Sample in sequencing-ready library out automated library preparation solution

Abstract

The present disclosure generally relates to cartridge-based methods and devices for preparing a sequencing nucleic acid library from a sample. In embodiments, a cartridge-based method comprises placing a sample in a self-contained cartridge having a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, utilizing the filter to isolate the nucleic acid from the sample; forming a library prep reaction mixture; causing the library prep reaction mixture to flow into the reaction vessel and preparing the sequencing nucleic acid library comprising the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid; isolating the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the library prep reaction mixture on to the filter and eluting each from the filter; and purifying and optionally size selecting the sequencing nucleic acid library on the filter.

Description

SAMPLE IN SEQUENCING-READY LIBRARY OUTAUTOMATED LIBRARY PREPARATION SOLUTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is being filed as a PCT International Patent Application which claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63 / 691,747, filed on September 6, 2024, the entire contents of which are hereby incorporated by reference in their entirety. To the extent appropriate, a claim of priority is made to the above-disclosed application.FIELD

[0002] The present invention relates generally to a cartridge-based methods, devices, and kits for preparing a sequencing nucleic acid library from a sample. The disclosed methods, devices, and kits overcome the hands-on and labor-intensive methods of preparing a sequencing nucleic acid library from a sample by, for example, compensating for insufficient staff, human errors, and time-consuming labor-intensive processes.INTRODUCTION

[0003] The creation of a sequencing nucleic acid library, e.g.. a Next Generation Sequencing (NGS) library, is hands-on, labor-intensive, and costly. Even for applications requiring less data, such as microbial whole genome sequencing (WGS) or RNA-seq, the majority' of the total sequencing cost can be attributed to library preparation, a critical upstream process that ultimately determines the success or failure of a sequencing run. A typical NGS library preparation begins with the extraction of genomic DNA (gDNA) followed by mechanical or enzymatic fragmentation. Sufficiently large yield from nucleic acid extraction is required to ensure high library complexity, while a fragmentation method that does not introduce bias is critical. The dsDNA fragments must then be ligated to indexed adapters or primers that will eventually enable hybridization to the NGS flow cell for cluster generation. To achieve this, the ends of the fragments are blunted, adenylated, and 5’ phosphorylated. A ligase enzyme is then able to attach the indexed adapters to the A- overhangs. This reaction generates unwanted by-products, known as adapter-dimers, when adapters are ligated to one another rather than to the DNA inserts. Adapterdimers can be problematic since they will readily hybridize and amplify on thesequencing flow cell, taking the place of DNA inserts and providing useless sequencing data. A post-ligation purification step is thus typically performed using solid phase reversible immobilization (SPRI), a method that employs paramagnetic beads (MBs) to selectively bind larger DNA fragments. Purification steps are sensitive and timeconsuming, requiring large-volume washes and pipette mixing. Following purification, adapter-ligated DNA fragments are amplified via a polymerase chain reaction (PCR) to generate input library concentrations of >5 ng / pL for the NGS flow cell. A post-PCR purification step is necessary to remove unwanted primer and adapter dimers, nucleotides, and any DNA fragments less than 200 bp in length.

[0004] It is clear that NGS sample preparation requires many sequential steps using specialized laboratory equipment. Because it is hands-on and labor-intensive it is error prone and susceptible to delay from insufficient staff, complicated workflows, and the requirement for numerous transitions between steps in a workflow. There are liquid handling robots from, e.g., Tecan®, Beckman Coulter® (e.g., BioMek®), or Perkin Elmer®; there are automated, batch-based nucleic acid extraction and library preparation instruments from Thermo Fisher® (e.g.. Ion Chef®); and there is the Miroculus Canvas electrowetting-based NGS library prep instrument. But, to date there is no solution to prepare a single sequencing library' on-demand and allow for scaling up to make up to 4, 16, or 80 libraries on a single instrument. A need exists for a streamlined workflow to compensate for insufficient staff, user errors, and. in some cases, to facilitate adoption of NGS at all.

[0005] The methods, devices, and kits presented herein achieve rapid creation of a sequencing nucleic acid library from a single sample, in some embodiments, using a closed and affordable instrument, e.g.. GeneXpert System from Cepheid®. The methods, devices, and kits of the present disclosure offer the only known solution for preparing a single sequencing library on-demand that allows for scaling, e.g., making up to 4, 16, or 80 libraries on a single instrument. Notably, methods disclosed herein provide complete control of the mixing and toggling process used during the creation of a sequencing nucleic library from a sample, e.g., via a GeneXpert System from Cepheid®. The control of the mixing and toggling conditions (e.g., speed, volume, viscosities) is important for optimization of yields, reduction of sample degradation, and generally a more desired sequencing nucleic acid library. This type of a control, and therefore a more desirable outcome, is not currently available with other available systems or methods.SUMMARY

[0006] The present disclosure presents cartridge-based methods, devices, and kits for preparing a sequencing nucleic acid library from a sample.

[0007] In one aspect, the present disclosure relates to a cartridge-based method for preparing a sequencing nucleic acid library' from a sample, comprising: (a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality- of chambers, a reaction vessel, and a filter disposed in a fluidic path betyveen the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of the nucleic acid and detection of signals generated during amplification; (b) utilizing the filter to isolate the nucleic acid from the sample; (c) forming a library prep reaction mixture comprising the nucleic acid and a library prep reagent that facilitates: i) generation of tagged nucleic acid fragments from the nucleic acid in the library prep reaction mixture, ii) generation of amplicons, wherein the amplicons are optionally tagged at one or both ends, and / or iii) attachment of oligonucleotide adapters to one or both ends of the tagged nucleic acid fragments or amplicons to form adapter-labeled nucleic acid; (d) causing the library prep reaction mixture to floyv into the reaction vessel and preparing the sequencing nucleic acid library comprising the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid; (e) isolating the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the library prep reaction mixture on to the filter and eluting each from the filter; and (f) purifying and optionally size selecting the sequencing nucleic acid library on the filter, yvherein a bubble bursting mechanism is utilized to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings of the reaction vessel.

[0008] In embodiments, releasing the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the filter during step (e) is during and / or after preparing the sequencing nucleic acid library’. In embodiments, a cartridge-based method for preparing a sequencing nucleic acid library from a sample further comprises eluting the nucleic acid from the filter following step (b). In embodiments, a cartridgebased method for preparing a sequencing nucleic acid library from a sample further comprises further comprising amplify ing the nucleic acid yvith at least one set of primers for amplify ing a target region of the nucleic acid within the reaction vessel following step (b).

[0009] In a second aspect, the present disclosure relates to a cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising: (a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a fdter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of nucleic acid and detection of signals generated during amplification; (b) utilizing the filter to isolate the nucleic acid from the sample; (c) fragmenting the nucleic acid to form nucleic acid fragments, wherein fragmenting is performed by mechanical fragmentation, chemical fragmentation, or enzymatic fragmentation; and / or amplifying the nucleic acid or nucleic acid fragments to form amplicons, wherein amplifying the nucleic acid or nucleic acid fragments is with at least one set of primers for amplifying a target region of the nucleic acid or nucleic acid fragments; (d) generating adapter-labeled nucleic acid comprising labeling the fragmented nucleic acid or amplicons at one or both ends the fragmented nucleic acid or amplicons; and (e) isolating the adapter labeled nucleic acid on to the filter to form the NGS nucleic acid library, wherein the cartridge-based method comprises reuse of the reaction vessel, the filter, and optionally one or more of the plurality of chambers with rinsing to remove or dilute carryover, wherein reuse of the reaction vessel comprises a bubble bursting mechanism to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings.

[0010] In embodiments of either the first or second aspect of the present disclosure, the sample in step (a) comprises intact cells, the method further comprises releasing nucleic acid from the intact cells by lysing the intact cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers and / or capturing the cells on the filter and lysing the cells by means of sonication, to release nucleic acid from the cells. In embodiments of either the first or second aspect of the present disclosure, the cartridge-based methods further comprise amplifying the adapter-labeled nucleic acid with at least one set of primers for amplifying and increasing adapter-labeled nucleic acid concentration following step (d) and / or further comprises purifying and size selecting the isolated adapter labeled nucleic acid to form the sequencing nucleic acid library and / or further comprises isolating and washing the nucleic acid on the filter, and eluting the nucleic acid from the filter prior to step (c). In aspects of the present disclosure, size selecting the isolated adapter labeled nucleic acid in step (e) comprises solid-phase reversible immobilization.

[0011] In embodiments of either the first or second aspect of the present disclosure, the cartridge-based methods further comprise using a reusable reagent for one or more steps, including lysing the cells in the sample, isolating the nucleic acid released from the cell, washing the nucleic acid, eluting the nucleic acid, library prep reagent, fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter labeled nucleic acid, amplifying the adapter labeled nucleic acid, isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purifying and size selecting the isolated adapter labeled nucleic acid.

[0012] In a third aspect, the present disclosure relates to a cartridge for preparing a sequence nucleic acid library from a sample, comprising: a cartridge body comprising a plurality of chambers in fluid communication therein; a filter disposed in a fluidic path between the plurality of chambers and a reaction vessel; the reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for amplification of nucleic acid and detection of a plurality of amplification products via real-time PCR, melt curve analysis, or a combination thereof, wherein the reaction vessel comprises: a planar frame defining a first fluidic path and a second fluidic path between a first planar substrate that encloses a first side of the planar frame and a second planar substrate that encloses a second side of the planar frame; a fluidic interface at one end of the planar frame, the fluidic interface comprising a first fluid inlet and a first fluid outlet of the first fluidic path, and a second fluid inlet and a second fluid outlet of the second fluidic path; a first reaction chamber arranged in the planar frame between the first and second substrates, the first reaction chamber being an enlarged portion of the first fluidic path disposed along the fluidic path between the first fluidic inlet and the first fluid outlet so as to allow a fluid sample introduced via the first fluid inlet to undergo amplification before exiting the first fluid outlet, wherein the second fluidic path further includes an optical chamber defined in the planar frame between the first and second substrate, the optical chamber including a planar optical- substrate adapted for detection of one or a plurality of amplification products adapted for detection of signals generated during amplification by an optical assembly, and wherein the optical chamber being in fluidic communication with the second fluidic inlet and the second fluidic outlet; a second reaction chamber arranged in the planar frame between the first and second substrates, the second reaction chamber being an enlarged portion of the second fluidic path disposed along the second fluidic pathbetween the second fluidic inlet and the optical chamber so as to allow a fluid sample introduced via the second fluid inlet to undergo amplification before filling the optical chamber, wherein the second reaction chamber includes a chamber exit in fluidic communication with the optical chamber entrance that is in fluidic communication with the optical chamber, and wherein the first reaction chamber together with the first fluid inlet are accessed by one the plurality of chambers and the second reaction chamber together with the second fluid inlet are accessed by a different one of the plurality of chambers.

[0013] In embodiments of the third aspect, the cartridge is configured to carry out non-isothermal amplification, optionally by thermal cycling or temperature oscillation. In additional embodiments of the third aspect, the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and a lysis chamber in fluidic communication with the sample chamber, wherein the lysis chamber is adapted for performing mechanical and chemical lysis to release nucleic acid from the biological sample, optionally wherein the sample chamber and lysis chamber are the same.

[0014] In embodiments of the third aspect, one or more lysis reagents comprise a chaotropic agent, a chelating agent, a buffer, and a detergent. The chaotropic agent may be selected from guanidinium thiocyanate, guanidinium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or a combination thereof. The one or more lysis reagents comprise a guanidinium compound, sodium hydroxide, EDTA, a buffer, and a detergent.

[0015] In embodiments of the third aspect, the filter is configured to bind the nucleic acid to be analyzed. The filter may comprise glass fibers and optionally a polymeric binder, or the glass fibers are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof. The filter may comprise a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron. The filter may be configured to bind unwanted material and allow the nucleic acid to pass through.

[0016] In embodiments of the third aspect, the cartridge further comprises a binding reagent, wash reagent, eluting reagent, or a combination thereof. The eluting reagent comprises ammonia or an alkali metal hydroxide. The eluting reagent maycomprise a poly anion, optionally selected from the group consisting of a carrageenan, a carrier nucleic acid, and i-carrageenan with KOH. In embodiments, the at least one of the plurality of chambers comprises one or more lyophilized reagents and in embodiments at least one of the first reaction chamber and the second reaction chamber comprises lyophilized reagents for amplification. The one or more lyophilized reagents is / are in the form of one or more beads. The one or more lyophilized reagents may be selected from primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof. In embodiments, the reagents and components in the reaction chambers are in solution.

[0017] In another aspect, the present disclosure relates to kits for carrying out the methods described herein, wherein in some cases the methods are carried out with devices described herein. Kits may include one or more reagents useful for practicing any of the disclosed methods. In some embodiments, a kit comprises one or more lysis reagents for releasing nucleic acid from the sample and / or one or more amplification reagents for amplifying nucleic acid from the sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1 A-1C show an overview of a sample cartridge with a valve assembly configured for performing differing sample processes, e.g., PCR and integrated nucleic acid analysis in accordance with some embodiments of the present disclosure. FIG. 1A shows the sample cartridge body with reaction vessel, FIG. IB shows an exploded view of the sample cartridge, and FIG. 1 C shows components of the valve assembly, in accordance with some embodiments.

[0019] FIG. ID shows a universal valve assembly, in accordance with some embodiments, that utilizes glass beads suited for mechanical lyses of certain types of targets, as compared to conventional valve assemblies in conventional sample cartridges.

[0020] FIG. 2 shows an overview of a reaction vessel embodiment of the present disclosure where the reaction vessel includes two chambers, each with an independent port: well I in liquid communication with port I and well 2 in liquid communication with port 2.

[0021] FIG. 3 shows a comparison of steps among 6 different commercially available library reparation protocols that are replaced by the methods, devices, and kits of the present disclosure.

[0022] FIG. 4 shows steps for an embodiment of a cartridge-based method for preparing a sequencing nucleic acid library from a sample.

[0023] FIG. 5 shows steps for an embodiment of a cartridge-based method for preparing a sequencing nucleic acid library from a sample.

[0024] FIG. 6 shows steps for an embodiment of bubble bursting optionally used during a cartridge-based method for preparing a sequencing nucleic acid library from a sample.

[0025] FIG. 7 shows steps for an embodiment of a cartridge-based method for preparing a sequencing nucleic acid library. The method includes unique molecular identifiers (UMIs) PCR, blocker displacement amplification (BDA) PCR, and adapter PCR completed within 5 hours or less.

[0026] FIG. 8A shows the sequence coverage of a library produced from Cartridge A according to an embodiment of the disclosure.

[0027] FIG. 8B shows the sequence coverage of a library produced from Cartridge A according to an embodiment of the disclosure.

[0028] FIG. 8C shows the sequence coverage of a library produced from Cartridge A according to an embodiment of the disclosure.

[0029] FIG. 9A shows the fragment size distribution of a library7created with a sample preparation workflow using an adapted Xpert® Carba-R assay with a TET sample preparation eluant, according to an embodiment of the disclosure.

[0030] FIG. 9B shows the fragment size distribution of a library created with a sample preparation workflow using an adapted Xpert® Carba-R assay with a TWB sample preparation eluant, according to an embodiment of the disclosure.

[0031] FIG. 10A shows the sequence coverage of a library produced from a sample preparation workflow using an adapted Xpert® Carba-R assay with a TET sample preparation eluant, according to an embodiment of the disclosure.

[0032] FIG. 10B shows the sequence coverage of a library produced from a sample preparation workflow using an adapted Xpert® Carba-R assay with a TWB sample preparation eluant, according to an embodiment of the disclosure.

[0033] FIG. 11 shows steps for an embodiment of a cartridge-based method for preparing a sequencing nucleic acid library. The nucleic acid library- is tagmented and amplifies to add indexes and adapters, and can be used on a commercially available sequencing platform such as Illumina®.DETAILED DESCRIPTION

[0034] The present disclosure presents cartridge-based methods, devices, and kits for preparing a sequencing nucleic acid library from a sample. The methods are readily automated using devices and kits of the present disclosure to achieve rapid creation of a sequencing nucleic acid library from a single sample, in some embodiments, using a closed and affordable instrument, e.g., GeneXpert System from Cepheid*.Definitions of Selected Terms

[0035] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0036] The term “nucleic acid" refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural as well as non-natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.

[0037] The term “nucleic acid” also includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA). which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA.

[0038] The term “nucleic acid” further encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triplestranded nucleic acid complexes, the nucleic acid strands need not be coextensive, i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands.

[0039] The term “nucleic acid” also encompasses any modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality7to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2’ -position sugar modifications. 5-position pyrimidine modifications, 8- position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5 -bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like. More particularly, in some embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides(containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e.g., Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7: 1817-195; Okamoto, et al.. (2002) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No. 2: 171-172), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entirety for their disclosure of LNAs.

[0040] The nucleic acid(s) can be derived from a completely chemical sy nthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0041] As used herein, the term “gene” encompasses coding sequences, introns, and any associated control sequences that participate in the expression of the coding sequences.

[0042] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two singlestranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the two single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0043] “Selective hybridization” or “selective annealing” refers to the binding of a nucleic acid to a target region in the absence of substantial binding to other nucleicacids present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated.

[0044] The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides, and any value in between. Typically, oligonucleotides are single-stranded DNA molecules.

[0045] The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from about 10 to about 30 nucleotides, or, in some embodiments, from about 10 to about 60 nucleotides in length. In some embodiments, primers can be, e g., about 15 to about 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.

[0046] A primer is said to “anneal to” or “hybridize to” another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence.

[0047] The term “primer pair” refers to a set of primers including a 5?“upstream primer” or “forward primer” that hybridizes with the complement of the 5’ end of the DNA sequence to be amplified and a 3’ "dow nstream primer” or “reverse primer” that hybridizes with the 3 ’ end of the sequence to be amplified. As will be recognized by those of skill in the art, the terms “upstream” and “downstream” or“forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments.

[0048] A “probe” is a nucleic acid capable of binding to a target region of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable moiety to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size.

[0049] As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target region under suitable annealing conditions.

[0050] The term “target” is used herein with reference to “target regions,” as well as “target organisms.” The former refers to nucleic acids to be detected, and the latter refers to organisms to be detected. The term, “target region” is generally used herein to refer to a segment of nucleic acid that is defined by a primer pair and that gives rise to an amplicon produced in an amplification reaction; the term “amplification target” is also used herein to refer to this type of target region. Primers and probes are also said to “target” nucleic acid sequences, and so these sequences can also be understood as “target regions.” Additionally, primers and probes are said to “target” or “be specific for” genes. In this usage, the primers and probes can be used to detect the presence of a particular gene by specifically hybridizing to a portion of the gene that indicates its presence. The meaning of “target” and “target regions” will be clear to one of skill in the art from the context in which the term is employed. In some embodiments, multiple target regions can be detected to detect a single target organism. In some embodiments, a single target region can be detected to detect a single target organism. In some embodiments, an assay can employ multiple target regions for one or more target organisms and single target regions for one or more different target organisms.

[0051] Amplification according to the present teachings encompasses any means by which at least a part of at least one target region is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniquesfor amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction-CCR), helicase-dependent amplification (HD A), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel, et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih, et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press. Totowa, N.J. (2002); Abramson, et al., Curr Opin Biotechnol., 1993 Feb.;4(l):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany, et al., PCT Publication No. WO 97 / 31256; Wenz, et al., PCT Publication No. WO 01 / 92579; Day, et al., Genomics, 29(1): 152-162 (1995), Ehrlich, et al., Science 252: 1643-50 (1991); Innis, et al, PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis, et al, Nature Biotechnology 18:561-64 (2000); and Rabenau, et al, Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com / geneticidproc / ussymp6proc / blegrad.html- ); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88: 188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997): Zirvi, et al, Nucl. Acid Res. 27:e40i-viii (1999); Dean et al, Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109: 1-11 (1991); Walker, et al, Nucl. Acid Res. 20: 1691-96 (1992); Polstra, et al, BMC Inf. Dis. 2: 18 (2002); Lage, et al, Genome Res. 2003 Feb.;13(2):294-307, and Landegren, et al, Science 241: 1077-80 (1988). Demidov, V., Expert Rev Mol Diagn. 2002 Nov.;2(6):542-8, Cook, et al., J Microbiol Methods. 2003 May;53(2): 165-74, Schweitzer, et al., Curr Opin Biotechnol. 2001 Feb.;12(l):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243, PCT Publication No. W02000 / 56927A3, and PCT Publication No. WO1998 / 03673A1.

[0052] In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary orsubstantially complementary sequences in at least one target region; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.

[0053] As used herein, the term “amplification conditions” refers to conditions that promote amplification of a target region in the presence of suitable primers.

[0054] As used herein, “in solution” means not immobilized on a substrate of any kind, for example, a bead or a surface in a cassette, such as a chamber wall.

[0055] A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0056] The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection.

[0057] A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.

[0058] The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0059] The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (e.g., a fluorescent signal).

[0060] The term “quencher,” as used herein generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0061] As used herein, the term “detecting” refers to “determining the presence of’ an item, such as a nucleic acid sequence.

[0062] The term “identifying’" as used herein refers to the action of recognizing a sample.

[0063] The term "‘sample” as used herein includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple aspirate, lymph (e.g.. disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e.. feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e g., harvested by random periareolar fine needle aspiration), any other bodily fluid, a tissue sample (e.g., tumor tissue) such as a biopsy of a tumor (e.g., needle biopsy) or a lymph node (e.g., sentinel lymph node biopsy), and cellular extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof such as plasma, serum, or a cell pellet.

[0064] The term “overlapping” or “staggered” hybridizing probes as used herein refers to two or more probes that are designed to span or ‘tile’ across genomic regions of interest, each having a nucleotide sequence (such as from about 2-20 nucleotides or from about 5-15 nucleotides) that is homologous with a portion of another probe located at or near its 3’ end or 5’ end. While the “overlapping” hybridizing probes can independently vary significantly in size, probes are generally designed to comprise about 20-60 nucleotides long and bind to nucleic acid template strand with an overlap “target” region of about 5-15 bp.

[0065] The term "‘about” as used herein in connection with a numerical value is meant to have its usual meaning in the context of the numerical value. Where necessary the word “about"’ may be replaced by the numerical value ±10%, or ±5%, or ±2%, or ±1%. any range in between these numerical values.

[0066] The term "‘sequential” hybridizing probes as used herein refers to two or more non-overlapping probes that are designed to span or ‘tile’ across genomic regions of interest. The sequential hybridizing probes may be end-to-end tiled with no bases separating them or they may be spaced farther apart.

[0067] The term “spaced out” hybridizing probes as used herein refers to two or more non-overlapping probes that are designed to span or ‘tile’ across genomic regions of interest. The spaced-out hybridizing probes are tiled with farther apart such that one or more bases separate them when hybridized to the genomic target of interest.

[0068] The term “wavelength” as used herein can refer to a specific, well- defined wavelength, for example representing a dominant or peak wavelength within arange of wavelengths. The term “wavelength” can also refer to a band of wavelengths (also termed a wavelength band) which can be, for example, up to a few tens of nanometers wide emitted by a fluorophore. Accordingly, the phrase “same wavelength” as used herein can refer to a single wavelength or two or more different wavelengths wi thin a wavelength band.

[0069] The term “SPRI beads” as used herein are known more generically as para magnetic beads. They are often supplied in a reagent of 20% PEG8000. 2.50 M NaCl, 10 mM tris / HCl, pH is 8.0, but can be supplied in different reagents. The para magnetic beads can range in size from 0. 1-10 pm mean diameter and in concentration depending on the application and sample attributes. In embodiments of the present disclosure, the para magnetic beads present in the PEG solution may have a mean diameter of about 1 pm and are at a concentration of 1 about mg / ml. This reagent is referred to as MAG / PEG8000 hence forth. MAG / PEG8000 when mixed with a DNA solution causes a precipitation of DNA onto the magnetic particles. The size of the DNA molecule that can precipitated on to the para magnetic beads depends upon the final concentration of PEG8000 and NaCl in the resulting mixture. As an example, a 1: 1 ratio solution of MAG / PEG8000 to DNA solution containing size fractionated DNA will result in the precipitation of DNA fragments greater 300 bp in size onto the para magnetic particles. A magnetic field can then be used to concentrate para magnetic beads which simultaneously provides a size selection as well as for a purification as DNA molecules shorter than 300 bp as well as non-precipitated material can be washed using 80% ethanol.Exemplary Methods

[0070] The methods described herein utilize a microfluidic cartridge to perform individual sequencing library preparations from a sample. As described herein, conventional methods of NGS sample and library preparation require many sequential steps using specialized laboratory equipment. Accordingly, integration of a sequencing sample and library preparation protocol in a single microfluidic cartridge presents several challenges and unknowns, including, but not limited to a) limitation in the number of chambers (wells) within the cartridge, b) the effects of reusing chambers, such as a reaction chamber, c) effects of sample preparation processes such as sonication and nucleic acid fragmentation on sequence quality', and d) recovery of nucleic acid, carrier beads, etc, within the cartridge. The methods developed and described herein provide novel sample processing, cell lysis, DNA extraction, PCRprocesses, and adds indexes and adapters, such as Illumina indexes and adapters in a single reaction contained entirely within the microfluidic cartridge. The disclosed methods successfully outputted individual sequencing library all in a single reaction within two hours, reduction in user variation resulting from in-cartridge sample processing and DNA isolation, simplified DNA extraction, and removal of the multiple enzymatic reactions and need for costly kits involved in standard library preparation. This technique obviates the need to split limited clinical samples; is readily incorporated into an existing microfluidic cartridge; and controls the maximum number of amplicons for cluster generation, ensuring the maximum amount of data per sample by limiting adapter-tailed primers. Further, generated libraries can be pooled with any standard library, allowing for batching even with other sample types.

[0071] Methods of the present disclosure include cartridge-based methods for preparing a sequencing nucleic acid library from a sample. Libraries for any of a number of sequencing technologies or sequencing assays can be prepared. In some aspects, the methods for preparing a sequencing nucleic acid library may be used for preparation of a Next Generation Sequencing library. The term '‘Next Generation Sequencing (NGS)” as used herein refers to sequencing methods that allow for massively parallel sequencing of clonally amplified molecules and of single nucleic acid molecules. Non-limiting examples of sequence assays that are suitable for use with the methods disclosed herein include nanopore sequencing (US Pat. Publ. Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 01 19259 and 2015 / 0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI- TOF / MS; Fu et al., Nature Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998), and NGS methods, including but not limited to sequencing by synthesis (e.g.. HiSeg™, MiSeq™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g.. SOLiD™, Life Technologies), ion semiconductor sequencing (e g., Ion Torrent™, Life Technologies), and SMRT® sequencing (e.g., Pacific Biosciences); quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (F1SSEQ), FISSEQbeads, wobble sequencing, multiplex sequencing, polymerized colony(POLONY) sequencing; nanogrid rolling circle sequencing (ROLON Y). allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (ROA) readout, ligated padlock probes, or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout), and the like. Commercially available sequencing technologies include: sequencing-by -hybridization platforms from Asymetrix Inc. (Sunnyvale, Calif), sequencing-by-synthesis platforms from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), sequencing-by -ligation platform from Applied Biosystems (Foster City, Calif). Other sequencing technologies include, but are not limited to. the Ion Torrent technology (ThermoFisher Scientific), and nanopore sequencing (Genia Technology from Roche Sequencing Solutions, Santa Clara, Calif); and Oxford Nanopore Technologies (Oxford, United Kingdom).

[0072] The cartridge-based methods for preparing a sequencing nucleic acid library from a sample have broad application, including for example: for use in diagnosing or prognosing whether a subject has cancer, has been successfully or unsuccessfully treated for cancer, or whether cancer in a subject has relapsed; for use in diagnosing or prognosing whether a subject has an infection, has been successfully or unsuccessfully treated for an infection, or whether an infection in a subject has relapsed; for use in detecting pathogen(s) in a subject, or detecting whether a pathogen has been successfully removed from a subject during or after treatment; for use in subtyping a disease, e.g., cancer; or for use in developing a course of treatment, including the identification of an effective therapeutic, dosing regimen, and / or length of treatment.

[0073] In an first embodiment, a cartridge-based method for preparing a sequencing nucleic acid library' from a sample, comprising: (a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of the nucleic acid and detection of signals generated during amplification; (b) utilizing the filter to isolate the nucleic acid from the sample; (c) forming a library prep reaction mixture comprising the nucleic acid and a library prep reagent that facilitates: i) generation of tagged nucleic acid fragments from thenucleic acid in the library' prep reaction mixture, ii) generation of amplicons, wherein the amplicons are optionally tagged at one or both ends, and / or iii) attachment of oligonucleotide adapters to one or both ends of the tagged nucleic acid fragments or amplicons to form adapter-labeled nucleic acid; (d) causing the library prep reaction mixture to flow into the reaction vessel and preparing the sequencing nucleic acid library comprising the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid; (e) isolating the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the library prep reaction mixture on to the filter and eluting each from the filter; and (f) purifying and optionally size selecting the sequencing nucleic acid library' on the filter.

[0074] FIG. 3 shows a comparison of steps among six different commercially available library reparation protocols that are replaced by the methods, devices, and kits of the present disclosure

[0075] FIG. 4 discloses a cartridge-based method 200 for preparing a sequencing nucleic acid library from a sample. Step (a) represents an embodiment of step 202 in FIG. 4, step (b) represents an embodiment of step 204 in FIG. 4, step (c) represents an embodiment of step 206 in FIG. 4, step (d) represents an embodiment of step 208 in FIG. 4, step (e) represents an embodiment of step 210 in FIG. 4 and step (!) represents an embodiment of steps 212 and 214 in FIG. 4.

[0076] In an embodiment of a cartridge-based method for preparing a sequencing nucleic acid library from a sample, a bubble bursting mechanism is utilized to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings of the reaction vessel. Notably, the ability' to reduce errant air bubbles increases in importance as a reaction vessel is filled multiple times and / or used for multiple reactions, e.g., during the preparation of a nucleic acid library.

[0077] The bubble bursting mechanism may comprise (i) heating the empty reaction vessel as disclosed in step 402 of FIG. 6, (ii) partially filling the reaction vessel with a first portion of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid as disclosed in step 404 of FIG. 6, (iii) heating the first portion of reaction mixture in the reaction vessel as disclosed in step 406 of FIG. 6, (iv) optionally discarding the first portion of reaction mixture as disclosed in step 408 of FIG. 6, and (v) filling the reaction vessel with a second portion of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid ornucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid as disclosed in step 410 of FIG. 6. In certain embodiments, bubble bursting further comprises repeating steps (i)-(v) with additional portions of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid. In certain embodiments, the bubble bursting mechanism may not comprise a partial fill, but instead includes heating the empty reaction vessel as disclosed in step 402 of FIG. 6 for a period of time.

[0078] For example, heating the empty reaction chamber may be to temperatures greater than 80°C, greater than 85°C, greater than 90°C, 95°C or greater, or about 95°C. The minimum time for heating the empty reaction vessel may be based on the chosen temperature but will be for at least 1 second, for at least 2 seconds, for at least 3 seconds, for at least 4 seconds, or for at least 5 seconds. The partial filing of the reaction vessel with a fist portion of the reaction mixture will depend on the volume of the reaction vessel, e.g., the partial filling will be with a volume that is at least 10% of the volume of the reaction vessel, at least 15% of the volume of the reaction vessel, at least 20% of the volume of the reaction vessel, or at least 25% of the volume of the reaction vessel. The volume should be sufficient to push air from the vessel port into the reaction vessel while, in certain embodiments, at the same time minimizing the amount of reagent subjected to heating during the bubble bursting steps in order to minimize degradation and / or decrease yield. Implementing an empty- or partial-tubefill bubble bursting protocol in the methods disclosed herein have been shown to enable multiple tubefills , and improved tube washing for removal of inhibitors.

[0079] In embodiments, releasing the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the filter during step (e) is during and / or after preparing the sequencing nucleic acid library. In certain embodiments, after utilizing the filter to isolate the nucleic acid from the sample in step (b), the nucleic acid is eluted from the filter following step (b), which in some embodiments may occur before step (c), during step (c), after step (c). or a combination thereof. In certain embodiments, a cartridge-based method for preparing a sequencing nucleic acid library from a sample further comprises amplifying the nucleic acid with at least one set of primers for amplify ing a target region of the nucleic acid within the reaction vessel following step (b) using amplification reagents and methods disclosed herein.

[0080] In a second embodiment, a cartridge-based method utilizes a fragmentation step. For example, a cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising: (a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of nucleic acid and detection of signals generated during amplification; (b) utilizing the filter to isolate the nucleic acid from the sample; (c) fragmenting the nucleic acid to form nucleic acid fragments, wherein fragmenting is performed by mechanical fragmentation, chemical fragmentation, or enzymatic fragmentation; and / or amplifying the nucleic acid or nucleic acid fragments to form amplicons, wherein amplifying the nucleic acid or nucleic acid fragments is with at least one set of primers for amplifying a target region of the nucleic acid or nucleic acid fragments; (d) generating adapter-labeled nucleic acid comprising labeling the fragmented nucleic acid or amplicons at one or both ends the fragmented nucleic acid or amplicons; and (e) isolating the adapter labeled nucleic acid on to the filter to form the NGS nucleic acid library.

[0081] In specific embodiments of the present disclosure a cartridge-based method further comprises reuse of the reaction vessel, the filter, and optionally one or more of the plurality of chambers with rinsing to remove or dilute carryover, wherein reuse of the reaction vessel comprises a bubble bursting mechanism to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings as discussed herein. One advantage of the presently disclosed methods and devices is that reaction vessels, filters, and one or more of the plurality of chambers in a cartridge may be reused multiple times thereby reducing human interaction, user error, improving efficiency, and allowing for optimization of yields, reduction of sample degradation, and generally a more desired sequencing nucleic acid librar .

[0082] FIG. 5 discloses a cartridge-based method 300 for preparing a sequencing nucleic acid library from a sample. Step (a) represents an embodiment of step 302 in FIG. 5, step (b) represents an embodiment of step 304 in FIG. 5, step (c) represents an embodiment of step 306 in FIG. 5, step (d) represents an embodiment of step 308 in FIG. 5, and step (e) represents an embodiment of step 310 in FIG. 5.

[0083] If the sample in step (a) of a method for preparing a sequencing nucleic acid library' from a sample comprises intact cells, the method further comprisesreleasing nucleic acid from the intact cells by lysing the intact cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers and / or capturing the cells on the filter and lysing the cells by means of sonication, to release nucleic acid from the cells. In other words, when a sample comprises intact cells, the nucleic acid can be released from the intact cells through a mechanical process, electrical process, optical process, chemical process, thermal process, acoustic process, or a combination thereof. Chemical processing may include a chemical treatment, a change in pH, or an enzymatic treatment. Optical processing may include exposure to UV or IR light. Electrical processing may include electroporation, electrophoresis, or isoelectric focusing. Mechanical processing may include mixing, filtering, pressurization, grinding, or cell disruption. Thermal processing may include heating and / or cooling from ambient temperature. Acoustical processing may include the use of ultrasound (e.g. ultrasonic lysis). Modulation of sonication parameters (time or amplitude) have been shown to improve coverage uniformity of libraries. Figures 8A-8C show sonication amplitude and time impacted fragmentation state of genomic DNA and may affect sequence depth of coverage of AT-rich regions according to embodiments of the disclosure. FIG. 8 A shows the sequence coverage of libraries produced from a cartridge with glass fiber filter with 40% and 15 second sonication. FIG. 8B shows the sequence coverage of libraries produced from a cartridge with a glass fiber filter with 40% and 10 second sonication. FIG. 8C shows the sequence coverage of libraries produced from a cartridge with a glass fiber filter with 30% and 15 second sonication. When Figures 8A-C are compared there is reduced coverage in certain AT-rich region for the 40% 15s sonication methodology'. This reduction can be seen as the arrows in FIG. 8A indicate the location of AT rich regions that are improved in coverage in FIG. 8B, and further improved in FIG. 8C, when FIG. 8A is compared to FIG. 8b and FIG. 8C. An example of a universal valve assembly, in accordance with some embodiments, that utilizes glass beads suited for mechanical lyses of certain types of targets, as compared to conventional valve assemblies in conventional sample cartridges is illustrated in FIG. ID. A cartridge with glass fiber filter is described in WO2023278798A1, which is hereby incorporated by reference. The cartridge utilizes a valve body platform that allows for detection of enveloped and free nucleic acid targets. The valve body includes a sample processing region or lysing chamber that provides for heat, mechanical, and / or chemical lysis. This allows a single cartridge to provide lysing for a multitude of differing types of target, thus, can beconsidered a “universal assay cartridge.'’ The filter is formed of glass fibers and is configured to accommodate glass beads to further facilitate mechanical lysis of hardy targets. The filter is also suitably sized (a pore size of about 0.7 urn) for receiving suitably sized glass beads for mechanical lysing. The device system can include a sonication hom engageable with the cartridge for processing biological material within the cartridge and operatively coupled with a controller configured to control sonication based at least in-part on a frequency providing a highest output amplitude as a resonant frequency. Typical power levels of 5-10 Watts are applied for between 15-30 seconds, which apply vibration at a frequency of about 30 kHz or greater, about 40 kHz or greater, such as about 50 kHz (e g. 50.5 kHz) to sufficiently lyse typical spore cells and releasing 50% of spore bound DNA into solution. In FIGs. 8A-8C, the time and amplitude are modulated to improve coverage uniformity. For example, 30% sonication for 15s provides a frequency below the typical lysis parameters, that is, less than 50 kHz.

[0084] A cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise eluting from the filter one or more of the nucleic acid released from the cells, nucleic acid fragments, tagged nucleic acid fragments, amplicons, tagged amplicons, and / or adapter labeled nucleic acid. As described herein, the filter may comprise glass fibers and optionally a polymeric binder, as in the GeneXpert® cartridge. Carrier beads or nucleic acid can get lodged onto the filter, such that single liquid aspirations are insufficient to remove all the beads or nucleic acid. Eluting from the filter may comprise toggling the nucleic acid released from the cells, nucleic acid fragments, tagged nucleic acid fragments, amplicons, tagged amplicons, and / or adapter labeled nucleic acid on the filter with an eluant. In certain embodiments, toggling can optionally comprise chemical processing, optical processing, electrical processing, mechanical processing, thermal processing, acoustical processing, or a combination thereof. In embodiments, toggling may also comprise a change in speed, volume, viscosity', or a combination thereof. Toggling is important for optimization yields and / or reduction in sample degradation. For example, small beads (3-5pm) may move with the fluid, whereas larger beads (e g., 8pm) may settle, requiring larger volumes or faster toggling flow rates. Notably, the cartridge-based methods for preparing a sequencing nucleic acid library from presently disclosed provide complete control of the mixing and toggling process, which are not the case in other automated library prep methods. Control of the mixing and toggling conditions(speed, volume, viscosities, etc.) is important for optimization of yields, reduction of sample degradation, and an overall better outcome.

[0085] In an embodiment, lysing the cells by sonication comprises exposing the cells to ultrasonic waves in a frequency range of approximately 5 kHz to approximately 120 kHz to lyse and release nucleic acid from the cells, or approximately 20 kHz to approximately 50 kHz to lyse and release nucleic acid from the cells.

[0086] A cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise isolating and washing the nucleic acid on the filter, and eluting the nucleic acid from the filter prior to step (c). In some examples, wash steps can include reversing the flow off of the filter. Then to obtain the target, sonication is applied and an elution is performed.

[0087] A cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise amplifying the adapter-labeled nucleic acid with at least one set of primers for amplify ing and increasing adapter-labeled nucleic acid concentration following step (d). Amplification is achieved using reagents and techniques discussed herein.

[0088] A cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise, purifying and size selecting the nucleic acid that had been isolated on the filter in step (e) during step (e). Size selecting the isolated adapter labeled nucleic acid in step (e) may comprise solid-phase reversible immobilization.

[0089] To address cartridge chamber limitations, reagent substitution and / or reusing chambers are considered. For example, the present disclosure considers reuse of library prep wash buffer for sample prep, however, the effect on library prep yield and sequence data quality must be accounted for. One advantage of the presently disclosed methods and devices is that both reagents and chambers may be reused while preparing a sequencing nucleic acid library7from a sample. Reuse of reagents removes human error, reduces the opportunity for external contamination, reduces cost, and increases efficiency. A cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise using a reusable reagent for one or more steps, including lysing the cells in the sample, isolating the nucleic acid released from the cell, washing the nucleic acid, eluting the nucleic acid, library prep reagent, fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter labeled nucleic acid, amplifying the adapter labeled nucleic acid,isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purifying and size selecting the isolated adapter labeled nucleic acid. In certain embodiments, a reusable agent is used for 2 or more steps, for 3 or more steps, for 4 or more steps, or for 5 or more steps. For example, a reusable agent may be used for washing the nucleic acid, purifying, and size selecting the isolated adapter labeled nucleic acid in certain embodiments.

[0090] Reusable reagents may include any reagent necessary or relied upon during a cartridge-based method for preparing a sequencing nucleic acid library. For example, in an embodiment, reusable reagent comprises a buffer, a salt, and polyethylene glycol (PEG). Figures 9A and 9B demonstrate embodiments of the disclosures where TWB can replace TET in sample preparation workflow in an adapted Xpert® Carba-R assay as the TWB sample preparation workflow adapted Xpert® Carba-R assay produced libraries of comparable yield and fragment size distribution as the control TET sample preparation adapted Xpert® Carba-R assay. For example, both FIG. 9A and 9B have peaks around 600 base pairs (bp as indicated on the x axis) with similar fluorescent intensity (FU, meaning fluorescent unit, as indicated on the y axes). In embodiments of the disclosure which have results shown in FIG. 9A and FIG. 9B, when sample prep workflow in an adapted Xpert® Carba-R assay with TWB was compared to a sample prep workflow in an adapted Xpert® Carba-R assay with TET both assays yielded a library at 1.90 ng / pl. Plasmids do show some differences from control in depth of coverage, especially for the smallest plasmid when TET is replaced by TWB in the sample prep workflow (data not shown). Using TWB as wash / eluant in sample prep may affect extraction efficiency of smaller DNA elements. However, a comparison of Figures 10A and 10B shows libraries generated from TWB eluates show improved uniformity in AT rich regions. FIG. 10A shows the library produced from a sample preparation workflow using an adapted Xpert® Carba-R assay with a TET sample preparation eluant, according to an embodiment of the disclosure. FIG. 10B shows the sequence coverage of a library produced from a sample preparation workflow using an adapted Xpert® Carba-R assay with a TWB sample preparation eluant, according to an embodiment of the disclosure. In the embodiment shown in Figures 10A and 10B, standard sonication parameters were used in both conditions (40% Amplitude, 15s). The arrows in FIG. 10A indicate the location of AT rich regions that are improved in uniformity in FIG. 10B when FIG. 10B is compared to FIG. 10A.Presence of 10X more Tween 20 in TWB compared to TET may affect the fragmentation of DNA during the sonication.

[0091] In embodiments, using the reusable reagent comprises: (i) drawing, from a first one of the plurality of chambers, a first portion of the reusable reagent and utilizing the first portion of reusable reagent, and (ii) drawing, from the first one of the plurality of chambers, a second portion of the reusable reagent and utilizing the second portion of reusable reagent. When desirable based on the sequencing reaction and sample, the least one of the first, second, third, and fourth reusable agents are different reusable agents. In embodiments, using the reusable reagent comprises recycling the reusable reagent after one or more uses.

[0092] In addition to reusing reagents, the disclosed invention also contemplates reusing chambers within the cartridge in order to remove human error, reduce the opportunity for external contamination, reduce cost, and increase efficiency. In certain embodiments, a cartridge-based method for preparing a sequencing nucleic acid library’ comprises reuse of at least one of the plurality’ of chambers, reuse of at least two of the plurality of chambers, reuse of at least three of the plurality of chambers, or reuse of at least four of the plurality of chambers. In embodiments, at least one of the plurality’ of chambers, the at least two of the plurality’ of chambers, the at least three of the plurality of chambers, or the at least four of the plurality of chambers for storing the reusable reagent is reused for collecting waste, fragmenting the nucleic acid, and / or generating the adapter-labeled nucleic acid. In certain embodiments, the at least one of the plurality’ of chambers is used to store a first reusable reagent prior to use, the at least two of the plurality of chambers is used to store a first and a second reusable reagent prior to use, the at least three of the plurality of chambers is used to store a first, a second, and a third reusable agent prior to use, or the at least four of the plurality of chambers is used to store a first, a second, a third, and fourth reusable agent prior to use. In an embodiment, the at least one of the plurality' of chambers for storing the reusable reagent is reused for a different purpose than another of the plurality of chambers for storing reusable reagent. When desirable based on the sequencing reaction, sample and reagent, using the reusable reagent comprises recycling the reusable reagent after one or more uses.

[0093] In embodiments, the reaction vessel of a cartridge-based method for preparing a sequencing nucleic acid library from a sample comprises one or more reaction chambers configured for amplification of nucleic acid and detection of signalsgenerated during amplification. Depending on the sample, complexity of the sequencing nucleic acid library, and the desired outcome of the reaction, in certain embodiments, the reaction vessel comprises one reaction chamber, the reaction vessel comprises two reaction chambers, the reaction vessel comprises three reaction chambers, or the reaction vessel comprises no more than one reaction chamber configured for amplification of nucleic acid and detection of signals generated during amplification. In certain embodiments, the disclosed methods are simplified and fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid are sequentially performed in a single reaction chamber while in other embodiments fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, and / or amplifying the adapter- labeled nucleic acid are sequentially performed in more than one reaction chamber. When appropriate, fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid comprises thermocycling, heating, and mixing.

[0094] In addition to contemplating the use of reusable reagents and reusable chambers, the presently disclosed methods also contemplate reusing filters, e.g., multiple uses for a filter. In embodiments, the reaction vessel of a cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising multiple uses of the filter including for capturing the cells in the sample, isolating the nucleic acid released from the cells, isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purifying and size selecting the isolated adapter labeled nucleic acid.

[0095] Regardless of whether a filter of the present disclosure is reused, the filter may comprise magnetic or polymeric beads, and in certain embodiments, the magnetic or polymeric beads are bound and / or adsorbed to the filter. While not necessary’ for the disclosed methods, methods and devices (e.g., cartridges) of the present disclosure may use magnetic beads via use of an external magnetic field to capture the magnetic beads, e.g., the magnetic field may be provided within the cartridge (e.g., within a chamber or the ultrasonic horn assembly) or within the device system itself. In specific embodiments, a small magnet (e g., neodymium) is affixed within a small cartridge chamber to effect adherence of the paramagnetic beads against the wall of a proximal chamber thereby allowing for use of these beads without needfor a filter, membrane, or matrix. The magnetic beads may be about 0.1 pm to about 10 pm, or about 0.5 pm to about 5 pm, or about 0.8 pm to about 3 pm. The magnetic beads may have a specific concentration of based upon the size of the magnetic beads, the pore size and / or composition of the filter, and / or the length of the nucleic acid fragments. In other specific embodiments, a polymeric matrix is included within a cartridge chamber, upstream or downstream the filter.

[0096] The magnetic or polymer beads or a polymer matrix may potentially have a plurality of moieties on their surface for the purpose of binding of specific molecular structures, or binding molecules of certain characteristics (charge, hydrophobicity), and / or antibodies or small molecules (e.g. streptavidin) thereby enabling increased binding. In certain embodiments, the magnetic or polymer beads or a polymer matrix are optionally modified with a polymeric binder, and / or are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.

[0097] In certain embodiments, tagmentation, i.e., unfragmented DNA is cleaved and tagged for analysis, is necessary for successfully preparing a sequencing nucleic acid library from a sample. When utilizing tagmentation, a cartridge-based method may further compromise (i) fragmenting the nucleic acid to form nucleic acid fragments in step (b) using enzymatic fragmentation by contacting the nucleic acid with atransposome solid support and / or free-floating transposome complexes, and generating tagged nucleic acid fragments by subjecting the nucleic acid and transposome solid support and / or free-floating transposome complexes to fragmentation conditions; (ii) forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and a) a set of primer pairs comprising adapters for amplifying the tagged nucleic acids fragments or b) ONT-specific adapters; and (iii) subjecting the adapter labeled reaction mixture to a) amplification conditions to amplify the tagged nucleic acid fragments or b) to click chemistry conditions to attach the ONT-specific adapters to the tagged nucleic acid. It may be desirable in certain embodiments for the generation of tagged nucleic acid fragments to occur in the reaction vessel in a first reaction vessel filling, and further comprise quenching generation of the tagged nucleic acid fragments in a second reaction vessel filling, isolating the tagged nucleic acid fragments on the filter, forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and a set of primer pairscomprising adapters for amplifying the tagged nucleic acids fragments; and subjecting the adapter labeled reaction mixture to amplification conditions to amplify the tagged nucleic acid fragments in a third reaction vessel filling. Quenching the generation of the tagged nucleic acid fragments may comprise adding SDS to the reaction vessel in the second reaction vessel filling.

[0098] In additional embodiments of the present disclosure, a cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid and a polymerase to form tagged amplicon; forming an adapter labeled reaction mixture comprising the tagged amplicons and ONT-specific adapters; and subjecting the adapter labeled reaction mixture to ligation conditions to attach the ONT-specific adapters to the tagged amplicons. If specific embodiments, the primers for amplifying the target region comprises 5 ’-phosphorylated primers and the polymerase lacks 3’-to-5’ exonuclease activity, resulting in a 5 ’-phosphorylated and 3’- A-tailed amplicon, and ligation comprises T4 ligase-mediated ligation of ONT-specific sequencing adapters.

[0099] In additional embodiments of the present disclosure, a cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise amplifying the nucleic acid with at least one set of unique molecular identifier (UMI) primers for amplifying a target region of the nucleic acid to form tagged amplicons in a first reaction vessel filling; amplifying the tagged amplicons via blocker displacement amplification to enrich for amplicons containing specific target region in a second reaction vessel filling; and amplifying the enriched tagged amplicons with at least one set of primers comprising adapters to form adapter labeled nucleic acid in a third reaction vessel filling.

[0100] In additional embodiments of the present disclosure, a cartridge-based method for preparing a sequencing nucleic acid library7from a sample may further comprise amplify ing the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid to form amplicons in a first reaction vessel filling, amplifying the amplicons with at least one set of internal primers for amplify ing a target region of the nucleic acid to form tiled amplicons in a second reaction vessel filling. As an alternative embodiment, a cartridge-based method for preparing a sequencing nucleic acid library from a sample may further comprise amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleicacid to form amplicons in a first reaction vessel filling; and enzymatically ligating ONT-specific adapters to the amplicons in a second reaction vessel filling.

[0101] In certain embodiments of a cartridge-based method for preparing a sequencing nucleic acid library from a sample that requires tagmentation, the generating tagged nucleic acid fragments happens in the reaction vessel in a first reaction vessel filling, and further comprises isolating the tagged nucleic acid fragments on the filter, forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and ONT-specific adapters; and subjecting the adapter labeled reaction mixture to click chemistry conditions to attach the ONT-specific adapters to the tagged nucleic acid. Sometimes it may be necessary for the methods to utilize click chemistry. In such embodiments, a cartridge-based method for preparing a sequencing nucleic acid library from a sample may comprise one or more click chemistry moieties on a 3’ end of nucleic acid fragment of the tagged nucleic acid fragment. The disclosed methods contemplate a wide variety of temperatures for successful use of click chemistry’. In an exemplary’ embodiment, the click chemistry conditions are at room temperature.

[0102] FIG. 11 illustrates an embodiment of the disclosure that uses an Illumina Seq Nano library’ prep kit (available from https: / / yvww.illumina.com / ) in a cartridge to obtain a sequencing ready WGS nucleic acid library from a bacterial culture.Automation and Systems

[0103] The automated sequencing nucleic acid library’ preparation systems provided herein perform rapid, simple, convenient, and affordable sequencing nucleic acid library' preparation.System Overview

[0104] In one aspect, the disclosure pertains to a sample cartridge that utilizes a valve body platform that alloyvs for detection of enveloped and free nucleic acids. In some embodiments, the valve body includes a sample processing region or lysing chamber that provides for either or both mechanical and chemical lysis. This allows a single cartridge to provide lysing for a multitude of differing types of targets. In some embodiments, the sample cartridge can perform processing and detection of targets requiring mechanical lysing and chemical lysing.

[0105] The sample cartridge device can be any device configured to perform one or more process steps relating to preparation and / or analysis of a biological fluid sample according to any of the methods described herein. In some embodiments, thesample cartridge device is configured to perform at least sample preparation. The sample cartridge can further be configured to perform additional processes, such as detection of a target region in a nucleic acid amplification test (NAAT), e.g., Polymerase Chain Reaction (PCR) assay, by use of a reaction vessel attached to the sample cartridge. The sample cartridge can further be configured to perform additional process, such as sequencing nucleic acid library preparing, by use of a reaction vessel attached to the sample cartridge. In some embodiments, the reaction vessel extends from the body of the cartridge. Preparation of a fluid sample generally involves a series of processing steps, which can include chemical, electrical, mechanical, thermal, optical or acoustical processing steps according to a specific protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, binding of analyte, and binding of unwanted material.

[0106] A sample cartridge suitable for use with the methods and kits disclosed herein, includes one or more transfer ports through which the prepared fluid sample can be transported into an attached reaction vessel for analysis. FIG. 1 A illustrates an exemplary assay cartridge 100 suitable for sample preparation and analytics testing by PCR when received in an instrument module in accordance with some embodiments. In the embodiment show n in FIG. 1 A, the sample cartridge is attached with a reaction vessel 116 (also referred to as a “reaction tube’' or “PCR tube”) adapted for analysis of a fluid sample processed within the sample cartridge 100. In some embodiments the reaction vessel extends from the cartridge body. Such a sample cartridge 100 includes various components including a main housing 102 having one or more chambers 108 for processing of the fluid sample, w hich typically include sample preparation before analysis. In these embodiments, the sample cartridge can be a fully integrated nucleic acid amplification and test system combining sample preparation, amplification, and detection together. The instrument module facilitates the processing steps needed to perform sample preparation and the prepared sample is transported through one of a pair of transfer ports into fluid conduit of the reaction vessel 116 attached to the housing of the sample cartndge 100. The prepared biological fluid sample is then transported into a reaction chamber of the reaction vessel where the biological fluid sample undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, concurrent with the amplification of the biological fluid sample, an excitation means, and an optical detection means of the module is used to detect optical emissions that indicate thepresence or absence of a target region of interest. It is appreciated that such a reaction vessel could include various differing chambers, conduits, or micro-well arrays for use in detecting the target analyte. The sample cartridge can be provided with means to perform preparation of the biological fluid sample before transport into the reaction vessel. Any chemical reagent required for cell lysis or means for binding or detecting an analyte of interest (e.g.. reagent beads) can be contained within one or more chambers of the sample cartridge, and as such can be used for sample preparation.

[0107] An exemplary use of a reaction vessel for analyzing a biological fluid sample is described in commonly assigned U.S. Patent No. 6,818,185, entitled “Cartridge for Conducting a Chemical Reaction,’" filed May 30, 2000, the entire contents of which are incorporated herein by reference for all purposes. Examples of the sample cartridge and associated modules are shown and described in U.S. Patent No. 6,374,684, entitled “Fluid Control and Processing System” filed August 25, 2000, and U.S. Patent No, 8,048,386, entitled “Fluid Processing and Control,” filed February 25, 2002, U.S. Provisional Application No. 63 / 217,672 entitled “Universal Assay- Cartridge and Methods of Use” filed July 1, 2021; U.S. Provisional Application No. 63 / 319,993 entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture” filed March 15, 2022; and U.S. Patent No. 10,562,030 entitled “Molecular Diagnostic Assay System"’ filed July 22, 2016; the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0108] Various aspects of the sample cartridge 100 can be further understood by referring to U.S. Patent No. 6,374,684 “the ’684 patent”), which described certain aspects of a sample cartridge in greater detail. Such sample cartridges can include a fluid control mechanism, such as a rotary fluid control valve assembly, that is fluidically connected to the chambers of the sample cartridge. The term “chamber” can be used interchangeably with the terms “well”, “tube”, and the like. Rotation of the rotary- fluid control valve permits fluidic communication between chambers and the valve so as to control flow of a biological fluid sample deposited in the cartridge into different chambers in which various reagents can be provided according to a particular protocol as needed to prepare the biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module comprises a motor such as a stepper motor that is ty pically coupled to a drive train that engages with a feature of the valve in the sample cartridge to control movement of the valve in coordination with movement of the syringe, thereby resulting movement of the fluid sample according tothe desired sample preparation protocol. The fluid metering and distribution function of the rotary valve according to a particular sample preparation protocol is demonstrated in the ’684 patent.Assay Cartridge and Valve AssembliesOverview

[0109] As shown in FIG. 1A, the test cartridge 100 comprises a cartridge body 102 containing a plurality of chambers 108 for reagents or buffers and sample processing. The chambers are disposed around a central syringe barrel 106 that is in fluid communication with a valve body 110 (see FIGS. IB and 1C) and that is sealed with a gasket 104. The valve body 110 can include a cap 112 and the entire cartridge body can be supported on a cartridge base 101. The valve body A pically contains one or more channels or cavities (chamber(s) 114) that can contain a filter as described herein that can function to bind and elute a nucleic acid. In some embodiments the cartridge further comprises one or more temperature-controlled channels or chambers that can, in certain embodiments, function as thermocycling chambers. A "plunger” not shown can be operated to draw fluid into the syringe barrel 106 and rotation of the valve body 110 provides selective fluid communication between the various reagent chambers 108 and channels, reaction chamber(s), mixing chambers, and optionally, any temperature-controlled regions. Thus, the various reagent chambers 108, reaction chambers, filter material(s), and temperature-controlled chambers or channels are selectively in fluid communication by rotation of the plunger and reagent movement (e.g., chamber loading or unloading) is operated by the “syringe” action of the plunger within the valve assembly. In other embodiments, the various reagent chambers, reaction chambers, filter material, and temperature-controlled chambers or channels are selectively in fluid communication by linear progression (e.g., by forced movement) of the reagents and sample from one chamber to the next. In some embodiments, the cartridge is a self-contained cartridge. In embodiments, the self-contained cartridge consists of 15 or less chambers, 12 or less chambers, or 11 or less chambers, not including the chamber(s) in the reaction vessel. In some embodiments, each chamber of the plurality of chambers independently has a volume capacity of 10 mL or less, 9 mL or less, 8 mL or less, 7 mL or less, 6 mL or less, 5 mL or less, 4 mL or less, 3 mL or less, 2 mL or less, or 1 mL or less. In some embodiments, one or more chambers have the same volume capacity. In embodiments, all chambers have the same volume capacity while in some embodiments chambers have different volume capacities.Reaction Modules

[0110] In certain embodiments the cartridge is configured for insertion into a reaction module. The module is configured to receive the cartridge therein. In certain embodiments the reaction module provides heating plates to heat the temperature- controlled chamber or channel. The module can optionally additionally include a fan to provide cooling where the temperature-controlled channel or chamber is a thermocycling channel or chamber. Electronic circuitry can be provided to pass information (e.g., optical information) to a computer for analysis. In certain embodiments the module can contain optical blocks to provide excitation and / or detection of one or more (e.g., 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, or more) optical signals representing, e.g., signal DNAs amplified for various PCR targets. In various embodiments an electrical connector can be provided for interfacing the module with a system e.g., system controller or with a discrete analysis / controller unit. In certain embodiments, the module also contains a controller that operates a plunger in the syringe barrel and the rotation of the valve body.Analytical System[OHl] In certain embodiments a system (e g., a processing unit) is provided. The system includes an enclosure that is configured to support and power multiple sample processing modules, where each processing module is configured to hold and operate a removable cartridge. In some embodiments, the system is configured to operate the sample processing modules to perform a PCR assay for one or more target region analytes and optionally to determine the level of one or more target RNA / DNA sequences within a corresponding removable sample cartridge. Typically, the processing on a sample within the corresponding removable sample cartridge involves operating the cartridge to perform a method as descnbed herein. In certain embodiments the system is configured to contain one sample processing module. In certain embodiments the system is configured to contain at least two or more sample processing modules (e g., at least 4, 8. 12, 16, 20, 24, 28, 32, 64, 128 or more) sample processing modules. In some embodiments, the system provides a user interface that allows the user input operational instructions and / or to monitor operation of the cartridges to determine the presence and / or quantity of one or more nucleic acids.

[0112] While the methods described herein are described primarily with reference to the GENEXPERT® cartridge by Cepheid Inc. (Sunnyvale, Calif), it will be recognized, that in view of the teachings provided herein the methods can beimplemented on other cartridge / microfluidic systems, including alternative cartridge designs having valve assemblies that involve multiple interfacing components, as well as cartridge body defined by multiple interfacing components to form the multiple chambers of the cartridges, for example, those described in Korean Application No. 102293717B1 and KR102362853B1, cartridges that utilizes ultrasonic waves to lyse cells in a biological sample, for example, those described in International Application No. WO2021 / 245390A1, cartridges and systems that utilizes an electrowetting grid for microdroplet manipulation and electrosensor arrays configured to detect analytes of interest, for example, those described in International Application No. WO2016 / 077341A2, cartridges that facilitate movement of nucleic acid from one chamber to the next chamber by opening a vent pocket, for example, those described in International Application No. WO2012 / 145730A2, multiplexed assay systems comprising a plurality of thermocycling units such that individual chambers can be heated, cooled, and / or compressed to mix fluid within the chamber or to propel fluid in the chamber into another chamber, for example, those described in International Application No. WO2015 / 138343A1, and as well as systems for rapid amplification of nucleic acids facilitated by flexible portions of the sample cartridge aligned to accomplish temperature cycling for nucleic acid amplification, for example, those described in International Application No. WO2017 / 147085A1. Such cartridge / microfluidic systems can include, for example microfluidic systems implemented using soft lithography, micro / nano-fabricated microfluidic systems implemented using hard lithography, and the like.

[0113] In certain embodiments, the cartridge for automated library preparation comprises a) a cartridge body comprising a plurality’ of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; b) a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and / or ii) detection and identification of a plurality of amplification products via real-time PCR; c) a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and d) a set of primer and a set of two or more probes for each of the at least two target regions,wherein each of the two or more probes in the set for a target region comprises a detectable label that emits light at the same wavelength.

[0114] The reaction vessel (also referred to herein as a reaction tube) comprises one or more reaction chambers for amplification and detection of the amplification products. In certain embodiments, the reaction vessel comprises one reaction chamber for amplification and detection of the amplification products. Accordingly, the nucleic acid, primers, and probes are present in a single reaction solution, and wherein generating and detecting the melt temperature signatures for the target regions are from the single reaction solution. In certain embodiments, the reaction vessel comprises more than one reaction chambers for amplification and detection of the amplification products, for example, two reaction chambers, three reaction chambers, or four reaction chambers. Each reaction chamber can be configured to detect a single amplification product or a plurality of amplification products.

[0115] The cartridge can be a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge. Additionally, it is appreciated that the assay methods described herein may further be realized in entirely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow testing cartridges, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0116] In various embodiments, the signal DNA(s) from PCR (nucleic acid amplification) reactions are amplified for detection and quantification. In certain embodiments, the amplification comprise any of a number of methods including, but not limited to polymerase chain reaction (PCR). ligase chain reaction (LCR). ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLP A), ligation followed by Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), rolling circle amplification (RCA), and the like.

[0117] In illustrative, but non-limiting embodiments, the amplification reaction may produce an optical signal that is proportional to the amount of amplified target region (e.g., signal DNA). Illustrative optical signals include, but are not limited to a fluorescent signal, a chemiluminescent signal, an electrochemiluminescent signal, a colorimetric signal, and the like. In certain embodiments the optical signal is afluorescent optical signal generated by a fluorescent indicator. In certain embodiments the fluorescent indicator is anon-specific intercalating dye that binds to doublestranded DNA products, while in certain other embodiments, the fluorescent indicator comprises a target sequence-specific probe (e.g., a TAQMAN® probe, a SCORPION® probe, a MOLECULAR BEACON®, and the like).

[0118] Single PCR reactions (nucleic acid amplification), or multiple PCR reactions (nucleic acid amplifications) run sequentially (or simultaneously in separate temperature controlled channels or chambers) can also use the same detectable label since sequentially run PCR signal DNAs are analyzed sequentially and the simultaneous PCR signal DNAs are distinguished by the occurrence in different temperature controlled channels or chambers. The signal produced by this amplification can be distinguished from other amplification products because it is not run at the same time and / or because it is run in a different reaction channel / chamber. However, where multiple nucleic acid amplifications are run simultaneously in the same chamber the reaction products of for each analysis are typically detected and / or quantified by the use of unique melt profiles and / or different and distinguishable labels.

[0119] In certain embodiments, amplification products (amplified nucleic acid from nucleic acid analysis) can be detected using methods well known to those of skill in the art. In certain embodiments the amplification is a straightforward simple PCR amplification reaction. In certain embodiments, however, a nested PCR reaction is used to amplify the nucleic acid from the nucleic acid analysis. In various embodiments, multiplexed PCR assays are contemplated, particularly where it is desired to analyze multiple products of the nucleic acid analysis in the same amplification reaction. In certain embodiments in such multiplexed amplification reactions, probe(s) for each specific target region has its own specific dye / fluorophore so that it is detectable independently of the other probes. In certain embodiments, typically, for signal generation, the probes used in various amplification reactions utilize a change in the fluorescence of a fluorophore due to a change in its interaction with another molecule or moiety brought about by changing the distance between the fluorophore and the interacting molecule or moiety for detection and / or quantification of the amplified product. Alternatively, other methods of detecting a polynucleotide in a sample, including, but not limited to. the use of radioactively labeled probes, are contemplated.Assay ConfigurationsReagents for Assay

[0120] As described herein, the nucleic acid can be bound to a nucleic acidbinding substrate, also referred to herein as a filter. In some examples, the filter comprises glass fibers and optionally a polymeric binder. The glass fibers may be modified with a nucleic acid binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof. In some examples, the filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron. In some aspects, the sample can be contacted with a binding reagent, wash reagent, or a combination during or after lysis. The binding reagent can promote binding of nucleic acids to the filter, facilitating the removal of non-target material. In some embodiments, the binding reagent can include a binding polymer such as polyacrylic acid (PAA), polyacry lamide (PAM), polyethylene glycol (PEG), poly(sulfobetaine), or a salt, or combinations thereof. In some embodiments, the filtering reagent and / or the washing reagent can include the binding reagent. For example, the binding reagent, the filtering reagent, and / or the washing reagent can include a binding polymer (e.g., PEG 200), buffer, inorganic salt(s), antioxidant and / or chelating agent, antifoam SEI 5, sodium azide, disaccharide or disaccharide derivative, carrier protein, a chaotropic agent (such as guanidium hydrochloride) detergent, DMSO, or a combination thereof. The binding polymer can be present in an amount of at least 10% v / v, at least 20% v / v, at least 30% v / v, and / or less than 60% v / v, less than 40% v / v, less than 30% v / v, less than 20% v / v, or less than 10% v / v or can fall within any range bounded by any of these values, e.g., from 10% to 60% v / v, of the binding reagent, filtering reagent, and / or the washing reagent. The buffer can be selected from the group consisting of Tris, 2-amino-2-hydroxymethyl- 1,3-propanediol, HEPES, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, and MES. The concentration of the buffer can range from about 5 mM to about 100 mM. such as from about 5 mM to about 50 mM. The salt, such as NaCl, KC1, or MgCh, can be present at a concentration from about 0.05 M to about 1 M, such as from about 0.1 M to about 0.5 M. The antioxidant and / or chelating agent comprises an agent selected from the group consisting of N-acetyl-L-cysteine. ethylenediaminetetraacetic acid (EDTA), di ethylene triamine pentaacetic acid (DTP A), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and a phosphonate chelating agent. In some embodiments the antioxidant and / or chelating agent comprises EDTA. In certain embodiments the antioxidant and / or chelating agent comprise 0.2% to about 5%, about 0.2% to about 3%, or about 0.5% to about 2%, or about 0.5% of the binding reagent, filtering reagent, and / or the washing reagent. In some embodiments the concentration of the antioxidant and / or chelating agent in the binding reagent, filtering reagent, or the washing reagent ranges from about 2 mM to about 50 mM or about 5 mM to about 20 mM. In some embodiments, the detergent is an ionic detergent or a non-ionic detergent. The detergent can be selected from an ionic detergent or a non-ionic detergent. In some examples, the detergent comprises a detergent selected from the group consisting of N-lauroylsarcosine, sodium dodecyl sulfate (SDS), cetyl methyl ammonium bromide (CT AB), TRITON®-X-100, n-octyl-|3-D-glucopyranoside, CHAPS, n-octanoylsucrose, n-octyl-P-D-maltopyranoside, n-octyl-P-D- thioglucopyranoside, PLURONIC® F-127, TWEEN® 20, Brij-35, and n-heptyl-P-D- glucopyranoside. The detergent can comprise about 0.1% to about 2% of the binding reagent, filtering reagent, and / or the washing reagent, and / or ranges from about 10 mM up to about 100 mM. The binding reagent, filtering reagent and / or the washing reagent can have a pH ranging from about pH 6.0 to about pH 8.0 (such as from about 6.5 to about 7.5).

[0121] In certain embodiments, the sample supernatant is then removed and the nucleic acid is eluted in an elution buffer such as a Tris / EDTA buffer. The elution buffer can comprise ammonia or an alkali metal hydroxide. In general, the elution buffer has a pH above about 9, above about 10, or above about 11. The elution buffer can further comprise a polyanion, optionally a carrageenan, a carrier nucleic acid, or i- carrageenan and KOH. The eluate may then be processed in the cartridge to detect target genes as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which are present in the cartridge as lyophilized particles. Particularly, the lyophilized particles can be in the form of beads and comprise primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof. The lyophilized can be present in the reaction vessel of the cartridge.

[0122] As would be appreciated by the skilled artisan, a Ct value is the number of cycles in a quantitative PCR experiment that are required for the fluorescent signal associated with the amplification of a specific target region to exceed a predeterminedthreshold value. As would be appreciated by the skilled artisan, this threshold value can be the background fluorescence levels measured in the experiment.Exemplary Cartridges

[0123] In embodiments, a cartridge for preparing a sequence nucleic acid library from a sample, comprises a cartridge body comprising a plurality of chambers in fluid communication therein; a filter disposed in a fluidic path between the plurality of chambers and a reaction vessel: the reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for amplification of nucleic acid and detection of a plurality of amplification products via real-time PCR, melt curve analysis, or a combination thereof.

[0124] In an embodiment, the reaction vessel comprises a planar frame defining a first fluidic path and a second fluidic path between a first planar substrate that encloses a first side of the planar frame and a second planar substrate that encloses a second side of the planar frame; a fluidic interface at one end of the planar frame, the fluidic interface comprising a first fluid inlet and a first fluid outlet of the first fluidic path, and a second fluid inlet and a second fluid outlet of the second fluidic path; a first reaction chamber arranged in the planar frame between the first and second substrates, the first reaction chamber being an enlarged portion of the first fluidic path disposed along the fluidic path between the first fluidic inlet and the first fluid outlet so as to allow a fluid sample introduced via the first fluid inlet to undergo amplification before exiting the first fluid outlet; and a second reaction chamber arranged in the planar frame betw een the first and second substrates, the second reaction chamber being an enlarged portion of the second fluidic path disposed along the second fluidic path betw een the second fluidic inlet and the optical chamber so as to allow a fluid sample introduced via the second fluid inlet to undergo amplification before filling the optical chamber. In some embodiments, each reaction chamber independently has a volume capacity of 100 pL or less, 80 pL or less, 70 pL or less, or 60 pL or less.

[0125] In an embodiment, the second fluidic path further includes an optical chamber defined in the planar frame between the first and second substrate, the optical chamber including a planar optical-substrate adapted for detection of one or a plurality of amplification products adapted for detection of signals generated during amplification by an optical assembly, and wherein the optical chamber being in fluidic communication with the second fluidic inlet and the second fluidic outlet. In an embodiment, the second reaction chamber includes a chamber exit in fluidiccommunication with the optical chamber entrance that is in fluidic communication with the optical chamber, and wherein the first reaction chamber together with the first fluid inlet are accessed by one of the plurality of chambers and the second reaction chamber together with the second fluid inlet are accessed by a different one of the plurality of chambers. In certain embodiments the reaction vessel of a presently disclosed cartridge is show n in FIG. 2.

[0126] In the embodiment shown in FIG. 2. the reaction vessel 116 includes a first chamber 502 and a second chamber 504, and the first chamber 502 and the second chamber 504 each have an independent port such that the first chamber 502 is in liquid communication with the first port 506 and the second chamber 504 is in liquid communication with the second part 508. A filter disposed in a fluidic path between the plurality of chambers and a reaction vessel can include more than one filter in the cartridge. In embodiments, the cartridge can have 2 filters, 3 filters, 4 filters, or more than 5 filters. In embodiments, a filter is disposed in more than one chamber of the cartridge. For example, a filter may be disposed in at least 2 chambers, at least three chambers, or at least 4 chambers. In certain embodiments, the filter in one chamber is the functionally the same as the filter in another chamber or the filter may be functionally different between chambers. For example, a filter may be functionalized with a chemical moiety, antibodies, magnetic particles, or any number of ways known in the art. In an embodiment, the filter comprises glass fibers and optionally a polymeric binder, or the glass fibers are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof. The purpose of functionalizing a filter is to use a filter for adhesion, absorption, isolation, etc. during sequencing library preparation. Accordingly, in embodiments, a cartridge of the present disclosure may have more than one filter where each filter has different functionality to serve a different purpose during sequencing library preparation.

[0127] In an embodiment, the filter is configured to bind the nucleic acid to be analyzed. The filter may be configured to bind unwanted material and allow the nucleic acid to pass through. In certain embodiments, a filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron. It should be appreciated that the filter may have a greater or lesser thickness and a larger orsmaller pore size based on the needs of the sample and sequencing steps for preparing a sequence nucleic acid library-.

[0128] The cartridge for preparing a sequence nucleic acid library from a sample may be configured to cany' out non-isothermal amplification, optionally bythermal cycling and / or temperature oscillation.

[0129] The plurality of chambers may include a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and a lysis chamber in fluidic communication with the sample chamber, wherein the lysis chamber is adapted for performing mechanical and chemical lysis to release nucleic acid from the biological sample. In an embodiment, the sample chamber and lysis chamber are the same. Mechanical lysis includes sonication to apply vibration frequency in a range from about 5 kHz to about 120 kHz or about 20kHz to about 50kHz. Chemical lysis may include a chemical treatment, a change in pH, an enzymatic treatment, or a combination thereof.

[0130] The one or more lysis reagents may include a chaotropic agent, a chelating agent, a buffer, a detergent, or a combination thereof. Example chaotropic agents include guanidinium thiocyanate, guanidinium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or a combination thereof. Example chelating agents include EDTA or any number of known agents that are capable of binding to metal ions. Example detergents are ionic detergents or a non-ionic detergents. In some examples, the detergent comprises a detergent selected from the group consisting of N- lauroylsarcosine, sodium dodecyl sulfate (SDS), cetyl methyl ammonium bromide (CTAB), TRITON®-X-100, n-octyl-P-D-glucopyranoside, CHAPS, n-octanoylsucrose, n-octyl-P-D-maltopyranoside, n-octyl-P-D-thioglucopyranoside, PLURONIC® F-127. TWEEN® 20, Brij-35, and n-heptyl-P-D-glucopyranoside. In an embodiment, the one or more lysis reagents comprise a guanidinium compound, sodium hydroxide, EDTA, a buffer, and a detergent.

[0131] The cartridge may further comprise a binding reagent, wash reagent, eluting reagent, or a combination thereof. The eluting reagent may comprise ammonia or an alkali metal hydroxide. The eluting reagent may comprise a polyanion, that may be selected from the group consisting of a carrageenan, a carrier nucleic acid, and i- carrageenan with KOH.

[0132] The least one of the plurality of chambers may comprise one or more lyophilized reagents, e.g., one or more lyophilized reagents selected from primers,probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof. In embodiments, at least one of the first reaction chamber and the second reaction chamber comprise lyophilized reagents for amplification, which may include lyophilized primers and probes. In certain embodiments, the one or more lyophilized reagents is / are in the form of one or more beads (e.g., magnetic beads).

[0133] It should be appreciated that while not necessary, in certain embodiments one or more (sometimes all) of the reagents and components in the reaction chambers are in solution.Samples for Sequencing

[0134] The biological sample can be selected from blood, plasma, serum, semen, a vaginal swab, a vaginal mucus sample, a vaginal tissue sample, a vaginal cell sample, spinal fluid, tissue, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, PCR reaction mixture, paraffin-embedded tissue sample, cell lysate, or in vitro nucleic acid modification reaction mixture. In specific embodiments, the nucleic-acid containing sample (biological sample) can be a fixed paraffin-embedded samples (e.g., from FFPET samples). In some embodiments, the nucleic acid containing sample can be a liquid biopsy sample for detection of cancer such as prostate, lung, breast, pancreas, colon, esophagus, ovary, bile duct, stomach, and liver cancers. In some embodiments, the nucleic acid containing sample can be a respirators’ sample for detection of an infectious disease. The nucleic acid-containing sample may comprise human, bacterial, fungal, animal, or plant material. In other embodiments, the nucleic acid-containing sample can be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction. In some examples, the biological sample comprises nucleic acid selected from genomic DNA, total RNA, short-DNA, small DNA, tumor- derived nucleic acid, methylated DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell free nucleic acid, or combinations thereof.

[0135] In some embodiments, the sample is bacterial culture. The bacterial culture may represent a biological sample collected from a subject, e.g., human. In some embodiments, the sample is a biological sample collected directly from a subject, e.g., a biopsy from a subject or a swab from a subject. In other embodiments, the sample is a sample that is not collected directly from a subject, such as, e.g., a wastewater sample or a sample from an air filter in a building. In embodiments, the sample is collected in order to determine a potential disease state of the subject.

[0136] The sample to be tested (e.g., sequenced) is, in some embodiments, fresh (i.e., never frozen). In other embodiments, the sample is a frozen specimen. In some embodiments, the sample is a tissue sample, such as a formalin-fixed paraffin embedded sample. In some embodiments, the sample is a liquid cytology sample.

[0137] In some embodiments, a sample to be tested is contacted with a buffer after collection. In some embodiments, the sample is contacted with buffer within about 5 minutes, within about 10 minutes, within about 30 minutes, within about I hour, or within about 2 hours of sample collection.

[0138] In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, or less than 0.75 ml of sample or buffered sample are used in the present methods. In some embodiments, from about 0. 1 ml to about 1 ml of sample or buffered sample is used in the present methods.Kits

[0139] Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. In some embodiments, a kit includes primer pairs for amplifying and / or detecting the nucleic acid targets described herein, with probes specific for these targets. In some embodiments, these kits can include a set of primer and a set of two or more probes for each target region, wherein each of the two or more probes in the set for a target region comprises a detectable label that emits light at the same wavelength. The kit may further comprise one or more lysis reagents for releasing nucleic acid from the biological sample.

[0140] A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or any other material useful in sample processing, washing, or conducting any other step of the assay.

[0141] Kits preferably include instructions for carrying out one or more of the library preparation methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user can be employed. Such media include, but are not limited to, electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "‘instructions” can include the address of an internet site that provides the instructions.

[0142] In some embodiments, the kit can include any of the reagents described above provided with or in one or more GENEXPERT® cartridge(s). See e.g., US Patents 5,958,349, 6,403,037, 6,440,725, 6,783.736, 6,818, 185; each of which is herein incorporated by reference for this description).

[0143] In certain embodiments, the kit can further contain instructional materials providing guidance to overcome problems that may occur. The instructional materials can also include information and / or instructions regarding the use of the lysis reagent and / or instructions for the collection, and / or storage, and / or shipping of a cell or tissue sample. In certain embodiments the kits additionally contain reagents and / or instructions teaching the use of the lysis buffer for isolation and recovery' of a nucleic acid.

[0144] Often and typically the instructional materials are provided in written form and can be printed on the kit components themselves (e.g. on the cover of a box, container, or on an envelope), or can be provided as an insert / instructional page or booklet. While the instructional materials ty pically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.EXAMPLESExample 1: In-cartridge Whole Genome Sequencing (WGS) library prep from bacterial culture.

[0145] This example demonstrates an embodiment of the present disclosure using an Illumina DNA Prep library' prep kit (available from https: / / www.illumina.com / ) in a cartridge to obtain a sequencing ready WGS nucleic acid library’ from a bacterial culture.

[0146] Protocol: A sample from a bacterial culture was placed into a GENEXPERT® cartridge by Cepheid Inc. The DNA was extracted from the bacterial culture sample by catching the bacteria on the cartridge filter, sonicating the cartridgeand filter, and eluting the DNA from the filter. The DNA was mixed with bead-linked- transposomes (BLTs), and the first tube fill was performed. The BLT tagmentation was stopped by mixing with SDS, performing the second tube fill, and incubating the reaction mixture. The BLTs were caught on the filter of the cartridge and washed off the filter. The cartridge chamber and tube were rinsed to recover any residual BLTs. The BLTs were caught on the filter and washed. BLT beads were recovered from the filter and mixed with Index PCR reagents. The third tube fill was performed, and Index PCR attached Illumina indexes and adapters. The sequencing library was purified and size-selected using solid-phase reversible immobilization (SPRI) beads. After the first tube fill, each subsequent tube fill included the bubble-bursting steps disclosed in Example 11.

[0147] Results. A sequencing ready WGS nucleic acid library from a bacterial culture was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 2: In-cartridge Whole Genome Sequencing (WGS) library prep from bacterial culture.

[0148] This example demonstrates an embodiment of the present disclosure using an Oxford Nanopore* Rapid PCR Barcoding kit (available from https: / / nanoporetech.com / ) in a cartridge to obtain a sequencing ready WGS nucleic acid library from a bacterial culture.

[0149] Protocol: A sample from a bacterial culture was placed into a GENEXPERT® cartridge by Cepheid Inc. The DNA was extracted from the bactenal culture sample by: mixing the bacteria culture sample, lysis buffer, and PEG200; binding the DNA to a RCC filter; washing the filter; and eluting the DNA from the filter. The first tubefill was performed, in which the DNA was fragmented and tagged with transposome complexes. The tagmented DNA was mixed with PCR reagents, including primers containing click chemistry moieties on their ends. The second tubefill w as performed for PCR amplification. The amplified material w as purified by PEG- NaCl size exclusion paired with the Cart A+ glass fiber filter. Finally, ONT-specific sequencing adapters were attached through room-temperature click chemistry reaction which resulted in a sequencing library. Results. A sequencing ready WGS nucleic acid library from a bacterial culture was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 2a: In-cartridge Whole Genome Sequencing (WGS) library prep from bacterial or fungal culture.

[0150] This example demonstrates an embodiment of the present disclosure using an Oxford Nanopore® Rapid PCR Barcoding kit (available from https: / / nanoporetech.com / ) in a cartridge to obtain a sequencing ready WGS nucleic acid library from a bacterial or fungal culture.

[0151] Protocol: A sample from a bacterial culture was placed into a GENEXPERT® cartridge by Cepheid Inc. The DNA was extracted from the bacterial culture sample by: capturing bacteria or fungi mixed with a lysis buffer on a Cart A+ glass fiber filter; washing the filter; lysing microbes by sonication; and eluting the DNA from the filter. The first tubefill was performed, in which the DNA was fragmented and tagged with transposome complexes. The tagmented DNA was mixed with PCR reagents, including primers containing click chemistry moieties on their ends. The second tubefill was performed for PCR amplification. The amplified material was purified by PEG-NaCl size exclusion paired with the Cart A+ glass fiber filter. Finally, ONT-specific sequencing adapters were attached through room-temperature click chemistry reaction which resulted in a sequencing library.

[0152] Results. A sequencing ready WGS nucleic acid library from a bacterial culture was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 2b: In-cartridge Whole Genome Sequencing (WGS) library prep from positive blood culture.

[0153] This example demonstrates an embodiment of the present disclosure using an Oxford Nanopore® Rapid PCR Barcoding kit (available from https: / / nanoporetech.com / ) in a cartridge to obtain a sequencing ready WGS nucleic acid library from a positive blood culture.

[0154] Protocol: A sample from a bacterial culture was placed into a GENEXPERT® cartridge by Cepheid Inc. The DNA was extracted from the bacterial culture sample by: mixing positive blood culture with lysis buffer outside of the cartridge, and incubating at room temperature; adding the blood culture / lysis mixture into the cartridge; capturing microbes mixed with a lysis buffer on a Cart A+ glass fiber filter; washing the filter; lysing microbes by sonication; and eluting the DNA from the filter. The first tubefill was performed, in which the DNA was fragmented and taggedwith transposome complexes. The tagmented DNA was mixed with PCR reagents, including primers containing click chemistry moieties on their ends. The second tubefill was performed for PCR amplification. The amplified material was purified by PEG- NaCl size exclusion paired with the Cart A+ glass fiber filter. Finally, ONT-specific sequencing adapters were attached through room-temperature click chemistry reaction which resulted in a sequencing library.

[0155] Results. A sequencing ready WGS nucleic acid library from a bacterial culture was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 3: In-cartridge targeted sequencing library prep from Melanoma sample.

[0156] This example demonstrates an embodiment of the present disclosure (FIG. 7) using a Nuprobe Varmap® NGS panel (available from https: / / nuprobe.com / ) in a cartridge to obtain a sequencing ready nucleic acid 11 bran from a melanoma sample. NuProbe’s original protocol (4 PCR steps, 5 bead cleanups, 7-8 hours) was condensed down to 3 PCR steps and 2 clean up steps.

[0157] Protocol: Unique Molecular Identifier (“UMI”) PCR was performed to attach unique molecular identifiers to input DNA in the first tube fill. Two cycles were about one hour. The UMI PCR was purified using PEG-NaCl size exclusion paired with an RCC glass fiber filter. Blocker displacement amplification (“BDA”) BDA-PCR was performed in the second tube fill to enrich the sample for amplicons containing mutations with blocker displacement amplification. Twenty three cycles were about two hours. A 1 : 100 dilution was performed. Adapter PCR was performed during the third tube fill to attach Illumina adapters. Twenty one cycles were about two hours. The sequencing library was purified and size-selected using PEG-NaCl size exclusion paired with a RCC glass fiber filter.

[0158] Results. A sequencing ready nucleic acid library from a melanoma sample was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 4: In-cartridge targeted sequencing library prep from genomic DNA.

[0159] This example demonstrates an embodiment of the present disclosure using an Oxford Nanopore® Ligation Sequencing kit (available fromhttps: / / nanoporetech.com / ) in a cartridge to obtain a targeted sequencing I i bran' from genomic DNA.

[0160] 0001 Protocol: Specific targets (2 amplicons) were amplified using 5’- phosphorylated primers and a polymerase lacking 3’-to-5’ exonuclease activity, resulting in a 5 ’-phosphorylated and 3 ’-A- tailed amplicon that was ready for enzy matic ligation. ONT-specific sequencing adapters were ligated using T4 ligase-mediated ligation. The sequencing library was purified and size-selected using solid-phase reversible immobilization (SPRI) beads.

[0161] Results. A sequencing ready nucleic acid library7from genomic DNA was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 5: StilPCR in-cartridge targeted sequencing library prep for TB drug resistance (TB culture, spiked and clinical sputum samples).

[0162] This example demonstrates an embodiment of the present disclosure using a targeted NGS library7method in a cartridge to obtain a sequencing ready nucleic acid library7from a TB culture, and spiked and clinical sputum samples.

[0163] In the experiment, the Xpert microfluidic cartridge’s sample processing, cell lysis, and DNA extraction, and PCR capabilities are used to perform individual sequencing library preparations, first from human spiked sputum specimens and then directly from clinical sputum across various smear grades, including smear-negative samples. A novel hemi-nested bedaquiline duplex PCR that amplifies the full-length bedaquiline resistance-conferring genes, rv0678 and atpE, as overlapping fragments (e.g., one forw ard and two reverse primers for rv0678) and adds Illumina indexes and adapters targeting the full-length rv0678 and atpE genes in a single reaction contained entirely within the microfluidic Xpert cartridge, and a publicly available pipeline (www.DrDx.Me) that performs alignment and variant calling.

[0164] Protocol: A M. tuberculosis (“MTB”) sample was placed into a GENEXPERT® cartridge by Cepheid Inc. The MTB DNA was extracted by: passing MTB culture sample, or sputum with sample reagent, over the cartridge filter; washing the filter; sonicating the cartage and filter; and eluting the DNA from the filter. For the first tube fill, PCR was performed to amplify two 600bp targets. For the second tube fill, PCR was performed to produce tiled amplicons by amplifying with internal primers. The sequencing library was purified and size-selected using solid-phasereversible immobilization (SPRI) beads. Sonification was modified to produce shorter or longer average DNA fragment sizes during the sample preparation.

[0165] Results. A sequencing ready nucleic acid library from TB culture, and spiked and clinical sputum samples was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 6: Nanoligation in-cartridge targeted sequencing library prep for TB drug resistance (TB culture, spiked and clinical sputum samples).

[0166] This example demonstrates an embodiment of the present disclosure using nanoligation (for ONT) to obtain a sequencing ready nucleic acid library from a TB culture, and spiked and clinical sputum samples.

[0167] Protocol: A.M. tuberculosis (“MTB ’) sample was placed into a GENEXPERT® cartridge by Cepheid Inc. The MTB DNA was extracted by: passing MTB culture sample, or sputum with sample reagent, over the cartridge filter; washing the filter; sonicating the cartridge and filter; and eluting the DNA from the filter. PCR was used to amplify 12 targets. Enzymatic ligation of ONT adapters was performed. The sequencing library' was purified and size-selected using solid-phase reversible immobilization (SPRI) beads.

[0168] Results. A sequencing ready nucleic acid library from TB culture, and spiked and clinical sputum samples was successfully created using an in-cartridge approach in a GENEXPERT® cartridge by Cepheid Inc.Example 7: In-cartridge magnetic bead purification for preparation of an NGS library.

[0169] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc. efficiently prepares mixtures of MAG / PEG8000 and DNA, e.g., an NGS library' PCR reaction, by toggling the mixture.

[0170] Protocol: There is a difference in phase density between a PCR solution and the PEG8000 bead solution. Accordingly, mixing between the PCR mixture and the PEG8000 / beads solution is more efficient when the system is used to toggle volumes. It was discovered that the preferred toggle volume is slightly less than the combined volume of the PCR mixture and the PEG8000 / beads solution. Toggling less than 50% of the mixed volume was not sufficient to mix the two phases. In addition, reducing the toggling rate below a threshold rate resulted in inefficient mixing of the PCR reaction since the MAG / PEG8000 solution is more dense that the PCR reaction.Thus, it requires more mixing to get a homogeneous mixture for efficient DNA precipitation. The preferred toggling speed is between 10 and 20 pl / second.

[0171] Results. In this example, a formula to determine the volume to toggle was determined: V(T) = V(PCR) + V(MAG / PEG8000) - ((V(PCR) + V(MAG / PEG8000) I 10)). Where V(T) = toggle volume, V(PCR) = volume of PCR reaction, V(MAG / PEG8000) = volume of MAG / PEG8000 reagents.Example 8: In-cartridge magnetic bead capture using filter.

[0172] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc. is used as a surrogate non-magnetic method to capture para magnetic beads.

[0173] Protocol / Results: Efficient capture of para magnetic beads by passing MAG / PEG8000 across the cartridge filter in the absence of a magnetic field was demonstrated. It was demonstrated that high concentrations of up to 4X paramagnetic beads (4 mg / ml) can be captured in the GENEXPERT® cartridge. It was demonstrated that para magnetic beads can be captured when the PEG8000 concentration in increased to 30 % and the NaCl concentration reduced to 2.0M.Example 9: In-cartridge magnetic bead size selection.

[0174] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc. efficiently captures para magnetic beads that are subsequently washed, and the bound DNA eluted to yield a size selected population of DNA.

[0175] Protocol / Results: It was demonstrated that the 80% ethanol wash could be substituted in the cartridge, with a wash buffer comprising 750 mM NaCl, 50 mM tris / HCl and 10% PEG8000, increasing manufacturability. The ratio of the MAG / PEG8000 reagent to DNA could be optimized in the cartridge such that DNA size fractionations could be achieved between 300 and 1500 bp.Example 10: In-cartridge magnetic functionality of magnetic beads.

[0176] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc., with a small magnet placed in a single chamber of the cartridge, efficiently captured para magnetic beads present in mixtures of MAG / PEG8000 and DNA.

[0177] Protocol / Results: It was demonstrated that the captured beads could be efficiently washed using the 750 mM NaCl. 50 mM tris / HCl and 10% PEG8000 washsolution. It was demonstrated that size fractionated DNA precipitated onto para magnetic beads could be eluted from para magnetic beads while captured on the magnet within the cartridge chamber.Example 11: In-cartridge DNA size-selection purification without magnetic beads.

[0178] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc. efficiently captures DNA, without using magnetic beads, that is subsequently washed, and the bound DNA eluted to yield a size selected population of DNA.

[0179] Protocol / Results: DNA was mixed with 25 ug of pectin precipitation reagent (in solution) (US20210324372A1, incorporated herein by reference). The DNA / pectin precipitation mixture was mixed with binding buffer (2.5M NaCl / 20% PEG-8000) and lyophilization buffer (BSA, tween-20, excipients), and incubated for 30 seconds. The resulting mixture was toggled (dispense 20 pL. aspirate 10 pL) over the Cart A+ filter to bind / precipitate DNA of desired size onto filter where a higher salt / PEG-8000 ratio results in greater retention of lower MW DNA species. The CartA+ filter with bound DNA was washed with FluA-B wash (200nM KCl / 10% PEG- 8000) by toggling over the filter (dispense 30 pL. aspirate 10 pL) to remove high salt / PEG, and unbound DNA fragments. The bound DNA was eluted with TET buffer (20 mM Tris-HCl) by toggling over the filter (dispense 20 pL, aspirate 10 pL).Example 12: In-cartridge bubble bursting procedure for multiple tube fills.

[0180] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc. is used to efficiently burst bubbles. In embodiments, this procedure may be performed for effective, bubble-free tubewashing to remove inhibitors from previous reactions, and for bubble-free tube fills.

[0181] Protocol / Results: An empty tube was heated to 95°C for a minimum of 5 seconds to burst existing bubbles in the tube. The tube was partially filled with 30pL to push any bubbles from the tube port into the tube, then heated to 95°C for 5 seconds to burst those bubbles before completely filling the tube with the rest of the solution. 30pL was sufficient to fill the tube 25%, and push air from the tube port into the tube. It w as also the minimal volume necessary to ensure the air from the tube port w as pushed into the tube, since there can be variability between cartridges. In addition, it minimized the amount of solution subjected to the 95°C heat, as the reaction components following the BLT (first tube fill) degrade in high heat, and heatdenaturing a large proportion of the volume would result in sample degradation, and a poor final yield.Example 13: In-cartridge toggling to recover magnetic beads.

[0182] This example demonstrates an embodiment of the present disclosure where a GENEXPERT® cartridge by Cepheid Inc. was used to efficiently recover magnetic beads.

[0183] Protocol: In cartridge with glass beads, it was observed that toggling <20 pl volumes over the filter path was insufficient to move the magnetic beads out of the VBA / filter and into the syringe. Magnetic beads of <5 pm tended to move with the liquid, but would at times be lodged into the edge of the filter in the VBA, and could be dislodged by toggling. Toggling at least 3X over the filter path with lOOuL at lOOuL / s was sufficient to recover the magnetic beads from the filter into the syringe.All publications, patents and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this disclosure pertains.All of the methods, devices, and kits disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the method, devices, and kits of this disclosure have been described in terms of the foregoing illustrative embodiments, it will be apparent to those of skill in the art that variations, changes, modifications, and alterations may be applied to the devices, kits, and methods, and in the steps or in the sequence of steps of the methods described herein, without departing from the true concept, spirit, and scope of the disclosure. More specifically, it will be apparent that certain agents, additives, and ingredients that are similar according to their physical, chemical, physiological, and / or gustative properties may be substituted for the agents, additives and ingredients described herein while the same or similar results would be achieved. In addition, it will be apparent that certain methods steps may be practiced in different sequences, including repeated sequences, or sometimes not at all, and the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the hereinafter appended claims.CLAUSESThe following numbered clauses define further example aspects and features of the present disclosure.1. A cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising:(a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of the nucleic acid and detection of signals generated during amplification;(b) utilizing the filter to isolate the nucleic acid from the sample;(c) forming a library prep reaction mixture comprising the nucleic acid and a library prep reagent that facilitates: i) generation of tagged nucleic acid fragments from the nucleic acid in the library prep reaction mixture, ii) generation of amplicons, wherein the amplicons are optionally tagged at one or both ends, and / or iii) attachment of oligonucleotide adapters to one or both ends of the tagged nucleic acid fragments or amplicons to form adapter-labeled nucleic acid;(d) causing the library prep reaction mixture to flow into the reaction vessel and preparing the sequencing nucleic acid library comprising the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid;(e) isolating the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the 11 bran prep reaction mixture on to the fdter and eluting each from the filter; and(f) purifying and optionally size selecting the sequencing nucleic acid library on the filter. wherein a bubble bursting mechanism is utilized to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings of the reaction vessel.2. The cartridge-based method of clause 1 , wherein releasing the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the filter during step (e) is during and / or after preparing the sequencing nucleic acid library.3. The cartridge-based method of clauses 1-2, further comprising eluting the nucleic acid from the filter following step (b).4. The cartridge-based method of clauses 1-3, further comprising amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid within the reaction vessel following step (b).5. A cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising:(a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of nucleic acid and detection of signals generated during amplification;(b) utilizing the filter to isolate the nucleic acid from the sample;(c) fragmenting the nucleic acid to form nucleic acid fragments, wherein fragmenting is performed by mechanical fragmentation, chemical fragmentation, or enzymatic fragmentation; and / or amplifying the nucleic acid or nucleic acid fragments to form amplicons, wherein amplifying the nucleic acid or nucleic acid fragments is with at least one set of primers for amplifying a target region of the nucleic acid or nucleic acid fragments;(d) generating adapter-labeled nucleic acid comprising labeling the fragmented nucleic acid or amplicons at one or both ends the fragmented nucleic acid or amplicons; and(e) isolating the adapter labeled nucleic acid on to the filter to form the NGS nucleic acid library, wherein the cartridge-based method comprises reuse of the reaction vessel, the filter, and optionally one or more of the plurality of chambers with rinsing to remove or dilute carry over, wherein reuse of the reaction vessel comprises a bubble bursting mechanism to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings.6. The cartridge-based method of clauses 1 or 5, wherein if the sample in step (a) comprises intact cells, the method further comprises releasing nucleic acid from the intact cells by lysing the intact cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers and / or capturing the cells on the filter and lysing the cells by means of sonication, to release nucleic acid from the cells.7. The cartridge-based method of clauses 5-6, further comprising amplifying the adapter-labeled nucleic acid w ith at least one set of primers for amplifying and increasing adapter-labeled nucleic acid concentration following step (d).8. The cartridge-based method of clauses 5-7, wherein step (e) further comprises purifying and size selecting the isolated adapter labeled nucleic acid to form the sequencing nucleic acid library.9. The cartridge-based method of clause 8, wherein size selecting the isolated adapter labeled nucleic acid in step (e) comprises solid-phase reversible immobilization.10. The cartridge-based method of any one of clauses 5-9, further comprising isolating and washing the nucleic acid on the filter, and eluting the nucleic acid from the filter prior to step (c).11. The cartridge-based method of any one of clauses 1-10, further comprising using a reusable reagent for one or more steps in clauses 1-10 including lysing the cells in the sample, isolating the nucleic acid released from the cell, washing the nucleic acid, eluting the nucleic acid, library prep reagent, fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter labeled nucleic acid, amplifying the adapter labeled nucleic acid, isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purify ing and size selecting the isolated adapter labeled nucleic acid.12. The cartridge-based method of clause 11, comprising using the reusable reagent for washing the nucleic acid, purifying and size selecting the isolated adapter labeled nucleic acid.13. The cartridge-based method of clauses 11 or 12, wherein the reusable reagent comprises a buffer, a salt, and polyethylene glycol (PEG).14. The cartridge-based method of any one of clauses 11-13, wherein using the reusable reagent comprises recycling the reusable reagent after one or more uses.15. The cartridge-based method of any one of clauses 1 1-13, wherein using the reusable reagent comprises:(i) drawing, from a first one of the plurality7of chambers, a first portion of the reusable reagent and utilizing the first portion of reusable reagent, and(ii) drawing, from the first one of the plurality of chambers, a second portion of the reusable reagent and utilizing the second portion of reusable reagent.16. The cartridge-based method of any one of clauses 1-15, comprising reuse of at least one of the plurality of chambers, reuse of at least two of the plurality7of chambers, reuse of at least three of the plurality7of chambers, or reuse of at least four of the plurality of chambers.17. The cartridge-based method of clause 16, wherein the at least one of the plurality of chambers is used to store a first reusable reagent prior to use. the at least two of the plurality of chambers is used to store a first and a second reusable reagent prior to use, the at least three of the plurality of chambers is used to store a first, a second, and a third reusable agent prior to use, or the at least four of the plurality of chambers is used to store a first, a second, a third, and fourth reusable agent prior to use.18. The cartridge-based method of clause 17, wherein at least one of the first, second, third, and fourth reusable agents are different reusable agents.19. The cartridge-based method of any one of clauses 16-18, wherein the at least one of the plurality of chambers, the at least two of the plurality of chambers, the at least three of the plurality of chambers, or the at least four of the plurality of chambers is reused for collecting waste, fragmenting the nucleic acid, or generating the adapter- labeled nucleic acid.20. The cartridge-based method of clause 19, wherein at least one of the plurality of chambers is reused for a different purpose than another of the plurality of chambers.21. The cartridge-based method of any one of clauses 1-20, wherein the reaction vessel comprises one or more reaction chambers configured for amplification of nucleic acid and detection of signals generated during amplification.22. The cartridge-based method of clause 21, wherein the reaction vessel comprises one reaction chamber, the reaction vessel comprises two reaction chambers, the reaction vessel comprises three reaction chambers, or the reaction vessel comprises no more than one reaction chamber configured for amplification of nucleic acid and detection of signals generated during amplification.23. The cartridge-based method of any one of clauses 1-22, wherein the self- contained cartridge consists of 15 or less chambers, 12 or less chambers, or 11 or less chambers, not including the reaction chamber(s).24. The cartridge-based method of any one of clauses 1-22, wherein each chamber of the plurality of chambers independently has a volume capacity of 10 mL or less, 9 mL or less, 8 mL or less, 7 mL or less, 6 mL or less, 5 mL or less, 4 mL or less, 3 mL or less, 2 mL or less, or 1 mL or less.25. The cartridge-based method of any one of clauses 1-22, wherein each reaction chamber independently has a volume capacity of 100 pL or less, 80 pL or less. 70 pL or less, or 60 pL or less.26. The cartridge-based method of any one of clauses 21-22, wherein fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid are sequentially performed in a single reaction chamber.27. The cartridge-based method of any one of clauses 21-22, wherein fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid are sequentially performed in more than one reaction chambers.28. The cartridge-based method of any one of clauses 21-27, wherein fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid comprises thermocycling, heating, and / or mixing.29. The cartridge-based method of any one of clauses 1-28, wherein the bubble bursting mechanism comprises:(i) heating the empty reaction vessel,(ii) partially filling the reaction vessel with a first portion of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid,(iii) heating the first portion of reaction mixture in the reaction vessel,(iv) discarding the first portion of reaction mixture, and(v) filling the reaction vessel with a second portion of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid.30. The cartridge-based method of clause 29, further comprising repeating steps (i)- (v) with additional portions of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid.31. The cartridge-based method of any one of clauses 1-30, comprising multiple uses of the filter selected from capturing the cells in the sample, isolating the nucleic acid released from the cells, isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purifying and size selecting the isolated adapter labeled nucleic acid.32. The cartridge-based method of any one of clauses 1-31, wherein the filter comprises magnetic beads, and optionally the magnetic beads are bound and / or adsorbed to the filter.33. The cartridge-based method of clause 32, wherein the magnetic beads are about 0. 1 pm to about 10 pm, or about 0.5 pm to about 5 pm, or about 0.8 pm to about 3 pm.34. The cartridge-based method of any one of clauses 32-33, wherein the magnetic beads have a specific concentration based upon the size of the magnetic beads, the pore size and / or composition of the filter, and / or the length of the nucleic acid fragments.35. The cartridge-based method of any one of clauses 32-33, wherein the magnetic beads are optionally modified with a polymeric binder, or are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.36. The cartridge-based method of any one of clauses 5-35, comprising:(i) fragmenting the nucleic acid to form nucleic acid fragments in step (b) using enzymatic fragmentation by contacting the nucleic acid with a transposome solid support and / or free-floating transposome complexes, and generating tagged nucleic acid fragments by subjecting the nucleic acid and transposome solid support and / or free-floating transposome complexes to fragmentation conditions;(ii) forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and a) a set of primer pairs comprising adapters for amplifying the tagged nucleic acids fragments or b) ONT-specific adapters; and(iii) subjecting the adapter labeled reaction mixture to a) amplification conditions to amplify the tagged nucleic acid fragments or b) to click chemistry conditions to attach the ONT-specific adapters to the tagged nucleic acid.37. The cartridge-based method of clause 36, wherein the generating tagged nucleic acid fragments happens in the reaction vessel in a first reaction vessel filling, and further comprises quenching generation of the tagged nucleic acid fragments in a second reaction vessel filling, isolating the tagged nucleic acid fragments on the filter, forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and a set of primer pairs comprising adapters for amplify ing the tagged nucleic acids fragments; and subjecting the adapter labeled reaction mixture to amplification conditions to amplify the tagged nucleic acid fragments in a third reaction vessel filling.38. The cartridge-based method of clause 37, wherein quenching generation of the tagged nucleic acid fragments comprises adding SDS to the reaction vessel in the second reaction vessel filling.39. The cartridge-based method of clause 36, wherein the generating tagged nucleic acid fragments happens in the reaction vessel in a first reaction vessel filling, and further comprises isolating the tagged nucleic acid fragments on the filter, forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and ONT-specific adapters; and subjecting the adapter labeled reaction mixture to click chemistry conditions to attach the ONT-specific adapters to the tagged nucleic acid.40. The cartridge-based method of clause 39, wherein the tagged nucleic acid fragments comprise one or more click chemistry moieties on a 3’ end of nucleic acid fragment.41. The cartridge-based method of any one of clauses 39-40, wherein the click chemistry conditions are at room temperature.42. The cartridge-based method of any one of clauses 1-35, comprising; amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid and a polymerase to form tagged amplicon; forming an adapter labeled reaction mixture comprising the tagged amplicons and ONT-specific adapters; and subjecting the adapter labeled reaction mixture to ligation conditions to attach the ONT-specific adapters to the tagged amplicons.43. The cartridge-based method of clause 42, wherein the primers for amplify ing the target region comprises 5 ’-phosphorylated primers and the polymerase lacks 3’-to- 5’ exonuclease activity, resulting in a 5 ’-phosphorylated and 3’-A-tailed amplicon, and ligation comprises T4 ligase-mediated ligation of ONT-specific sequencing adapters.44. The cartridge-based method of any one of clauses 1-35, comprising: amplifying the nucleic acid with at least one set of unique molecular identifier (UMI) primers for amplifying a target region of the nucleic acid to form tagged amplicons in a first reaction vessel filling; amplify ing the tagged amplicons via blocker displacement amplification to enrich for amplicons containing specific target region in a second reaction vessel filling; andamplifying the enriched tagged amplicons with at least one set of primers comprising adapters to form adapter labeled nucleic acid in a third reaction vessel filling.45. The cartridge-based method of any one of clauses 1-35, comprising: amplifying the nucleic acid with at least one set of primers for amplify ing a target region of the nucleic acid to form amplicons in a first reaction vessel filling, amplifying the amplicons with at least one set of internal primers for amplifying a target region of the nucleic acid to form tiled amplicons in a second reaction vessel filling.46. The cartridge-based method of any one of clauses 1-35, comprising: amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid to form amplicons in a first reaction vessel filling; and enzymatically ligating ONT-specific adapters to the amplicons in a second reaction vessel filling.47. The cartridge-based method of any one of clause 1-46, further comprising eluting from the filter one or more of the nucleic acid released from the cells, nucleic acid fragments, tagged nucleic acid fragments, amplicons, tagged amplicons, and / or adapter labeled nucleic acid.48. The cartridge-based method of any one of clause 1-47, wherein eluting from the filter comprises toggling the nucleic acid released from the cells, nucleic acid fragments, tagged nucleic acid fragments, amplicons, tagged amplicons, and / or adapter labeled nucleic acid on the filter with an eluant.49. The cartridge-based method of clause 48, wherein toggling optionally comprises chemical, optical, electrical, mechanical, thermal, acoustical processing, or a combination thereof, and wherein toggling optionally comprises a change in speed, volume, viscosity, or a combination thereof, and wherein the toggling is important for optimization yields and / or reduction in sample degradation.50. The cartridge-based method of any one of clause 1-48, wherein lysing the cells by sonication comprises exposing the cells to ultrasonic waves in a frequency range of approximately 5 kHz to approximately 120 kHz to lyse and release nucleic acid from the cells, or approximately 20 kHz to approximately 50 kHz to lyse and release nucleic acid from the cells.51. A cartridge for preparing a sequence nucleic acid library from a sample, comprising:a cartridge body comprising a plurality7of chambers in fluid communication therein; a filter disposed in a fluidic path between the plurality of chambers and a reaction vessel; the reaction vessel fluidically coupled to the plurality7of chambers of the cartridge body and configured for amplification of nucleic acid and detection of a plurality of amplification products via real-time PCR. melt curve analysis, or a combination thereof, wherein the reaction vessel comprises: a planar frame defining a first fluidic path and a second fluidic path between a first planar substrate that encloses a first side of the planar frame and a second planar substrate that encloses a second side of the planar frame; a fluidic interface at one end of the planar frame, the fluidic interface comprising a first fluid inlet and a first fluid outlet of the first fluidic path, and a second fluid inlet and a second fluid outlet of the second fluidic path; a first reaction chamber arranged in the planar frame between the first and second substrates, the first reaction chamber being an enlarged portion of the first fluidic path disposed along the fluidic path between the first fluidic inlet and the first fluid outlet so as to allow a fluid sample introduced via the first fluid inlet to undergo amplification before exiting the first fluid outlet, wherein the second fluidic path further includes an optical chamber defined in the planar frame between the first and second substrate, the optical chamber including a planar optical-substrate adapted for detection of one or a plurality7of amplification products adapted for detection of signals generated during amplification by an optical assembly, and wherein the optical chamber being in fluidic communication with the second fluidic inlet and the second fluidic outlet; a second reaction chamber arranged in the planar frame between the first and second substrates, the second reaction chamber being an enlarged portion of the second fluidic path disposed along the second fluidic path between the second fluidic inlet and the optical chamber so as to allow a fluid sample introduced via the second fluid inlet to undergo amplification before filling the optical chamber,wherein the second reaction chamber includes a chamber exit in fluidic communication with the optical chamber entrance that is in fluidic communication with the optical chamber, and wherein the first reaction chamber together with the first fluid inlet are accessed by one the plurality' of chambers and the second reaction chamber together with the second fluid inlet are accessed by a different one of the plurality of chambers.52. The cartridge of clause 51. wherein the cartridge is configured to carry out nonisothermal amplification, optionally by thermal cycling or temperature oscillation.53. The cartridge of any one of clauses 51-52, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and a lysis chamber in fluidic communication with the sample chamber, wherein the lysis chamber is adapted for performing mechanical and chemical lysis to release nucleic acid from the biological sample, optionally wherein the sample chamber and lysis chamber are the same.54. The cartridge or cartridge-based method of any one of clauses 1-53, wherein the one or more lysis reagents comprise a chaotropic agent, a chelating agent, a buffer, and a detergent.55. The cartridge or cartridge-based method of any one of clauses 1-54, wherein the chaotropic agent is selected from guanidinium thiocyanate, guanidinium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or a combination thereof.56. The cartridge or cartridge-based method of any one of clauses 1-55, wherein the one or more lysis reagents comprise a guanidinium compound, sodium hydroxide, EDTA, a buffer, and a detergent.57. The cartridge or cartridge-based method any one of clauses 1-56, wherein the filter is configured to bind the nucleic acid to be analyzed.58. The cartridge or cartridge-based method of any one of clauses 1-57, wherein the filter comprises glass fibers and optionally a polymeric binder, or the glass fibers are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety', an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.59. The cartridge or cartridge-based method of any one of clauses 1-58, wherein the filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron.60. The cartridge or cartridge-based method of any one of clauses 1-59, wherein the filter is configured to bind unwanted material and allow the nucleic acid to pass through.61. The cartridge or cartridge-based method of any one of clauses 1-60, wherein the cartridge further comprises a binding reagent, wash reagent, eluting reagent, or a combination thereof.62. The cartridge or cartridge-based method of any one of clauses 1-61, wherein the eluting reagent comprises ammonia or an alkali metal hydroxide.63. The cartridge or cartridge-based method of any one of clauses 1-62, wherein the eluting reagent comprises a polyanion, optionally selected from the group consisting of a carrageenan, a carrier nucleic acid, and i-carrageenan with KOH.64. The cartridge or cartridge-based method of any one of clauses 1-63, wherein at least one of the plurality of chambers comprises one or more lyophilized reagents.65. The cartridge or cartridge-based method of any one of clauses 1-64, wherein at least one of the first reaction chamber and the second reaction chamber comprises lyophilized reagents for amplification.66. The cartridge or cartridge-based method of any one of clauses 1-65, wherein the one or more lyophilized reagents is / are in the form of one or more beads.67. The cartridge or cartridge-based method of any one of clauses 1-66, wherein the one or more lyophilized reagents are selected from primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof.68. The cartridge or cartridge-based method of any one of clauses 1-67, wherein the one or more lyophilized reagents comprise lyophilized primers and probes.69. The cartridge or cartridge-based method of any one of clauses 1-68, wherein reagents and components in the reaction chambers are in solution.

Claims

CLAIMSWhat is claimed:

1. A cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising:(a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of the nucleic acid and detection of signals generated during amplification;(b) utilizing the filter to isolate the nucleic acid from the sample;(c) forming a library prep reaction mixture comprising the nucleic acid and a library prep reagent that facilitates: i) generation of tagged nucleic acid fragments from the nucleic acid in the library prep reaction mixture, ii) generation of amplicons, wherein the amplicons are optionally tagged at one or both ends, and / or iii) attachment of oligonucleotide adapters to one or both ends of the tagged nucleic acid fragments or amplicons to form adapter-labeled nucleic acid;(d) causing the library prep reaction mixture to flow into the reaction vessel and preparing the sequencing nucleic acid library comprising the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid;(e) isolating the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the library prep reaction mixture on to the fdter and eluting each from the filter; and(f) purifying and optionally size selecting the sequencing nucleic acid library on the filter, wherein a bubble bursting mechanism is utilized to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings of the reaction vessel.

2. The cartridge-based method of claim 1, wherein releasing the tagged nucleic acid fragments, amplicons, and / or adapter labeled nucleic acid from the filter during step (e) is during and / or after preparing the sequencing nucleic acid library.

3. The cartridge-based method of claims 1 -2, further comprising eluting the nucleic acid from the filter following step (b).

4. The cartridge-based method of claims 1-3, further comprising amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid within the reaction vessel following step (b).

5. A cartridge-based method for preparing a sequencing nucleic acid library from a sample, comprising:(a) placing a sample comprising nucleic acid in a self-contained cartridge having, in fluid communication, a plurality of chambers, a reaction vessel, and a filter disposed in a fluidic path between the plurality of chambers and the reaction vessel, wherein the reaction vessel is configured for amplification of nucleic acid and detection of signals generated during amplification;(b) utilizing the filter to isolate the nucleic acid from the sample;(c) fragmenting the nucleic acid to form nucleic acid fragments, wherein fragmenting is performed by mechanical fragmentation, chemical fragmentation, or enzymatic fragmentation; and / or amplifying the nucleic acid or nucleic acid fragments to form amplicons, wherein amplifying the nucleic acid or nucleic acid fragments is with at least one set of primers for amplifying a target region of the nucleic acid or nucleic acid fragments;(d) generating adapter-labeled nucleic acid comprising labeling the fragmented nucleic acid or amplicons at one or both ends the fragmented nucleic acid or amplicons; and(e) isolating the adapter labeled nucleic acid on to the fdter to form the NGS nucleic acid library, wherein the cartridge-based method comprises reuse of the reaction vessel, the filter, and optionally one or more of the plurality of chambers with rinsing to remove or dilute carryover, wherein reuse of the reaction vessel comprises a bubble bursting mechanism to reduce errant air bubbles forming in the reaction vessel caused by multiple fillings.

6. The cartridge-based method of claims 1 or 5, wherein if the sample in step (a) comprises intact cells, the method further comprises releasing nucleic acid from the intact cellsby lysing the intact cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers and / or capturing the cells on the filter and lysing the cells by means of sonication, to release nucleic acid from the cells.

7. The cartridge-based method of claims 5-6, further comprising amplifying the adapter-labeled nucleic acid with at least one set of primers for amplifying and increasing adapter-labeled nucleic acid concentration following step (d).

8. The cartridge-based method of claims 5-7, wherein step (e) further comprises purifying and size selecting the isolated adapter labeled nucleic acid to form the sequencing nucleic acid library.

9. The cartridge-based method of claim 8, wherein size selecting the isolated adapter labeled nucleic acid in step (e) comprises solid-phase reversible immobilization.

10. The cartridge-based method of any one of claims 5-9, further comprising isolating and washing the nucleic acid on the filter, and eluting the nucleic acid from the filter prior to step (c).

11. The cartridge-based method of any one of claims 1-10, further comprising using a reusable reagent for one or more steps in claims 1-10 including lysing the cells in the sample, isolating the nucleic acid released from the cell, washing the nucleic acid, eluting the nucleic acid, library prep reagent, fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter labeled nucleic acid, amplifying the adapter labeled nucleic acid, isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purifying and size selecting the isolated adapter labeled nucleic acid.

12. The cartridge-based method of claim 11, comprising using the reusable reagent for washing the nucleic acid, purifying and size selecting the isolated adapter labeled nucleic acid.

13. The cartridge-based method of claims 11 or 12, wherein the reusable reagent comprises a buffer, a salt, and polyethylene glycol (PEG).

14. The cartridge-based method of any one of claims 11-13, wherein using the reusable reagent comprises recycling the reusable reagent after one or more uses.

15. The cartridge-based method of any one of claims 11-13, wherein using the reusable reagent comprises:(i) drawing, from a first one of the plurality of chambers, a first portion of the reusable reagent and utilizing the first portion of reusable reagent, and(ii) drawing, from the first one of the plurality of chambers, a second portion of the reusable reagent and utilizing the second portion of reusable reagent.

16. The cartridge-based method of any one of claims 1-15, comprising reuse of at least one of the plurality of chambers, reuse of at least two of the plurality of chambers, reuse of at least three of the plurality of chambers, or reuse of at least four of the plurality of chambers.

17. The cartridge-based method of claim 16, wherein the at least one of the plurality of chambers is used to store a first reusable reagent prior to use, the at least two of the plurality of chambers is used to store a first and a second reusable reagent prior to use, the at least three of the plurality of chambers is used to store a first, a second, and a third reusable agent prior to use, or the at least four of the plurality of chambers is used to store a first, a second, a third, and fourth reusable agent prior to use.

18. The cartridge-based method of claim 17, wherein at least one of the first, second, third, and fourth reusable agents are different reusable agents.

19. The cartridge-based method of any one of claims 17-18, wherein the at least one of the plurality of chambers, the at least two of the plurality of chambers, the at least three of the plurality of chambers, or the at least four of the plurality of chambers for storing the reusablereagent is reused for collecting waste, fragmenting the nucleic acid, or generating the adapter- labeled nucleic acid.

20. The cartridge-based method of claim 19, wherein at least one of the plurality of chambers for storing the reusable reagent is reused for a different purpose than another of the plurality of chambers for storing reusable reagent.

21. The cartridge-based method of any one of claims 1-20, wherein the reaction vessel comprises one or more reaction chambers configured for amplification of nucleic acid and detection of signals generated during amplification.

22. The cartridge-based method of claim 21, wherein the reaction vessel comprises one reaction chamber, the reaction vessel comprises two reaction chambers, the reaction vessel comprises three reaction chambers, or the reaction vessel comprises no more than one reaction chamber configured for amplification of nucleic acid and detection of signals generated during amplification.

23. The cartridge-based method of any one of claims 1-22, wherein the self-contained cartridge consists of 15 or less chambers, 12 or less chambers, or 11 or less chambers, not including the reaction chamber(s).

24. The cartridge-based method of any one of claims 1-22, wherein each chamber of the plurality of chambers independently has a volume capacity of 10 mL or less, 9 mL or less, 8 mL or less, 7 mL or less, 6 mL or less, 5 mL or less, 4 mL or less, 3 mL or less, 2 mL or less, or 1 mL or less.

25. The cartridge-based method of any one of claims 1-22, wherein each reaction chamber independently has a volume capacity of 100 pL or less, 80 pL or less, 70 pL or less, or 60 pL or less.

26. The cartridge-based method of any one of claims 21-22, wherein fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter- labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid are sequentially performed in a single reaction chamber.

27. The cartridge-based method of any one of claims 21-22, wherein fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter- labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid are sequentially performed in more than one reaction chambers.

28. The cartridge-based method of any one of claims 21-27, wherein fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter- labeled nucleic acid, and / or amplifying the adapter-labeled nucleic acid comprises thermocycling, heating, and mixing.

29. The cartridge-based method of any one of claims 1-28, wherein the bubble bursting mechanism comprises:(i) heating the empty reaction vessel,(ii) partially fdling the reaction vessel with a first portion of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid,(iii) heating the first portion of reaction mixture in the reaction vessel,(iv) discarding the first portion of reaction mixture, and(v) filling the reaction vessel with a second portion of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter- labeled nucleic acid, or amplifying the adapter-labeled nucleic acid.

30. The cartridge-based method of claim 29, further comprising repeating steps (i)-(v) additional portions of reaction mixture for fragmenting the nucleic acid, amplifying the nucleic acid or nucleic acid fragments, generating the adapter-labeled nucleic acid, or amplifying the adapter-labeled nucleic acid.

31. The cartridge-based method of any one of claims 1-30, comprising multiple uses of the filter for capturing the cells in the sample, isolating the nucleic acid released from the cells, isolating the tagged nucleic acid fragments, isolating the amplicons, isolating the adapter labeled nucleic acid, and / or purifying and size selecting the isolated adapter labeled nucleic acid.

32. The cartridge-based method of any one of claims 1-31, wherein the filter comprises magnetic beads, and optionally the magnetic beads are bound and / or adsorbed to the filter.

33. The cartridge-based method of claim 32, wherein the magnetic beads are about 0.1 pm to about 10 pm, or about 0.5 pm to about 5 pm, or about 0.8 pm to about 3 pm.

34. The cartridge-based method of any one of claims 32-33, wherein the magnetic beads have a specific concentration based upon the size of the magnetic beads, the pore size and / or composition of the filter, and / or the length of the nucleic acid fragments.

35. The cartridge-based method of any one of claims 32-33, wherein the magnetic beads are optionally modified with a polymeric binder, or are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.

36. The cartridge-based method of any one of claims 5-35, comprising:(i) fragmenting the nucleic acid to form nucleic acid fragments in step (b) using enzymatic fragmentation by contacting the nucleic acid with a transposome solid support and / or free-floating transposome complexes, and generating tagged nucleic acid fragments by subjecting the nucleic acid and transposome solid support and / or free-floating transposome complexes to fragmentation conditions;(ii) forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and a) a set of primer pairs comprising adapters for amplifying the tagged nucleic acids fragments or b) ONT-specific adapters; and(iii) subjecting the adapter labeled reaction mixture to a) amplification conditions to amplify the tagged nucleic acid fragments or b) to click chemistry conditions to attach the ONT- specific adapters to the tagged nucleic acid.

37. The cartridge-based method of claim 36, wherein the generating tagged nucleic acid fragments happens in the reaction vessel in a first reaction vessel filling, and further comprises quenching generation of the tagged nucleic acid fragments in a second reaction vessel filling, isolating the tagged nucleic acid fragments on the filter, forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and a set of primer pairs comprising adapters for amplifying the tagged nucleic acids fragments; and subjecting the adapter labeled reaction mixture to amplification conditions to amplify the tagged nucleic acid fragments in a third reaction vessel filling.

38. The cartridge-based method of claim 37, wherein quenching generation of the tagged nucleic acid fragments comprises adding SDS to the reaction vessel in the second reaction vessel filling.

39. The cartridge-based method of claim 36, wherein the generating tagged nucleic acid fragments happens in the reaction vessel in a first reaction vessel filling, and further comprises isolating the tagged nucleic acid fragments on the filter, forming an adapter labeled reaction mixture comprising the tagged nucleic acid fragments and ONT-specific adapters; and subjecting the adapter labeled reaction mixture to click chemistry conditions to attach the ONT-specific adapters to the tagged nucleic acid.

40. The cartridge-based method of claim 39, wherein the tagged nucleic acid fragments comprise one or more click chemistry moi eties on a 3’ end of nucleic acid fragment.41 . The cartridge-based method of any one of claims 39-40, wherein the click chemistry conditions are at room temperature.

42. The cartridge-based method of any one of claims 1-35, comprising; amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid and a polymerase to form tagged amplicon; forming an adapter labeled reaction mixture comprising the tagged amplicons and ONT- specific adapters; and subjecting the adapter labeled reaction mixture to ligation conditions to attach the ONT- specific adapters to the tagged amplicons.

43. The cartridge-based method of claim 42, wherein the primers for amplifying the target region comprises 5 ’-phosphorylated primers and the polymerase lacks 3’-to-5’ exonuclease activity, resulting in a 5 ’-phosphorylated and 3’-A-tailed amplicon, and ligation comprises T4 ligase-mediated ligation of ONT-specific sequencing adapters.

44. The cartridge-based method of any one of claims 1-35, comprising: amplifying the nucleic acid with at least one set of unique molecular identifier (UMI) primers for amplifying a target region of the nucleic acid to form tagged amplicons in a first reaction vessel filling; amplifying the tagged amplicons via blocker displacement amplification to enrich for amplicons containing specific target region in a second reaction vessel filling; and amplifying the enriched tagged amplicons with at least one set of primers comprising adapters to form adapter labeled nucleic acid in a third reaction vessel filling.

45. The cartridge-based method of any one of claims 1-35, comprising: amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid to form amplicons in a first reaction vessel filling, amplifying the amplicons with at least one set of internal primers for amplifying a target region of the nucleic acid to form tiled amplicons in a second reaction vessel filling.

46. The cartridge-based method of any one of claims 1 -35, comprising: amplifying the nucleic acid with at least one set of primers for amplifying a target region of the nucleic acid to form amplicons in a first reaction vessel filling; and enzymatically ligating ONT-specific adapters to the amplicons in a second reaction vessel filling.

47. The cartridge-based method of any one of claim 1-46, further comprising eluting from the filter one or more of the nucleic acid released from the cells, nucleic acid fragments, tagged nucleic acid fragments, amplicons, tagged amplicons, and / or adapter labeled nucleic acid.

48. The cartridge-based method of any one of claim 1-47, wherein eluting from the filter comprises toggling the nucleic acid released from the cells, nucleic acid fragments, tagged nucleic acid fragments, amplicons, tagged amplicons, and / or adapter labeled nucleic acid on the filter with an eluant.

49. The cartridge-based method of claim 48, wherein toggling optionally comprises chemical, optical, electrical, mechanical, thermal, acoustical processing, or a combination thereof, and wherein toggling optionally comprises a change in speed, volume, viscosity, or a combination thereof, and wherein the toggling is important for optimization yields and / or reduction in sample degradation.

50. The cartridge-based method of any one of claim 1-49, wherein lysing the cells by sonication comprises exposing the cells to ultrasonic waves in a frequency range of approximately 5 kHz to approximately 120 kHz to lyse and release nucleic acid from the cells, or approximately 20 kHz to approximately 50 kHz to lyse and release nucleic acid from the cells.

51. A cartridge for preparing a sequence nucleic acid library from a sample, comprising: a cartridge body comprising a plurality of chambers in fluid communication therein;a filter disposed in a fluidic path between the plurality of chambers and a reaction vessel; the reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for amplification of nucleic acid and detection of a plurality of amplification products via real-time PCR, melt curve analysis, or a combination thereof, wherein the reaction vessel comprises: a planar frame defining a first fluidic path and a second fluidic path between a first planar substrate that encloses a first side of the planar frame and a second planar substrate that encloses a second side of the planar frame; a fluidic interface at one end of the planar frame, the fluidic interface comprising a first fluid inlet and a first fluid outlet of the first fluidic path, and a second fluid inlet and a second fluid outlet of the second fluidic path; a first reaction chamber arranged in the planar frame between the first and second substrates, the first reaction chamber being an enlarged portion of the first fluidic path disposed along the fluidic path between the first fluidic inlet and the first fluid outlet so as to allow a fluid sample introduced via the first fluid inlet to undergo amplification before exiting the first fluid outlet, wherein the second fluidic path further includes an optical chamber defined in the planar frame between the first and second substrate, the optical chamber including a planar optical-substrate adapted for detection of one or a plurality of amplification products adapted for detection of signals generated during amplification by an optical assembly, and wherein the optical chamber being in fluidic communication with the second fluidic inlet and the second fluidic outlet; a second reaction chamber arranged in the planar frame between the first and second substrates, the second reaction chamber being an enlarged portion of the second fluidic path disposed along the second fluidic path between the second fluidic inlet and the optical chamber so as to allow a fluid sample introduced via the second fluid inlet to undergo amplification before filling the optical chamber, wherein the second reaction chamber includes a chamber exit in fluidic communication with the optical chamber entrance that is in fluidic communication with the optical chamber, andwherein the first reaction chamber together with the first fluid inlet are accessed by one the plurality of chambers and the second reaction chamber together with the second fluid inlet are accessed by a different one of the plurality of chambers.

52. The cartridge of claim 51, wherein the cartridge is configured to carry out nonisothermal amplification, optionally by thermal cycling or temperature oscillation.

53. The cartridge of any one of claims 51-52, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and a lysis chamber in fluidic communication with the sample chamber, wherein the lysis chamber is adapted for performing mechanical and chemical lysis to release nucleic acid from the biological sample, optionally wherein the sample chamber and lysis chamber are the same.

54. The cartridge or cartridge-based method of any one of claims 1-53, wherein the one or more lysis reagents comprise a chaotropic agent, a chelating agent, a buffer, and a detergent.

55. The cartridge or cartridge-based method of any one of claims 1-54, wherein the chaotropic agent is selected from guanidinium thiocyanate, guanidinium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or a combination thereof.

56. The cartridge or cartridge-based method of any one of claims 1-55, wherein the one or more lysis reagents comprise a guanidinium compound, sodium hydroxide, EDTA, a buffer, and a detergent.

57. The cartridge or cartridge-based method any one of claims 1-56, wherein the fdter is configured to bind the nucleic acid to be analyzed.

58. The cartridge or cartridge-based method of any one of claims 1-57, wherein the filter comprises glass fibers and optionally a polymeric binder, or the glass fibers are optionallymodified with a DNA binding ligand such as an alkylamine, a cycloalkyl amine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.

59. The cartridge or cartridge-based method of any one of claims 1-58, wherein the filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron.

60. The cartridge or cartridge-based method of any one of claims 1-59, wherein the filter is configured to bind unwanted material and allow the nucleic acid to pass through.

61. The cartridge or cartridge-based method of any one of claims 1-60, wherein the cartridge further comprises a binding reagent, wash reagent, eluting reagent, or a combination thereof.

62. The cartridge or cartridge-based method of any one of claims 1-61, wherein the eluting reagent comprises ammonia or an alkali metal hydroxide.

63. The cartridge or cartridge-based method of any one of claims 1-62, wherein the eluting reagent comprises a polyanion, optionally selected from the group consisting of a carrageenan, a carrier nucleic acid, and i-carrageenan with KOH.

64. The cartridge or cartridge-based method of any one of claims 1-63, wherein at least one of the plurality of chambers comprises one or more lyophilized reagents.

65. The cartridge or cartridge-based method of any one of claims 1-64, wherein at least one of the first reaction chamber and the second reaction chamber comprises lyophilized reagents for amplification.

66. The cartridge or cartridge-based method of any one of claims 1-65, wherein the one or more lyophilized reagents is / are in the form of one or more beads.

67. The cartridge or cartridge-based method of any one of claims 1-66, wherein the one or more lyophilized reagents are selected from primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof.

68. The cartridge or cartridge-based method of any one of claims 1-67, wherein the one or more lyophilized reagents comprise lyophilized primers and probes.

69. The cartridge or cartridge-based method of any one of claims 1-68, wherein reagents and components in the reaction chambers are in solution.

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