show that the desired targets were captured from the whole blood.

[0193] B. Multiplex Complete Capture Protocol Oligomers The following nested PCR2 primers (all supplied by IDT; "52-Bio" represents the two biotin groups on the 5'-end of the oligomer) were used to amplify each of the designated targets: 16s rRNA target P3F (SEQ ID NO: 18): 5'-AAAACGAGACATGCCGAGCATCCGCTTTAAGTCCCGCAACGAGCGCAA-3' P3R (SEQ ID NO: 19): / 52-Bio / ACCGTGCTGCCTTGGCTTCATTGTGGTCTTGACGTCATCCCCACCTTCCTC-3' 23s rRNA target P31F1 (SEQ ID NO: 20): 5'-AAAACGAGACATGCCGAGCATCCGCCGCATGTGTAGGATAGGTGGGAG-3' P31F2 (SEQ ID NO:21): 5'-AAAACGAGACATGCCGAGCATCCGCCGCATGTACAGGATAGGTAGGAG-3' P31R (SEQ ID NO: 22): / 52-Bio / GAGACCGCCCCAGTCAAACT-3' CTX-M group 1 target P48F (SEQ ID NO: 23): / 52-Bio / AAAACGAGACATGCCGAGCATCCGCTGTTAGGAAGTGTGCCGCTG-3' P48R (SEQ ID NO: 24): 5'-ACCGTGCTGCCTTGGCTTCATTGTGGTCTCCCGACTGCYGCTCTAAT-3' ("Y" is a mixture of C and T)

[0194] Protocol / reaction conditions 1000 genomic copies of E. coli genomic DNA (gDNA) were spiked into PCR2 singleplex reactions of P3F and P3R, P31F1, P31F2 and P31R, and P48F and P48R. Reactions were prepared in 0.2 mL tubes according to the recipes shown in Table 1.

[0195] [Table 3]

[0196] PCR2 amplification was carried out using the following thermal protocol, with steps b, c and d repeated for 40 cycles: a. 98°C for 30 seconds, b. 98°C for 5 seconds, c. 58°C for 10 seconds, d. 72°C for 30 seconds, e. hold at 4°C.

[0197] Reaction Pool - 40 μL of each singleplex PCR2 reaction was pooled.

[0198] Complete capture of PCR products -Streptavidin beads (MyOne C1, ThermoFisher Scientific) were resuspended at 8.33 mg / mL in bead resuspension buffer [1.50 M NaCl (Invitrogen), 10 mM Tris-HCl (pH 7.5) (Invitrogen), 0.10% Tween 20 (ThermoFisher Scientific)].

[0199] Binding of biotinylated target DNA to streptavidin beads - Equal volumes (120 µL) of resuspended beads and PCR2 product were combined with pipette mixing in a 0.2 mL tube. The tube was incubated at room temperature (approximately 20-26 °C) for 5 min with gentle mixing by vortexing, after which the beads were collected using a magnetic rack.

[0200] Washing of streptavidin beads with bound template - The beads were then washed twice by pipette mixing with 200 μL of wash buffer (1 M NaCl, 5 mM Tris-HCl (pH 7.5), 0.05% Tween 20, 0.5 mg / mL BSA), discarding the supernatant between each wash.

[0201] Elution with NaOH - 50 μL of 40 mM NaOH was added to a 0.2 mL tube, vortexed for 10 seconds, and left for 30 seconds. After 2 minutes of bead collection using a magnetic rack, the eluate was transferred to a 0.2 mL tube.

[0202] Results and Conclusions The target DNA was enriched using a PCR reaction, and the resulting biotinylated PCR2 products were pooled and captured using streptavidin beads, followed by elution of the ssDNA with NaOH. Elution of the desired ssDNA from the three NaOH eluates was confirmed on a TBE (Tris-Borate-EDTA) gel at 200V until the reference dye reached the bottom of the gel. The gel was then stained with 1x SYBR gold for at least 20 minutes and visualized on a UV station as shown in Figure 51.

[0203] C. Target Polynucleotide Enrichment Using Multiplex Polymerase Chain Reaction (PCR) Oligomers (all supplied by IDT; "Y" is a mixture of C and T, "W" is a mixture of A and T, "S" is a mixture of C and G, and "R" is a mixture of A and G)

[0204] The following polymerase chain reaction (PCR) 1 primers were used to amplify each of the designated targets: 16s rRNA target P1 (SEQ ID NO: 25): 5'-TGTAGCGGTGAAATGCGYAGA-3' P1 (SEQ ID NO: 26): 5'-CGGTCGACTTAACGCGTTAGCT-3' P1 (SEQ ID NO: 27): 5'-CGGAGTGCTTAATGCGTTWGCT-3' P2 (SEQ ID NO: 28): 5'-CGCAAGGTTGAAACTCAAAGGAATTG-3' P2 (SEQ ID NO: 29): 5'-CCGCAAGGTTAAAACTCAAATGAATTG-3' P2 (SEQ ID NO: 30): 5'-GGGACTTAACCCAACATYTCAC-3' P29 (SEQ ID NO: 31): 5'-CCTGGCTCAGAATGAACGCT-3' P29 (SEQ ID NO: 32): 5'-CCTGGCTCAGGACGAACGCT-3' P29 (SEQ ID NO: 33): 5'-GAGTCTGGACCGTGTCTCAGT-3' P29 (SEQ ID NO: 34): 5'-GAGTCTGGGCCGTGTCTCAGT-3' P3 (SEQ ID NO: 35): 5'-CGTGTGTAGCCCAGGTCATAAGG-3' P3 (SEQ ID NO: 36): 5'-CACGTGTGTAGCCCAAATCATAAGG-3' P3 (SEQ ID NO: 37): 5'-TGTGTAGCCCTGGTCGTAAGG-3' P3 (SEQ ID NO: 38): 5'-TCAGCTCGTGTCGTGAGATGTT-3' P3 (SEQ ID NO: 39): 5'-CGTCAGCTCGTGTTGTGAAATGTT-3' P30 (SEQ ID NO: 40): 5'-CTCCTACGGGAGGCAGCAGT-3' P30 (SEQ ID NO: 41): 5'-CCTCCGTATTACCGCGGCTG-3' 23S rRNA target P31 (SEQ ID NO: 42): 5'-GAAAGACCCCGTGAACCTTTACT-3' P31 (SEQ ID NO: 43): 5'-GAAAGACCCCGTGGAGCTTTACT-3' P31 (SEQ ID NO: 44): 5'-CCTTCGTGCTCCTCCGTTAC-3' P31 (SEQ ID NO: 45): 5'-CCTTTGAGCGCCTCCGTTAC-3' P4 (SEQ ID NO: 46): 5'-ACACAGGTCTCTGCTAAACCGTAAG-3' P4 (SEQ ID NO: 47): 5'-ACACAGGTCTCTGCAAAATCGTAAG-3' P4 (SEQ ID NO: 48): 5'-ACACAGCACTGTGCAAACACGAAAG-3' P4 (SEQ ID NO: 49): 5'-TACCCGACAAGGAATTTCGCTACC-3' Internal control target P25 (SEQ ID NO: 50): 5'-TGGCAGCTTCACTTTCTCTTGC-3' P25 (SEQ ID NO: 51): 5'-CCAGCTCCAATCACACCAACA-3' SHV target P26 (SEQ ID NO: 52): 5'-CAGCTGCTGCAGTGGATGGT-3' P26 (SEQ ID NO: 53): 5'-CCGGSGTATCCCGCAGATA-3' KPC target P28 (SEQ ID NO: 54): 5'-AACCATTCGCTAAACTCGAACAGG-3' P28 (SEQ ID NO: 55): 5'-CCTTGAATGAGCTGCACAGTGG-3' mecC target P32 (SEQ ID NO: 56): 5'-GCCGTAATAGTACCTGGTTTGAA-3' P32 (SEQ ID NO: 57): 5'-GCCYTTYGGGTGTTTTGTTAGG-3' MCR-1 target P33 (SEQ ID NO: 58): 5'-TCTGCAACACCAATCCTTATAACG-3' P33 (SEQ ID NO: 59): 5'-CATCATATCGCTTAAAATACGCAGGC-3' NDM target P34 (SEQ ID NO: 60): 5'-AGATTGCCGAGCGACTTGGC-3' P34 (SEQ ID NO: 61): 5'-CAACTTTGGCCCGCTCAAGG-3' OXA-23-like target P35 (SEQ ID NO: 62): 5'-ACAGAATATGTGCCAGCCTCTACA-3' P35 (SEQ ID NO: 63): 5'-CATGGCTTCTCCTAGTGTCATGTCT-3' OXA-48-like target P36 (SEQ ID NO: 64): 5'-GCGGTAGCAAAGGAATGGCA-3' P36 (SEQ ID NO: 65): 5'-TGCTTGGTTCGCCCGTTTA-3' OXA-51-like P37 (SEQ ID NO: 66): 5'-AACGAAGCACACACTACGGGTGT-3' P37 (SEQ ID NO: 67): 5'-TGCTCAAGGCCGATCAAAGCATT-3' gyrA targeting P39 (SEQ ID NO: 68): 5'-GCAATGACTGGAACAAAGCCTA-3' P39 (SEQ ID NO: 69): 5'-ACCAGCATGTAACGCAGCGA-3' mecA target P40 (SEQ ID NO: 70): 5'-CATGAAAAATGATTATGGCTCAGGTAC-3' P40 (SEQ ID NO: 71): 5'-TGGAACTTGTTGAGCAGAGGTTC-3' vanA target P41 (SEQ ID NO: 72): 5'-GGCTGCGATATTCAAAGCTCAG-3' P41 (SEQ ID NO: 73): 5'-CTGAACGCGCCGGCTTAAC-3' vanB target P42 (SEQ ID NO: 74): 5'-GTATGGAAGCTATGCAAGAAGCC-3' P42 (SEQ ID NO: 75): 5'-CATGCAAAACCGGGAAAGCCA-3' TEM_E104K target P45 (SEQ ID NO: 76): 5'-GCGGTATTATCCCGTGTTGACG-3' P45 (SEQ ID NO: 77): 5'-TCACTCATGGTTATGGCAGCA-3' TEM_G238S target P46 (SEQ ID NO: 78): 5'-GATAAAGTTGCAGGACCACTTCTG-3' P46 (SEQ ID NO: 79): 5'-CCCCGTCRTGTAGATAACTACGA-3' CTX-M group 1 target P48 (SEQ ID NO: 80): 5'-CGGCARCCGTCACGCTGT-3' P48 (SEQ ID NO: 81): 5'-CATCAGCACGATAAAGTATTTGCGA-3' CTX-M group 2 targets P49 (SEQ ID NO: 82): 5'-TGCATGCGCAGRCGAACA-3' P49 (SEQ ID NO: 83): 5'-CCTTACTGGTACTGCACATCGC-3' P49 (SEQ ID NO: 84): 5'-TTGCTGGTGCTGCACATCGC-3' CTX-M group 8-25 targets P50 (SEQ ID NO: 85): 5'-TACCACCACGCCRTTAGCGA-3' P50 (SEQ ID NO: 86): 5'-ACAACCCACGATGTGGGTAG-3' CTX-M group 9 targets P51 (SEQ ID NO: 87): 5'-GTGCTTTATCGCGGTGATGAAC-3' P51 (SEQ ID NO: 88): 5'-GTTAACCAGATCGGCAGGCT-3' 28S rRNA H13-20 target P52 / 53 (SEQ ID NO: 89): 5'-ACTGTACTTGTGCGCTATCGGT-3' P52 / 53 (SEQ ID NO: 90): 5'-TCCTCAGTAACGGCGAGTGAAGC-3' 28S rRNA H26-31 P54 (SEQ ID NO: 91): 5'-CCGTCTTGAAACACGGACCA-3' P54 (SEQ ID NO: 92): 5'-GTTTCCTCTGGCTTCACCCTATTC-3' 28S rRNA H45-46 target P56 (SEQ ID NO: 93): 5'-AACAACTCACCGGCCGAATG-3' P56 (SEQ ID NO: 94): 5'-ATGGAACCTTTCCCCACTTCAGT-3' 28S rRNA H78-79 target P57 (SEQ ID NO: 95): 5'-CCCTGTTGAGCTTGACTCTAGTTTGA-3' P57 (SEQ ID NO: 96): 5'-CTGCGTTATGGTTTAACAGATGTGC-3' IMP group identification Reg1 target P59 (SEQ ID NO: 97): 5'-GACGCCTATCTGATTGAYACTCCA-3' P59 (SEQ ID NO: 98): 5'-CATTTGTTAATTCAGATGCATAYGTGG-3' P59 (SEQ ID NO: 99): 5'-GAGGCTTACCTAATTGACACTCCA-3' P59 (SEQ ID NO: 100): 5'-CTGAAGCTTATCTAATTGACACTCCA-3' P59 (SEQ ID NO: 101): 5'-CTGATGCCTATATAATTGACACTCCA-3' P59 (SEQ ID NO: 102): 5'-CATTAGTTAATTCAGACGCATACGTGG-3' IMP group identification Reg2 target P60 (SEQ ID NO: 103): 5'-GCAAATTTAGAAGCTTGGCCAAAGTCY-3' P60 (SEQ ID NO: 104): 5'-GCCTTTACTTTCATTTAGCCCTTTAA-3' P60 (SEQ ID NO: 105): 5'-AAATGTTGAAGCATGGCCACATTCG-3' P60 (SEQ ID NO: 106): 5'-GCCTTTTGCTTTCATTAAGCCCTTTTA-3' VIM group target P61 (SEQ ID NO: 107): 5'-GGTGTTTGGTCGCATATCGCAAC-3' P61 (SEQ ID NO: 108): 5'-GCGATCGTCATGAAAGTGCGT-3' gyrB target P62 (SEQ ID NO: 109): 5'-TCCTATAAAGTGTCCGGCGGTC-3' P62 (SEQ ID NO: 110): 5'-TCTCGCCGGTAACCGCCA-3' P63 (SEQ ID NO: 111): 5'-AACCAGGCGATTCTGCCG-3' P63 (SEQ ID NO: 112): 5'-GCAGCTTGTCCGGGTTGTA-3' P64 (SEQ ID NO: 113): 5'-GCACCATTTAGTGTGGGAAATTGTCG-3' P64 (SEQ ID NO: 114): 5'-TAACTTCGACAGCTGGACGT-3' P65 (SEQ ID NO: 115): 5'-GGCGGTGGCGGATACAAAGTAT-3' P65 (SEQ ID NO: 116): 5'-ACCTGTCTTATCAGTTGTGCCAAC-3'

[0205] The following nested PCR2 primers were used to amplify each of the designated targets: 16S rRNA target P1 (SEQ ID NO: 117): 5'-TAGAACACCGATGGCGAAGGC-3' P1 (SEQ ID NO: 118): 5'-TCGTGGACTACCAGGGTATCTA-3' P2 (SEQ ID NO: 119): 5'-TTTCGATGCAACGCGAAGAACCT-3' P2 (SEQ ID NO: 120): 5'-TACGAGCTGACGACAGCCATG-3' KPC target P28 (SEQ ID NO: 121): 5'-CTTTGGCGGCTCCATCGG-3' P28 (SEQ ID NO: 122): 5'-CTCCTCAGCGCGGTAACTTAC-3' MCR-1 target P33 (SEQ ID NO: 123): 5'-CGGTATGCTCGTTGGCTTAGATG-3' P33 (SEQ ID NO: 124): 5'-GTGATTGCCCATTTGGTGCAG-3' vanA target P41 (SEQ ID NO: 125): 5'-TTGTATGGACAAATCGTTGACATACA-3' P41 (SEQ ID NO: 126): 5'-GTAGCTGCCACCGGCCTAT-3' 28S rRNA H45-46 target P56 (SEQ ID NO: 127): 5'-AATGGATGGCGCTCAAGCGT-3' P56 (SEQ ID NO: 128): 5'-ACTGCCACCAAGATCTGCACTAG-3'

[0206] Protocol / reaction conditions One hundred genome copies of each organism were spiked into "PCR1 Master Mix" (see Table 2 for formulation).

[0207] [Table 4]

[0208] PCR1 - PCR amplification was performed using the following three-step thermocycle, with steps b and c repeated for 25 cycles: a. 98°C for 30 seconds (initial denaturation step), b. 98°C for 5 seconds (denaturation step), c. 65°C for 25 seconds (annealing / extension step).

[0209] Sample dilution - Samples were then diluted 40-fold using molecular grade water (ThermoFisher Scientific) to reduce off-target PCR amplicon levels.

[0210] Add PCR2 Reagents - 10 μL of diluted PCR1 material was added to 40 μL of "PCR2 Master Mix" to make a final dilution of 1:200 (see Table 3 for PCR2 Master Mix formulation). In this example, the PCR2 Master Mix contains 1.50 μM of each primer pair (P1, P28, P33, and P56).

[0211] [Table 5]

[0212] PCR2 - PCR2 amplification was carried out using the following three-step thermal protocol, with steps b and c repeated for 40 cycles: a. 98°C for 30 seconds (initial denaturation step), b. 98°C for 5 seconds (denaturation step), c. 65°C for 25 seconds (annealing / extension step).

[0213] The PCR2 products were confirmed using Bioanalyzer sizing and quantification.

[0214] Results and Conclusions Two multiplex PCR reactions were used to enrich the target DNA. The resulting PCR2 products were separated and quantified using Bioanalyzer quantification. Table 4 shows the concentrations obtained from the PCR2 targets after successful PCR amplification.

[0215] [Table 6]

[0216] In conclusion, 100 genome copies of each organism were enriched using multiplex PCR to obtain the desired dsDNA.

[0217] D. Target Nucleic Acid Enrichment Using Multiplex Polymerase Chain Reaction (PCR) and Integral Capture Oligomers (all supplied by IDT; "52-Bio" represents two biotin groups on the 5'-end of the oligomer; "Y" is a mixture of C and T; "W" is a mixture of A and T)

[0218] The following PCR1 primers were used to amplify specific targets: 16s rRNA target P1 (SEQ ID NO: 25): 5'-TGTAGCGGTGAAATGCGYAGA-3' P1 (SEQ ID NO: 26): 5'-CGGTCGACTTAACGCGTTAGCT-3' P1 (SEQ ID NO: 27): 5'-CGGAGTGCTTAATGCGTTWGCT-3' P2 (SEQ ID NO: 28): 5'-CGCAAGGTTGAAACTCAAAGGAATTG-3' P2 (SEQ ID NO: 29): 5'-CCGCAAGGTTAAAACTCAAATGAATTG-3' P2 (SEQ ID NO: 30): 5'-GGGACTTAACCCAACATYTCAC-3' vanA target P41 (SEQ ID NO: 72): 5'-GGCTGCGATATTCAAAGCTCAG-3' P41 (SEQ ID NO: 73): 5'-CTGAACGCGCCGGCTTAAC-3'

[0219] The following nested PCR2 primers were used to amplify each of the 16s rRNA targets: P2 (SEQ ID NO: 129): 5'-AAAACGAGACATGCCGAGCATCCGCTTTCGATGCAACGCGAAGAACCT-3' P2 (SEQ ID NO: 130): 5'- / 52-Bio / TACGAGCTGACGACAGCCATG-3'

[0220] Protocol / reaction conditions One hundred genome copies of each organism were added to the "PCR1 Master Mix." The PCR1 Master Mix was prepared in a 0.2 mL tube according to the formulation shown in Table 5.

[0221] [Table 7]

[0222] PCR1 - PCR amplification was performed using the following three-step thermal protocol, with steps b and c repeated for 25 cycles: a. 98°C for 30 seconds (initial denaturation step), b. 98°C for 5 seconds (denaturation step), c. 65°C for 25 seconds (annealing / extension step).

[0223] Sample dilution - Samples were then diluted 40-fold using molecular grade water (ThermoFisher Scientific) to reduce off-target PCR amplicon levels.

[0224] Addition of PCR2 Reagents - 10 μL of diluted PCR1 material was added to 40 μL of "PCR2 Master Mix" to make a final dilution of 1:200. The PCR2 Master Mix was prepared according to the formula shown in Table 6. In this example, the PCR2 Master Mix contains 1.50 μM primer mix (P2 shown in

[0487] ).

[0225] [Table 8]

[0226] PCR2 - PCR2 amplification was carried out using the following three-step thermal protocol, with steps b and c repeated for 45 cycles: a. 98°C for 30 seconds (initial denaturation step), b. 98°C for 5 seconds (denaturation step), c. 65°C for 25 seconds (annealing / extension step).

[0227] The PCR2 products were confirmed using Bioanalyzer sizing and quantification.

[0228] Complete capture of PCR products -Streptavidin beads (MyOne C1, ThermoFisher Scientific) were resuspended at 8.33 mg / mL in bead resuspension buffer [1.50 M NaCl (Invitrogen), 10 mM Tris-HCl (pH 7.5) (Invitrogen), 0.10% Tween 20 (ThermoFisher Scientific)].

[0229] Binding of biotinylated target DNA to streptavidin beads - Equal volumes (120 µL each) of resuspended beads and PCR2 product were combined in a 0.2 mL tube. The tube was incubated at room temperature (approximately 20-26 °C) for 10 min, mixed gently by vortexing, and then the beads were allowed to bind on a magnetic rack.

[0230] Washing of streptavidin beads with bound template - The beads were then washed three times with 200 μL of wash buffer [1 M NaCl, 5 mM Tris-HCl (pH 7.5), 0.05% Tween 20, 0.5 mg / mL BSA], discarding the supernatant between each wash.

[0231] Elution with NaOH: 50 μL of 40 mM NaOH was added to a 0.2 mL tube, vortexed for 10 seconds, and left for 30 seconds. After immobilization on a magnetic rack for 2 minutes, the eluate was transferred to a new 0.2 mL tube.

[0232] Results and Conclusions The ssDNA elution from the NaOH eluate was checked on a TBE gel at 200 V until the reference dye reached the bottom of the gel. The gel was then stained with 1× SYBR gold for at least 20 minutes and visualized on a UV station as shown in FIG.

[0233] In conclusion, DNA from three target organisms was amplified in PCR1, and then one target was amplified in PCR2. Total capture of the PCR2 product was then performed to generate the desired ssDNA for hybridization with primers bound to the surface of a semiconductor chip.

[0234] E. Targeted enrichment (PCR1 and nested PCR2) followed by copy control Oligomers (all supplied by IDT; "iSP18" is a hexaethylene glycol (HEG) internal spacer; 3BiodT is a 3' biotin molecule linked to a terminal dT nucleotide; 56-FAM / is a 5' linkage of 6-FAM (fluorescein); / ZEN / is a proprietary IDT ZEN quencher molecule; / 3IABkFQ / is a 3' Iowa Black FQ quencher). The following polymerase chain reaction (PCR) 1 primers were used for the E. Faecium (EFM) target: P41F (SEQ ID NO: 131): 5'-GGCTGCGATATTCAAAGCTCAG-3' P41R (SEQ ID NO: 132): 5'-CTGAACGCGCCGGCTTAAC-3' The following nested PCR2 primers were used to target the EFM amplicon from PCR1: P41F (SEQ ID NO: 133): 5'-AAAACGAGACATGCCGAGCATCCGCTTGTATGGACAAATCGTTGACATACA-3' P41R (SEQ ID NO: 134): 5'-ACCGTGCTGCCTTGGCTTCATTGTGGTCGTAGCTGCCACCGGCCTAT-3' The following hairpin oligo sequence (SEQ ID NO: 135) was used: 5'-CGCGCGAAAAAAAAAAAAAAAAAAAAA / iSp18 / TTTTTTTTTTTTTTTCGCGCGAAAAACTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC-3'

[0235] An intervening oligo was used between the hairpin oligo and the beads: PolydT oligo sequence (SEQ ID NO: 136): TTTTTTTTTTTTTTTTTTTT / 3BiodT /

[0236] For quantitative PCR (qPCR), the following primers were used: RPA1F (SEQ ID NO: 137): 5'-AAAACGAGACATGCCGAGCATC-3' RPA1outerR (SEQ ID NO: 138): 5'-TCGCGCGAAAAACTCCTCTGG-3' FAM probe (SEQ ID NO: 139): 5'- / 56-FAM / TGCTGGGAT / ZEN / AGCTACTCCCGCCTTT TGG / 3IABkFQ / -3'

[0237] Standard curve control sequence (SEQ ID NO: 140): 5'-AAAACGAGACATGCCGAGCATCCGCTTGTATGGACAAATCGTTGACATACATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAATAAAGATGATAGGCCGGTGGCAGCTACGACCACAATGAAGCCAAGGCAGCACGGTGCCAGAGGAGTTTTTCGCGCGA-3'

[0238] Protocol / reaction conditions Sample dilution - EFM genomic DNA (gDNA) was diluted to 1000 copies / μL

[0239] Add PCR1 Reagents - 10 μL of diluted EFM gDNA was added to 40 μL of PCR1 Master Mix. The PCR1 Master Mix was prepared according to the formulation shown in Table 7.

[0240] [Table 9]

[0241] PCR1 - PCR amplification was carried out using the following three-step thermal protocol, with steps b and c repeated for 30 cycles: a. 98°C for 30 seconds (initial denaturation step), b. 98°C for 5 seconds (denaturation step), c. 65°C for 25 seconds (annealing / extension step).

[0242] Sample dilution - 2.5 μL of PCR1 reaction was diluted with 97.5 μL of water to make a 1:40 dilution.

[0243] Addition of PCR2 Reagents - 5 μL of diluted PCR1 material was added to 20 μL of PCR2 Master Mix to make a final dilution of 1:200. The PCR2 Master Mix was prepared according to the formulation shown in Table 8.

[0244] [Table 10]

[0245] PCR2 - PCR2 amplification was carried out using the following thermal protocol, repeating steps b, c and d for 45 cycles. a. 98°C for 30 seconds (initial denaturation step), b. 98°C for 5 seconds (denaturation step), c. 55°C for 10 seconds (annealing step), d. 72°C for 30 seconds (extension step), e. increasing from 65°C to 95°C at a rate of 0.5°C per cycle (melting step).

[0246] The PCR2 product was confirmed using Bioanalyzer sizing and quantification. The stock concentration was found to be 800ng / μL, which correlates to 1.88×10^13 copies per 40μL. This 40μL sample was used as the "neat" condition.

[0247] PCR2 sample dilution - Samples were diluted 1:10 for a separate "1:10" input, resulting in 1.88x10^12 copies per 40μL.

[0248] Addition of Extension Master Mix - 60 μL of Extension Master Mix was added to 40 μL of either "neat" or "1:10" PCR2 output material, mixed with a pipette, followed by rapid vortexing and rapid spin. The Extension Master Mix was prepared according to the formulation shown in Table 9.

[0249] [Table 11]

[0250] Extension - Extension was carried out using the following thermal protocol: a. 92°C for 2 minutes, b. 64°C for 2 minutes, c. 68°C for 10 minutes.

[0251] Add Hybridization Mix - 50 μL of Hybridization Mix was added to the completed extension reaction and mixed with a pipette. The Hybridization Mix was prepared according to the formula shown in Table 10.

[0252] [Table 12]

[0253] Preparation and addition of streptavidin-conjugated magnetic beads: Streptavidin particles (prepared in-house) - 0.2 mg were washed 3 times with 1x wash buffer (Table 11) and resuspended in 50 μL of 1x wash buffer in a 0.2 mL tube. 50 μL of conjugated beads were then added to the extension / hybridization mix and mixed with a pipette.

[0254] [Table 13]

[0255] The reaction was incubated at 25° C. for 2 minutes and then the tube was placed on a magnetic stand for 30 seconds.

[0256] The supernatant was aspirated and the beads mixed with 1× wash buffer (Table 5) by pipette and placed back on the magnetic stand until clear.

[0257] The wash was repeated as above for a total of three times.

[0258] After the third wash, the supernatant was removed and the beads were resuspended in 30 μL of ultrapure water.

[0259] The tubes were incubated at 70°C for 1 minute, flicked to mix, spun, and returned to 70°C for 1 minute.

[0260] The eluate was removed and quantified using qPCR, and 2 μL of input was added to 13 μL of PCR quantification master mix prepared according to the formulation shown in Table 12. All reactions were performed in triplicate. Standard curve control sequences were run at 2×10^7, 2×10^6, 2×10^5 and 2×10^4 copies.

[0261] [Table 14]

[0262] The qPCR reaction was carried out using the following thermal protocol, where steps b, c and d were repeated for 40 cycles: a. 95°C for 60 seconds, b. 95°C for 10 seconds, c. 64°C for 25 seconds, d. 72°C for 10 seconds.

[0263] Results and Conclusions The target DNA was concentrated using PCR reactions. The output of PCR2 was quantified using a bioanalyzer and found to be 80.0ng / μL, which corresponds to 1.88×10^13 copies per 40μL (used as the "neat" condition). The sample was also diluted 1:10 for a separate "1:10" input, which was 1.88×10^12 copies per 40μL.

[0264] The copy control process normalized the outputs of two samples with input concentrations differing by 10-fold to nearly equal values ​​(only a 6% difference), demonstrating the ability of this process to normalize a wide range of input concentrations to the desired values, as shown in Table 13.

[0265] [Table 15]

[0266] F. Capture of a Predetermined Amount of Amplicon by a Capture Oligomer Comprising a Capture Sequence and Its Complement Oligomers PCR to amplify a segment from the E. coli uidA gene was performed using the primers: Ec_uidA_F (SEQ ID NO: 141): GTATCAGCGCGAAGTCTTTATACC Ec_uidA_R (SEQ ID NO: 142): GGCAATAACATACGGAGTGACATC

[0267] The primers were designed to generate an amplicon having the following sequence (SEQ ID NO:143): GTATCAGCGCGAAGTCTTTATACCGAAAGGTTGGGCGGGCCAGCGTATTGTACTGCGTTTCGATGCGGTCACTCATTACGGCAAAGTGTGGGTAAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACTCCGTATGTTATTGCC

[0268] A capture oligomer, designated uidA_PA_1.2, was prepared having the following sequence (SEQ ID NO: 144): AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTCTA / iSp18 / TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGACGCAAGCTACTGGTGATTTGGCAATAACATACGGAGTGACATCGGCTTC (iSp18 = hexaethylene glycol (HEG) internal spacer (IDT))

[0269] In this oligomer, the 5' polyA sequence is the capture sequence. CCTCTA is the linker sequence. iSp18 is an internal extension blocker. The polyT sequence following iSp18 is the complement of the capture sequence. AGACGCAAGCTACTGGTGATTT is a fourth additional sequence. The target hybridizing sequence (THS) is GGCAATAACATACGGAGTGACATCGGCTTC, which specifically hybridizes to a segment of the uidA gene sequence in the target amplicon. In this example, the THS is longer than the reverse PCR primer (see sequence above) which it overlaps, to increase the Tm of the THS and give it a competitive advantage over the reverse primer in hybridizing to the target.

[0270] A secondary capture reagent was used with the following sequence: dT 20 -Biotin (SEQ ID NO: 145): TTTTTTTTTTTTTTTTTTTT / 3'Biotin

[0271] For quantitative PCR (qPCR) analysis of copy control products, the following primers and probes were used: Ec_uidA_F (SEQ ID NO: 141): GTATCAGCGCGAAGTCTTTATACC uidA_Probe (SEQ ID NO: 146): 56-FAM / TAGCCGCCCTGATGCTCCATCACTTCCTG / 3'Iowa Black TQ_R (SEQ ID NO: 147): AGACGCAAGCTACTGGTGAT

[0272] Protocol / reaction conditions (1) PCR amplicons were generated for target uidA using the primers above. The amplicons were purified using AMPure XP (Beckman Coulter) using the manufacturer's recommended protocol and quantified by qPCR using the uidA forward and reverse primers with uidA_Probe.

[0273] (2) Capture oligomer annealing and amplicon strand extension - Purified uidA amplicons were diluted 2, 10 or 100 fold. A 20 μL aliquot of each dilution of the amplicon was added to 0.07 U / μl SDPol (Bioron), 1× SDPol reaction buffer, 0.17 mM dNTPs, 3 mM MgCl 2 , 1mg / ml BSA and 5×10 10 Oligomer annealing / extension reactions consisting of 100 copies of capture oligomer were captured in a final volume of 30 μL. The capture oligomer was annealed to the 3' end of the complementary strand of the uidA amplicon and the amplicon strand was extended using a thermal cycler according to the following thermal profile: 92°C for 2 min, 54°C for 2 min, 68°C for 10 min, 54°C for 2 min, followed by a controlled ramp down (0.3°C / sec) to 20°C. In this example, the 3' end of the capture oligomer was also extended.

[0274] (3) Hybridization of the Complement of the Capture Sequence of the Capture Oligomer—The entire capture oligomer / amplicon extension reaction mixture was added to 10 μL of secondary capture reagent at a 4x concentration to give a final concentration of 125 mM NaCl, 0.25 mg / ml BSA, and 10 μl of the complement of the capture sequence. 7 , 10 8 or 10 9 copies (i.e., three different amounts were tested). Hybridization was performed by incubating the reaction mixture at room temperature (20-24 °C) for 15 min.

[0275] (4) Capture of amplicon and capture oligomer extension product / capture oligomer complexes - A 5 μL aliquot (50 μg) of MyOneC1 streptavidin beads (ThermoFisher Scientific) in 250 mM NaCl and 1 mg / ml BSA was added to the hybridization mixture (step 3 above) to capture oligomer / amplicon extension product / complement of the capture sequence complex onto the beads, and the beads were washed according to the manufacturer's recommendations.

[0276] (5) Elution - After the final wash was completed and the wash buffer removed, 10 μL of water was added to the bead pellet, the beads resuspended and incubated for 2 minutes at 70° C. The beads were pelleted with a magnet and the eluate removed.

[0277] (6) Quantification - The amount of elution product as well as the captured oligomer extension product (see step 2 above) was quantified by qPCR using primers targeting the uidA_F primer site and the TQ primer-adapter site together with the uidA_Probe.

[0278] Results and conclusions: 2-fold, 10-fold and 100-fold dilutions of the amplicons produced in the targeted enrichment step (see step 1 above) were 7 , 10 8 or 10 9 Each PCR dilution was subjected to a copy control process (see steps 2-5 above) using a capture oligomer of 10 copies. As shown in Table 14, the amount of amplicon recovered was proportional to the amount of capture oligomer added for each PCR dilution (approximately in the expected ratio; see the "Ratio" column in the table). The variability between replicates at each data point was low (see the "Standard Deviation" column).

[0279] [Table 16]

[0280] Furthermore, despite a 50-fold difference in amplicon input, the variation in output was 10 7 , 10 8 and 10 9 The copy number for the capture oligomer was 2.36-fold, 3.11-fold, and 3.44-fold, respectively (Table 1). As shown above, the variation between replicates was low.

[0281] These data demonstrate that the capture oligomers described herein can be used to obtain a given amount of target output from a range of different input target amounts.

[0282] Additional experiments were performed essentially as described above, but in a multiplex format using capture oligomers whose THS regions were designed to target the uidA gene of E. coli, the nuc gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, or the rpb7 gene of Candida albicans (i.e., four capture oligomers were used per reaction). The uidA, nuc, and vanA genes were amplified separately using PCR as described above, using the primers shown above for uidA and additional primers designed for the nuc and vanA genes, respectively. The resulting amplicons were diluted 10-fold or 100-fold, and 20 μL aliquots of each dilution of each individual target were amplified with 5 × 10 of each of the four capture oligomers described above. 10 A separate capture oligomer annealing and extension of the amplicon strand reaction containing 1 copy each (i.e., 4-plex capture oligos, but with only one target present) was added. The reaction conditions were the same as described, except for the following thermal profile: 92°C for 2 min, 64°C for 2 min, 68°C for 10 min, 98°C for 2 min, 57°C for 2 min, followed by a controlled ramp-down (0.3°C / sec) to 20°C. After this reaction was completed, 5 x 10 of the complement of the capture sequence of the capture oligomer was added. 8 A copy was added to each reaction, followed by the capture, wash, elution and quantification steps described above.

[0283] The output amounts after capturing amplicons with input amounts that differed by 10-fold are shown in Table 15. The results show that for each of the three individual target amplicons tested in multiplex format, the 10-fold difference in input amplicon levels was reduced to a difference in average output levels after copy control of 1.4-fold or less. Furthermore, the range of average output levels after copy control spanned approximately 1.6-fold across all three target amplicons, although their input levels spanned more than 270-fold.

[0284] [Table 17]

[0285] Further experiments were performed in a singleplex format essentially as above, but using a capture oligomer in which THS anneals to a universal binding site in a tag sequence that was incorporated into the target of interest during the PCR amplification step. Primers were designed to target the bacterial 23S rRNA gene and PCR amplicons were generated from bacterial genomic DNA. The reverse primer contained a universal sequence tag that was incorporated into the amplicon during PCR. Neat (i.e., no dilution; approximately 9 × 10 12 copy) or 50-fold dilution (approximately 2 × 10 11 Prepare a 40 μL aliquot of 1000 copies of the PCR product by mixing with 0.02 U / μL SD polymerase (Bioron), 0.4× SD polymerase reaction buffer (Bioron), 0.012 mM 4dNTPs, 1.8 mM MgCl 2 , 0.6 mg / mL BSA and 5 x 10 in a final reaction volume of 100 µL 10 Capture oligomer annealing and amplicon strand extension were performed as described above, except that 10 copies of the capture oligomer were added to the reaction mixture containing the capture oligomer. The thermal profile used was 92°C for 2 min, 54°C for 2 min, 68°C for 10 min, 54°C for 2 min, followed by a controlled ramp down to 20°C (0.3°C / sec). The total volume of the reaction was adjusted to 1 mg / ml BSA, 125 mM NaCl, and 5 x 10 8The 50 μL volume of 3× annealing mix containing 1× secondary capture reagent was added. Annealing was performed by incubating the reaction mixture on a thermal block at 25° C. for 10 min. A 50 μL volume (200 μg in this experiment) of MyOneC1 streptavidin beads (ThermoFisher Scientific) in 4× wash buffer consisting of 4 M NaCl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.20% Tween 20, 2 mg / mL BSA was added to the 150 μL reaction described above. The resulting complex containing the capture oligomer, amplicon extension product and the complement of the capture sequence was captured on the beads, and the beads were washed according to the manufacturer's recommendations (in this case, the use of 1× wash buffer; see 4X formulation immediately above). A volume of 20 μL of water was used for elution (protocol and others the same as above). qPCR was performed using a specific forward primer and a reverse primer targeting the universal tag.

[0286] Despite a 50-fold difference in amplicon input levels, the output only varied 2.5-fold after annealing the capture oligomer and extending the amplicon strand step, and only 1 (i.e., perfectly normalized) after contact with the secondary capture reagent and isolation of the resulting complex (see Table 16). These results further demonstrate an embodiment of the present disclosure in which THS binds to a universal tag sequence.

[0287] [Table 18]

[0288] Further experiments were performed essentially as described above (singleplex, universal THS), but in a multiplex format using a capture oligomer that anneals to a universal tag sequence that the THS incorporated into each of the targets of interest during the PCR amplification step. Eight different amplicons targeting regions in the bacterial 16S rRNA gene, 23S rRNA gene, and the antibiotic resistance marker KPC were generated from K. pneumonia genomic DNA in eight separate singleplex reactions. One amplicon targeting a synthetic internal control (IC) DNA was also generated for a total of nine individual amplicons. Equal amounts of all nine amplicons were pooled and diluted neat (i.e., no dilution; approximately 1 × 10 13 copy) or 10-fold dilution (approximately 1 × 10 12 54 μL aliquots of 5 × 10 copies were added to separate the annealing of the capture oligomer and the extension of the amplicon strand reaction mixture (final volume of 100 μL each). 11 Copy capture ol...

Claims

1. 1. A method for analyzing a target in a sample, comprising: Introducing a sample into the cartridge; introducing the cartridge into an instrument operable to manipulate the sample, automatically isolating a target nucleic acid from the sample; amplifying the isolated target nucleic acid; sequencing the amplified target nucleic acid using next generation sequencing; Including, the sample remains in the cartridge throughout the isolating, amplifying, and sequencing steps; A method for analyzing a target in a sample.

2. 2. The method of claim 1, wherein the isolating, amplifying and sequencing steps are performed within 8 hours after introducing the sample into the cartridge.

3. 2. The method of claim 1, wherein the target nucleic acid comprises a fungal nucleic acid present in the sample at levels as low as 3 copies.

4. 2. The method of claim 1, wherein the target nucleic acid comprises a bacterial nucleic acid present in the sample at levels as low as 3 copies.

5. 2. The method of claim 1, wherein the target nucleic acid comprises a viral nucleic acid present in the sample at levels as low as a single copy.

6. The method of claim 1 , wherein the cartridge has an external volume of about 3 liters or less.

7. The method of claim 6 , wherein the cartridge has an external volume of about 2.5 liters or less.

8. The method of claim 7 , wherein the cartridge has an external volume of about 2.1 liters or less.

9. The method of claim 6 , wherein the cartridge has a longest linear dimension of about 200 mm or less.

10. The method of claim 9 , wherein the cartridge has a longest linear dimension of about 160 mm or less.

11. 2. The method of claim 1, wherein the sample is selected from the group consisting of a biological sample, a clinical sample, an environmental sample and a food sample.

12. The method of claim 11 , wherein the sample is a biological sample obtained from a subject and is unprocessed prior to introduction into the cartridge.

13. The method of claim 1 , wherein the isolating step comprises digesting proteins in the sample.

14. 14. The method of claim 13, comprising digesting the protein using proteinase K.

15. 10. The method of claim 1, wherein the isolating step comprises lysing an organism to release the target nucleic acid.

16. The method of claim 15 , wherein the lysing comprises mechanical lysing.

17. 17. The method of claim 16, wherein the mechanical lysis comprises flowing the sample through a lysis chamber in the cartridge and rotating a paddle within the lysis chamber.

18. 20. The method of claim 17, wherein the mechanical melting further comprises adding zirconium beads to the melting chamber prior to rotating the paddle within the melting chamber.

19. The method of claim 1 , wherein the isolating step comprises denaturing the target nucleic acid.

20. 20. The method of claim 19, wherein the denaturation comprises heat denaturation.

21. The isolating step comprises: annealing a target capture oligonucleotide to the target nucleic acid to form a complex; binding the complex onto a solid support; removing unbound material from the solid support; The method of claim 1 , comprising capturing the target nucleic acid by

22. 22. The method of claim 21, wherein removing unbound material comprises washing the solid support-bound complex with a wash reagent.

23. 22. The method of claim 21 , wherein the amplifying step is performed on a target nucleic acid bound to the solid support.

24. 22. The method of claim 21, further comprising eluting the target nucleic acid from the washed solid support to prepare the isolated target nucleic acid.

25. 24. The method of claim 23, wherein the amplifying step is performed directly on the eluted target nucleic acid without any intervening steps.

26. 21. The method of claim 20, wherein the isolating step automatically isolates the target nucleic acid from a sample having a volume of about 1 mL to about 25 mL.

27. 10. The method of claim 1, wherein the isolating step comprises only one purification step.

28. The method of claim 1 , wherein the isolated nucleic acid is amplified without quantification.

29. The step of amplifying performing a first amplification of the isolated target nucleic acid using a first primer set to produce a first amplification product; diluting and aliquoting the first amplification product into a plurality of aliquots; performing a second amplification of the target nucleic acid in a plurality of aliquots using a plurality of second primer sets to produce a plurality of second amplification products; pooling the second amplification products; The method of claim 1 , comprising:

30. 30. The method of claim 29, wherein one or more primers in the first primer set are identical to one or more primers in the plurality of second primer sets.

31. 31. The method of claim 30, wherein the amplifying step further comprises purifying the pooled second amplification products to produce the amplified target nucleic acid.

32. 31. The method of claim 30, wherein one or more of the first and second amplifications comprises a PCR amplification.

33. 31. The method of claim 30, wherein the plurality of aliquots comprises at least 10 separate aliquots.

34. 31. The method of claim 30, wherein the first PCR amplification and the second PCR amplification are performed without quantification.

35. 31. The method of claim 30, wherein one or more of the plurality of second primer sets is nested with respect to the first primer set.

36. 2. The method of claim 1, wherein the amplifying step comprises performing copy control on the amplified target nucleic acid prior to the sequencing step.

37. The method of claim 1 , wherein the amplifying step comprises only one purification step.

38. The method of claim 1 , wherein the amplified target nucleic acid is sequenced without quantification.

39. The step of sequencing comprises: immobilizing the amplified target nucleic acid onto a semiconductor surface within the cartridge that contains an ion-sensitive field effect transistor (ISFET) sensor; The method of claim 1 , comprising:

40. 40. The method of claim 39, wherein all products of the amplifying step flow over the semiconductor surface without an intervening step.

41. 40. The method of claim 39, wherein the amplified target nucleic acid is immobilized by a capture oligomer bound above the ISFET sensor, the capture oligomer hybridizing to a portion of the target nucleic acid.

42. 42. The method of claim 41, wherein the surface comprises an array of ISFET sensors having wells disposed thereon.

43. 42. The method of claim 41, wherein at least one of the wells is positioned above a plurality of ISFET sensors in the array of ISFET sensors.

44. 43. The method of claim 42, wherein one or more of the wells contain a surface-bound forward primer that hybridizes to a portion of the target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, and the sequencing step comprises paired-end sequencing.

45. 43. The method of claim 42, wherein one or more of the wells, or gaps between one or more of the wells, contain a plurality of binding-inactive oligomers that do not hybridize to the target nucleic acid.

46. 40. The method of claim 39, wherein the amplified target nucleic acid is immobilized by a universal capture oligomer bound above the ISFET sensor, the universal capture oligomer hybridizing to a universal binding site.

47. 47. The method of claim 46, wherein the amplifying step comprises amplifying the isolated target nucleic acid using a primer that includes the universal binding site.

48. 47. The method of claim 46, wherein the amplifying step comprises ligating an adaptor comprising the universal binding site to the isolated target nucleic acid.

49. 47. The method of claim 46, wherein the sequencing step comprises clonal amplification of the immobilized target nucleic acid.

50. 50. The method of claim 49, wherein said clonal amplification comprises recombinase polymerase amplification.

51. 50. The method of claim 49, wherein said clonal amplification comprises rolling circle amplification.

52. 50. The method of claim 49, wherein the clonal amplification comprises bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification.

53. A sample cartridge comprising: Sample input, a sample preparation unit operable to receive a sample from the sample input and to isolate a target nucleic acid from the sample; a library preparation unit operable to receive the isolated target nucleic acid from the sample preparation unit and to amplify the isolated target nucleic acid; and a sequencing unit operable to receive the amplified target nucleic acid from the library preparation unit and to sequence the amplified target nucleic acid; A sample cartridge comprising:

1. An apparatus comprising: an apparatus including a cartridge interface including physical and electronic connections operable to drive movement of the sample and reagents within the cartridge and to communicate with the sequencing unit; Including, the system.

54. 54. The system of claim 53, wherein one or more reagents necessary for isolating the target nucleic acid, amplifying the isolated target nucleic acid, and sequencing the amplified nucleic acid are dry reagents, and the device is operable to reconstitute the one or more reagents.

55. one or more reagent cartridges containing one or more reagents necessary to isolate the target nucleic acid, amplify the isolated target nucleic acid, and sequence the amplified nucleic acid; the device is operable to transfer reagents from the one or more reagent cartridges to the sample cartridge; 54. The system of claim 53.

56. the sample cartridge and the one or more reagent cartridges include a sealed pneumatic interface (SPI) port; 56. The system of claim 55, wherein the device is operable to transfer the one or more reagents from the one or more reagent cartridges to the sample cartridge via the SPI port using one or more pipettes.

57. 57. The system of claim 56, wherein the apparatus includes a three degree of freedom pipette gantry operable to transfer the one or more reagents.

58. 54. The system of claim 53, operable to isolate, amplify and sequence target fungal nucleic acid in the sample at levels as low as 3 copies.

59. 54. The system of claim 53, operable to isolate, amplify and sequence target bacterial nucleic acid in the sample at levels as low as three copies.

60. 54. The system of claim 53, which is operable to isolate, amplify, and sequence target viral nucleic acid present in the sample at levels as low as a single copy.

61. 54. The system of claim 53, wherein the cartridge has an external volume of about 3 liters or less.

62. 62. The system of claim 61, wherein the cartridge has an external volume of about 2.5 liters or less.

63. 63. The system of claim 62, wherein the cartridge has an external volume of about 2.1 liters or less.

64. 62. The system of claim 61, wherein the cartridge has a longest linear dimension of about 200 mm or less.

65. 65. The system of claim 64, wherein the cartridge has a longest linear dimension of about 160 mm or less.

66. 54. The system of claim 53, wherein the device has a volume of about 150 liters or less.

67. 67. The system of claim 66, wherein the device has a volume of about 135 liters or less.

68. 54. The system of claim 53, wherein the device has a longest linear dimension of about 700 mm or less.

69. 70. The system of claim 68, wherein the device has a longest linear dimension of about 650 mm or less.

70. 54. The system of claim 53, wherein the sample cartridge is operable to accept a biological sample, a clinical sample, an environmental sample, and a food sample.

71. 54. The system of claim 53, wherein the sample cartridge is operable to receive an unprocessed biological sample.

72. 54. The system of claim 53, wherein isolating the target nucleic acid comprises digesting proteins in the sample.

73. 73. The system of claim 72, wherein the device is operable to expose the sample to proteinase K in the sample preparation unit.

74. 54. The system of claim 53, wherein the sample preparation unit is operable to lyse an organism to release the target nucleic acid.

75. 75. The system of claim 74, wherein the sample preparation unit comprises a lysis chamber including a rotating paddle, and the device is operable to interface with the sample cartridge to flow the sample into the lysis chamber and rotate the rotating paddle to mechanically lyse organisms in the sample.

76. 76. The system of claim 75, further comprising zirconium beads in the lysis chamber.

77. 54. The system of claim 53, wherein isolating the target nucleic acid comprises denaturing the target nucleic acid.

78. 78. The system of claim 77, wherein the device is operable to supply thermal energy to the sample preparation unit to denature nucleic acids therein.

79. The apparatus, Exposing the sample to a target capture oligonucleotide and a solid support in the sample preparation unit, allowing the target capture oligonucleotide to anneal to the target nucleic acid to form a complex and bind the complex to the solid support.

54. The system of claim 53, operable to:

80. 80. The system of claim 79, wherein the device is further operable to introduce a wash buffer to the solid support-bound complex to separate the solid support-bound complex from unbound sample.

81. 81. The system of claim 80, wherein the apparatus is operable to transfer the separated solid support-bound complexes to the library preparation unit and amplify the solid support-bound target nucleic acids.

82. 80. The system of claim 79, wherein the device is operable to introduce an elution buffer to the separated solid support bound complexes to elute the target nucleic acids from the solid support and transfer the eluted target nucleic acids to the library preparation unit for amplification.

83. 80. The system of claim 79, wherein the device is operable to introduce amplification reagents to the solid support bound complex and amplify the target nucleic acid within the sample preparation unit.

84. 54. The system of claim 53, operable to automatically accommodate a sample received by the sample input having a volume of between about 1 mL and about 25 mL.

85. The device interfaces with the library preparation unit of the sample cartridge to introduce necessary reagents and provide thermal energy; performing a first amplification of the isolated target nucleic acid using a first primer set to produce a first amplification product; diluting and aliquoting the first amplification product into a plurality of aliquots; performing a second amplification of the target nucleic acid in the plurality of aliquots using a plurality of second primer sets to produce a plurality of second amplification products; pooling the second amplification products; 54. The system of claim 53, operable to:

86. 86. The system of claim 85, wherein one or more primers in the first primer set are identical to one or more primers in the plurality of second primer sets.

87. 86. The system of claim 85, wherein the device is further operable to purify the pooled second amplification products to produce the amplified target nucleic acid.

88. 86. The system of claim 85, wherein one or more of the first and second amplifications comprises a PCR amplification.

89. 86. The system of claim 85, wherein the plurality of aliquots comprises at least 10 separate aliquots.

90. 54. The system of claim 53, further operable to perform copy control on one or more of the isolated target nucleic acid and the amplified target nucleic acid to control the number of output copies transferred to the library preparation unit or the sequencing unit, respectively.

91. 54. The system of claim 53, wherein the sequencing unit comprises a semiconductor surface including an array of ion-sensitive field effect transistor (ISFET) sensors, each having a well disposed thereover, the device is operable to immobilize the amplified target nucleic acid above the array of ISFET sensors, and the array of ISFET sensors electronically connects with the device via the electronic connection of the cartridge interface when a sample cartridge is disposed therein.

92. 92. The system of claim 91, wherein the apparatus is operable to direct all output from the library preparation unit to the wells on the semiconductor surface.

93. 92. The system of claim 91, comprising capture oligomers bound onto the array of ISFET sensors, the capture oligomers configured to hybridize to a portion of the target nucleic acid.

94. 92. The system of claim 91, wherein at least one of the wells is positioned above a plurality of ISFET sensors in the array of ISFET sensors.

95. 92. The system of claim 91, wherein one or more of the wells contain a surface-bound forward primer that hybridizes to a portion of the target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, and the device is operable to perform paired-end sequencing.

96. 92. The system of claim 91, wherein one or more of the wells and the gaps between the wells contain a plurality of binding-inactive oligomers that do not hybridize to the target nucleic acid.

97. 92. The system of claim 91, comprising a universal capture oligomer bound onto the array of ISFET sensors, the universal capture oligomer configured to hybridize to a universal binding site.

98. 98. The system of claim 97, wherein the apparatus is operable to interface with the library preparation unit to amplify the isolated target nucleic acid using a primer comprising the universal binding site.

99. 98. The system of claim 97, wherein the apparatus is operable to ligate an adaptor to the isolated target nucleic acid in the sample preparation unit or the library preparation unit, the adaptor comprising the universal binding site.

100. 98. The system of claim 97, wherein the device is operable to interface with the sequencing unit to perform clonal amplification of the immobilized target nucleic acid.

101. The system of claim 100, wherein the clonal amplification comprises recombinase polymerase amplification.

102. 101. The system of claim 100, wherein the clonal amplification comprises rolling circle amplification.

103. 101. The system of claim 100, wherein the clonal amplification comprises bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification.

104. 54. The system of claim 53, wherein the physical and electrical connections include a pneumatic system for driving fluid movement within the sample cartridge.

105. 54. The system of claim 53, wherein the cartridge interface further comprises physical and electronic connections by which the device operatively communicates with one or more of the sample preparation unit and the library preparation unit.

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