Systems and methods for sequencing

Abstract

The sequencing system includes an automated sequencing instrument adapted to determine variant calls of one or more extracted polynucleotide samples using a targeted assay with one DNA pool per sample and an average amplicon size ranging from 100 to 120 bases, with a performance of at least 98.5% raw read accuracy and a runtime ranging from 5 hours to 14 hours to determine variant calls of four extracted polynucleotide samples.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 890,003, filed August 21, 2019, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to systems and methods for manipulating and analyzing nucleic acids. [Background technology]

[0003] Biological and medical research is increasingly turning to nucleic acid sequencing to enhance biological research and medicine. For example, biologists and zoologists are turning to sequencing to study animal migration, species evolution, and the origins of traits. The medical community is using sequencing to study the origins of disease, drug susceptibility, and the origins of infection. Thus, sequencing has broad applicability to many aspects of biology, therapeutics, diagnostics, forensics, and research.

[0004] Nevertheless, the use of sequencing can be limited by assay availability, sequencing run time, preparation time, and cost. Additionally, high-quality sequencing has historically been an expensive process, thus limiting its implementation.

[0005] Therefore, improved sequencing systems would be desirable.

[0006] The present disclosure may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings. [Brief explanation of the drawings]

[0007] [Figure 1] 1 includes an illustration of an exemplary sequencing system. [Figure 2]1 includes an illustration of an exemplary system including a sensor array. [Figure 3] 1 includes an illustration of an exemplary sensor and associated well. [Figure 4] 1 includes an illustration of an exemplary method for preparing a sequencing device. [Figure 5] 1 illustrates an exemplary schema for preparing bead assemblies. [Figure 6] 1 includes an illustration of an exemplary sequencing system. [Figure 7] 1 includes an illustration of an exemplary device. [Figure 8] 1 includes an illustration of an exemplary device deck. [Figure 9] 1 includes an illustration of a reagent storage compartment of an exemplary device. [Figure 10] 1 includes an illustration of an exemplary consumable item. [Figure 11] 1 includes an illustration of an exemplary cartridge system. [Figure 12] 1 includes an illustration of an exemplary cartridge system. [Figure 13] 1 includes an illustration of an exemplary docking station for receiving cartridges. [Figure 14] 1 includes an illustration of an exemplary sensor device. [Figure 15] 1 includes an illustration of an exemplary sensor device. [Figure 16] 1 includes an illustration of an exemplary fluid coupler. [Figure 17] 1 includes an illustration of an exemplary fluid coupler. [Figure 18] 1 includes an illustration of an exemplary fluid coupler. [Figure 19] 1 includes an illustration of an exemplary interconnection between a sensor device and a fluid coupler. [Figure 20] 1 includes an illustration of an exemplary interconnection between a sensor device and a fluid coupler. [Figure 21] 1 includes an illustration of an exemplary mechanical system for interacting with a sensor device. [Figure 22]1 includes an illustration of an exemplary mechanical system for interacting with a sensor device. [Figure 23] 1 includes an illustration of an exemplary slide mechanism for use in a mechanical system. [Figure 24] 1 includes an illustration of an exemplary slide mechanism for use in a mechanical system. [Figure 25] 1 includes an illustration of an exemplary mechanical assembly for providing a fluid connection between a fluid coupler and a sensor device. [Figure 26] 1 includes an illustration of an exemplary fluid manifold. [Figure 27] 1 includes an illustration of an exemplary fluid manifold. [Figure 28] 1 includes a block flow diagram of an exemplary method for interacting with a sensor device using a mechanical system. [Figure 29] 1 includes a schematic representation of an exemplary magnetic loading system. [Figure 30] 1A and 1B schematically illustrate the movement of a solution containing magnetic beads relative to a magnetic package at a first speed. [Figure 31] 10A and 10B schematically illustrate movement of a solution containing magnetic beads relative to a magnetic package at a second speed. [Figure 32] 10A and 10B illustrate schematic illustrations of the reverse movement of a solution containing magnetic beads relative to a magnetic package. [Figure 33] 1 illustrates a microchip loaded with beads. [Figure 34] 1 illustrates a schematic diagram of a magnetic loading model. [Figure 35] 1 includes an illustration of an exemplary loading device. [Figure 36] 1 includes an illustration of an exemplary loading device. [Figure 37] 1 includes an illustration of an exemplary loading device. [Figure 38] 1 includes an illustration of an exemplary loading device. [Figure 39] FIG. 1 is an exploded view generally illustrating a fluid multiplexer block of the present teachings. [Figure 40] 2 is an isometric view generally illustrating a fluid multiplexer unit of a fluid multiplexer block such as the fluid multiplexer block of FIG. 1. [Figure 41] 1 is a schematic representation generally illustrating fluidic integration between a fluidic multiplexer unit and selected lanes of a multi-lane sensor device of the present teachings. [Figure 42] 1 is a rear isometric view generally illustrating a fluid multiplexer block clamp assembly including a fluid multiplexer block clamp having a fluid multiplexer block assembly mounted thereto. FIG. [Figure 43] 10A-10C are cross-sectional views illustrating the general integration of electrodes into a fluidic multiplexer unit. [Figure 44] 1 is a front isometric view generally illustrating a fluid multiplexer block clamp assembly including a fluid multiplexer block clamp having a fluid multiplexer block assembly attached thereto. FIG. [Figure 45] 1 is a cross-sectional view generally illustrating an assembly of a fluid multiplexer block of a fluid multiplexer block clamp attached to a multi-lane sensor device positioned in a sensor device mounting assembly. [Figure 46] FIG. 1 is an enlarged isometric view generally illustrating the attachment of a fluidic multiplexer block to a multi-lane sensor array device. [Figure 47] 1 is a schematic representation generally illustrating a fluid system of a sequencing system of the present teachings. [Figure 48] 1 includes an illustration of an exemplary electronic interface. [Figure 49] 1 shows a schematic diagram of server system components. [Figure 50] FIG. 1 is a block diagram of an analysis pipeline. [Figure 51] FIG. 1 is a schematic diagram of generating an assay definition file. [Figure 52] FIG. 1 is a schematic diagram of an exemplary assay definition file package. [Figure 53]1 includes an illustration of a schema for seeding a support. [Figure 54] 1 includes an illustration of a schema for seeding a support. [Figure 55] 1 includes an illustration of a schema for seeding a support. [Figure 56] 1 includes an illustration of a schema for seeding a support. [Figure 57] 1 includes an illustration of a schema for seeding a support. [Figure 58] 1 includes an illustration of a schema for seeding a support. [Figure 59] 1 includes an illustration of a schema for seeding a support. [Figure 60] Included is a graph showing the total number of reads resulting from various amplification methods. [Figure 61A] Included are graphs illustrating parameters corresponding to template copy number. [Figure 61B] Included are graphs illustrating parameters corresponding to template copy number. [Figure 61C] Included are graphs illustrating parameters corresponding to template copy number. [Figure 61D] Included are graphs illustrating parameters corresponding to template copy number. [Figure 61E] Included are graphs illustrating parameters corresponding to template copy number. [Figure 61F] Included are graphs illustrating parameters corresponding to template copy number.

[0008] The use of the same reference symbols in different drawings indicates similar or identical items. DETAILED DESCRIPTION OF THE INVENTION

[0009] In one embodiment, the sequencing system comprises an automated sequencing device adapted to determine sequences and variant calls of polynucleotides from a set of sample polynucleotides, which utilizes a targeted assay to generate a library of sequenced amplicons or target polynucleotides and can provide an aligned sequence list and, optionally, variant calls, within a desirable timeframe.

[0010] An embodiment of an automated sequencing device includes a preparation deck for preparing a library of target polynucleotides. In an example, the target polynucleotides are seeded onto a substrate, such as polymer beads or hydrogel beads. The automated sequencing device can further include a loading device for applying the seeded substrate onto a sensor device, and can include, for example, a sequencer for performing a sequencing-by-synthesis reaction and detecting nucleotide incorporation. The automated sequencing device can further include a computing device for determining base calls, aligned reads, and variant calls using data from the sequencer. In addition, the system can include a user interface or network interface for communicating reports associated with the base calls, aligned reads, or variant calls to a user.

[0011] definition As used herein, the term "nucleic acid" and variations thereof are used interchangeably with the term "polynucleotide" and refer to a polymer of nucleotides, including, for example, deoxyribonucleic acid and ribonucleic acid. Nucleic acids include, but are not limited to, DNA, cDNA, RNA, chimeric RNA / DNA, and nucleic acid analogs.

[0012] As used herein, a primer is any single-stranded nucleic acid molecule (e.g., an oligonucleotide) that can prime or initiate nucleic acid synthesis upon hybridization to a complementary nucleic acid sequence. Typically, such nucleic acid synthesis occurs in a template-dependent manner, with nucleotides polymerized onto at least one end of the primer during such nucleic acid synthesis. Primers usually have a free 3' hydroxyl, but in some embodiments, the primer end is blocked (e.g., to prevent extension from the 3' end) or the primer is a fusion primer designed so that different portions of the primer bind to different partners. In reactions involving primer extension (e.g., preseeding amplification), the blocking portion at the 3' end of a blocked fusion primer can reduce the level of primer-dimer formation. In some embodiments, a blocked or unblocked primer is a tailed primer, whose 5' end contains a sequence that is non-complementary to the target nucleic acid to which the remainder of the primer is complementary. This 5' tail can be used as a template for primer extension. In various embodiments of the methods provided herein, the nucleic acid molecule comprises a first primer binding sequence and, optionally, a second primer binding sequence. In some embodiments, the reactions described herein comprise a population of first primers and, optionally, a population of second primers, each binding a forward primer binding sequence and a reverse primer binding sequence, or vice versa. In some embodiments, the first and second primers are referred to as a primer pair. In some embodiments, the first primer or the second primer is a universal primer. The first primer can bind to either the forward primer binding sequence or the reverse primer binding sequence, and the second primer can bind to either the forward primer binding sequence or the reverse primer binding sequence. Thus, the terms "first" and "second" when used herein with respect to primers are relative terms, and each can refer to a forward primer or a reverse primer depending on the context in which they are used.

[0013] As used herein, nucleic acid amplification refers to the process by which a new strand of nucleic acid is synthesized by nucleotide polymerization, involving one or more cycles of: separation of double-stranded nucleic acid into single strands (e.g., denaturation or dissociation), annealing of a primer to the single strand of the separated double-stranded nucleic acid (e.g., hybridization), and extension of the hybridized primer. As used herein, the term "primer extension" and its variants refer to any method for catalyzing nucleotide incorporation into the end of a nucleic acid molecule. In some embodiments, an amplification cycle includes (a) partial, incomplete, or complete denaturation or dissociation of a strand of double-stranded nucleic acid, (b) hybridization or annealing of a primer to the partially or completely single-stranded nucleic acid, and (c) primer extension to form an extended primer strand. In some embodiments, an amplification cycle optionally includes (a) hybridization of a first primer to a template nucleic acid strand, (b) primer extension to form a first extended nucleic acid strand, and (c) partial or incomplete denaturation of the extended strand from the template strand. Optionally, the denatured portion of the template strand from step (c) is free to hybridize with a different primer in the next amplification cycle. In some embodiments, primer extension in an amplification cycle involves displacement of one strand of the duplex nucleic acid from the other strand of the duplex, or displacement of the first extended strand from the template strand. A second primer can be included that hybridizes to the 3' end of the first extended strand.

[0014] Many methods of nucleic acid amplification are known in the art. Some examples include recombinase-polymerase amplification (RPA), template walking, and polymerase chain reaction (PCR) amplification. In an RPA reaction, a recombinase, a polymerase, and optionally a recombinase accessory protein are used to amplify a nucleic acid molecule in the presence of primers and nucleotides. The recombinase and optionally the recombinase accessory protein dissociate at least a portion of the double-stranded template nucleic acid molecule, allowing the primer to hybridize so that the polymerase can then bind and initiate replication. An example of a recombinase accessory protein is single-stranded binding protein (SSB), which prevents rehybridization of dissociated nucleic acid molecules. Typically, RPA reactions are isothermal and are performed at isothermal temperatures. In some cases, the RPA reaction can be performed in an emulsion. In a template walking reaction, a polymerase is used to amplify a template nucleic acid molecule in the presence of primers and nucleotides under reaction conditions that allow at least a portion of the double-stranded template nucleic acid molecule to dissociate so that the primers can hybridize and then the polymerase can bind and initiate replication. In PCR, the double-stranded template nucleic acid molecule is typically dissociated by thermal cycling. After cooling, the primers bind to complementary sequences and are available for replication by the polymerase. In some embodiments of the methods provided herein, the preseeding or templated reaction is carried out in a reaction mixture formed with components necessary for amplifying the template nucleic acid molecule. In any of the disclosed aspects, the reaction mixture includes some or all of the following: a population of template nucleic acid molecules, a polymerase, one or more supports or surfaces (e.g., solid supports) having a population of first primers attached, nucleotides, or cofactors such as divalent cations. In some embodiments, the reaction mixture further includes a second primer and, optionally, a diffusion-limiting agent. In some embodiments, the population of template nucleic acid molecules comprises template nucleic acid molecules joined to at least one adapter sequence that hybridizes to a first or second primer.In some embodiments, the reaction mixture forms an emulsion, such as in emulsion RPA or emulsion PCR. In reactions involving RPA, the reaction mixture includes a recombinase and, optionally, a recombinase accessory protein. The various components of the reaction mixture are discussed in further detail herein.

[0015] As used herein, the terms "identity" and "identical," and variations thereof, when used in reference to two or more nucleic acid sequences, refer to the sequence similarity of two or more sequences (e.g., nucleotide or polypeptide sequences). In the context of two or more homologous sequences, the percent identity or homology of a sequence or subsequence thereof refers to the percentage of all monomeric units (e.g., nucleotides or amino acids) that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity). The percent identity can occur in a specified region when compared and aligned for maximum correspondence within a comparison window or specified region. Sequences are said to be "substantially identical" if there is at least about 80%, or at least about 85%, identity at the amino acid level or nucleotide level. In some cases, sequences are "substantially identical" if there is at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity at the amino acid or nucleotide level. Preferably, the identity exists over a region that is at least about 20, 25, 50, or 100 residues in length, or over the entire length of at least one comparison sequence. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent hybridization conditions.

[0016] As used herein, the terms "complementary" and "complement," as well as variations thereof, refer to any two or more nucleic acid sequences (e.g., part or all of a template nucleic acid molecule, target sequence, or primer) that can undergo cumulative base pairing at two or more individual corresponding positions in an antiparallel orientation, as in a hybridized duplex. Such base pairing can proceed according to any set of established rules, for example, according to the Watson-Crick base pairing rules. Optionally, there can be "complete" or "total" complementarity between a first nucleic acid sequence and a second nucleic acid sequence, in which each nucleotide in the first nucleic acid sequence can undergo stabilized base pairing interactions with a nucleotide in a corresponding antiparallel position on the second nucleic acid sequence. "Partial" complementarity describes a nucleic acid sequence in which at least 20% but less than 100% of the residues in one nucleic acid sequence are complementary to the residues in another nucleic acid sequence. In some embodiments, at least 50% but less than 100% of the residues in one nucleic acid sequence are complementary to the residues in the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95%, or 98%, but less than 100%, of the residues of one nucleic acid sequence are complementary to the residues in another nucleic acid sequence. Sequences are said to be "substantially complementary" if at least 85% of the residues of one nucleic acid sequence are complementary to the residues in another nucleic acid sequence. In some embodiments, two complementary or substantially complementary sequences can hybridize to each other under standard or stringent hybridization conditions. "Non-complementary" refers to a nucleic acid sequence in which less than 20% of the residues of one nucleic acid sequence are complementary to the residues in another nucleic acid sequence. A sequence is said to be "substantially non-complementary" if less than 15% of the residues of one nucleic acid sequence are complementary to the residues in another nucleic acid sequence. In some embodiments, two non-complementary or substantially non-complementary sequences cannot hybridize to each other under standard or stringent hybridization conditions. A "mismatch" occurs at any position in a sequence where the two opposing nucleotides are not complementary. Complementary nucleotides include those that are efficiently incorporated by DNA polymerases opposite each other during DNA replication under physiological conditions.

[0017] As used herein, the term "monoclonal" and variations thereof, when used in reference to one or more polynucleotide populations, refers to a population of polynucleotides in which approximately 50-99%, or up to 100%, or 100% of the members of the population share at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least 99% identity, or about 100% identity, or 100% identity at the nucleotide sequence level. As used herein, the phrase "substantially monoclonal" and variations thereof, when used in reference to one or more polynucleotide populations, refers to one or more polynucleotide populations in which one polynucleotide molecule, e.g., an amplified template polynucleotide molecule, is the single most predominant polynucleotide in the population. Thus, all members of a monoclonal or substantially monoclonal population need not be completely identical or complementary to one another. For example, different portions of a polynucleotide template can be amplified or replicated to produce members of the resulting monoclonal population; similarly, a certain number of "errors" or incomplete extensions may occur during amplification of the original template, thereby generating a monoclonal or substantially monoclonal population in which individual members may exhibit sequence variability among themselves. In some embodiments, low or minute levels of heterologous polynucleotides may occur during nucleic acid amplification reactions; thus, a substantially monoclonal population may contain a small number of one or more polynucleotides (e.g., less than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, 0.1%, or 0.001% of the diverse polynucleotides). In certain examples, at least 90% of the polynucleotides in the population are at least 90% identical to the original single template used as the basis for amplification to produce the substantially monoclonal population.In certain embodiments, amplification of the template polynucleotide produces a population of polynucleotides, wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the members of the population of polynucleotides share at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the template nucleic acid from which the population was generated. In certain embodiments, a sufficiently large fraction of the polynucleotides produce a population of polynucleotides that share sufficient sequence identity to allow sequencing of at least a portion of the amplified templates using a high-throughput sequencing system.

[0018] In some embodiments, at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% of the members of the nucleic acid molecules attached to the templated support will share greater than 90%, 95%, 97%, 99%, or 100% identity with the template nucleic acid molecule. In some embodiments, members of the nucleic acid population produced using any of the amplification methods hybridize to each other under high stringency hybridization conditions.

[0019] In some embodiments, the amplification methods provided herein, including, for example, the amplification processes used for nucleic acid templating, as well as the instruments, devices, systems, compositions, and kits associated with these methods, generate substantially monoclonal populations of nucleic acid molecules that contain sufficiently low levels of polyclonal contaminants to allow successful sequencing in high-throughput sequencing methods. For example, the amplification methods can generate substantially monoclonal populations of nucleic acid molecules that provide for the production of a signal (e.g., a sequencing signal, a nucleotide incorporation signal, etc.) that can be detected using a particular sequencing system. Such signals include any detectable signal indicative of nucleotide polymerization, including, but not limited to, optical or optically detectable signals, non-optical signals (or signals detectable by non-optical detection techniques), changes in ion (e.g., hydrogen ion) concentration, pH, electrical signals, voltage, and any such signal fluctuations. Optionally, the signal can then be analyzed to accurately determine the sequence or identity of any one or more nucleotides or bases present in any nucleic acid molecule of the population. Examples of sequencing systems suitable for detecting or analyzing such signals include, but are not limited to, systems that include an ion sensor, such as a field effect transistor (FET), e.g., a chemFET or ISFET. A "chemFET" or chemical field effect transistor is a type of field effect transistor that acts as a chemical sensor. A chemFET has a structural analogue of a MOSFET transistor, in that the charge on the gate electrode is applied by a chemical process. An "ISFET" or ion-sensitive field effect transistor is used to measure the concentration of ions in a solution, and can measure the ion concentration (H +A change in the capacitance (e.g., the capacitance of the transistor) causes a corresponding change in the current through the transistor. Non-limiting examples of systems that include FET sensors include Ion Torrent sequencing systems, such as the Ion Torrent PGM™ sequencing systems, including the 314, 316, and 318 systems, the Ion Torrent Proton™ sequencing systems, including the Proton I (Thermo Fisher Scientific, Waltham, MA), and the Ion Torrent Proton™ sequencing systems, including the Ion S5 and S5XL (Thermo Fisher Scientific, Waltham, MA). In one embodiment, an ISFET-based sequencing system for signal detection or analysis is described in detail herein. In some embodiments, a substantially monoclonal nucleic acid population allows for accurate sequencing of at least five contiguous nucleotide residues on a system incorporating an FET sensor, such as an Ion Torrent sequencing system.

[0020] As used herein, the term "clonal amplification" and variations thereof refer to any process by which a monoclonal or substantially monoclonal population of polynucleotides is produced through amplification of polynucleotides. In some embodiments of clonal amplification, two or more polynucleotides are amplified to produce at least two monoclonal or substantially monoclonal populations of polynucleotides.

[0021] As used herein, the term "preseeding," also referred to herein as "seeding," refers to a process involving the attachment of polynucleotides to a surface or support. In some embodiments, preseeding involves the attachment of one or more nucleic acids to a surface or support or to one or more sites on a surface or support. The preseeded surface or support is used, for example, for further manipulation or analysis of the attached nucleic acids, such as nucleic acid amplification (including, for example, amplification in a templated process), sequencing, or other processes. In some embodiments, the preseeding process generates one or more surfaces or supports having one or more nucleic acid molecules attached. In some embodiments, preseeding generates one or more surfaces or supports having a single polynucleotide attached. The one or more surfaces or supports having one or more nucleic acid molecules attached can be included in a population, a plurality, or a set of two or more surfaces or supports, where some, a few, a majority, or substantially all of the surfaces or supports have one or more nucleic acid molecules attached. In some embodiments, the one or more nucleic acid molecules attached to different surfaces or supports or at different sites on the surfaces or supports are different. In some embodiments, in the preseeding process, a large number (or a plurality) of substantially identical copies of a nucleic acid molecule (or substantially monoclonal nucleic acid) are attached to a surface or support, or a large number (or a plurality) of different nucleic acids are attached to one or more sites on a surface or support. In some embodiments, a limited number of substantially identical copies of a polynucleotide (or substantially monoclonal nucleic acid) are attached to a surface or support in the preseeding process to generate a population of monoclonal or substantially monoclonal nucleic acids. In some embodiments, preseeding of a surface or support is a process that does not involve nucleic acid amplification, and involves attachment of nucleic acids to the surface or support, for example, by hybridization of the nucleic acid to a complementary polynucleotide attached to the support. In some embodiments, preseeding of a surface or support involves nucleic acid amplification, e.g., one or more cycles of nucleic acid amplification (e.g., PCR) or isothermal amplification.For example, nucleic acid amplification may be used in a preseeding process to generate one or more copies of a nucleic acid that can be attached to a surface or support (e.g., by hybridization). Typically, preseeding involves nucleic acid amplification to generate a surface or support having two or more nucleic acids, or multiple copies of a nucleic acid attached thereto. Surfaces or supports generated by a preseeding or seeding process as provided herein are referred to as "preseeded" or "seeded" supports.

[0022] As used herein, "limited number" when referring to the number of nucleic acids (or substantially identical copies or substantially monoclonal nucleic acids) attached to a surface or support in a preseeding or template method typically refers to the number of nucleic acids that is controlled for various purposes. The limited number of copies of nucleic acids can be sufficient to provide a crowding effect in subsequent larger-scale amplification (e.g., templated) of nucleic acids on a surface or support to generate a larger, substantially monoclonal population of nucleic acids, for example, by preventing or reducing template transfer between reaction sites, thereby preventing or reducing the formation of a polyclonal population. Such a limited number of template copies can be limited, for example, by using a relatively short nucleic acid amplification time to prevent or reduce template transfer between reaction sites, but to generate a sufficient number of template copies to provide a crowding effect in subsequent amplification.

[0023] As used herein, the term "templated" refers to the process of generating two or more, or a plurality, or a population of substantially identical polynucleotides, or the process of generating a substantially monoclonal population of nucleic acids that can be used as templates in nucleic acid analysis methods, including nucleic acid sequencing, such as sequencing by synthesis of polynucleotides. The polynucleotides generated in the templated process are typically referred to as nucleic acid templates. In some embodiments, templated involves attaching a polynucleotide template to a surface or support. In some embodiments, templated involves generating two or more, or a plurality, of distinct surfaces or supports, or discrete sites on a surface or support, each having attached thereto two or more, or a plurality, or a population of substantially identical polynucleotides, or a substantially monoclonal population of polynucleotides. In some embodiments, templated involves generating one or more surfaces or supports, or discrete sites on a surface or support, to which a substantially monoclonal population of polynucleotides is attached. In some embodiments, templating is performed using at least 50,000, 75,000, 100,000, 125,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 10 6In some embodiments, templating produces one or more surfaces or supports having a substantially monoclonal population of about 50,000 to 500,000 template nucleic acid molecules attached to each templated surface or support, or, for example, produces surfaces or supports having about 50,000 to 400,000 template nucleic acid molecules, about 50,000 to 300,000 template nucleic acid molecules, about 50,000 to 200,000 template nucleic acid molecules, or about 50,000 to 100,000 template nucleic acid molecules attached to each templated support. In some embodiments, templating generates one or more templated surfaces or supports having a substantially monoclonal population of about 100,000 to 400,000 template nucleic acid molecules attached to each templated surface or support, or, for example, about 100,000 to 300,000 template nucleic acid molecules, about 100,000 to 200,000 template nucleic acid molecules, or about 150,000 to 300,000 template nucleic acid molecules attached to each templated support. In some embodiments, templating is performed starting from one or more preseeded or seeded surfaces or supports. In such embodiments, the templates are at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1000-fold, at least 2500-fold, at least 5000-fold, at least 10,000-fold, at least 25,000-fold, at least 50,000-fold, at least 100,000-fold, at least 250,000-fold, at least 5,000,000-fold, or at least 10 6One or more templated surfaces or supports can be generated, including a plurality of pre-seeded nucleic acid molecules, each of which can be 1-fold or more. In some embodiments, only about one or only one nucleic acid molecule is present on the pre-seeded support. In some embodiments, at least 50,000, 75,000, or 100,000 substantially monoclonal template nucleic acid molecules, or about 25,000 to 1,000,000 substantially monoclonal template nucleic acid molecules, are present on the pre-seeded surface or support, e.g., a solid surface or support, for example, about 25,000 to 500,000, about 25,000 to 250,000, about 25,000 to 125,000, or about 25,000 to 100,000 substantially monoclonal template nucleic acid molecules.

[0024] In some embodiments, the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, include a support, e.g., a solid or semi-solid support, for entrapment, concentration, sequestration, isolation, localization, amplification, or transfer of nucleic acids that can be used in analytical methods. A solid surface or support may comprise a polymeric, glass, or metallic material. Examples of solid supports include membranes, planar surfaces, microtiter plates, beads, filters, test strips, slides, coverslips, and test tubes. A solid surface or support refers to any solid phase material onto which oligomers are synthesized, attached, ligated, or otherwise immobilized. Supports can optionally include "resins," "phases," "surfaces," and "supports." Supports may be composed of organic polymers, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Supports may also be inorganic, such as glass, silica, controlled pore glass (CPG), or reverse-phase silica. The support configuration may be, for example, in the form of beads, spheres, particles, granules, gels, or surfaces. The surface may be, for example, planar, substantially planar, or non-planar, as well as concave, convex, or any combination thereof. The support may be porous, semi-porous, or non-porous, and may have swelling or non-swelling properties. The support may be shaped to include one or more wells, depressions, or other vessels, receptacles, features, or locations. One or more supports may be configured in an array at various locations. The support is optionally addressable (e.g., for robotic delivery of reagents) or by detection means including scanning with laser illumination and confocal or polarized light collection. The support (e.g., beads) may be disposed within or on another support (e.g., within the wells of a second support). Examples of bead materials include, but are not limited to, gels, hydrogels, or acrylamide polymers.In some embodiments, the support is an ionosphere particle (Thermo Fisher Scientific, Waltham, MA). Examples of solid supports include "microparticles," "beads," "microbeads" (although optionally not necessarily spherical in shape), spheres, filters, flow cells, wells, grooves, channel reservoirs, gels, or the inner walls of capillaries. In some embodiments, the support comprises texture (e.g., etchings, hollowing, pores, three-dimensional scaffolds, or ridges). Support sizes include, but are not limited to, supports having a minimum cross-sectional length (e.g., diameter) of 50 microns or less, 10 microns or less, 3 microns or less, approximately 1 micron or less, approximately 0.5 microns or less, e.g., approximately 0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers). Surfaces or solid supports also include magnetic or paramagnetic beads (e.g., magnetic or paramagnetic nanoparticles or microparticles). For example, paramagnetic microparticles include paramagnetic beads with streptavidin attached (e.g., Dynabeads™ M-270 from Invitrogen, Carlsbad, CA). Particles can have an iron core or can be hydrogel or agarose (e.g., Sepharose™). Microparticles (e.g., Dynabeads from Dynal, Oslo, Norway) can be made from a variety of inorganic or organic materials, including, but not limited to, glass (e.g., controlled pore glass), silica, zirconia, cross-linked polystyrene, polyacrylate, polymethylmethacrylate, titanium dioxide, latex, polystyrene, etc. Magnetization can facilitate collection and concentration of microparticle-attached reagents (e.g., polynucleotides and ligase) after amplification and can also facilitate additional steps (e.g., washing, reagent removal, etc.). The bead surface can be functionalized to attach one or more, or a plurality, or population of primers. In some embodiments, the beads are of any size that can fit into the reaction chamber.For example, one bead can fit into the reaction chamber. In some embodiments, two or more beads fit into the reaction chamber. In some embodiments, the methods provided herein, as well as the instruments, devices, systems, compositions, and kits for carrying out these methods, include a support or surface to which one, two or more, a plurality, or a population of oligonucleotides (e.g., primers) are attached. The support or surface can be coated with an acrylamide compound, a carboxylic acid compound, or an amine compound to attach nucleic acid molecules (e.g., a first primer or a second primer). For example, an amino-modified nucleic acid molecule (e.g., a primer) can be attached to a carboxylic acid-coated support. A primer can be attached to the acrylamide compound coating on the surface. The particle can be coated with an avidin-like compound (e.g., streptavidin) to bind biotinylated nucleic acids. In some embodiments, the oligonucleotides attached to the support or surface are substantially identical or contain primer sequences that are substantially identical in all oligonucleotides. In some embodiments, two or more different oligonucleotides are attached to the support or surface. In some embodiments, a surface has a population of first primers attached thereto, the first primers of the population sharing a common first primer sequence. In some embodiments, a surface has a population of first primers and a population of second primers attached thereto, the first primers of the population sharing a common first primer sequence and the second primers of the population of second primers sharing a common second primer sequence. In some embodiments, the population of first primers is immobilized on the surface. In other embodiments, the population of first primers and the population of second primers are immobilized on the surface.

[0025] overview 1 , a system 100 including a fluidics circuit 102 is connected by inlets to at least two reagent reservoirs (104, 106, 108, 110, or 112), a waste reservoir 120, and a biosensor 134 by a fluid pathway 132 that connects a fluidics node 130 to an inlet 138 of the biosensor 134 for fluid communication. Reagents from the reservoirs (104, 106, 108, 110, or 112) can be driven into the fluidics circuit 102 by a variety of methods, including pressure, a pump such as a syringe pump, gravity feed, etc., and are selected by control of a valve 114. Reagents from the fluidics circuit 102 can be driven by the valve 114 receiving a signal from a control system 118 to a waste container 120. Reagents from the fluidics circuit 102 can also be driven by the biosensor 134 to a waste container 136. The control system 118 includes a controller for the valve 114 that generates signals to open or close via an electrical connection 116 .

[0026] The control system 118 also includes controllers for other components of the system, such as a wash solution valve 124 and a reference electrode 128, which are connected to the control system 118 by electrical connection 122. The control system 118 also includes control and data acquisition functions for the biosensor 134. In one mode of operation, the fluidics circuit 102 sequentially delivers selected reagents 1, 2, 3, 4, or 5 to the biosensor 134 under the programmed control of the control system 118, such that the fluidics circuit 102 is primed and washed, and the biosensor 134 is cleaned during the selected reagent flow. Fluid entering the biosensor 134 exits through an outlet 140 and collects in a waste container 136 via control of a pinch valve regulator 144. The valve 144 is in fluid communication with the sensor fluid output 140 of the biosensor 134.

[0027] Devices including a dielectric layer defining wells formed from first and second accesses and exposing sensor pads find particular use in detecting chemical reactions and by-products, such as detecting the release of hydrogen ions in response to nucleotide incorporation, useful in genetic sequencing, among other applications. In certain embodiments, a sequencing system includes a flow cell having a sensory array disposed thereon, communication circuitry in electronic communication with the sensory array, and a reservoir and fluid control device in fluid communication with the flow cell. In an example, FIG. 2 shows an enlarged cross-sectional view of a flow cell 200, illustrating a portion of a flow chamber 206. A reagent flow 208 flows across the surface of the well array 202, and the reagent flow 208 flows over the open ends of the wells of the well array 202. The well array 202 and the sensor array 205 may together form an integrated unit that forms the lower wall (or floor) of the flow cell 200. A reference electrode 204 may be fluidly coupled to the flow chamber 206. Additionally, the flow cell cover 230 encapsulates the flow chamber 206 to contain the reagent flow 208 within a limited area.

[0028] FIG. 3 illustrates a close-up view of well 301 and sensor 314, as illustrated at 210 in FIG. 2. The well's volume, shape, aspect ratio (such as the ratio of base width to well depth), and other dimensional characteristics may be selected based on the nature of the reaction to be performed, as well as the reagents, byproducts, or labeling techniques, if any, used. Sensor 314 may be a chemical field-effect transistor (chemFET), more specifically, an ion-sensitive FET (ISFET) having a floating gate 318 with a sensor plate 320, optionally separated from the well interior by a material layer 316. Sensor 314 may respond to (and generate an output signal related to) the amount of charge 324 present on material layer 316 opposite sensor plate 320. Material layer 316 may be a ceramic layer such as an oxide of zirconium, hafnium, tantalum, aluminum, or titanium, or a nitride of titanium, among others. Alternatively, material layer 316 may be formed of a metal such as titanium, tungsten, gold, silver, platinum, aluminum, copper, or a combination thereof. In examples, material layer 316 may have a thickness in the range of 5 nm to 100 nm, such as in the range of 10 nm to 70 nm, in the range of 15 nm to 65 nm, or even in the range of 20 nm to 50 nm.

[0029] While material layer 316 is illustrated as extending beyond the boundaries of the illustrated FET components, material layer 316 can extend along the bottom of well 301 and, optionally, along the walls of well 301. Sensor 314 can respond to (and generate an output signal related to) the amount of charge 324 present on material layer 316 opposite sensor plate 320. Changes in charge 324 can cause a change in current between the source 321 and drain 322 of the chemFET. The chemFET can then be used directly to provide a current-based output signal, or indirectly using additional circuitry to provide a voltage-based output signal. Reactants, wash solutions, and other reagents can enter or exit the well by diffusion mechanism 340.

[0030] The well 301 can be defined by a wall structure, which can be formed from one or more layers of material. In examples, the wall structure can have a thickness extending from the lower surface to the upper surface of the well in the range of 0.01 micrometers to 10 micrometers, such as in the range of 0.05 micrometers to 10 micrometers, the range of 0.1 micrometers to 10 micrometers, the range of 0.3 micrometers to 10 micrometers, or the range of 0.5 micrometers to 6 micrometers. In particular, the thickness can be in the range of 0.01 micrometers to 1 micrometer, such as in the range of 0.05 micrometers to 0.5 micrometers or the range of 0.05 micrometers to 0.3 micrometers. The wells 301 of the array 202 can have a characteristic diameter defined as four times the square root of the cross-sectional area (A) divided by pi (e.g., sqrt(4*A / π)) that is 5 micrometers or less, such as 3.5 micrometers or less, 2.0 micrometers or less, 1.6 micrometers or less, 1.0 micrometers or less, 0.8 micrometers or less, or even 0.6 micrometers or less. In an example, the wells 301 can have a characteristic diameter of at least 0.01 micrometers. In a further example, the wells 301 can define a volume in the range of 0.05 fL to 10 pL, such as in the range of 0.05 fL to 1 pL, in the range of 0.05 fL to 100 fL, in the range of 0.05 fL to 10 fL, or even in the range of 0.1 fL to 5 fL.

[0031] In one embodiment, the reaction performed in well 301 can be an analytical reaction to identify or determine a feature or characteristic of an analyte of interest. Such a reaction can directly or indirectly produce by-products that affect the amount of charge adjacent to sensor plate 320. If such by-products are produced in small amounts, decay rapidly, or react with other components, multiple copies of the same analyte can be analyzed simultaneously in well 301 to increase the output signal generated. In one embodiment, multiple copies of the analyte can be attached to a solid support 312 either before or after deposition in well 301. The solid support 312 can be solid or porous, including microparticles, nanoparticles, beads, gels, etc. For simplicity and ease of description, the solid support 312 is also referred to herein as a particle or bead. For nucleic acid analytes, multiple linked copies can be created by rolling circle amplification (RCA), exponential RCA, or similar techniques to produce amplicons without the need for a solid support.

[0032] In particular, a solid support, such as a bead support, can contain copies of a polynucleotide. In a specific example illustrated in Figure 4, polymer particles can be used as a support for a polynucleotide during a sequencing procedure. For example, such hydrophilic particles can immobilize a polynucleotide for sequencing using a fluorescent sequencing procedure. In another example, hydrophilic particles can immobilize multiple copies of a polynucleotide for sequencing using an ion-sensing procedure. Alternatively, the above-mentioned treatment can improve the binding of the polymer matrix to the surface of the sensor array. The polymer matrix can capture an analyte, such as a polynucleotide, for sequencing.

[0033] Bead supports may be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Supports may also be inorganic, such as glass, silica, controlled pore glass (CPG), or reverse-phase silica. Support configurations may be in the form of beads, spheres, particles, granules, gels, or surfaces. Supports may be porous or non-porous and may have swelling or non-swelling properties. In some embodiments, the support is an ionic sphere particle. Examples of bead supports are disclosed in U.S. Pat. No. 9,243,085, entitled "Hydrophilic Polymeric Particles and Methods for Making and Using Same," and U.S. Pat. No. 9,868,826, entitled "Polymer Substrates Formed from Carboxy Functional Acrylamide," each of which is incorporated herein by reference.

[0034] In some embodiments, the solid support is a "microparticle," "bead," "microbead," or the like (optionally, but not necessarily, spherical) having a minimum cross-sectional dimension (e.g., diameter) of 50 microns or less, preferably 10 microns or less, 3 microns or less, approximately 1 micron or less, approximately 0.5 microns or less, e.g., approximately 0.1, 0.2, 0.3, or 0.4 microns or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers). In examples, the support is at least 0.1 micron. Microparticle (or bead support) can be made of a variety of inorganic or organic materials, including, but not limited to, glass (e.g., controlled pore glass), silica, zirconia, cross-linked polystyrene, polyacrylate, polymethyl methacrylate, titanium dioxide, latex, polystyrene, and the like. Magnetization can facilitate collection and concentration of microparticle-attached reagents (e.g., polynucleotides or ligase) after amplification and can also facilitate additional steps (e.g., washing, reagent removal, etc.). In certain embodiments, populations of microparticles with different shapes, sizes, or colors are used. The microparticles can optionally be encoded, for example, with quantum dots, such that each microparticle or group of microparticles can be individually or uniquely identified.

[0035] Magnetic beads (e.g., Dynabeads from Dynal, Oslo, Norway) can have a size ranging from 1 micron to 100 microns, such as 2 microns to 100 microns. Magnetic beads can be formed from inorganic or organic materials, including, but not limited to, glass (e.g., controlled pore glass), silica, zirconia, cross-linked polystyrene, polystyrene, or combinations thereof.

[0036] In some embodiments, the bead support is functionalized to attach a population of first primers. In some embodiments, the beads are of any size that can fit into the reaction chamber. For example, one bead can fit into the reaction chamber. In some embodiments, two or more beads fit into the reaction chamber. In some embodiments, the minimum cross-sectional length (e.g., diameter) of the beads is about 50 microns or less, or about 10 microns or less, or about 3 microns or less, about 1 micron or less, about 0.5 microns or less, e.g., about 0.1, 0.2, 0.3, or 0.4 microns, or even smaller (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers).

[0037] Generally, polymer particles can be engineered to contain biomolecules, including nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, sugars, polysaccharides, lipids, or derivatives or analogs thereof. For example, polymer particles can be bound or attached to biomolecules. The termini or any internal portion of the biomolecule can be bound or attached to the polymer particle. The polymer particles can be bound or attached to biomolecules using linking chemistries. Linking chemistries include covalent or non-covalent bonds, including ionic bonds, hydrogen bonds, affinity bonds, dipole-dipole bonds, van der Waals bonds, and hydrophobic bonds. Linking chemistries include affinities between binding partners, e.g., an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunologically reactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigen antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement.

[0038] 4, a plurality of bead supports 404 can be placed in solution with a plurality of polynucleotides 402 (target or template polynucleotides). The plurality of bead supports 404 can be activated or otherwise prepared to bind to the polynucleotides 402. For example, the particles 404 can include oligonucleotides (capture primers) complementary to a portion of a polynucleotide in the plurality of polynucleotides 402. In another example, the bead supports 404 can be modified with the target polynucleotides 402 using techniques such as biotin-streptavidin binding.

[0039] In some embodiments, the template nucleic acid molecule (template polynucleotide or target polynucleotide) can be derived from a sample, which can be derived from natural or non-natural sources. The nucleic acid molecules in the sample can be derived from an organism or cell. Any nucleic acid molecule can be used; for example, the sample can include genomic DNA covering part or all of the genome, mRNA, or miRNA from an organism or cell. In other embodiments, the template nucleic acid molecule can be synthetic or recombinant. In some embodiments, the sample contains nucleic acid molecules with substantially identical sequences or a mixture of different sequences. Exemplary embodiments are typically performed using nucleic acid molecules within and produced by living cells. Such nucleic acid molecules are typically isolated directly from natural sources, such as cells or body fluids, without in vitro amplification. Thus, the sample nucleic acid molecules are used directly in subsequent steps. In some embodiments, the nucleic acid molecules in the sample can include two or more nucleic acid molecules with different sequences.

[0040] This method can optionally include a target enrichment step before, during, or after library preparation and before the pre-seeding reaction. Target nucleic acid molecules containing target loci or regions of interest can be enriched, for example, by multiplex nucleic acid amplification or hybridization. Multiplex nucleic acid amplification, such as multiplex PCR, can be performed to generate amplicons using a variety of methods, and a variety of methods can be used in one embodiment. Enrichment by any method can be followed by a universal amplification reaction before adding template nucleic acid molecules to the pre-seeding reaction mixture. Any of the embodiments of the present teachings can include enriching for a plurality of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 target nucleic acid molecules, target loci, or regions of interest. In any of the disclosed embodiments, the target locus or region of interest can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length and can comprise a portion or the entire template nucleic acid molecule. In other embodiments, the target locus or region of interest can be about 1 to 10,000 nucleotides in length, e.g., about 2 to 5,000 nucleotides, about 2 to 3,000 nucleotides, or about 2 to 2,000 nucleotides in length.In any of the embodiments of the present teachings, multiplex nucleic acid amplification can include generating at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 copies of each target nucleic acid molecule, target locus, or region of interest.

[0041] In some embodiments, after library preparation and optional enrichment steps, the library of template nucleic acid molecules can be templated onto one or more supports. One or more supports can be templated in two reactions: a seeding reaction to generate pre-seeded solid supports, and a templated reaction using one or more pre-seeded supports to further amplify the attached template nucleic acid molecules. The pre-seeding reaction is typically an amplification reaction and can be carried out using a variety of methods. For example, the pre-seeding reaction can be carried out by an RPA reaction, a template walking reaction, or PCR. In an RPA reaction, a template nucleic acid molecule is amplified in the presence of primers and nucleotides using a recombinase, a polymerase, and optionally a recombinase accessory protein. The recombinase and optionally a recombinase accessory protein dissociate at least a portion of the double-stranded template nucleic acid molecule, allowing the primer to hybridize so that the polymerase can bind and initiate replication. In some embodiments, the recombinase accessory protein can be a single-stranded binding protein (SSB), which prevents rehybridization of dissociated template nucleic acid molecules. Typically, RPA reactions can be performed at isothermal temperatures. In a template walking reaction, a polymerase is used to amplify a template nucleic acid molecule in the presence of primers and nucleotides under reaction conditions that allow at least a portion of the double-stranded template nucleic acid molecule to dissociate so that the primers can hybridize and then the polymerase can bind and initiate replication. In PCR, the double-stranded template nucleic acid molecule is dissociated by thermal cycling. After cooling, the primers bind to complementary sequences and are available for replication by the polymerase. In any of the aspects of the present teachings, the preseeding reaction can be performed in a preseeding reaction mixture formed with components necessary for amplifying the template nucleic acid molecule. In any of the disclosed aspects, the preseeding reaction mixture includes some or all of the following: a population of template nucleic acid molecules, a polymerase, one or more solid supports having a population of attached first primers, nucleotides, and cofactors such as divalent cations.In some embodiments, the preseeding reaction mixture can further include a second primer and, optionally, a diffusion limiting agent. In some embodiments, the population of template nucleic acid molecules includes template nucleic acid molecules joined to at least one adapter sequence capable of hybridizing to the first or second primer. In some embodiments, the reaction mixture can form an emulsion, as in emulsion RPA or emulsion PCR. In preseeding reactions performed by RPA reactions, the preseeding reaction mixture can include a recombinase and, optionally, a recombinase accessory protein. The various components of the reaction mixture are discussed in further detail herein.

[0042] In certain seeding embodiments, the hydrophilic particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). In an example, particles 404 include a capture primer complementary to a portion of a template polynucleotide 402. The template polynucleotide can hybridize to the capture primer. The capture primer can be extended to form beads 406 that include an attached target polynucleotide. Other beads can remain unattached to a target nucleic acid, and other template polynucleotides can float freely in solution.

[0043] In an example, a bead support 406 containing a target polynucleotide can be attached to a magnetic bead 410 to form a bead assembly 412. In particular, the magnetic bead 410 is attached to the bead support 406 by a double-stranded polynucleotide bond. In an example, an additional probe containing a linker moiety can hybridize to a portion of the target polynucleotide on the bead support 406. The linker moiety can be attached to a complementary linker moiety on the magnetic bead 410. In another example, a template polynucleotide used to form the target nucleic acid attached to the bead 406 can include a linker moiety attached to the magnetic bead 410. In another example, a template polynucleotide complementary to the target polynucleotide attached to the bead support 406 can be generated from a linker-modified primer attached to the magnetic bead 410.

[0044] The linker moiety attached to the polynucleotide and the linker moiety attached to the magnetic bead can be complementary to each other and can be attached to each other. In examples, the linker moieties have affinity and can include an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunologically reactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigen antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement. In a particular example, the linker moiety attached to the polynucleotide includes biotin, and the linker moiety attached to the magnetic bead includes streptavidin.

[0045] The bead assembly 412 can be deposited over a substrate 416 of a sequencing device that includes a well 418. In an example, a magnetic field can be applied to the substrate 416 to attract the magnetic beads 410 of the bead assembly 412 toward the well 418. The bead support 406 enters the well 418. For example, a magnet can be moved parallel to the surface of the substrate 416, resulting in the deposition of the bead support 406 into the well 418.

[0046] The bead assembly 412 can be denatured to remove the magnetic beads 410, leaving the bead support 406 in the well 418. For example, the hybridized double-stranded DNA of the bead assembly 412 can be denatured using thermal cycling or an ionic solution to release the magnetic beads 410 and the template polynucleotide with a linker moiety attached to the magnetic beads 410. For example, the double-stranded DNA can be treated with an aqueous solution with low ion content, such as deionized water, to denature and separate the strands. In an example, foam washing can be used to remove the magnetic beads.

[0047] Optionally, while in the well 418, the target polynucleotide 406 can be amplified, referred to herein as templating, to provide a bead support 414 bearing multiple copies of the target polynucleotide. In particular, the beads 414 bear a monoclonal population of the target polynucleotide. Such an amplification reaction can be performed using polymerase chain reaction (PCR) amplification, recombinant polymerase amplification (RPA), or a combination thereof. Alternatively, amplification can be performed before the bead support 414 is deposited into the well.

[0048] In certain embodiments, an enzyme such as a polymerase is present on, attached to, or in proximity to a particle or bead. In examples, the polymerase is present in a solution or in a well to facilitate polynucleotide replication. A variety of nucleic acid polymerases can be used in the methods described herein. In exemplary embodiments, the polymerase can comprise an enzyme, fragment, or subunit thereof capable of catalyzing polynucleotide replication. In other embodiments, the polymerase can be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fused or otherwise engineered polymerase, a chemically modified polymerase, a synthetic molecule, or an analog, derivative, or fragment thereof. Examples of enzymes, solutions, compositions, and amplification methods can be found in WO 2019 / 094,524, entitled "METHODS AND COMPOSITIONS FOR MANIPULATING NUCLEIC ACIDS," which is incorporated herein by reference in its entirety.

[0049] Although the polynucleotides of the beaded support 414 are illustrated as being on the surface, the polynucleotides can extend within the beaded support 414. Hydrogels and hydrophilic particles with a low concentration of polymer to water can contain polynucleotide segments within and throughout the beaded support 414, or the polynucleotides can reside in pores and other openings. In particular, the beaded support 414 can allow diffusion of enzymes, nucleotides, primers, and reaction products used to monitor reactions. A larger number of polynucleotides per particle produces a better signal.

[0050] In an exemplary embodiment, the bead support 414 can be utilized in a sequencing device. For example, the sequencing device 416 can include an array of wells 418.

[0051] In an example, a sequencing primer can be added to the well 418, or the bead support 414 can be pre-exposed to a primer before being placed in the well 418. In particular, the bead support 414 can include an attached sequencing primer. The sequencing primer and the polynucleotide form a nucleic acid duplex comprising a polynucleotide (e.g., a template nucleic acid) hybridized to the sequencing primer. The nucleic acid duplex is an at least partially double-stranded polynucleotide. An enzyme and nucleotides can be provided in the well 418 to facilitate a detectable reaction, such as nucleotide incorporation.

[0052] Sequencing can be performed by detecting nucleotide addition. Nucleotide addition can be detected using methods such as fluorescence or ion detection. For example, a set of fluorescently labeled nucleotides can be provided to the system 416, and the set can be transferred to the well 418. Excitation energy can also be provided to the well 418. When a nucleotide is captured by the polymerase and added to the end of the extending primer, the label on the nucleotide will fluoresce, indicating which type of nucleotide has been added.

[0053] Alternatively, solutions containing a single type of nucleotide can be sequentially applied. In response to the nucleotide addition, the pH in the local environment of well 418 can change. Such a pH change can be detected by an ion-sensitive field-effect transistor (ISFET). Thus, the pH change can be used to generate a signal indicative of the order of nucleotides complementary to the polynucleotides in particle 410.

[0054] In particular, the sequencing system can include a well or multiple wells disposed on a sensor pad of an ion sensor, such as a field-effect transistor (FET). In embodiments, the system includes one or more polymer particles loaded into a well disposed on the sensor pad of the ion sensor (e.g., FET), or one or more polymer particles loaded into multiple wells disposed on the sensor pad of the ion sensor (e.g., FET). In embodiments, the FET can be a chemFET or an ISFET. A "chemFET" or chemical field-effect transistor is a type of field-effect transistor that acts as a chemical sensor. A chemFET has a structural analogue of a MOSFET transistor, in which a charge on the gate electrode is applied by a chemical process. An "ISFET" or ion-sensitive field-effect transistor can be used to measure the concentration of an ion in a solution; a change in the concentration of an ion (e.g., H+) causes a corresponding change in the current through the transistor.

[0055] In some embodiments, the FET may be a FET array. As used herein, an "array" is a planar organization of iodine, such as sensors or wells. The array may be one-dimensional or two-dimensional. A one-dimensional array may have one column (or row) of elements in the first dimension and multiple columns (or rows) in the second dimension. The number of columns (or rows) in the first and second dimensions may be the same or different. A FET or array may be 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or more FETs.

[0056] In embodiments, one or more microfluidic structures can be fabricated above the FET sensor array to provide suppression or limitation of biological or chemical reactions. For example, in one implementation, the microfluidic structure can be configured as one or more wells (or wells, or reaction chambers, or reaction wells, these terms are used interchangeably herein) disposed above one or more sensors of the array such that the one or more sensors disposed above a given well detect and measure the presence, level, or concentration of an analyte in the given well. In some embodiments, there can be a 1:1 correspondence between FET sensors and reaction wells.

[0057] 4, in another example, wells 418 of the array of wells can be operably connected to a measurement device. For example, for fluorescence, wells 418 can be operably coupled to a light detection device. For ion detection, the underside of wells 418 can be disposed over a sensor pad of an ion sensor, such as a field effect transistor.

[0058] An exemplary system involving sequencing via detection of ionic by-products of nucleotide incorporation is the Ion Torrent PGM™, Proton™, or S5™ sequencer (Thermo Fisher Scientific), an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions generated as by-products of nucleotide incorporation. Typically, hydrogen ions are released as by-products of nucleotide incorporation during template-dependent nucleic acid synthesis by a polymerase. The Ion Torrent PGM™, Proton™, or S5™ sequencer detects nucleotide incorporation by detecting hydrogen ion by-products of nucleotide incorporation. The Ion Torrent PGM™, Proton™, or S5™ sequencer can contain multiple template polynucleotides to be sequenced, with each template disposed in a respective sequencing reaction well in the array. Each well of the array can be coupled to at least one ion sensor capable of detecting the release of H+ ions generated as by-products of nucleotide incorporation or changes in solution pH. The ion sensor includes a field-effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H+ ions or changes in solution pH. The ion sensor can provide an output signal indicative of nucleotide incorporation, which can be expressed as a voltage change whose amplitude correlates with the H+ ion concentration in the respective well or reaction chamber. Different nucleotide types can be sequentially flowed into the reaction chamber and incorporated into the extending primer (or polymerization site) by the polymerase in an order determined by the template sequence. Each nucleotide incorporation can be accompanied by the release of H+ ions in the reaction well, along with a concomitant change in localized pH. The release of H+ ions is registered by the sensor's FET, thereby generating a signal indicative of the occurrence of nucleotide incorporation. Nucleotides not incorporated within a particular nucleotide flow may not generate a signal.The amplitude of the signal from the FET can also correlate with the number of nucleotides of a particular type that are incorporated into the extending nucleic acid molecule, thereby enabling homopolymeric regions to be resolved. Thus, during a sequencer run, flowing multiple nucleotides into a reaction chamber and monitoring incorporation across overlapping wells or reaction chambers can enable the instrument to simultaneously resolve many nucleic acid templates.

[0059] Seeding of the bead support and capture by the magnetic beads can be accomplished in a variety of ways. For example, turning to 502 in FIG. 5 , a template polynucleotide (B'-A) ​​can be captured by a capture probe (B) attached to a bead support 510. The capture probe (B) can be extended to a complementary position to the template polynucleotide. Optionally, the resulting double-stranded polynucleotide can be denatured to remove the template nucleic acid (B'-A), leaving a single strand (B-A') attached to the bead support 510. As illustrated in 504, a primer (A) modified with a linker moiety such as biotin can hybridize to portion (A') of the nucleic acid (B-A') attached to the bead support 510. Optionally, the primer (A) can be extended to form a complementary nucleic acid (A-B').

[0060] As illustrated in 506, magnetic beads 512 can be introduced into the solution. The magnetic beads 512 can include a linker complementary to the linker portion attached to the primer (A). For example, the linker attached to the primer (A) can be biotin, and the magnetic beads 512 can be coated with streptavidin. As described above, the magnetic beads 512 can be used to wash the solution and assist in the deposition of the bead support 510 and the attached nucleic acid (B-A') into the well of a sequencing device. As illustrated in 508, the double-stranded polynucleotide can be denatured to result in dehybridization of the nucleic acid (B'-A) ​​from the nucleic acid (B-A') attached to the bead support 510. Thus, the bead support 510 is deposited into the well of the sequencing device, bearing the single-stranded target nucleic acid (B-A'). Alternatively, the linker-modified probe (A) need not be extended to form a complementary polynucleotide having the length of the polynucleotide (B-A'). The extension reaction can be carried out using the polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), or other amplification reaction.

[0061] Device 6 includes an illustration of an exemplary sequencing apparatus system 600 including a controller 602 in communication with a preparation deck 604, a loading station 606, and a sequencing station 608. The preparation deck 604 can include a pipetting robot 612 that can access samples 614, reagents and solutions 616, a thermocycler 618, and other devices 620, such as a magnetic separator or centrifuge. Target sequences prepared on the preparation deck 604 can be provided to the loading station 606. For example, the preparation deck 604 can provide seeded substrates containing target sequences that are provided to the loading station 606 for loading onto a sensor device.

[0062] Once loaded, the sensor device containing the target sequence can be transported to the sequencing station 608 utilizing a sliding mechanism 610. The sequencing station 608 can include fluidics and electronic interfaces to interact with the sensor device to sense the addition of nucleotides during the sequencing-by-synthesis reaction. Data collected from the sensing device can be provided to a sequencing computer 622, which can perform base calling, read alignment, and variant calling.

[0063] The controller 602 may further communicate with a user interface 624, such as a monitor, keyboard, mouse, touch screen, or any combination thereof, among other interfaces. Additionally, the controller 602 may communicate with a network interface that may access a local area network, a wide area network, or a global network. The network interface 626 may be a wired or wireless interface using various standard communication protocols. Additionally, the system may be powered by a power supply 628.

[0064] 7 includes an illustration of an exemplary device 700 incorporating a three-axis pipetting robot. In an example, the device 700 may be a sequencer incorporating a sample preparation platform. For example, the device 700 may include an upper portion 102 and a lower portion 104. The upper portion may include a door 706 for accessing a deck 710 where samples, reagent containers, and other consumables are located. The lower portion may include a cabinet for storing additional reagent solutions and other portions of the device 700. Additionally, the device may include a user interface, such as a touchscreen display 708.

[0065] In certain examples, the device 700 may be a sequencing device. In some embodiments, the sequencing device includes an upper section, a display screen, and a lower section. In some embodiments, the upper section may include a deck that supports components and consumables of the sequencing device, including a sample preparation section, sequencing chips, and reagent strip tubes and carriers. In some embodiments, the lower section may house reagent bottles and waste containers used for sequencing.

[0066] In some embodiments, one or more cameras mounted on a cabinet in the upper section of the device are aimed at the deck to monitor which items are being placed in preparation for the sequencing run. The cameras can capture video or images at timed intervals. For example, images may be captured at intervals of 1 to 4 seconds or any suitable interval. In another example, frames of the video stream can be sampled at intervals ranging from 0.5 to 4 seconds. A computer or processor analyzes the images to detect the user's completion of a task. The computer or processor may provide feedback and instructions for the next task in preparation via a display screen. The display screen may present graphical representations of device components and consumables to illustrate instructions to the user.

[0067] An exemplary device deck 710 is illustrated in FIG. 8 as device deck 800. Deck 800 is housed in the upper section of the device and is in the field of view of one or more cameras. The sample preparation deck may include multiple locations configured to accept reagent strips, supplies, sequencing chips, and other consumables. As used herein, consumables are components used by the device that are periodically replaced as they are used. For example, consumables include reagent and solution strips or containers, pipette tips, microwell arrays, and flow cells, as well as associated sensors, among other disposable components that are not part of the device's permanent components.

[0068] In the example, system 800 includes a pipetting robot 802 that accesses various reagent strips and containers, pipette tips, microwell arrays, and other consumables to perform tests. Additionally, the system can include a mechanism for running the tests 804. Exemplary mechanism 804 includes a mechanical conveyor or slide and fluidic system.

[0069] In the example, deck 800 includes trays 806 or 808 for receiving solution or reagent strips of a particular configuration. In the example of a sequencing device, tray 806 can be used for appropriately configured strips of library and template solutions, and tray 808 can receive appropriately configured library and template reagents.

[0070] Additionally, the device can be configured to accept microwell arrays 810 and 812 at specific locations on the deck. For example, a sample can be provided in an array of wells, such as microwell array 812. In another example, the system can be configured to accept additional reagents 814 in a different strip configuration. In another example, reagent solutions can be provided in array 816. In a further example, container array 820 can be provided in conjunction with instrumentation, such as a thermocycler. Additionally, the system can include other devices, such as a centrifuge, that can be provided with consumables, such as tubes. Additionally, a tray can be provided to accept pipetting tips 822.

[0071] The proper provision of consumables at each of these locations can be monitored by a vision system including one or more cameras. The deck may also be equipped with one or more cameras to track the supply and maintenance of reagents and other consumables. The user can be prompted via a user interface if a reagent to be used to execute a plan is low or if a reagent consumable is in a used state.

[0072] 9 includes an illustration of a reagent storage cabinet 704 for storing larger volume reagent and solution containers. For example, cabinet storage 704 includes an interface 902 for receiving a reagent cartridge. In another example, storage 704 can provide space for containers 904 or 906. In a further example, storage 704 can include space for waste container 808.

[0073] An exemplary consumable, such as a solution or reagent strip, configured to fit into slot 1002 is illustrated in FIG. 10. In the example, the strip includes a base 1002 and a top 1004 coupled to base 1002. Top 1004 includes a window 1006 that provides access to well 1010 or 1012. Optionally, top 1004 can provide a window 1008 to provide access to a tube 1014 inserted into a tube receptacle in base 1002.

[0074] The top can further include a grip 1016. For example, the grip 1016 can be used to hold the reagent container 1000 when inserting or removing the reagent container 1000 from an analytical device. Additionally, the top 1004 can define end structures 1018 or 1020 configured to engage complementary structures on the device and to restrict the orientation of the strip relative to its position within the device. Additionally, a code 1022, such as a barcode or QR code, can be present on the top 1004.

[0075] Flow through initialization In an exemplary system for preparing a reagent solution on a sequencing device, a source of an initial solution can be connected to a reservoir in the cartridge. The initial solution can include salts, surfactants, and preservatives.

[0076] The cartridge can include a container that stores a concentrate of a reagent used in a sequencing reaction. For example, the container can contain concentrated nucleotides, concentrated modified nucleotides, or a blend of nucleotides. In another example, the container can contain cofactors and enzymes useful for sequencing.

[0077] The concentrated nucleotides can be blended with the initial solution to produce a nucleotide solution that is stored in a separate container. In particular, the reagent storage container can be part of the system illustrated in FIG. 1, having reagent storage containers 104, 106, 108, 110, or 112.

[0078] The source can be a pressurized system to induce flow through the cartridge. Alternatively, the solution can be pumped from the source into the cartridge. In another example, a vacuum can be applied to the container to draw the solution through the cartridge and into the container. In another example, flow can be induced by both vacuum and pressure or pumping.

[0079] 11 illustrates an exemplary cartridge system including a cartridge 1102 and a docking station 1104. The cartridge 1102 can fit into a docking area 1122 of a primary platform 1110 of the docking station 1104. In particular, the docking station 1104 can include guides such as rails 1116 that cooperate with other guides, such as rails 1118 on the cartridge 1102, to position the cartridge 1102 in the docking station 1104, as illustrated in FIG. 12 . The cartridge 1102 can further include a handle 1120 to assist in inserting and removing the cartridge 1102 from the docking station 1104.

[0080] The docking station 1104 includes a second platform 1112 that is movable relative to the first platform 1110. For example, the platform 1112 can be driven relative to the platform 1110 by a driver 1124, such as a motor mechanism or a screw mechanism. In particular, the platform 1112 can be guided up and down by guides 1126.

[0081] The platform 1112 is attached to a tube on its upper surface and includes a fluid coupler 1114 that can interface with the receptacle 1106 of the cartridge 1102. Once the cartridge 1102 is inserted into the dock 1104 and secured by the platform 1110, the platform 1112 can be driven downward or toward the platform 1110 by a driver 1124 so that the fluid coupler 1114 can engage with the receptacle 1106 of the cartridge 1102. In particular, a guide 1126 can ensure the positioning of the fluid coupler 1114 relative to the cartridge 1102.

[0082] Cartridge 1102 includes a plurality of containers 1106. For example, the cartridge can include a number of containers 1106 at least equal to the number of nucleotides (i.e., four). As illustrated, cartridge 1102 has five containers, although more or less than five may be included. Cartridge 1102 can further include additional reagent concentrate containers containing, for example, combinations of nucleotides, other ionic compositions, enzymes, or surfactants.

[0083] The cartridge 1102 also includes guides 1118, illustrated as rails, for guiding the cartridge 1102 when engaging the dock 1104. The cartridge may also include a handle 1120 to assist in inserting and removing the cartridge from the docking station 1104. Additionally, the cartridge may include positioning features, such as indentations, that are useful for ensuring that the fluid coupler 1114 is properly positioned to engage the container 1106.

[0084] 13, the container 1106 includes a receiver, a clip, and a seal. The seal includes a central bore 1310 and at least one peripheral bore 1308 positioned radially outward from the central bore 1310.

[0085] In cross section, the container 1106 can further include a frit 1312 disposed within the receiver cavity. The frit 1312 can include a central bore aligned with the central bore 1310 of the seal. The cavity can also be in fluid communication with the peripheral openings 1308. A fluid can be applied to the central bore 1310, and the fluid flows through the frit 1312 and down into the cavity. The fluid then flows from the outer holes 1308 into the channels of the seal.

[0086] Frit 1312 can include protrusions on its upper surface configured to engage with the central bore of the seal. In particular, the protrusions of the frit can enter the central bore of the seal at the protrusions of the seal. The frit can be fluid-permeable. For example, frit 1312 can be formed of a porous material, such as a porous ceramic or metallic material. In another example, frit 1312 can be formed of a porous polymeric material or a water-permeable polymer or fibrous material. Frit 1312 can include a central bore that extends at least partially into or completely through frit 1312. In addition, frit 1312 includes a larger surface area at the top that allows solution to flow through the frit to remove the concentrated nucleotide solution or reagent solution from the frit to the receiver cavity.

[0087] 13 , when cartridge 1102 is inserted into dock 1104 and platform 1112 is positioned adjacent to platform 1110, fluid coupler 1114 engages with a seal of a vessel, specifically vessel 1106. A central tube of fluid coupler 1114 enters central bore 1310 of the seal. An outer concentric ring 1360 engages with the seal radially outward of the channel, enclosing the channel. Fluid coupler 1114 further includes openings for engaging tubes 1364 and 1366. In the example, opening 1364 is positioned at the axial center of fluid coupler 1114, and second opening 1366 is disposed radially outward from the central axis of fluid coupler 1114.

[0088] In a particular example, fluid can flow into opening 1364 and be driven up tube 1360 into the central bore 1310 of the seal. Fluid flows through frit 1312 into the receiver cavity. Fluid can flow through opening 1308 into the channel enclosed by fluid coupler 1114. Fluid can exit the channel through hole 1368 connected to opening 1366. Alternatively, the flow can be reversed.

[0089] Tip and Slide Mechanism In examples, biosensors and flow cells are examples of sensor devices. Figures 14 and 15 illustrate an exemplary sensor device 1400, such as a microchip including a flow cell. For example, the sensor device 1400 includes a substrate 1402 having a die 1404 with a plurality of microwells in fluid communication with a sensor array. A flow cell 1406 is fixed on the substrate and provides a volume above the die 1404.

[0090] In the example, the flow cell 1406 includes a set of fluid inlets 1408 and a set of fluid outlets 1410. In particular, the flow cell 1406 can be divided into lanes 1412. Each lane is individually accessed by a respective fluid inlet 1408 and fluid outlet 1410.

[0091] As illustrated, the sensor device 1400 includes four lanes 1412. Alternatively, the sensor device 1400 can include fewer or more than four lanes. For example, the sensor device 1400 can include 1 to 10 lanes, such as 2 to 8 lanes, or 4 to 6 lanes. The lanes 1412 can be fluidically isolated from one another. Thus, the lanes 1412 can be used at separate times, in parallel, or simultaneously, depending on the run design.

[0092] The sensor device 1400 may further include guide structures 1414, for example formed as part of the flow cell 1406, for engaging complementary structures on the fluidic coupler. Such guide structures 1414 assist in aligning the fluid inlet 1408 and fluid outlet 1410 with associated ports on the fluidic coupler.

[0093] 16, 17, and 18 include illustrations of an exemplary fluid coupler 1600. The fluid coupler 1600 includes a body 1614 that defines a fluid pathway between a set of ports. Additionally, the fluid coupler 1600 can include a connector section 1612 for engaging a mechanical assembly and assisting in positioning the fluid coupler 1600 relative to the mechanical assembly. In another example, the fluid coupler 1600 can include wings 1616 that define reference holes 1610 for engaging with guide rods of the mechanical assembly, further assisting in positioning the fluid coupler 1600 relative to the sensor device.

[0094] The body 1614 of the fluidic coupler 1600 can define an opening 1602 in fluid communication with a first set of ports 1604. The opening 1602 can be sized to receive the end of a pipette tip and allow pipetting of a fluid composition into the opening 1602. The opening 1602 is in fluid communication with the set of ports 1604 that are configured to engage with the inlet 1408 of the sensor device 1400 ( FIG. 14 ). The fluidic coupler 1600 can further define a second set of ports 1606 that can engage and provide fluid communication with the outlet 1410 of the sensor device 1400 ( FIG. 14 ).

[0095] 18, the system can further include a third set of ports 1818 in fluid communication with the second set of ports 1606. The third set of ports 1818 can engage a fluid manifold of a mechanical assembly, such as the fluid manifold 2540 illustrated in FIGS. 26 and 27. Optionally, the fluid coupler 1600 can include a fourth set of ports 1820 that can connect with the fluid manifold or can be closed depending on the configuration of the mechanical assembly.

[0096] The ports 1604, 1606, 1818, or 1820 can be formed from a resilient material, such as rubber or an elastomeric polymer. In an example, the ports can be formed as an overmold using the resilient material.

[0097] Returning to FIG. 16, the body 1614 of the fluid coupler 1600 may further include guide features 1608 that are complementary to the guide features 1414 of the sensor device 1400 (FIG. 14).

[0098] 19 and 20 , a fluid coupler 1600 can engage with a sensor device 1400. The body 1614 of the fluid coupler 1600 can be aligned with the substrate 1402 of the sensor device 1400, allowing a first set of ports 1604 of the fluid coupler 1600 to be in fluid communication with an inlet 1408 of the sensor device 1400. Additionally, a second set of ports 1606 can be in fluid communication with an outlet 1410 of the sensor device 1400. For example, the guide structures 1414 and 1608 can engage to align the ports with the inlet and outlet. Optionally, a third set of ports 1818 can be in fluid communication with a manifold. In a further example, a set of fluid ports 1820, optionally in fluid communication with the second set of ports 1608, can be in fluid communication with a fluid manifold.

[0099] Thus, a fluid composition can be pipetted into openings 1602 in fluid communication with first set of ports 1604, which provide the fluid composition to flow cell 1406 of sensor device 1400 via inlet 1408 of flow cell 1406. After processing, the remainder of the fluid composition can be drawn from outlet 1410 of sensor device 1400 through second set of ports 1606 and third set of ports 1818 to the fluid manifold.

[0100] 21 and 22 include illustrations of an exemplary mechanical system 2100 for moving sensor devices between various stations within the system. For example, a sliding mechanism 2102 can move along rails 2104 to guide a sensor device (e.g., sensor device 1400 of FIG. 14 ) between stations 2106, 2108, and 2110. For example, a sensor device can be inserted into the sliding mechanism 2102 at station 2106. In an example, the sensor device can be inserted in a vertical orientation, with the inlet and outlet facing to the side rather than upward. A sensor 2112 can detect the presence of the sensor device and enable the sliding mechanism 2102 to move if the sensor device is present. For example, the sliding mechanism 2102 can move the sensor device to station 2108, where the sensor device can be loaded with a sample, such as by the magnetic loading method described above in connection with FIG. 4 . In particular, the fluidic coupler can be inserted into a space 2116 provided by the mechanical assembly 2114, which presses the fluidic coupler against the sensor device and engages the fluidic coupler with the manifold. Once the magnetic loading procedure is complete, the mechanical assembly 2114 can detach from the fluidic coupler and the slide 2102 can move the sensor device to a subsequent station 2110, such as a fluidic station that provides reagents and other conditions for sequencing.

[0101] As illustrated in FIG. 22, a drive 2218, such as a screw drive, can include a screw 2220 that engages a clutch 2222 to move the slide 2102, and thus the sensor device, between stations 2106, 2108, and 2110.

[0102] 23, the slide 2102 can be positioned at station 2106 where stop post 2332 engages the slide 2102 at feature 2354. Further, the slide 2102 can be moved forward from station 2106 to station 2108 where it can engage solenoid stop post 2334, and the slide can be moved rearward (to the left as illustrated) to engage solenoid stop 2334 with feature 2354. Additionally, the slide 2102 can be moved along rail 2104 to engage forward stop 2436, illustrated in FIG. 24, aligning the slide and sensor receptacle 2324 with station 2110. To return the slide 2102 to station 2106, the solenoid stop post 2334 can be disengaged to allow the slide 2102 to pass.

[0103] When a sensor device is inserted into receptacle 2324 at station 2106, sensor 2112 can sense the presence of the sensor device in receptacle 2324 through opening 2356. For example, sensor device 2112 can be an optical sensor that optically detects the presence of the sensor device in receptacle 2324 through opening 2356.

[0104] Clutch 2222 can be used to provide both a rearward force (illustrated as towards the left) against stop 2324 or 2334 and a forward force (illustrated as towards the right) against stop 2436. For example, clutch system 2222 includes a nut 2326 for engaging screw 2220 of screw drive mechanism 2218. Nut 2326 engages with coupling 2328 having a central bore that allows screw 2220 to pass through coupling 2328. Coupling 2328 is attached to nut 2326 using pin and spring system 2350. The pin is movably connected to the nut 2326 such that when a spring in pin and spring system 2350 is compressed, the pin moves through nut 2326.

[0105] Coupling 2328 is also connected to connector plate 2330 using pin and spring system 2352. The pins of pin and spring system 2352 can be configured to move through connector plate 2330 when the springs of pin and spring system 2352 are compressed. Alternatively or additionally, the pins of pin and spring system 2352 can be configured to move through coupling 2328.

[0106] The connector plate 2330 is coupled to the slide 2102 and moves back and forth in response to rotation of the screw 2220. When the slide 2102 is moved backward against the stops 2332 or 2334 (illustrated as left in FIG. 23 ), the spring of the pin and spring system 2352 can be compressed and the pin can move through either the connector plate 2330 or the coupling 2328. Thus, rotation of the screw 2220 provides a known force backward against the rod 2332 or 2334, providing precise positioning of the sensor device within the receptacle 2324. In a further example, when the slide 2102 is moved forward to engage the stop 2436, the slide 2102 stops moving. Further rotation of the screw 2220 moves the nut further forward (illustrated as right in FIG. 23 ). The spring of pin and spring system 2350 is compressed and the pin of pin and spring system 2350 moves through nut 2326, providing a known force for slide 2102 against forward stop 2436. Such force and positioning provides a precise location of sensor device receptacle 2324 and sensor device in station 2110.

[0107] FIG. 25 includes an illustration of an exemplary mechanical assembly 2114 for providing fluid coupling between a sensor device and a fluid coupler. When the slide is in place, space 2538 for the sensor device is provided. Additionally, space 2116 for the fluid coupler is provided. When the mechanical assembly 2114 is engaged, the fluid coupler is pushed into fluid communication with the sensor device and the manifold 2540. For example, a drive mechanism 2542 utilizing a screw drive having a screw 2544 can utilize a clutch system having a nut 2546 and coupled to the frame 2548 by a pin 2564 and spring 2566 to move the frame 2548 of the mechanical assembly 2114 forward (illustrated as up in FIG. 25) and rearward (illustrated as down in FIG. 25). The pin 2564 can be movably coupled to the nut 2546 such that a head 2578 of the pin 2564 is positioned against the nut 2546 until the manifold 2540 is pressed against the fluid coupler. Further forward movement of the nut 2546 moves the pin 2564 through the nut 2546, allowing the spring 2566 to be compressed.

[0108] The frame components 2548 can move back and forth together in response to movement of the nut 2546. A lever 2550 is rotatably coupled to the frame 2548 with a fastener 2552. When the assembly is in the rearward position, an adjustment screw 2586 attached to the lever 2550 engages a stop 2584, causing the opposite side of the lever 2550 to pivot rearward (downward in the illustrated example). As the nut 2546 moves forward, the adjustment screw 2586 gradually disengages from the stop 2584, causing the opposite side of the lever 2550 to pivot forward, biased, for example, by a spring 2582. Pivoting the lever 2550 moves a guide plate 2556 forward relative to the frame 2548, which is also moving forward (upward in the illustrated example). Guide plate 2556 is connected to guide rods 2558 and 2560, which move forward with guide plate 2556 to engage reference holes in the fluid couplers. Guide rod 2560 may be further guided by guide 2562, which engages a portion of manifold 2540. As guide rod 2560 moves forward, it may disengage from sensor 2576, indicating that guide rod 2560 is engaged with a reference hole in the fluid coupler.

[0109] Once manifold 2540 and guide rods 2558 and 2560 are engaged with the fluid coupler, nut 2546 can continue to move forward while frame 2546 remains stationary. Pin 2564 can move through nut 2546, and spring 2566 is compressed, providing a known force against the fluid coupler and a sensor device in fluid communication with the fluid coupler. Such force provides the desired leak-free fluid coupling between the sensor device and the fluid coupler.

[0110] The circuit board 2570, including the sensors 2572 and 2574, can be connected to the movable frame 2548 and can move with the frame 2548. The flag 2568 can be connected to the nut 2546. From the rearward position to a second position where the manifold 2540 and frame 2548 connect with the fluid coupler, the position of the flag 2568 remains constant relative to the position sensor 2572. When the manifold 2540 is positioned against the fluid coupler and sensor device, the nut 2546 moves forward relative to the frame 2548. Thus, the flag 2568 moves forward toward the sensor 2572. When the flag 2568 is detected by the sensor 2572, the forward drive of the nut 2546 can be stopped. Thus, a known compression of the spring 2566 is achieved, applying a known force against the frame 2548, the manifold 2540, and the fluid coupler.

[0111] As the nut 2546 is moved rearward from its forward position, the flags 2568 disengage from the sensor 2572 until the pins 2564 on their heads 2578 lock against the nut 2546. As the nut 2546 continues to move rearward, the frame 2548 and manifold 2540 are pulled rearward along with the sensor circuit board 2570. The adjustable screws 2586 engage the stops 2584, withdrawing the guide rods 2558 and 2560 from the reference holes in the fluid couplers. As the guide rods are withdrawn from the fluid couplers, the reference rod 2560 engages the sensor 2576, indicating that the guide rods have been withdrawn from the reference holes in the fluid couplers. The sensor 2574 continues to move rearward with the sensor circuit board 2570 attached to the frame 2548 until the sensor 2574 engages the flag 2580, indicating that the nut 2546 is in its rearmost position. The fluid coupler can be disengaged and removed from the mechanical assembly 2114. Additionally, the sliding mechanism 2102 can move the sensor device to the next station 2110.

[0112] 26 and 27 include illustrations of an exemplary manifold 2540 for use with the mechanical assembly 2114. The manifold 2540 can include a slot 2642 on its front surface for receiving a fluid coupler. The slot 2642, along with rest structures 2644 and 2646, can set the vertical position of a fluid coupler, such as the fluid coupler 1600 illustrated in FIG. 16 . The connector section 1612 of the fluid coupler 1600 can extend beyond the manifold 2540 toward the back surface of the manifold. The manifold 2540 can further include reference holes 2650 and 2648 that align with the reference hole 1610 of the fluid coupler 1600. The reference hole 2650 can be sized to receive a guide rod 2558. The reference hole 2648 can be sized to receive a guide rod 2560 and, optionally, a guide 2562.

[0113] In particular, the manifold 2540 includes a set of fluid openings 2652 for engaging with the third port 1818 of the fluid coupler 1600 (illustrated in FIG. 18 ). The set of openings 2652 is in fluid communication with a set of ports 2654 disposed on the back surface of the fluid manifold 2540. Such ports 2654 can be connected to a vacuum to allow fluid to be drawn through the ports 2654, the openings 2652, the third set of ports 1818 of the fluid coupler 1600, and the second set of ports 1606 of the fluid coupler 1600. Optionally, an additional set of fluid openings and flow ports can be provided to connect with the fourth set of fluid ports 1820 of the fluid coupler 1600.

[0114] 28 includes a block flow diagram illustrating a method 2800 for fluidly engaging a sensor device. For example, as illustrated in block 2802, a sensor device can be inserted into a holder or receptacle for the slide when the slide is in a first position. Optionally, a detector can determine whether the sensor device is properly positioned in the holder or receptacle before allowing the slide to be moved to a second position.

[0115] The sensor device and slide can be moved to a second position, as illustrated in block 2804. In an exemplary system, the second position can represent a position where a sample is loaded onto the sensor device. For example, a fluidic coupler can be pressed against a flow cell of the sensor device, as illustrated in block 2806. The fluidic coupler can include openings that allow a fluid composition to be pipetted into the openings and through a port of the fluidic coupler that is engaged with an inlet of the flow cell of the sensor device.

[0116] For example, a pipette can withdraw an aliquot of a fluid composition, as illustrated in block 2808. The aliquot can be applied to an opening of the fluidic coupler, as illustrated in block 2810. The aliquot can pass through the opening of the fluidic coupler, through the first set of ports, through an inlet of the sensor device, and into a flow cell of the sensor device.

[0117] In an example, the fluid composition can be processed in a flow cell, as illustrated in block 2812. For example, magnetic loading techniques can be applied to load the sample into the wells of the sensor device.

[0118] As illustrated in block 2814, the remainder of the fluid composition can be drawn from the flow cell of the sensor device. For example, a vacuum chamber pressed against the fluidic coupler and attached to a manifold in fluid communication with the outlet of the flow cell can draw the remainder of the fluid composition from the flow cell. The process of pipetting an aliquot of the fluid composition, applying the aliquot, processing the fluid composition, and drawing the remainder of the fluid composition can be repeated, for example, to apply additional sample or to wash the flow cell.

[0119] Once the loading process is complete, the sensor device can be released from the mechanical assembly, as illustrated in block 2816. For example, the mechanical assembly can be retracted to a rear position, releasing the sensor device and fluid coupler, and moving the sensor device to a subsequent station.

[0120] The slide and sensor device can be moved to a third location, as illustrated in block 2818. For example, the sensor device can be moved to a sequencing section of the system.

[0121] Magnetic loading Figure 29 is a schematic representation of an exemplary magnetic loading system. Specifically, Figure 29 shows a substrate 2900 that supports a chip surface 2910 and a flow cell 2920. A magnetic package 2950 is disposed on a tray 2960 proximal to the substrate 2900.

[0122] Magnetic package 2950 is shown with two magnets 2952 and 2954. While the embodiment of FIG. 29 shows magnets 2952 and 2954, the disclosed principles are not so limited and may include more or fewer magnets than shown in FIG. 29 . Magnets 2952, 2954 may be separated by inert material 2953. Inert material 2953 may function as a non-conductive insulator. In certain embodiments, magnets 2952 and 2954 may be positioned such that the north pole of magnet 2952 is directly opposite the south pole of magnet 2954. In this positioning, substrate 2900 is simultaneously exposed to the north and south poles of magnets 2952 and 2954. In other embodiments, magnets 2952 and 2954 may be positioned such that substrate 2900 is exposed only to the north or south pole of the magnets.

[0123] Substrate 2900 may include any material configured to receive microchip 2910 (interchangeably, chip or sensor device). Microchip 2910 may include a top surface having a plurality of receptacles, such as microwells, cavities, divots, dimples, or other receptacles, configured to receive one or more sequencing beads. In one embodiment, chip 2900 may include microwells configured to receive sequencing beads. One such exemplary microchip is offered by Ion Torrent® as the Ion 541 Chip™. An ​​exemplary microchip is discussed with reference to FIG. 14.

[0124] Flow cell 2920 is positioned above the top surface of microchip 2910 to allow fluid communication to the surface of the microchip. Fluid may be transferred through ports 2922 and 2924 formed on chip 2910. Magnetic beads and sequencing beads (not shown), along with one or more reagents, may be transferred to the surface of microchip 2910 through ports 2922 and 2924. Once the sequencing beads are loaded onto the surface of microchip 2910, wash reagents may be transferred through ports 2922 and 2924 to remove unwanted particles or reagents.

[0125] The tray 2960 (and magnetic package 2950) may move relative to the substrate 2900, as indicated by arrow 2962. While the movement and orientation of the substrate is illustrated as horizontal, in the alternative, the substrate may be oriented vertically and the movement may be up and down. The movement may be moderated by an actuator 2970 in combination with a programmable processor or controller 2980 that specifies the speed and direction of movement of the tray 2960. The actuator 2970 may include, for example, a motor or solenoid controlled by the controller 2980 having one or more processor circuitry and memory circuitry. The controller 2980 may be a programmable controller. In one embodiment of the present disclosure, the controller 2980 may be configured to receive input information 2982 from an auxiliary source indicating when to move the tray 2962 relative to the substrate 2900 (which may be stationary). The information 2982 may also include data related to the speed of movement of the tray 2960 as a function of the type of particles loaded on the chips. Such data may be stored in one or more memory circuitry associated with the controller 2980.

[0126] FIG. 30 schematically illustrates the movement of a solution containing magnetic beads at a first speed relative to a magnetic package. In FIG. 30, the top surface of a microchip 3010 is exposed to a reagent (or solution) 3050. The reagent 3050 may include magnetic beads as well as sequencing beads. The magnetic beads may include any beads that have an affinity for or are responsive to a magnetic field. In one embodiment, the size of the magnetic beads is selected to prevent the magnetic beads from entering microwells, cavities, or divots formed in the surface of the microchip. Exemplary magnetic beads may be substantially spherical with a diameter of approximately 1 μm to 100 μm.

[0127] Magnets 3052 and 3054 are separated by inert material 3053 to form a magnetic package. Arrow 3059 indicates the direction of movement of magnetic package 3050 relative to microchip 3010. Reagent 3050 is disposed on top of microchip 3010. Reagent 3050 may include one or more magnetic beads bound to sequencing beads. Reagent 3050 may be a liquid, a gel, or any material with thixotropic viscosity for movement over a solid surface. Multiple magnetic beads (not shown) may be disposed in reagent 3050 in a manner such that the magnetic beads can move or rotate freely relative to one another.

[0128] FIG. 31 schematically illustrates the movement of a solution containing magnetic beads at a second speed relative to the magnetic package. FIG. 31 also illustrates faster magnet movement (as indicated by arrow 3060) compared to that of FIG. 31. The shape of reagent 3050 indicates a relatively wide dispersion of reagent 3050 (containing magnetic beads), while the shape of reagent 3056 suggests a narrower, more densely packed reagent (containing magnetic beads). FIGS. 30 and 31 also illustrate that when the relative movement is slow, the leading edge of the reagent / beads aligns with the inner or leading edge of the lagging magnet. When the relative movement is fast, the reagent / bead pile lags behind the leading edge of the lagging magnet.

[0129] Figure 32 shows a schematic representation of the movement of a solution containing magnetic beads relative to the direction of reversal of the magnetic package. Arrow 3062 indicates the reversal of the magnet's direction of movement. As seen in Figure 32, when the magnet switches direction of movement, the reagent / bead pile remains in the same place until it is picked up by the inner edge of the new lagging magnet (3054). Reversing the direction of magnet movement can be useful for loading beads into microwells or for allowing multiple sweeps of the reagent pile across the surface of an array on a microchip.

[0130] In an example, the magnet can be cycled for 5-50 sweeps (back and forth), such as 5-35 sweeps or 10-30 sweeps. In an example, each sweep takes 1-5 minutes, such as 1-3 minutes. Once the bead supports are loaded into the wells, the bead assemblies can be denatured and the surface foam washed to remove the magnetic beads.

[0131] Once mounted on the microchip, a suspension containing the bead complex is deposited in a flow cell on the microchip surface. Figure 33 illustrates a microchip loaded with magnetic beads according to one embodiment of the present disclosure. More specifically, Figure 33 shows a microchip 3302 with a flow cell 3312 positioned thereon. The flow cell 3312 includes ports 3322 and 3324 for receiving and discarding reagents. The flow cell 3312 may have more than two ports, as illustrated in Figure 14, for example. The microchip 3302 is disposed on a substrate 3310. One or more magnets (not shown) are disposed below the substrate 3310. The magnets generate a magnetic field that causes a line of magnetic beads 3350 to form on the surface of the microchip 3302. Movement of the magnet causes movement of the line 3350 (i.e., the magnetic beads) along the surface of the microchip 3302. As the magnetic beads move along the surface, the sequencing beads bound to the magnetic beads in the reagent enter wells or cavities on the surface of the microchip 3302.

[0132] FIG. 34 schematically illustrates a magnetic bead loading model. In FIG. 34, a microchip surface 3402 is shown with multiple microwells 3410. A stream 3420 contains, among other things, sequencing beads 3432, 3434 attached to magnetic beads 3430. As illustrated in FIG. 34, the sequencing beads 3432 and 3434 can have a smaller diameter than the magnetic beads 3430. The microwells 3410 are sized to accept the sequencing beads 3432, 3434. Each microwell 3410 can be configured to receive at least one sequencing bead 3432, 3434 and exclude the magnetic beads 3430. Although not shown, each microwell 3410 can be coupled to sensing circuitry including one or more electrodes, as well as electronic circuitry configured to detect the presence of an analyte in the microwell 3410. The analyte can be bound to the sequencing beads or released as a result of one or more reactions within the well. Surface 3450 illustrates schematically the surface of a flow cell having input and output ports (not shown).

[0133] The sequencing beads may have different sizes. In one embodiment, the sequencing beads 3432, 3434 are selected so that at least one sequencing bead can enter the microwell. In other words, the diameter of the sequencing bead may be selected to be smaller than the opening of the microwell. Although the microwell 3410 is shown with tapered sidewalls, claimed embodiments are not so limited, and the microwell may have different shapes and configurations without departing from the disclosed principles.

[0134] As shown, stream 3420 may contain multiple beads. Magnetic beads 3430 may have magnetic properties. In certain embodiments, stream 3420 may contain other reagents in addition to beads. Magnetic beads 3430 may include Dynabeads® M-270 or Dynabeads® M-280 supplied by Thermo Fisher Scientific, which have a bead diameter of approximately 2.8 μm. Each magnetic bead 3430 may have, for example, a biotinylated nucleic acid, antibody, or other biotinylated ligand and streptavidin for coupling to the target. Magnetic beads 1530 can be attached to sequencing beads 3432, 3434 using such biotin / streptavidin binding.

[0135] Such loading methods can be implemented in hardware having a horizontal or vertical configuration. For example, the hardware can hold a substrate on which beads are deposited horizontally. In another example, the hardware can hold a substrate vertically when the plane of the substrate is approximately parallel to gravity. As used herein, vertical refers to an orientation in which the plane of the substrate's major surface is closer to parallel to gravity than perpendicular to gravity. In the example illustrated in Figures 35, 36, 37, and 38, a magnetic loading system 3500 includes a plate 3502 and a magnet holder 3504 that guides a magnet along the plate 3502. In the illustrated example, the plate 3502 is fixed to a vertical structure 3514, which is fixed to a horizontal structure 3516. The magnet holder 3504 can move the magnet up and down along the plate 3502 to facilitate loading of bead supports, such as sequencing beads, into wells of a substrate disposed on the opposite side of the plate 3502.

[0136] In certain examples, the drive mechanism 3506 can facilitate up and down movement of the magnet holder 3504 along the plate 3502. For example, the drive mechanism 3506 can rotate a threaded screw 3518 to drive the connector plate 3510 up and down along the screw 3518. The connector plate 3510 is connected to the magnet holder 3504. Optionally, the connector plate 3510 can be coupled to a guide plate 3508. The guide plate 3508 can slide along rails 3512 to provide stability to the movement of the connector plate 3510 and the magnet holder 3504.

[0137] As illustrated in Figure 36, substrate holder 3620 (e.g., the manifold of Figure 26 or Figure 27) provides space 3622 for a substrate, or sensor device such as a microchip with a flow cell, to be inserted and held against plate 3502. As a magnet attached to holder 3504 moves up and down along the vertical surface of plate 3502, bead supports attached to magnetic beads in solution are deposited into the wells of the substrate. In an example, the substrate is a sequencing chip with a flow cell into which a solution is disposed.

[0138] 37, plate 3502 can optionally include a recess for receiving heater 3724. Heater 3724 can be utilized to control the temperature of plate 3502 and, optionally, a substrate positioned adjacent the surface of plate 3502. Alternatively, heater 3724 can be utilized to facilitate melting of double-stranded nucleic acids or to control the temperature of amplification.

[0139] The magnet holder 3504 can include one or more magnets. For example, as illustrated in Figure 38, the magnet holder 3504 can include a magnet 3828 and a magnet 3830. The magnets 3828 or 3830 can be separated by air. Alternatively, the magnets can be separated by a paramagnetic or insulating material.

[0140] In an example, the magnets are configured such that different poles of the magnets are positioned against plate 3502. For example, magnet 3828 may be configured with a north pole positioned adjacent plate 3502, and magnet 3830 can be configured with a south pole positioned adjacent plate 3502. Alternatively, the south pole of magnet 3828 and the north pole of magnet 3830 can be positioned adjacent plate 3502. In a further alternative, the same pole of each magnet can be positioned adjacent plate 3502.

[0141] The system may further include a sensor 3826 that detects the position, e.g., the lower boundary, of the magnet. As illustrated in Figure 38, the guide plate 3508 may interfere with the optical sensor 3826 when the magnet is in its lower position. Alternatively, other sensors may be used to determine the position of the plate and associated magnet.

[0142] After loading the beads into the wells of the sensor device or microchip, the polynucleotides on the sequencing beads can be amplified to form a monoclonal population of polynucleotides on the sequencing beads, which can be sequenced, for example, using ion-based sequencing techniques.

[0143] Multi-Lane Fluidics FIG. 39 illustrates an exploded view of a fluidic multiplexer block 39100, which can generally serve as a component of the fluidic system of an integrated next-generation sequencing system and provide distribution of various solutions used during an analysis to a multi-lane sensor array device. According to the present teachings, various embodiments of the fluidic systems disclosed herein are configured to perform a series of fluidic operations to sequentially deliver various solutions to a multi-lane sensor array device over the course of a next-generation sequencing analysis. Exemplary fluidic operations include washing, priming, and nucleotide reagent delivery through a fluidic multiplexer block, such as the fluidic multiplexer block 39100 of FIG. 39 . Such a fluidic multiplexer block is configured to provide independent fluid distribution to each lane of a multi-lane sensor array device used for detection during the analysis. According to the present teachings, any number or combination of lanes can be used during an analysis, such that a single lane at any location can be used alone during a run, all four lanes can be used simultaneously during a run, or any combination of lanes can be used simultaneously during a run. The use of a fluidic multiplexer block of the present teachings for fluid distribution to a multi-lane sensor array device during a series of fluidic operations can avoid cross-contamination of solutions used during an analysis in various fluid compartments and provide sharp transitions between reagent fluid streams during an analysis. Additionally, various embodiments of the fluidic system of the present teachings provide a constant electrolyte fluid environment for the reference electrode, thereby providing a constant and stable reference voltage for the multi-lane sensor array device.

[0144] As depicted in FIG. 39 , the fluidic multiplexer block 4100 includes fluidic multiplexer units 4200A-4200D and a first end cover 4105A and a second end cover 4105B. According to the present teachings, each fluidic multiplexer unit has fluidic multiplexer circuitry formed within the body of the fluidic multiplexer unit. Thus, as depicted in FIG. 39 , each of the fluidic multiplexer units 4200A-4200D has fluidic multiplexer circuitry 4215A-4215D formed within the body of the respective fluidic multiplexer unit. As disclosed in more detail herein, each fluidic multiplexer unit in the fluidic multiplexer block 4100 is in independently controllable fluidic communication with a respective one of the flow cell lanes of a multi-lane sensor array device. Thus, a first lane of a multi-lane sensor array device may be fluidically integrated with fluidic multiplexer unit 4200A, while a second lane may be fluidically integrated with fluidic multiplexer unit 4200B, a third lane may be fluidically integrated with fluidic multiplexer unit 4200C, while a fourth lane may be fluidically integrated with fluidic multiplexer unit 4200D. Moreover, any number or combination of lanes may be used during an analysis, such that during analysis setup, an end user may select one lane in any position to be used alone during a run, all four lanes to be used simultaneously during a run, or any combination of lanes to be used simultaneously during a run.

[0145] FIG. 40 generally illustrates one embodiment of a fluidic multiplexer unit 4200 that contains four input reagents and a calibration solution in each of five fluidic branches, as well as a distribution channel for a wash solution. The fluidic circuit 4215 is formed in a substrate 4205 having a first surface 4201 and an opposing second surface 4203. As depicted in FIG. 40, the first surface 4201 and the opposing second surface 4203 are substantially parallel to one another. The fluidic multiplexer unit 4200 can have a first fluidic interface side 4202 with an opposing second fluidic interface side 4204. As depicted in FIG. 40, a third fluidic interface side 4206 joins the first and second interface edges on one side, while a fourth fluidic interface side 4208 joins the first and second interface edges on the opposite side. The substrate 4205 can be constructed of a variety of materials, such as glass, ceramic, and plastic. Exemplary polymeric materials include polycarbonate, polymethyl methacrylate, polyetherimide, and polyimide. Reagent inlet ports 4210, 4216, 4222, and 4228 and calibration solution inlet port 4234 are in fluid communication with inlet channels 4211, 4217, 4223, 4229, and 4235, respectively. Inlet channels 4211, 4217, 4223, 4229 are in fluid communication with each of the five fluid branches' curved channels 4213, 4219, 4225, 4231, and 4235, respectively. Finally, wash solution inlet port 4240 is in fluid communication with wash solution channel 4242.

[0146] As depicted in Figure 40, each inlet channel forms a T-junction with each curved channel, such that each curved channel consists of two branches. Such a T-junction forming two branches is depicted in Figure 40, where inlet channel 4211 forms a T-shape with curved channel 4213, forming first branch channel 4212 and second branch channel 4214. Similarly, inlet channel 4217 forms a T-shape with curved channel 4219, forming first branch channel 4218 and second branch channel 4220, while inlet channel 4223 forms a T-shape with curved channel 4225, forming first branch channel 4224 and second branch channel 4226. Additionally, inlet channel 4229 forms a T-shape with curved channel 4231, forming first branch channel 4230 and second branch channel 4232. Finally, inlet channel 4235 forms a T-shape into curved channel 4237, forming first branch channel 4236 and second branch channel 4238. Of the five fluid branches, first branch channels 4212, 4218, 4224, 4230, and 4236 of curved channels 4213, 4219, 4225, 4231, and 4237, respectively, are in fluid communication with central channel 4250. As depicted in FIG. 40 , central channel 4250 is in fluid communication with sensor interface inlet connector port 4260, which is in fluid communication with a sensor inlet port (not shown). Additionally, wash solution inlet port 4240 is in fluid communication with wash solution channel 4242, which is also in fluid communication with sensor interface inlet connector port 4260. The sensor interface outlet connector port 4262 is in fluid communication with a sensor outlet port (not shown) as well as a sensor waste channel 4244. The sensor waste channel 4244 is in fluid communication with a sensor waste receptacle (not shown), which is connected to the fluid multiplexer unit 4200 through a sensor waste port outlet 4264.Each of the second branch channels 4214, 4220, 4226, 4232, and 4238 of the curved channels 4213, 4219, 4225, 4231, and 4237 is fluidly connected to a main waste channel 4246, which is fluidly connected to a main waste receptacle (not shown), which is connected to the fluid multiplexer unit 4200 through a main waste outlet port 4266.

[0147] FIG. 41 generally illustrates a schematic representation of the fluidic integration of a fluidic multiplexer unit 4200 of the fluidic multiplexer block 4100 of FIG. 39 with a multi-lane sensor device.

[0148] With regard to fluid delivery and control for performing various analyses on a multi-lane sensor array device, such as the sensor array device 10 of FIG. 41, the fluidic circuitry 4215 of the fluidic multiplexer unit 4200 may be in fluid communication with one flow cell lane of the sensor array device 10. For illustrative purposes, one fluidic multiplexer unit is shown fluidically integrated with one flow cell lane in FIG. 41. However, because each lane is fluidically integrated with one of the fluidic multiplexer units, such as the fluidic multiplexer units 4200A-4200D of FIG. 39, an end user may select any number or combination of lanes during an analysis. As a non-limiting example, a first flow cell lane, such as flow cell lane 4A of the sensor array device 10 of Figure 41, can be fluidically integrated with a first fluidic multiplexer, such as fluidic multiplexer unit 4200A of Figure 39, while a second flow cell lane, such as flow cell lane 4B of the sensor array device 10 of Figure 41, can be fluidically integrated with a second fluidic multiplexer, such as fluidic multiplexer unit 4200B of Figure 39. Similarly, a third flow cell lane, such as flow cell lane 4C of the sensor array device 10 of Figure 41, can be fluidically integrated with a third fluidic multiplexer, such as fluidic multiplexer unit 4200C of Figure 39, while a fourth flow cell lane, such as flow cell lane 4D of the sensor array device 10 of Figure 41, can be fluidically integrated with a fourth fluidic multiplexer, such as fluidic multiplexer unit 4200D of Figure 39. In that regard, what is described herein for purposes of illustration in FIG. 41 generally discloses how each fluidic multiplexer unit 41200 is fluidically integrated with each of the corresponding flow cell lanes of a multi-lane sensor device.

[0149] Thus, during analysis setup, an end user can select one lane at any location to be used alone during a run, all four lanes to be used simultaneously during a run, or any combination of lanes to be used simultaneously during a run. As disclosed in more detail below, the fluidic system of a sequencer apparatus of the present teachings can include multiple solution containers that provide various solutions for use during the analysis. For example, the various solutions can include various nucleotide reagents, calibration solutions, diluent (wash) solutions, and cleaning solutions used in the analysis. The various solutions used during the analysis can be in controllable fluid communication with any flow cell lane of the sensor array device 10 via a flow cell inlet, such as flow cell inlet 3A of flow cell lane 4A in FIG. 41. The fluidic system of a sequencer apparatus of the present teachings can include reagent fluid lines from each reagent container, which can be selectively fluidly connected to inlet channels of fluidic circuit 4215, such as inlet channels 4211, 4217, 4223, 4229, 4235, and 4242. Additionally, as depicted in Figure 41, each of the various solutions used in the course of an analysis may have their fluid flow controlled by valves, such as fluid line valves V1-V6, in each of the reagent fluid lines L1-L4, as well as in the calibration solution line L5 and wash solution line L6, respectively. Note that the calibration fluid line valve V5 is generally in a closed position except during a calibration sequence before a run is initiated to calibrate the sensor array selected for use in the run. Thus, the calibration fluid line valve V5 is closed during a sequencing run.

[0150] In conjunction with the controllable flow of various solutions used in the course of an analysis, the fluidic multiplexer unit 4200 of FIG. 41 can perform fluidic operations including, but not limited to, providing selected reagent delivery to flow cell lanes of the sensor array device 10, washing the fluidic multiplexer circuit 4215 and a flow cell, such as flow cell lane 4A of the sensor array device 10, and priming the fluidic multiplexer circuit 4215 with a selected reagent. Such fluidic operations can provide cross-contamination-free delivery of reagents to flow cells, such as flow cell lane 4A of FIG. 41, can provide sharp transitions between reagent fluid streams, and can provide a constant electrolyte fluid environment for a reference electrode 4275 shown in FIG. 41, which is in fluid communication with the central channel 4250 and thereby provides a constant, stable reference voltage for the sensor array device 10.

[0151] For example, the fluidic multiplexer unit 4200 of FIG. 41 can selectively provide fluid communication between any of the reagent fluid lines L1-L4 and the first flow cell inlet 3A of the first flow cell lane 4A, thereby providing selective reagent flow through the first flow cell lane 4A of the sensor array device 10. A non-limiting exemplary reagent solution fluid path of the present teachings is provided by a reagent delivery operation in which the wash solution fluid line valve V6 is in a closed state and one of the reagent fluid line valves V1-V4 is in an open state, provided that one of the selected reagents is in fluid communication with the fluidic multiplexer circuit 4215. Under such conditions, the selected reagent can flow through the fluidic multiplexer circuit 4215 and then through the waste channel 4246 to waste. Additionally, the selected reagent can flow through the fluid multiplexer circuit 4215 to the first flow cell inlet 3A of the first flow cell lane 4A, then through the first flow cell lane 4A to the first outlet port 5A, and finally through the flow cell outlet line (see Figure 40) to the flow cell waste container.

[0152] With regard to fluidic control of the wash solution, the fluidic multiplexer unit 4200 of FIG. 41 can selectively provide fluidic communication between the wash solution and the first flow cell lane 4A. Thus, with wash solution line valve V6 in an open state, wash solution line L6 can be in fluidic communication with the fluidic multiplexer waste channel 4246, as well as the flow cell waste channel (see FIG. 39), provided that washing of the fluidic multiplexer circuit 4215 and the first flow cell lane 4A occurs. A non-limiting exemplary wash solution fluid path of the present teachings is provided by a wash operation in which wash solution fluid line valve V6 is in an open state and each of the reagent fluid line valves V1-V4 is in a closed state, provided that wash solution can flow through wash solution fluid channel 4242 to the T-junction with the central channel 4250. Central channel 4250 is in fluid communication with waste channel 4246 through fluidic multiplexer circuit 4215 so that wash solution can flow to the fluidic multiplexer waste through waste channel 4246. As disclosed in more detail herein, wash solution can flow from first inlet port 3A through first flow cell lane 4A to first outlet port 5A and then to a flow cell waste container.

[0153] According to the present teachings, priming of the fluidic multiplexer circuit 4215 of the fluidic multiplexer unit 4200 can be performed sequentially with selected reagents, for example, after a wash operation and before the selected reagents are brought into fluid communication with the first flow cell lane 4A of FIG. 41 . A non-limiting example illustrating reagent priming is provided by a reagent priming operation in which solution fluid line valve V6 is in an open state and one of the reagent fluid line valves V1-V4 is in an open state, provided that one of the selected reagents is in fluid communication with the fluidic multiplexer circuit 4215 of the fluidic multiplexer unit 4200. Under such operation, the flow rate of the wash solution relative to the flow rate of the reagent is selected such that the wash solution flows through the wash channel 4242 and through the first flow cell lane 4A of the sensor array device 10 to chip waste. Under such conditions, the selected reagent circulates through the fluidic multiplexer circuit 4215 because it is blocked from flowing through the sensor array device 10 by the flow of wash solution through the device. Thus, the selected reagent flows through fluidic multiplexer circuit 4215 and to the main waste through waste channel 4246. Thus, when a reagent delivery operation as previously described herein is initiated, the reagent selected in the reagent priming operation is in direct flow communication with the first flow cell inlet 3A.

[0154] Thus, various embodiments of the fluidic system of the present teachings are configured to perform a series of operations for sequentially delivering various solutions to a sensor array device during a next-generation sequencing analysis. For example, the series of operations can include washing, priming, and delivering nucleotide reagents to the sensor array device through a fluidic multiplexer block unit, as depicted in FIG. 41 . Using the fluidic multiplexer block of the present teachings for fluid distribution to the sensor array device during a series of fluidic operations can avoid cross-contamination of reagents in various fluidic compartments and provide sharp transitions between reagent fluid streams. Additionally, as disclosed in more detail herein, various embodiments of the fluidic system of the present teachings provide a constant electrolyte fluid environment for the reference electrode, thereby providing a constant and stable reference voltage for the sensor array device.

[0155] FIG. 42 is a rear isometric view generally illustrating the fluid multiplexer block clamp assembly 4150. As depicted in FIG. 42, the fluid multiplexer block clamp assembly 4150 includes a fluid multiplexer block clamp 4400 with the fluid multiplexer block assembly 4110 mounted therein. The fluid multiplexer block clamp 4400 may include an electrode connection mounting plate 4410 mounted on a side 4342 of the electrode adapter fluid interface block 4340. The electrode connection mounting plate 4410 allows for connection of an electrical lead 4412 and a ground lead 4414 to the electrode adapter fluid interface block 4340 of the fluid multiplexer block clamp assembly 4150. The fluid multiplexer block clamp 4400 also includes shoulder screws 4420, 4422, and 4424, as well as a fourth shoulder screw disposed below the shoulder screw 4424 and the opposite shoulder screw 4422. The force applied to the shoulder screws of the fluid multiplexer block clamp 4400 is set to provide 4 degrees of movement to the fluid multiplexer block attached to the fluid multiplexer block clamp 4400 to provide flexibility for docking the fluid multiplexer block to a multi-lane sensor array device.

[0156] 42 , with respect to the fluid multiplexer block assembly 4110 attached to the fluid multiplexer block clamp 4400, the orientation of the fluid multiplexer block 4100 and fluidic block connections shows the fluid interface block 4312 attached to the fluid multiplexer block 4100 at the top of the fluid multiplexer block clamp assembly 4150, while the fluid interface block 4302 is attached to the fluid multiplexer block 4100 at the bottom of the fluid multiplexer block clamp assembly 4150. As depicted in FIG. 42 , the orientation of the first and second flexible tubing sets 4314 and 4316 is such that they similarly protrude from the top of the fluid multiplexer block clamp assembly 4150, while the first and second flexible tubing sets 4304 and 4306 similarly protrude from the bottom of the fluid multiplexer block clamp assembly 4150. Similarly, the fluid interface block 4332 is attached to the fluid multiplexer block 4100 at the back of the fluid multiplexer block clamp assembly 4150 and below the electrode adapter fluid interface block 4340. As depicted in FIG. 42 , the orientation of the first and second flexible tubing sets 4334 and 4336 is such that they similarly protrude from the back of the fluid multiplexer block clamp assembly 4150. Finally, the fluid interface block 4322 is attached to the fluid multiplexer block 4100 at the back of the fluid multiplexer block clamp assembly 4150 and above the electrode adapter fluid interface block 4340. As depicted in FIG. 42 , the orientation of the first and second flexible tubing sets 4324 and 4326 is such that they similarly protrude from the top of the electrode adapter fluid interface block 4340.

[0157] 43 is a cross-sectional view generally illustrating the orientation of the fluidic multiplexer block 4100 when mounted in a fluidic multiplexer block clamp assembly, as well as the integration of electrodes into the fluidic multiplexer unit. A first face 4102 of the fluidic multiplexer block is depicted with a fluidic interface block 4302 mounted on the first face 4102 and first and second flexible tubing sets 4304 and 4306 connected to the fluidic interface block 4302, while a second face 4104 of the fluidic multiplexer block is depicted with a fluidic interface block 4312 mounted on the second face 4104 and first and second flexible tubing sets 4314 and 4316 connected to the fluidic interface block 4312. Similarly, the third side 4106 of the fluidic multiplexer block is depicted with a fluidic interface block 4332 mounted on the third side 4106 and first and second flexible tubing sets 4334 and 4336 connected to the fluidic interface block 4332. Additionally, the third side 4106 of the fluidic multiplexer block is depicted with an electrode adaptor fluidic interface block 4340 mounted on the third side 4106. As depicted in FIG. 43 , the fluidic interface block 4322 is mounted to the electrode adaptor fluidic interface block 4340 such that the first and second flexible tubing sets 4324 and 4326 connected to the fluidic interface block 4332 are in fluid communication with the electrode adaptor fluidic interface block inlet channels 4342 and 4344, respectively. In that regard, the electrode adapter fluid interface block 4340 is attached to the third face 4106 of the fluid multiplexer block such that the electrode adapter fluid interface block inlet channels 4342 and 4344 are coupled to and sealed to the sensor waste outlet port 4264 and the washing solution inlet port 4240, respectively.43, the fourth side 4108 of the fluidic multiplexer block has a sensor interface inlet connector port 4260 and a sensor interface outlet connector port 4262. As disclosed earlier herein, the fourth side 4108 of the fluidic multiplexer block has a corresponding set of sensor interface inlet connector ports and sensor interface outlet connector ports for each fluidic multiplexer unit of the fluidic multiplexer block 4100. As disclosed in more detail later herein, the sensor interface inlet connector port 4260 and the sensor interface outlet connector port 4262 are coupled to and sealed to the inlet and outlet ports, respectively, of the multi-lane sensor device.

[0158] With regard to providing electrode connections to each fluidic multiplexer unit that provide a constant, stable reference electrode voltage for the multi-lane sensor array device, FIG. 43 depicts a cross-sectional view of an electrode adapter fluidic interface block 4340 with an electrode connection mounting plate 4410 mounted thereon. FIG. 43 depicts an electrode 4275 in an enlarged bore in a section of the electrode adapter fluidic interface block inlet channel 4344, which provides fluid passage through the electrode adapter fluidic interface block inlet channel 4344 in fluid communication with the wash solution channel 4242. The electrode 4275 is electrically coupled to a voltage source connected to the electrode connection mounting plate 4410 through an electrical lead 4412 and a ground lead 4414 (see FIG. 42 ). As previously disclosed herein, the second flexible tubing set 4326 is in fluid communication with a source of wash solution of stable electrolyte composition. Thus, the electrode 4275 is in a fluidic environment that provides a constant, stable reference electrode voltage for the multi-lane sensor array device.

[0159] 44 is a front isometric view generally illustrating a fluidic multiplexer block clamp assembly 4150 including a fluidic multiplexer block clamp 4400 with a fluidic multiplexer block assembly 4110 mounted therein. As depicted in FIG. 44 , a fourth face 4108 of the fluidic multiplexer block of the fluidic multiplexer block 4100 has sensor interface inlet connector ports 4260A-4260D and sensor interface outlet connector ports 4262A-4262D for each of the fluidic multiplexer units 4200A, 4200B, 4200C, and 4200D, respectively. A first alignment notch 4107A of the first fluidic manifold unit 4200A and a second alignment notch 4107B of the fourth fluidic manifold unit 4200D are configured to assist in the alignment and sealing process of the fluidic multiplexer block clamp assembly 4150 to the multi-lane sensor array device. As previously disclosed herein, fluid multiplexer block clamp 4400 provides four degrees of movement to a fluid multiplexer block attached to fluid multiplexer block clamp 4400 to provide flexibility for docking the fluid multiplexer block to the multi-lane sensor array device. Additionally, first alignment notch 4107A and second alignment notch 4107B are configured to provide self-alignment of the multi-lane sensor array device to the multi-lane sensor array device, thereby enabling sealing of sensor interface inlet connector ports and sensor interface outlet connector ports, such as sensor interface inlet connector ports 4260A-4260D and sensor interface outlet connector ports 4262A-4262D, to the respective inlet and outlet ports of the multi-lane sensor array device.

[0160] FIG. 45 is a cross-sectional view generally illustrating a fluidic multiplexer block assembly 4110 mounted to a multi-lane sensor device 10, such as the multi-lane sensor device 10 depicted schematically in FIG. 41. As depicted in FIG. 45, the multi-lane sensor device 10 is mounted to a sensor device mounting and positioning assembly 4450. The locations of the fluidic interface blocks 4302, 4312, 4322, and 4332 of the fluidic multiplexer block assembly 4110 are also apparent in the cross-sectional view of FIG. 45. When the fluidic multiplexer block assembly 4110 is mounted to the multi-lane sensor device 10, each sensor interface inlet connector port and each sensor interface outlet connector port are coupled and sealed to a corresponding sensor array inlet port and each sensor array outlet port, respectively, for each lane of the multi-lane sensor array device. In that regard, FIG. 46 is an enlarged isometric view generally illustrating the mounting and sealing of the fluidic multiplexer block to the multi-lane sensor array. As depicted in Figure 46, the sensor array device 10 has lanes 4A-4D, each having an inlet port and an outlet port, as illustrated for lane 4A, which has inlet port 3A and outlet port 5A. In Figure 46, the juxtaposition of the first alignment pin 12A of the sensor array device 10 with the first alignment notch 4107A of the fluidic multiplexer block 4100 shows how the complementary pair engages for alignment of the sensor array device 10 and the fluidic multiplexer block 4100. In addition, Figure 46 shows how each inlet port and each outlet port of the sensor array device 10 can be coupled to and sealed to a corresponding sensor interface inlet connector port and each sensor interface outlet connector port of the fluidic multiplexer block 4100.In Figure 46, this is illustrated for lane 4A, where the juxtaposition of the first inlet port 3A to the sensor interface inlet connector port 4260A and the first outlet port 5A to the sensor interface outlet connector port 4262A can each be coupled and sealed to the sensor array device 10 once the fluid multiplexer blocks 4100 are fully engaged with each other.

[0161] 47 is a schematic representation generally illustrating a fluidic system 41000 of a sequencing system of the present teachings. As depicted in FIG. 47, the fluidic system 41000 has a pneumatic control system 4500 and a liquid handling control system, such as a solution handling manifold 4600 and a reagent distribution manifold assembly 4700.

[0162] In that regard, pneumatic control system 4500 is in fluid communication with a first wash solution container 4520 through a first pneumatic inlet line controlled via valve 4501, a second wash solution container 4522 through a second pneumatic inlet line 4504 controlled via valve 4503, and a cleaning solution container 4524 through a third pneumatic inlet line 4506 controlled via valve 4505. Similarly, pneumatic control system 4500 is in fluid communication with a reagent container assembly 4670 through a fourth pneumatic inlet line 4508 controlled via valve 4507. With respect to fluidic system control, pneumatic control system 4500 is in fluid communication with a solution processing manifold 4600 through a fifth pneumatic inlet line 4510 controlled via valve 4509. Finally, the pneumatic control system 4500 is in fluid communication with the pinch manifold 4800 via a sixth pneumatic inlet line 4512, which is controlled via a valve 4511. As disclosed in more detail herein, the pneumatic control system 4500 and the pinch manifold 4800, in combination with the flow sensor 4610, provide system regulation and control between the solution input sources and the waste. In accordance with the present teachings, the fluidic system regulation and control of the fluidic system 41000 provides a defined, controllable pressure differential between the solution input sources, such as the first wash solution container 4520, the second wash solution container 4522, the cleaning solution container 4524, and the reagent container assembly 4670, and the waste container 4550. Thus, the defined and controlled pressure differential provides a defined and controlled flow rate of the various solutions used in the course of an analysis through the various liquid circuits of the fluidic system 41000. Flow rates may include approximately 15 μl / sec for single-lane chip flow, 45 μl / sec for single-lane chip and main waste flow, 180 μl / sec for full chip and main waste flow, or greater than 300 μl / sec during system cleaning operations.

[0163] Solution processing manifold 4600 provides control of the distribution of various solutions used for cleaning and filling. As depicted in FIG. 47, solution processing manifold 4600 has solution processing manifold lines 4620 in fluid communication with flow sensors 4610. In accordance with the present teachings, flow sensors 4610 provide dynamic input to pneumatic control system 4500 to calibrate defined flow rates of the various liquid circuits of fluid system 41000 using pinch manifold 4800 to provide defined volumetric flows when filling nucleotide containers. With respect to the liquid input source, the first cleaning solution container 4520 is fluidly connected to the solution processing manifold 4600 through the first cleaning solution outlet line 4530, while the second cleaning solution container 4522 is fluidly connected to the solution processing manifold 4600 via the second cleaning solution outlet line 4532, and the cleaning solution container 4524 is fluidly connected to the solution processing manifold 4600 through the calibration solution outlet line 4534.

[0164] To provide sufficient reagent volume for the massively parallel processing performed during next-generation sequencing, the fluidics system 41000 is configured to provide significant volumes of various solutions used in the course of analysis. In that regard, the reagent concentrate cartridge 4660 is in fluid communication with the wash solution containers 4520 and 4522. As used herein, a wash solution is an aqueous-based solution of stable electrolyte composition that can be used as a solvent in the preparation of calibration solutions and sequencing reagents for wash operations as previously described herein, as well as to continuously refresh the electrolyte solution around the reference electrode. The reagent concentrate cartridge 4660 can include a calibration solution concentrate container 4661, while the remaining concentrate is a nucleotide reagent concentrate. For example, the first nucleotide reagent concentrate container 4663 can contain a deoxyguanosine triphosphate (dGTP) reagent concentrate, while the second nucleotide reagent concentrate container 4665 can contain a deoxycytidine triphosphate (dCTP) reagent concentrate, the third nucleotide reagent concentrate container 4667 (e.g., the cartridge of FIG. 11 ) can contain a deoxyadenosine triphosphate (dATP) reagent concentrate, while the fourth nucleotide reagent concentrate container 4669 can contain a deoxythymidine triphosphate (dTTP) reagent concentrate. The reagent concentrate cartridge 4660 is also in fluid communication with a reagent container assembly 4670. Each container of the reagent container assembly 4670 holds a significant volume of calibration solution and dNTP reagents used in the high-throughput next-generation sequencing-by-synthesis analysis system of the present teachings. Each reagent container can have a capacity of approximately 225 ml to support a dilution solution volume of approximately 150 ml. In preparing bulk calibration solutions and bulk nucleotide reagents, the concentrated reagents are diluted approximately 100-fold when mixed in the reagent container.

[0165] Bulk preparation of calibration solutions and nucleotide reagents can be performed before the start of a sequencing run. For bulk preparation of calibration solutions, with valves 4602 and 4621 of solution processing manifold 4600 open and all other solution processing manifold valves closed, wash solution in wash solution container 4520 can flow through wash solution outlet line 4530 into solution processing manifold line 4620, through calibration concentrate line 4631 into calibration solution concentrate container 4661, and then through calibration solution inlet line 4641 into calibration solution container 4671. Wash solution can continue to flow through and into calibration solution concentrate container 4661 for a predetermined fill volume of calibration solution container 4671, at which point calibration solution concentrate container 4661 is effectively depleted of calibration solution concentrate.

[0166] Next, bulk preparation of the first nucleotide solution can be performed by opening valves 4602 and 4623 of solution processing manifold 4600 and closing all other solution processing manifold valves, so that the washing solution in washing solution container 4520 can flow into solution processing manifold line 4620, through first nucleotide reagent concentrate line 4633 into first nucleotide reagent concentrate container 4663, and then through first nucleotide reagent inlet line 4643 into first nucleotide reagent container 4673 until first nucleotide reagent container 4673 is filled.

[0167] After bulk preparation of the first nucleotide solution is completed, bulk preparation of the second nucleotide solution can be performed by opening valves 4602 and 4625 of the solution processing manifold 4600 and closing all other solution processing manifold valves, so that the washing solution in the washing solution container 4520 can flow through the washing solution outlet line 4530 into the solution processing manifold line 4620, through the second nucleotide reagent concentrate line 4635 into the second nucleotide reagent concentrate container 4665, and then through the second nucleotide reagent inlet line 4645 into the second nucleotide reagent container 4675 until the second nucleotide reagent container 4675 is filled.

[0168] Following bulk preparation of the second nucleotide reagent, bulk preparation of a third nucleotide solution can be performed by opening valves 4602 and 4627 of solution processing manifold 4600 and closing all other solution processing manifold valves, so that the washing solution in washing solution container 4520 can flow through washing solution outlet line 4530 into solution processing manifold line 4620, through third nucleotide reagent concentrate line 4637 into third nucleotide reagent concentrate container 4667, and then through third nucleotide reagent inlet line 4647 into third nucleotide reagent container 4677 until third nucleotide reagent container 4677 is filled.

[0169] Finally, bulk preparation of the fourth nucleotide solution can be performed by opening valves 4602 and 4629 of the solution processing manifold 4600 and closing all other solution processing manifold valves, so that the washing solution in the washing solution container 4520 can flow through the washing solution outlet line 4530 into the solution processing manifold line 4620, through the fourth nucleotide reagent concentrate line 4639 into the fourth nucleotide reagent concentrate container 4669, and then through the fourth nucleotide reagent inlet line 4649 into the fourth nucleotide reagent container 4679 until the fourth nucleotide reagent container 4679 is filled.

[0170] With sufficient calibration solutions and nucleotide reagents prepared for a high-throughput next-generation sequencing-by-synthesis run, the reagent distribution manifold assembly 4700 can control the distribution of various solutions in the reagent container assembly 4670 to the sensor array device 10 through the fluid multiplexer block 4100 and ultimately to waste 4530 through the chip waste line 4324 or main waste line 4334 (see, e.g., Figure 43).

[0171] 47, the reagent distribution manifold assembly 4700 can include a reagent distribution manifold 4702, a reagent distribution manifold 4712, and a reagent distribution manifold 4722. The heater block 4750 of FIG. 47 can contact the flexible tubing sets 4304, 4306, 4314, 4316, 4326, and 4336 to ensure uniform temperatures of the solutions and reagents before flowing through the fluidic multiplexer block 4100 and the sensor array device 10.

[0172] The reagent distribution manifold 4702 can have a first set of valves in a valve block 4704 that can individually control fluid communication between the solution process manifold lines 4620 and the flexible tubing set 4326. As depicted for the fluidic multiplexer block assembly 43110 in FIG. 43, each tube of the flexible tubing set 4326 is connected to a corresponding wash solution inlet port of a corresponding fluidic multiplexer unit, such as wash solution inlet port 40240 of the fluidic multiplexer unit 40200 in FIG. 40. In addition, the reagent distribution manifold 4702 can have a second set of valves in a valve block 4706 that can individually control fluid communication between the calibrator solution line outlets 4651 and the flexible tubing set 4336. As depicted for the fluid multiplexer block assembly 4110 in FIG. 43, each tube of the flexible tubing set 4336 is connected to a corresponding calibration solution inlet port of a corresponding fluid multiplexer unit, such as the calibration solution inlet port 4234 of the fluid multiplexer unit 4200 in FIG. 40.

[0173] To provide a flow of wash solution through one or more selected lanes of the multilane sensor array device, either valve 4602 or 4604 of solution processing manifold 4600 can be opened while all other valves of solution processing manifold 4600 are closed. Either one valve, all four valves, or any combination of valves in valve block 4704 of reagent distribution manifold 4702 can be opened so that wash solution from reservoirs 4520 and 4522 flows through either wash solution outlet line 4530 or 4532, respectively, into solution processing manifold line 4620 and can be distributed by the corresponding fluid multiplexer block unit to one lane, all four lanes, or any combination of lanes, depending on the selection of valves in valve block 4704. To provide a flow of calibration solution through one or more selected lanes of the multilane sensor array device, all valves of solution processing manifold 4600 are closed. Either one valve, all four valves, or any combination of valves in the valve block 4706 of the reagent distribution manifold 4702 can be opened so that the calibration solution from the calibration solution container 4671 flows into the calibration solution line outlet 4651 and can be distributed by the corresponding fluid multiplexer block unit to one lane, all four lanes, or any combination of lanes depending on the selection of the valves in the valve block 4706.

[0174] The reagent distribution manifold 4712 can have a first set of valves in a valve block 4714 that can individually control fluid communication between the first nucleotide reagent outlet line 4653 and the flexible tubing set 4304. As depicted for the fluidic multiplexer block assembly 4110 in FIG. 43, each tube in the flexible tubing set 4304 is connected to a corresponding first nucleotide reagent inlet port of a corresponding fluidic multiplexer unit, such as first nucleotide reagent inlet port 4210 of the fluidic multiplexer unit 4200 in FIG. 40. In addition, the reagent distribution manifold 4712 can have a second set of valves in a valve block 4716 that can individually control fluid communication between the second nucleotide reagent outlet line 4655 and the flexible tubing set 4306. As depicted for the fluid multiplexer block assembly 4110 in FIG. 43, each tube of the flexible tubing set 4306 is connected to a corresponding second nucleotide reagent inlet port of a corresponding fluid multiplexer unit, such as the second nucleotide reagent inlet port 4216 of the fluid multiplexer unit 4200 in FIG. 40.

[0175] All valves of solution handling manifold 4600 are closed to provide flow of the first nucleotide reagent through one or more selected lanes of the multilane sensor array device. Either one valve, all four valves, or any combination of valves in valve block 4714 of distribution manifold 4712 can be opened so that the first nucleotide reagent from first nucleotide reagent container 4673 flows into first nucleotide reagent outlet line 4653 and can be distributed by the corresponding fluidic multiplexer block unit to one lane, all four lanes, or any combination of lanes, depending on the selection of valves in valve block 4714. All valves of solution handling manifold 4600 are closed to provide flow of the second nucleotide reagent through one or more selected lanes of the multilane sensor array device. Either one valve, all four valves, or any combination of valves in valve block 4716 of distribution manifold 4712 can be opened so that second nucleotide reagent from second nucleotide reagent container 4675 flows into second nucleotide reagent outlet line 4655 and can be distributed by the corresponding fluid multiplexer block unit to one lane, all four lanes, or any combination of lanes depending on the selection of valves in valve block 4716.

[0176] The reagent distribution manifold 4722 can have a first set of valves in a valve block 4724 that can individually control fluid communication between the third nucleotide reagent outlet line 4657 and the flexible tubing set 4314. As depicted for the fluidic multiplexer block assembly 4110 in FIG. 43, each tube in the flexible tubing set 4314 is connected to a corresponding third nucleotide reagent inlet port of a corresponding fluidic multiplexer unit, such as third nucleotide reagent inlet port 40222 of the fluidic multiplexer unit 40200 in FIG. 40. In addition, the reagent distribution manifold 4722 can have a second set of valves in a valve block 4726 that can individually control fluid communication between the fourth nucleotide reagent outlet line 4659 and the flexible tubing set 4316. As depicted for the fluid multiplexer block assembly 4110 in FIG. 43, each tube of the flexible tubing set 4316 is connected to a corresponding fourth nucleotide reagent inlet port of a corresponding fluid multiplexer unit, such as the fourth nucleotide reagent inlet port 4228 of the fluid multiplexer unit 4200 in FIG. 40.

[0177] All valves of solution handling manifold 4600 are closed to provide flow of the third nucleotide reagent through one or more selected lanes of the multilane sensor array device. Either one valve, all four valves, or any combination of valves in valve block 4724 of distribution manifold 4722 can be opened so that third nucleotide reagent from third nucleotide reagent container 4677 flows into third nucleotide reagent outlet line 4657 and can be distributed by the corresponding fluidic multiplexer block unit to one lane, all four lanes, or any combination of lanes, depending on the selection of valves in valve block 4724. All valves of solution handling manifold 4600 are closed to provide flow of the fourth nucleotide reagent through one or more selected lanes of the multilane sensor array device. Either one valve, all four valves, or any combination of valves in valve block 4726 of distribution manifold 4722 can be opened so that fourth nucleotide reagent from fourth nucleotide reagent container 4679 flows into fourth nucleotide reagent outlet line 4659 and can be distributed by the corresponding fluid multiplexer block unit to one lane, all four lanes, or any combination of lanes depending on the selection of the valves in valve block 4726.

[0178] A scheduled cleaning of all fluidic components of the fluidic system 41000 can be performed. Such cleaning is typically performed after all four lanes of a chip have been sequenced or before a new chip is installed in the system. Cleaning can be performed with the depleted reagent concentration cartridge and the used multilane sensor array device in place. When valve 4606 is opened, each of valves 4623-4629 in the solution handling manifold 4600 can be sequentially opened, which can then open all of the valves in the set of valves in the corresponding valve block of the distribution manifold assembly 4700. As previously described, with such sequential flow paths for the calibration solution and each of the nucleotide reagents, cleaning solution from the cleaning solution reservoir 4524 can flow sequentially through every fluidic component of the fluidic system 41000 and into the waste reservoir 4550. Finally, a drying procedure is performed to prepare the system for its next use. In the drying procedure, valves 4602, 4604, and 4606 are closed, all other valves in the solution handling manifold 4600 are open, and all valves in the reagent distribution manifold assembly 4700 are open. In that configuration, clean, dry air is passed through the liquid handling components of the fluidic system 41000, driving any remaining liquid into the waste container 4550.

[0179] As previously disclosed herein, the pneumatic control system 4500 and pinch manifold 4800, in combination with the flow sensor 4610, provide system regulation and control between the solution input source and the waste. The pinch manifold 4800 includes eight pinch regulators, each constructed as described in U.S. Pat. No. 9,375,716. These devices essentially operate as three-port pressure followers having an input fluid port, an output fluid port, and a control air pressure port. When the waste line fluid resistance defined between the pinch regulator output fluid port and the waste container 4550 is connected, the pressure at the output fluid port will approximately equal the pressure at the pinch regulator control air pressure port, regardless of the pressure at the input fluid port. The flow rate through the pinch regulator is equal to the output fluid port pressure divided by the waste line fluid resistance. To accurately calibrate the flow rate, the fluid control system 41000 is configured to allow cleaning solution to flow to the desired pinch regulator on the pinch manifold 4800. A set of known air pressures is applied to the air control port of each pinch regulator, and flow sensor 4610 measures the exact flow rate corresponding to the air control pressure. A table of flow rate versus air control pressure for each pinch regulator is then stored and can be utilized by the device software to accurately deliver any desired flow rate.

[0180] FIG. 48 includes an illustration of an exemplary electronic interface 4802 for interfacing with a sensor device. For example, the electronic interface 4802 can include pins for interacting with the electronic interface of the sensor device. Optionally, the interface 4802 can include a mechanism 4804 for disengaging the sensor device from the electronic interface 4802. For example, when a fluid manifold is pressed against the sensor device (as illustrated in FIG. 46 ), the mechanism 4804 can be depressed. When the fluid manifold is disconnected from the sensor apparatus, the mechanism 4804 can push the sensor device away from the electronic interface 4802. Optionally, the system can further include a mechanism for controlling temperature, such as a heat sink 4806. The heat sink 4806 can include fins. Alternatively, the heat sink 4806 can be a liquid-cooled heat sink.

[0181] In particular, a fluidics system and electronic interface are used to detect nucleotide incorporation during the sequencing-by-synthesis reaction, and data is collected and provided to a sequencing device server system.

[0182] Sequencer Software In some embodiments, the nucleic acid sequencing device can be interfaced with a server system for controlling various components of the sequencing device and processing data output from sequencing runs on the sequencing device. The server system software can include web applications, databases, and analysis pipelines to support connections from the sequencing device (e.g., Figure 6). The server system software can provide the following major functionalities and application program interfaces (APIs):

[0183] 1. APIs for user authentication, reagent tracking, run information, and run tracking / logging. Supported devices may include sequencing and extraction devices.

[0184] 2. API for LIMS (Laboratory Information Management System) to create samples, libraries, plan runs and retrieve plan run status.

[0185] 3. Support for sample and run data management.

[0186] 4. Support for assay configuration and execution of analytical pipelines for data analysis and reporting.

[0187] 5. Interface to a software update server for software updates and maintenance.

[0188] 6. Supports configuration to connect to annotation and reporting systems, such as Thermo Fisher Scientific's Ion Reporter, deployed in cloud-based systems or local systems, and establishes a secure, authenticated connection with the cloud-based system to transfer mapped or unmapped BAM files.

[0189] 7. Supports configuration for connecting to resource systems in cloud computing environments such as Thermo Fisher Cloud, and establishes a secure, authenticated connection with the cloud resource system to download software and system content and transmit telemetry data.

[0190] Figure 49 shows a schematic diagram of the server system components. In some embodiments, the basic software architecture may include a web interface, a remote monitoring agent, a database, an API to the device, an analysis pipeline, containerization of the analysis pipeline (e.g., using Docker), an annotation and reporting system (e.g., Ion Reporter from Thermo Fisher Scientific), and connectivity to a cloud-based support and resource system (e.g., Thermo Fisher Cloud). The cloud-based support and resource system may be implemented in a cloud computing and storage system. The cloud-based support and resource system stores content including assay definition files. A server of the cloud computing and storage system may download content such as assay definition files to a local server system. The cloud-based support and resource system may receive telemetry data from the local server system. In this specification, the terms server system, local server system, and user's server system are used interchangeably.

[0191] In some embodiments, the user interface (UI) may be implemented via web application software. The UI may provide a sample management page. The sample management UI page allows users to input sample information into the system. Sample information includes a unique sample identifier (ID), sample name, and sample preparation reagent tracking information. Validation logic is built into the sample management flow that locks the sample preparation steps to a predefined assay workflow. The UI may provide an assay management page. The assay management UI page allows users to view and create assays. The assays lock the workflow to predefined parameters for each step of the process. Validation logic may be built into the assay configuration to ensure assay configuration. The UI may provide a run planning and monitoring page. The run planning and monitoring UI page allows users to plan runs and monitor ongoing runs. The UI may provide an output data page. The output data UI page allows users to view analytical results along with quality control (QC) metric evaluations, logs, and audit trails of the generated results. The UI may provide a configuration page. The configuration UI page allows users to view and configure the system.

[0192] In some embodiments, application programming interfaces (APIs) may be provided through a Java platform, which may include, for example, a Tomcat server that may be used to build Web ARchive (WAR) files for web-based applications.

[0193] The code modules for the various steps of an analysis pipeline may be referred to as actors in the context of the Kepler workflow engine. For example, the code modules for an analysis step may be implemented by the binary code of a Java program contained in an actor jar. The Kepler workflow engine defines the processing components of a workflow as "actors" and chains together the steps of an algorithm or analysis pipeline execution by a processor. (https: / / kepler-project.org) For example, the Kepler workflow engine may be used to compose the workflow of the analysis pipeline in Figure 49.

[0194] The server system may include one or more databases. For example, the server system may include a relational database for storing sample data, run data, and system / user configuration. The relational database may include two separate databases: an assay development database and a Dx database. The assay development database may store sample data, run data, and system / user configuration for the RUO mode of operation, i.e., the assay development mode of operation. The Dx database may store sample data, run data, and system / user configuration for the IVD mode of operation, i.e., the Dx mode of operation.

[0195] The server system may include an annotation database, AnnotationDB, for storing annotation source data. For example, the annotation database may be implemented as a NoSQL database or a non-relational database, such as a MongoDB database. Each annotation source may be stored as a JSON (JavaScript Object Notation) string with meta information indicating the source name and version. Each annotation source may include a list of annotations keyed to an annotation ID. The server system may include a variome database, VariomeDB, for storing variant information. For example, the variome database may be implemented as a NoSQL database or a non-relational database, such as a MongoDB database. VariomeDB may store a collection of variant call results for a particular sample. For example, a JSON-formatted record may include meta information for identifying the sample.

[0196] For example, the AnnotationDB database may store one or more of the following annotation sources:

[0197] 1.RefGene model: hg19_refgene_63, version 63

[0198] 2. RefGene Functional Canonical Transcript Scores: hg19_refgeneScores_4, Version 4

[0199] 3.dbSNP:dbsnp_138, version 138

[0200] 4. Canonical RefSeq transcript: hg19_refgene_63, version 63

[0201] 5.5000Exomes:hg_esp6500_1, version 1

[0202] 6. ClinVar: clinvar_1, version 1

[0203] 7.DGV:dgv_20130723, version 20130723

[0204] 8. OMIM:omim_03022014, version 03022014

[0205] Other annotation sources may be included. Other versions of the above annotation sources may be included. Annotation sources may provide public or proprietary annotation information content.

[0206] For each call in the variome database, each annotation source can be queried for annotations that match the variant, and the matching annotations can be stored in the variome database as key-value pairs along with the variant. The annotated variants can be included in a user's results file, such as an annotated VCF file. A VCF file is a tab-delimited text file used to store variants of gene sequences. In some embodiments, annotation methods for use with the present teachings can include one or more features described in U.S. Patent Application Publication No. 2016 / 0026753, published January 28, 2016, which is incorporated herein by reference in its entirety.

[0207] In some embodiments, the server system can include an analysis pipeline for processing sequencing data generated during a sequencing run of an assay performed by a sequencing device. The sequencer transfers sequencing data files and experiment log files to the server system memory, for example, raw .dat files, processed .dat files that generate block-based 1.wells files, and thumbnail data. The analysis pipeline accesses the data files from memory and begins data analysis of the run.

[0208] In some embodiments, Docker containers and Docker images may be used to package the analysis pipeline and operating system-specific binaries. Docker is a tool used to create, deploy, and run applications by using containers. Containers allow applications to have all the parts they need bundled together as one package, such as libraries and other dependencies. This allows the application software to use the same Linux kernel as the host system. A Docker image file may be packaged with the libraries and binaries required by the analysis pipeline code. Docker may be used to adapt an application or algorithm to newer or different versions of an operating system (OS) to create a Docker image for the application that is compatible with the OS version.

[0209] In some embodiments, the server system may include a crawler service for transferring data from the sequencing device to the analysis pipeline. The crawler is an event-based service that can be developed using the JAVA NIO watcher API (application programming interface). NIO (Non-Blocking I / O) is a collection of Java programming language APIs that provide features for intensive input / output (I / O) operations. The crawler may monitor an FTP directory configured for the sequencing device to transfer run data from the sequencing device to the analysis pipeline.

[0210] Figure 50 is a block diagram of an analysis pipeline, according to one embodiment. The sequencing device generates raw data files (.DAT or .dat files) during a sequencing run of an assay. Signal processing may be applied to the raw data to generate incorporation signal measurement data in files, such as 1.wells files, which are transferred to an FTP location on a server along with run log information. The signal processing step may derive background signals corresponding to the wells. The background signals may be subtracted from the measured signals of the corresponding wells. The remaining signals may be fitted by an incorporation signal model to estimate the incorporation of each nucleotide in each well at each flow. The output from the above signal processing is signal measurements per well and per flow, which may be stored in a file, such as the 1.wells file.

[0211] In some embodiments, the base calling step may perform phase estimation, normalization, and run a solver algorithm to identify the best partial sequence match and make the base call. The base sequences of the sequence reads are stored in an unmapped BAM file. The base calling step may generate the total number of reads, the total number of bases, and the average read length as QC measurements to indicate base call quality. Base calling may be performed by analyzing any suitable signal characteristics (e.g., signal amplitude or intensity). Signal processing methods for use with the present teachings may include one or more features described in U.S. Patent Application Publication No. 2013 / 0090860, published April 11, 2013; U.S. Patent Application Publication No. 2014 / 0051584, published February 20, 2014; and U.S. Patent Application Publication No. 2012 / 0109598, published May 3, 2012, each of which is incorporated herein by reference in its entirety.

[0212] Once the base sequence for the sequence read is determined, the sequence read can be provided to an alignment step, for example, in an unmapped BAM file. The alignment step maps the sequence read to a reference genome to determine aligned sequence reads and associated mapping quality parameters. The alignment step can generate a percentage of mappable reads as a QC indicator to indicate alignment quality. The alignment results are stored in a mapped BAM file. A method for aligning sequence reads for use with the present teachings can include one or more features described in U.S. Patent Application Publication No. 2012 / 0197623, published August 2, 2012, which is incorporated herein by reference in its entirety.

[0213] The structure of the BAM file format is described in the "Sequence Alignment / Map Format Specification" (https: / / github.com / samtools / hts-specs) dated September 12, 2014. As used herein, a "BAM file" refers to a file compatible with the BAM format. As used herein, an "unmapped" BAM file refers to a BAM file that does not contain aligned sequence read information or mapping quality parameters, and a "mapped" BAM file refers to a BAM file that contains aligned sequence read information and mapping quality parameters.

[0214] In some embodiments, the variant calling step may include detecting single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), multi-nucleotide polymorphisms (MNPs), and complex block substitution events. In various embodiments, the variant caller can be configured to communicate the called variants for a sample genome as *.vcf, *.gff, or *.hdf data files. Any file format can be used to communicate the called variant information, as long as it can be parsed or extracted for analysis. Variant detection methods for use with the present teachings may include one or more features described in U.S. Patent Application Publication No. 2013 / 0345066, published December 26, 2013; U.S. Patent Application Publication No. 2014 / 0296080, published October 2, 2014; U.S. Patent Application Publication No. 2014 / 0052381, published February 20, 2014; and U.S. Patent No. 9,953,130, issued April 24, 2018, each of which is incorporated herein by reference in its entirety. In some embodiments, the variant calling step may be applied to molecularly tagged nucleic acid sequence data. Variant detection methods for molecularly tagged nucleic acid sequence data may include one or more features described in U.S. Patent Application Publication No. 2018 / 0336316, published November 22, 2018, which is incorporated herein by reference in its entirety.

[0215] In some embodiments, the analysis pipeline may include a fusion analysis pipeline for fusion detection. The fusion detection method may include one or more features described in U.S. Patent Application Publication No. 2016 / 0019340, published January 21, 2016, which is incorporated herein by reference in its entirety. In some embodiments, the fusion analysis pipeline may be applied to molecularly tagged nucleic acid sequence data. The fusion detection method for molecularly tagged nucleic acid sequence data may include one or more features described in U.S. Patent Application Publication No. 2019 / 0087539, published March 21, 2019, which is incorporated herein by reference in its entirety.

[0216] In some embodiments, the analysis pipeline may include a copy number variation analysis pipeline for detecting copy number variations. Methods for detecting copy number variations may include one or more features described in U.S. Patent Application Publication No. 2014 / 0256571, published September 11, 2014, U.S. Patent Application Publication No. 2012 / 0046877, published February 23, 2012, and U.S. Patent Application Publication No. 2016 / 0103957, published April 14, 2016, each of which is incorporated herein by reference in its entirety.

[0217] In some embodiments, the server system software may support encapsulated assay configurations, including assay names, assay types, panels, hotspot files (if any), reference names, control names (if any), quality control QC thresholds, assay descriptions (if any), data analysis parameters and values, instrument run script names, and other configurations that define an assay. The entire set of information is referred to as an assay definition. Assay configuration content and corresponding workflows may be delivered to users as modular software components in assay definition files (ADFs). The server system software may import assay definition files containing assay configurations. The import process may be initiated by importing a zip file containing an encrypted Debian file and triggering the installation process. The user interface may provide a page for users to select an ADF to import. The cloud-based support and resource system's application store stores ADFs supporting various assays, panels, and workflows available for user selection for download to the user's local server system.

[0218] An Assay Definition File (ADF) is an encapsulated file that defines the configuration of a molecular test or assay, including the assay name, technology platform configuration (e.g., Next-Generation Sequencing (NGS), chip type, chemistry type), workflow steps (sample preparation, instrument script, analysis, report), analytical algorithm, regulatory labeling (e.g., Research Use Only (RUO), In Vitro Diagnostic (IVD), Central European In Vitro Diagnostic (CE-IVD), Internal Use Only (IUO)), etc.), target markers (panel), reference genome version, consumables, controls, QC thresholds, report genes, and variants. The ADF provides a modular approach for building assay capabilities on local sequencing devices. Assay software can be provided by the ADF separately from the sequencing device's platform software.

[0219] Advantages of using ADF in assay configuration include: Encapsulation of assay workflow and analysis Single-click installation Modular software structure implemented with Docker, allowing for decoupling from the platform software, so assay configurations do not require revalidation after software updates Multi-layer encryption for secure delivery Streamlined support for original equipment manufacturer (OEM) assay configuration Streamlined customization of reporting Support for local regulatory requirements Plug-and-play format supports technology-agnostic workflows Enable rapid expansion of molecular testing menu and laboratory adoption of assays

[0220] In some embodiments, an assay definition file (ADF) may include software code modules for one or more of the following steps: 1) library preparation, 2) templating, 3) sequencing, 4) analysis, 5) variant interpretation, and 6) report generation. For the library preparation and templating workflow steps, the ADF may include scripts for library preparation, templating, and enrichment of templated beads. For the sequencing and analysis workflow steps, the ADF may include a Docker image package of algorithm binary code and parameters for the analysis pipeline described with respect to Figure 50. For the variant interpretation workflow step, the ADF may include a list of annotation sources that can be used to analyze and annotate variants. For the report generation workflow step, the ADF may include report templates and image files for use in generating the report.

[0221] The ADF may include instrument scripts for controlling workflow steps for the sequencing instrument. For example, the scripts may include parameters controlling the amount of pipetting and robotic control. Instrument scripts may be customized for specific assays.

[0222] For example, for the sequencing and analysis steps, the ADF may contain a Docker image of the end-to-end analysis pipeline. The Docker image may contain OS-specific libraries and binaries for the algorithms for each step of the analysis pipeline. The algorithm binaries may include analysis pipeline steps including signal processing, base calling, alignment, and variant calling, such as those described with respect to Figure 50. In another example, the ADF Debian file may package specific code modules for a particular assay, such as code modules for signal processing, base calling, and RNACounts.

[0223] The ADF may include scripts for configuring reagent kits. These scripts support calculations of consumables needed for a sequencing run. The configuration scripts included in the ADF may include one or more of the following: Barcode Set and Chip Library kits and consumables, including the ability to associate sample control configurations (e.g., sample in-line controls) with their QC parameters Templated kits and consumables, including the ability to correlate internal controls with QC parameters Sequencing kits that include the ability to correlate internal controls with QC parameters

[0224] An ADF may include one or more reference genome files. Examples of reference genomes include hg19 and GRCH38. The reference genome files may be packaged into the main ADF along with workflow information. Alternatively, the reference genome files may be packaged into a separate ADF that supplements the main ADF.

[0225] The ADF may include code modules for the workflow of fusion panels and fusion target region panels. The ADF may include fusion target region reference files and hotspot files for analysis.

[0226] The ADF may include assay parameters at various points in the workflow that can be configured by the user. Configurable parameters may be displayed in the user interface for adjustment by the user. New parameters may be added at any actor level. Configurable parameters may be passed to the analysis pipeline. Input formats for configurable assay parameters may include one or more of single string text, Boolean, multi-line text, floating point, radio button, dropdown, and file upload. For example, file upload may use file formats such as .properties and .json.

[0227] The ADF can include QC parameters used for quality control and assay performance thresholds at various points in the workflow. For example, types of QC parameters include run QC parameters, sample QC parameters, internal control QC parameters, and assay-specific QC parameters. QC parameters can be defined by one or more data types (e.g., integer, floating point), lower limits, upper limits, and default values.

[0228] The ADF may include designated data tab columns for result representation selected from the database for a given assay. The selected data tab columns support configuration of the user interface display of results and columns to include in a PDF report for the assay. The ADF may include image files for result display of a given assay. The ADF may include support for multiple languages ​​for PDF reports. The ADF may include a download file list for any files generated by the analysis pipeline for a given assay. A file list for a sample or run may be displayed in the user interface. The ADF may include a gene list. The gene list may be used to display a list of known genes for a given cancer type in a PDF report in the user interface.

[0229] An ADF may contain a set of plugins to be used for a given assay. The ADF may specify a set of plugins and their versions. If the ADF does not specify a plugin version, the latest version of the plugin installed on the server system may be used for the given assay.

[0230] The ADF may include new workflow templates to support the creation of custom assays. The new workflow templates may include a set of sequential assay chevron steps. The parameters of the steps may be displayed.

[0231] The ADF may include a list of annotation sources and sets to support the construction of new annotation sets. The ADF may include a filter chain to be applied to variants detected by the analysis pipeline for a given assay. The ADF may include a rule set for the annotation of variants.

[0232] ADFs can be configured to support different types of assays. Examples include, but are not limited to, oncology-related assays (e.g., Oncomine assays from Thermo Fisher Scientific), immuno-oncology-related assays (e.g., T cell receptor (TCR), microsatellite instability (MSI), and tumor mutation load (TML)), infectious disease-related assays (e.g., microbiome), reproductive health-related assays, and exome-related assays. ADFs can also be configured for custom assays.

[0233] Figure 51 is a schematic diagram of generating an assay definition file, according to one embodiment. An assay definition can be generated by build.sh, debscripts, and makedeb.sh, which initiate file copying and database population of assay information to form a Debian file. The content of an assay definition can include seed data, including assay parameters, a BED file (a Browser Extensible Data File—BED file—defines a chromosomal location or region), a panel file, a gene list, a hotspot file (a BED or VCF file that typically defines regions within genes containing variants), and allowed reagents. The content of an assay definition can include localized versions of the assay name, description, and report messages to support display of assay information in different languages. The assay definition file can support packaging of new analysis pipelines. The ADF can include optional post-processing scripts that can be executed for variant calling, fusion calling, and CNV calling based on the type of assay. The ADF can include optional Docker container images of updates to specific analysis pipeline binaries. Docker container images may be packaged with the ADF so that changes to the platform, such as the operating system or third-party libraries, do not affect the results of the assay or the functionality of this system.

[0234] The Debian file may be serialized to prevent unauthorized modification. The serialized assay definition may be further encrypted using the Advanced Encryption Standard (AES), a symmetric key algorithm. A text file containing the assay meta information may also be encrypted using AES and the same encryption key. The encrypted assay definition file may be compressed into a zip format along with the encrypted meta information file. Other encryption formats may also be applied to the serialized assay definition information. For example, the meta information may include one or more of the following: Analysis pipeline version, Reference genome path for the reference genome file location, Assay Unique Name - the internal name of the assay to check for unique occurrences in this system, Docker image name - used to launch the assay and install assay-dependent file references. Any dependency package names required to launch the analysis pipeline.

[0235] 52 is a schematic diagram of an example of assay definition file packaging. A compressed assay definition file in compressed format 40 may include serialized and encrypted assay definition Debian packaging 41, serialized and encrypted meta information text file 42, and optional serialized and encrypted Docker image Debian packaging 43. The server system may decrypt both the meta information text file 42 and the assay definition serialized file 41 before installing the assay definition Debian file.

[0236] The server system and modular software components can be configured to control multiple functional modes, including RUO, or AD, mode, and IVD, or Dx, mode. Referring to FIG. 1 , a Tomcat Server can be configured to include a web archive (WAR) file for RUO mode and a WAR file for IVD mode. The server system can be configured to include an RUO variome database for variants detected by RUO assays and an IVD variome database for variants detected by IVD assays. The server system can be configured to include a Kepler workflow engine associated with separate analytical pipelines for RUO mode and IVD mode. An RUO Docker image file for RUO assays can be configured as a separate file from an IVD Docker image file for IVD assays. The relational database can be configured to have separate databases: an assay development (AD) database for RUO mode and a Dx database for IVD mode. A server system that initially supports only RUO mode can be configured to support RUO mode and IVD mode through a software update.

[0237] ADFs can be generated separately for RUO and IVD mode assays. RUO mode ADFs can contain assay definitions for assays used in research. RUO mode ADFs can be developed by third parties. IVD mode ADFs contain assay definitions for assays that comply with local regulatory requirements for diagnostic use.

[0238] Assay Automated sequencing devices can be adapted for use in a variety of target assays. For example, target assays can utilize chemistries such as Ion Ampliseq, Ion Ampliseq HD, among other chemistries. For example, automated sequencing devices can be adapted for use in assays such as RNA-seq, Diff-seq, or S1-seq, among other library preparation assays. Other exemplary assays include, among other Oncomine cancer assays, such as OCAv3, Oncomine focus assay, or Oncomine TCR beta-LR assay.

[0239] This assay can be used with nucleic acids sourced from swabs, blood, FFPE tissue samples, cfTNA, among other sources. The nucleic acids can be in the form of DNA or RNA, and can optionally be converted to cDNA.

[0240] An assay can have a large number of primer pairs, for example, in the range of 10 to 24,000. In an example, the number of primer pairs can be in the range of 100 to 1,000, such as in the range of 100 to 500 or in the range of 150 to 300. In another example, the number of primer pairs is in the range of 300 to 5,000, such as in the range of 400 to 4,000.

[0241] The assay can produce libraries with average amplicon sizes in the range of 50 to 500, such as in the range of 50 to 200 or in the range of 75 to 125. In another example, the amplicon size can be in the range of 200 to 500, such as in the range of 200 to 400 or 200 to 300.

[0242] The assay can be performed with a single pool, or can utilize multiple pools. For example, the assay can use a single DNA pool. In another example, the assay utilizes two DNA pools. In a further example, the assay utilizes two RNA pools. In a particular example, the assay utilizes two DNA pools and two RNA pools.

[0243] Preseeding (or Seeding) and Templating Generally, before analyzing nucleic acids (e.g., sequencing), the amount of nucleic acid is increased for optimal detection of the signal generated in the analysis. Typically, many copies of nucleic acids, called amplicons, are generated by amplification. These copies are often referred to as analytical templates. For example, in sequencing by synthesis, amplicons serve as templates for nucleotide polymerization, which are detected and provide data for sequence determination. In some cases, two or more different nucleic acid sequences can be simultaneously amplified and then simultaneously sequenced. For example, in some methods of simultaneously amplifying different nucleic acids, amplification is performed using a pair of universal primers that are complementary to the sequences present in each of the different nucleic acids in the amplified nucleic acid population, while the remaining sequences of the different nucleic acids in the amplified population are different for each nucleic acid (i.e., a polyclonal population of nucleic acids). In such cases, the monoclonality of the amplicons generated from each different nucleic acid is often desirable because the different characteristics of the various nucleic acid molecules in the polyclonal population can complicate the interpretation of assay data, such as sequencing.

[0244] In some embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, the nucleic acid to be analyzed is amplified prior to analysis. In some embodiments, the nucleic acid to be analyzed is clonally amplified to generate a monoclonal or substantially monoclonal population of nucleic acid amplicons, e.g., in a nucleic acid-templated process. In some embodiments, different nucleic acids within a polyclonal population of multiple nucleic acids are clonally amplified to generate a separated monoclonal or substantially monoclonal population of different nucleic acids.

[0245] In some embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, nucleic acid molecules in a sample are used to prepare a collection or library of nucleic acid molecules suitable for downstream sequencing. Some embodiments include a target enrichment step before, during, or after library preparation. Target nucleic acid molecules containing target loci or regions of interest can be enriched, for example, by multiplex nucleic acid amplification or hybridization. Multiplex nucleic acid amplification, such as multiplex PCR, can be performed using a variety of methods to generate amplicons and can be used in any of the method embodiments. Enrichment by any method can be followed by a universal amplification reaction.

[0246] In some embodiments of the methods provided herein, as well as the instruments, devices, systems, compositions, and kits for carrying out these methods, one or more nucleic acids to be analyzed (e.g., sequenced) are amplified in a templated process to generate a population of monoclonal or substantially monoclonal template nucleic acids. The templated methods combined with the instruments, devices, systems, compositions, and kits provided herein incorporate processes and compositions for generating, containing, isolating, transporting, replicating, or manipulating a substantially monoclonal population of nucleic acids in a rapid, efficient, and cost-effective manner, while providing a significant increase in the production of high-quality nucleic acid sequencing reads or longer nucleic acid sequencing reads with a reduced number of duplicate, uninformative, erroneous, or blank reads compared to existing methods. In some embodiments, the templated process generates one or more monoclonal or substantially monoclonal populations of template nucleic acids, in which the nucleic acids are attached to one or more surfaces or supports, such as solid supports or surfaces. Template nucleic acid molecules can be immobilized to a surface or support by any method, including, but not limited to, physical adsorption, by the formation of ionic or covalent bonds, or a combination thereof. In some embodiments, sites on a support or surface are preseeded with a single nucleic acid molecule from a collection of nucleic acids (e.g., a nucleic acid library, an amplified target, or a portion of a nucleic acid library or sample), or a limited number of primarily substantially identical or monoclonal nucleic acid molecules, to provide individual supports or sites (e.g., attachment sites) attached to a single nucleic acid molecule or a localized, substantially monoclonal population of nucleic acids that can be unambiguously analyzed by downstream sequencing methods.Such preseeded supports or surfaces are used as clean, confined, contained, or isolated sources of distinct single nucleic acid molecules, or of two or more substantially identical or monoclonal nucleic acid molecules, that can be easily manipulated and clonally amplified, e.g., in a templated reaction, to generate a collection of relatively pure, confined, contained, or isolated nucleic acid templates for use in high-throughput sequencing workflows to improve sequencing results. Thus, in some embodiments or aspects of the methods of manipulating nucleic acids provided herein, a preseeding reaction is performed before or simultaneously with the templated reaction, e.g., in a high-throughput sequencing workflow.

[0247] In some embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, amplification of a template nucleic acid on a surface or support, e.g., a solid surface or support, is carried out in two or more reactions, including, for example, one or more preseeding reactions that generate a substantially monoclonal population of one or more preseeded support or surface sites to which a single template nucleic acid molecule is attached, followed by one or more templated reactions on the preseeded support or site to generate copies (e.g., at least 10-fold or more copies) of the attached template nucleic acid molecule or molecules on the one or more supports or sites. Thus, in some embodiments, the templated reaction mixture includes one or more preseeded supports or surfaces. One advantage of performing preseeding and templated reactions is that this workflow produces higher quality sequencing reads in high-throughput sequencing reactions. In some embodiments, nucleic acid molecules are amplified directly on a support, such as multiple sites, beads or microparticles, or sites on a support comprising an array of reaction chambers. For example, nucleic acid molecules can be preseeded onto supports in reaction sites or chambers (e.g., wells or microwells), or preseeded onto supports in bulk in solution and then distributed to reaction sites, e.g., on a solid support or surface. The different sites are optionally members of an array of sites. The array can comprise a two-dimensional array of sites on a surface (e.g., a flow cell, electronic device, transistor chip, reaction chamber, channel, etc.) or a three-dimensional array of sites within a matrix or other medium (e.g., a solid, semi-solid, liquid, fluid, etc.). In some embodiments, wells or reaction sites contain one preseeded support per well or reaction site. In some embodiments, template nucleic acids attached to the preseeded supports distributed into the wells or reaction sites then undergo one or more templated reactions, in which the templates are amplified on the supports to generate a monoclonal or substantially monoclonal population of template nucleic acids.Alternatively, nucleic acids can be localized, deposited, or positioned at different sites prior to the pre-seeding reaction. In one example, supports can be distributed into an array of wells, and nucleic acid molecules can be pre-seeded onto the solid supports while the solid supports are held in place in the array of wells. In some embodiments, the template nucleic acids pre-seeded onto the supports in the wells or reaction sites then undergo one or more templated reactions, and the templates are amplified on the supports to generate a monoclonal or substantially monoclonal population of template nucleic acids. In some embodiments, the method for nucleic acid amplification includes one or more, or two or more, or multiple surfaces or supports, or populations of surfaces or supports.

[0248] In some embodiments, the preseeding (or seeding) methods provided herein involve hybridizing a nucleic acid molecule (e.g., a single-stranded nucleic acid) to a complementary nucleic acid, such as an oligonucleotide or primer, bound to or immobilized on a support or surface. Such methods are typically performed under annealing conditions for a short period of time. In some embodiments, the seeding method involves hybridizing a support, e.g., a solid support such as a bead, particle, or site on a surface, under conditions in which only one nucleic acid molecule is attached to the support. Such conditions include, for example, contacting a population of nucleic acids (e.g., an amplified target from a library, or a portion of a nucleic acid library, or a sample of nucleic acids) under annealing conditions with a substantial excess of supports or sites relative to the number of nucleic acid molecules. For example, in some embodiments, the support-to-nucleic acid molecule ratio (or site-to-nucleic acid molecule) is selected to optimize the percentage of supports bearing a single template polynucleotide molecule, or a monoclonal or substantially monoclonal population of template nucleic acid molecules attached to the support. For example, preseeding can be performed at a support-to-nucleic acid molecule (or site-to-nucleic acid molecule) ratio of at least about 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, or 100:1. In some embodiments, the preseeding (or seeding) method further includes manipulation (e.g., conversion) of the library or sample nucleic acid prior to or simultaneously with hybridization of the template nucleic acid to the primer immobilized on the support. Such manipulation can include, for example, the addition of one or more adaptor nucleotide sequences or nucleic acid amplification.

[0249] In some embodiments, one or more preseeded supports are formed during the preseeding (or seeding) reaction using a preseeding reaction mixture. In some embodiments, the preseeding reaction mixture includes some or all of the following: a population of nucleic acid molecules, one or more supports or surfaces, a polymerase, a population of first primers, nucleotides, a population of second primers, divalent cations, or a diffusion-limiting agent. In some embodiments, the preseeding reaction mixture includes one or more, or at least two, nucleic acid molecules with the same or different sequences, a population of first primers (which may or may not be in solution, or some of which may be in solution, or some of which may be immobilized on a support), a population of second primers (which may or may not be in solution), and optionally one or more supports or surfaces. In some embodiments, the one or more supports or surfaces are introduced into the reaction mixture at a specific time during the preseeding reaction, for example, after one or more cycles of amplification of the nucleic acid molecules attached to the support or surface. In some embodiments, one or more supports or surfaces have a population of first primers or a population of sequences contained in the first primers attached thereto. In some embodiments, at least one, some, or all of the supports contain a population of first primers or a population of sequences contained in the first primers that are substantially identical to each other. In some embodiments, all of the primers on the support are substantially identical to each other, or all contain substantially identical first primer sequences or sequences contained in the first primers. In some embodiments, at least one of the supports contains two or more different primers attached thereto. For example, at least one support can contain a population of first primers or a population of sequences contained in the first primers and a population of second primers. The support can be attached to a universal primer.The universal primer optionally hybridizes (or can hybridize) to all or substantially all of the template nucleic acid molecules in the reaction mixture. The reaction mixture can include a first support covalently attached to a first target-specific primer and a second support covalently attached to a second target-specific primer, where the first and second target-specific primers are different from each other. Optionally, the first target-specific primer is substantially complementary to the first target nucleic acid sequence, and the second target-specific primer is substantially complementary to the second target nucleic acid sequence, where the first and second target nucleic acid sequences are different. In some embodiments, the reaction mixture includes multiple different supports or surfaces; for example, the pre-seeding reaction mixture includes one or more beads (particles, nanoparticles, microparticles, etc.), where at least two different template nucleic acid molecules are attached to different beads, thereby forming at least two different beads, each of which is attached to a different template nucleic acid molecule. In some embodiments, the preseeding reaction mixture comprises a single surface (e.g., a planar surface, a flow cell, or an array of reaction chambers) in which at least two different template nucleic acid molecules are amplified onto two different regions, sites, or locations on the surface, thereby forming a single surface attached to two or more template nucleic acid molecules. In some embodiments, the support or surface comprises multiple instances of a second primer, and the method comprises hybridizing at least one extended first primer strand to the second primer on the support or surface, as in bridge PCR. In some embodiments, the preseeding reaction mixture comprises one or more nucleic acid molecules, a plurality of nucleic acid molecules, or a population of nucleic acid molecules, e.g., double-stranded or single-stranded nucleic acids, and one or more or multiple supports or surfaces with multiple first primers, or multiple sequences contained in the attached first primers, wherein the nucleic acid molecules contain a sequence of nucleotides substantially complementary to or substantially identical to the first primer (i.e., first primer binding sequence), and the preseeding reaction mixture optionally comprises a polymerase and nucleotides.In some embodiments, the preseeding reaction mixture further includes a second primer, which may be in solution. The nucleic acid molecule can optionally contain a second primer binding sequence. In some embodiments, the reaction mixture includes a population of first primers and a population of second primers that bind to a sequence within the template nucleic acid molecule. The population of first primers can be identical, substantially identical copies, or different sequences. The population of second primers can be identical, substantially identical copies, or different sequences. In some embodiments, the first primers and the optional population of second primers are universal primers (or contain the sequence of a universal primer), and all copies of the first primers are identical, and all copies of the second primers are identical. Thus, in some embodiments, both the population of first primers and the population of second primers are universal primers (or contain the sequence of a universal primer) that bind to a universal primer binding sequence on the template nucleic acid molecule. In some embodiments, the preseeding mixture includes a recombinase and, optionally, a recombinase accessory protein.

[0250] In some embodiments, the preseeding or templated reaction uses one or more enzymes capable of catalyzing the polymerization of nucleotides. In any of the embodiments provided herein, the one or more enzymes capable of polymerization include at least one polymerase. In some embodiments, the reaction is performed with one type or a mixture of polymerases or ligases. In some embodiments, the at least one polymerase includes a thermostable or thermolabile polymerase. In some embodiments, the at least one polymerase includes a biologically active fragment of a DNA or RNA polymerase, or a mutant version thereof, that maintains sufficient catalytic activity to polymerize or incorporate at least one nucleotide under any suitable conditions. The polymerase can optionally have or lack exonuclease activity. In some embodiments, the polymerase has 5' to 3' exonuclease activity, 3' to 5' exonuclease activity, or both. In some embodiments, the polymerase lacks any one or more of such exonuclease activities. Examples of polymerases include Taq DNA polymerase, T7 DNA polymerase, Sau DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, Klenow, and fragments and derivatives thereof. In some embodiments, the polymerase has strand displacement activity. An exemplary polymerase is Bst DNA polymerase (exonuclease minus), a 67 kDa Bacillus stearothermophilus DNA polymerase protein (large fragment), exemplified by accession number 2BDP_A, which has 5' to 3' polymerase activity and strand displacement activity but lacks 3' to 5' exonuclease activity.Other polymerases include Taq DNA polymerase I from Thermus aquaticus (exemplified by accession number 1TAQ), Eco DNA polymerase I from Escherichia coli (accession number P00582), Aea DNA polymerase I from Aquifex aeolicus (accession number 067779), or functional fragments or variants thereof. Another exemplary polymerase is Bsu DNA polymerase (large fragment (NEB)). Bsu DNA polymerase I, large fragment, retains the 5' to 3' polymerase activity of Bacillus subtilis DNA polymerase I (1), but lacks the 5' to 3' exonuclease domain. In certain embodiments, the large fragment of Bsu DNA polymerase lacks 3' to 5' exonuclease activity. In certain embodiments, for example, when the reaction includes RPA, the one or more enzymes capable of polymerization include T5 or T7 DNA polymerase. In some embodiments, the one or more enzymes capable of polymerization include T5 or T7 DNA polymerase with one or more amino acid mutations that reduce its 3' to 5' exonuclease activity. In some embodiments, the T5 or T7 DNA polymerase with one or more amino acid mutations that reduce its 3' to 5' exonuclease activity does not contain amino acid mutations that disrupt the processivity of the T5 or T7 DNA polymerase. In some embodiments, the T5 or T7 DNA polymerase contains one or more amino acid mutations that eliminate detectable 3' to 5' exonuclease activity, and the one or more amino acid mutations do not disrupt the processivity of the T5 or T7 DNA polymerase. In certain exemplary embodiments, the preseeding or templating reaction mixture includes Sau polymerase, T7 DNA polymerase with reduced 3' to 5' exonuclease activity, Bsu polymerase, or a combination thereof, which are particularly well suited for RPA reactions.

[0251] Preseeding reaction mixtures, as well as other amplification reactions in the methods provided herein, including templated reaction mixtures, can contain a source of nucleotides or their analogs used by the polymerase as substrates for the extension reaction. In some embodiments, the preseeding reaction mixture contains nucleotides (dNTPs) for chain extension of the template nucleic acid molecule, resulting in a single or substantially monoclonal population of template nucleic acid molecule sequences attached to one or more supports. In some embodiments, the nucleotides are not exogenously labeled. For example, the nucleotides can be naturally occurring nucleotides or synthetic analogs that do not contain fluorescent moieties, dyes, or other exogenous optically detectable labels. Optionally, the nucleotides do not contain groups that terminate nucleic acid synthesis (e.g., dideoxy groups, reversible terminators, etc.). In other embodiments, the nucleotides contain a label or tag. In some embodiments, the preseeding reaction mixture or templated reaction mixture contains one or more cofactors. Cofactors include, for example, compositions that enhance or modulate the activity of another reaction component, such as an enzyme. In some embodiments, the cofactors include one or more divalent cations. Examples of divalent cations include magnesium, manganese, and calcium. In various embodiments, the preseeding or templated reaction mixture comprises a buffer containing one or more divalent cations. In exemplary embodiments, the buffer contains magnesium or manganese ions. In some embodiments, the preseeding or templated reaction is initiated by the addition of a cofactor, particularly a divalent cation. In some embodiments, the preseeding or templated reaction mixture used herein for nucleic acid amplification may include at least one cofactor for recombinase assembly on nucleic acids or for homologous nucleic acid pairing.

[0252] In some embodiments, the preseeding reaction includes a plurality of solid supports (e.g., included at the beginning of the reaction or at any other time during the reaction) on which are immobilized (i) a plurality of nucleic acid molecules comprising a target sequence and one or more adapter sequences (e.g., universal adapter sequences) at one or both ends, (ii) a plurality of soluble forward primers, (iii) a plurality of soluble reverse primers (which may be blocked primers or tail primers), and (iv) capture primers that hybridize to the adapter sequences or to the complements of the sequences contained within the reverse primers. In some embodiments, the preseeding or templated reaction is performed in a single reaction mixture with a plurality of nucleic acid molecules having the same or different target sequences. In some embodiments, each nucleic acid molecule generated from a sample includes a target sequence joined at its end to at least one universal adapter sequence (e.g., an A adapter or P1 adapter sequence). In some embodiments, the nucleic acid molecule is a double-stranded molecule having complementary top and bottom strands. In some embodiments, a preseeding reaction is performed to amplify a single, monoclonal, or substantially monoclonal copy of a nucleic acid molecule using forward and reverse soluble primers hybridized to adapter sequences and attach it to a solid support (see, for example, Figure 4). While Figure 4 depicts a series of reactions for a single double-stranded nucleic acid molecule and a single bead in a single reaction mixture, the same single reaction mixture can contain multiple double-stranded nucleic acid molecules and multiple beads that undergo the same series of reactions to generate at least two beads, each with one template nucleic acid or a monoclonal or substantially monoclonal template population attached. In addition, beads can be attached to multiple B capture primers. Furthermore, while the bottom of Figure 53 depicts biotinylated primer extension products binding to the B capture primer, non-biotinylated primer extension products can bind to the B capture primer. Also, a mixture of biotinylated and non-biotinylated primer extension products can be attached to multiple B capture primers that are attached to beads.In some embodiments, a single reaction mixture contains multiple nucleic acid molecules, each nucleic acid molecule having the same or different target sequence attached to at least one universal adaptor sequence.

[0253] Referring to an exemplary method as depicted in Figure 53, a nucleic acid molecule having an upper strand and a lower strand is denatured, and the separated upper and lower strands are used in a primer extension reaction using a soluble primer (e.g., a soluble A primer) and a soluble block-tailed primer (e.g., a soluble block-tailed P1 / B primer) to generate multiple primer extension products with adapter sequences that can bind to immobilized B primers during a preseeding or templating reaction. The primer extension reaction generates different products for the two strands due to the different sequences and orientations of the upper and lower strands and the different primers used. In the first primer extension reaction, a soluble primer that binds the A primer binding site is used to generate the complement of the lower strand. For purposes of illustration in Figure 53 (left), the soluble A' primer is complementary to the A adapter sequence. In some embodiments, a mixture of soluble A' primers of various lengths is used for the first primer extension reaction. The mixture of soluble A' primers can vary in length at the 5' end, the 3' end, or both the 5' and 3' ends of these primers. For example, Primer Mix S (a mixture of A' primers that can include 5' non-complementary sequences of various lengths, with or without 5' biotin adducts (depicted as "Bio" in Figure 53)) can be used in a first primer extension reaction to generate one of several possible first extension products, depending on which soluble A' primer is used in the first primer extension reaction (Figure 53, left). For example, the first extension product contains, in the 5' to 3' direction, a complementary A adapter sequence (depicted as A' in Figure 53, left), a complementary bottom sequence (depicted as BOTTOM' in Figure 53, left), and a complementary P1 sequence (depicted as P1' in Figure 53, left). In some embodiments, in the second primer extension reaction, the newly synthesized P1' sequence of the first extension product can bind to a soluble P1 primer, allowing primer extension to occur from the 3' end of the P1' sequence of the first primer extension product. The soluble P1 primer can be a tail primer.The soluble P1 primer can carry a blocking moiety at its 3' end, which can inhibit primer extension from the 3' end of the primer. The blocked primer can prevent the formation of primer-dimer amplicons during the preseeding reaction, which can result in low-quality sequencing reads and a reduced amount of sequencing reads from the template nucleic acid molecule. The soluble P1 primer can be a reverse-tailed P1 primer that includes an attached 5' B adapter sequence such that primer extension of the first extension product results in the addition of the complement of the B sequence (shown as B' on the left in Figure 53) to the 3' end using the tailed primer P1 as a template. In an exemplary embodiment, the soluble P1 primer can have a 3' blocked end (shown as a circled "X" on the left in Figure 53) to prevent extension from the 3' end of the soluble P1 primer (Figure 53, left). The second primer extension reaction can generate multiple second extension products of varying lengths, depending on which soluble A' primer was used in the first extension reaction. The multiple second extension products contain, in the 5' to 3' direction, a complementary A adapter sequence (shown as A' on the left in Figure 53), a complementary bottom strand sequence (shown as BOTTOM' on the left in Figure 53), a complementary P1 sequence (shown as P1' on the left in Figure 53), and a complementary B adapter sequence (shown as B' on the left in Figure 53). The second primer extension product can contain or lack a 5' biotin adduct (Figure 53, left). The second extension reaction can generate multiple second extension products of different lengths, which can contain or lack a biotin adduct, and which contain a B' adapter sequence. Any of these second extension products can bind / hybridize to a B capture sequence immobilized on a solid surface (bead). The immobilized B primer can undergo a third primer extension reaction, thereby generating a third extension product that is immobilized on the bead and complementary to the second extension product (Figure 53, bottom). The right side of Figure 53 depicts the double-stranded nucleic acid undergoing denaturation and a series of reactions on the top strand.In some embodiments, the P1' adapter sequence of the top strand can bind to a soluble P1 primer and undergo a first primer extension reaction to generate a first extension product (Figure 53, right). In some embodiments, the soluble P1 primer is a tailed primer. The soluble P1 primer can carry a blocking moiety at the 3' end of the soluble P1 primer, which can inhibit primer extension from the 3' end of the primer (Figure 53, right). The soluble P1 primer can be a tailed P1 primer that includes an attached 5' B adapter sequence such that primer extension results in the addition of the complement of the B sequence (B') to the 3' end of the first extension product using the tailed primer P1 as a template (Figure 53, right). In an exemplary embodiment, the soluble P1 primer can have a 3' blocked end (shown as a circled "X" in Figure 53, right) to prevent extension from the 3' end of the soluble P1 primer (Figure 53, right). The first primer extension reaction generates multiple first extension products containing, in the 5' to 3' direction, the A' adapter sequence, the top strand sequence, the P1' adapter sequence, and the B' adapter sequence (Figure 53, right). The first extension products containing the B' adapter sequence can bind / hybridize to a B capture sequence immobilized on a solid surface (bead). The immobilized B primer can undergo a second primer extension reaction, thereby generating second extension products that are immobilized on beads and complementary to the first extension products (Figure 53, bottom).

[0254] In some embodiments, a pre-seeding (or seeding) reaction can be performed as illustrated in Figure 54. In this example, a target polynucleotide B-A' and its complement, a template polynucleotide (A-B'), are amplified in the presence of a beaded support having a capture primer. The target polynucleotide has a capture portion (B) that is the same as or substantially similar to the sequence of the capture primer bound to the beaded support. A substantially similar sequence is one in which the complement can hybridize to each of the substantially similar sequences. The beaded support can have a capture primer that is the same as or substantially similar to the sequence of the B portion of the target polynucleotide, thereby enabling hybridization of the complement of the capture portion (B) of the target polynucleotide with this capture primer attached to the beaded support. Optionally, the target polynucleotide can include a second primer location (P1) adjacent to the capture portion (B) of the target polynucleotide and can further include a target region adjacent to the primer and in which the complement portion (A') borders the sequencing primer portion (A) of the target polynucleotide. When amplified in the presence of a bead support containing a capture primer, a template polynucleotide complementary to the target polynucleotide can hybridize with the capture primer (B). The target polynucleotide can remain in solution. The system can undergo extension, in which the capture primer B is extended complementary to the template polynucleotide, resulting in a target sequence bound to the bead support. One or more additional amplifications can be performed at this stage in the presence of the support with the capture primer. One or more additional amplifications can be performed in the presence of the free primer (B), the bead support, and a free modified sequencing primer (A) with an attached linker portion (L). The primer (B) and the modified primer (L-A) can interfere with the free target polynucleotide and template polynucleotide, preventing them from binding to the bead support and each other. In particular, the modified sequencing primer (A) with the attached linker portion can hybridize with the complementary portion (A') of the target polynucleotide attached to the bead support.Optionally, the linker-modified sequencing primer LA hybridized to the target polynucleotide can be extended to form a linker-modified template polynucleotide. Such linker-modified template polynucleotides hybridized to the target nucleic acid attached to the bead support can then be captured by magnetic beads and used to magnetically isolate (concentrate) the bead-attached target polypeptides and load them into a sequencing device. Amplification or extension can be performed using polymerase chain reaction (PCR) amplification, recombinase polymerase amplification (RPA), or other amplification techniques. In a specific example, each step of the scheme illustrated in FIG. 5 is performed using PCR amplification. While FIG. 54 depicts a series of reactions on a single double-stranded nucleic acid molecule and a single bead in a single reaction mixture, the same single reaction mixture can contain multiple double-stranded nucleic acid molecules and multiple beads that undergo the same series of reactions to generate at least two beads, each bearing a single template nucleic acid or a population of monoclonal or substantially monoclonal templates.

[0255] In some embodiments, a pre-seeding (or seeding) reaction can be performed as illustrated in Figure 55. In this example, an alternative scheme includes a target polynucleotide (P1-A') and its complementary template polynucleotide (A-P1'). The target polynucleotide and template polynucleotide are amplified in a solution containing a linker-modified sequencing primer (LA) and a truncated P1 primer (trP1) having a portion with the sequence of a capture primer (B). In examples, the truncated P1 primer (trP1) contains a subset of the sequence of P1, or all of the sequence P1. During subsequent amplification in the presence of the linker-modified sequencing primer (LA) and the truncated P1 primer (trP1-B), the seed contains a linker-modified template polynucleotide (LA-B') operable to hybridize to a bead support bearing the capture primer (B). Thus, the linker-modified template polynucleotide (LA-B') hybridizes with the capture primer (B) on the bead and extends to form a target polynucleotide (B-A') attached to the bead support. The linker-modified template polynucleotide hybridized to the target polynucleotide-attached bead can be used to attach to magnetic beads, for example, to implement magnetic loading of beads into a sequencing device or to enrich nucleic acids attached to the beads. The linker portion of the linker-modified template polynucleotide can take various forms, such as biotin, which can bind to a linker portion attached to a magnetic bead, such as streptavidin. Each amplification reaction can be performed using polymerase chain reaction (PCR), recombinase-polymerase amplification (RPA), or other amplification techniques. In the example illustrated in Figure 55, this scheme can be implemented using two or three cycles of PCR. Such a series of PCR reactions results in a greater percentage of bead supports with a single target polynucleotide attached. As a result, a larger population of monoclonals can be generated, eg, in a templated reaction, eg, in the wells of a sequencing device.Although Figure 55 depicts a series of reactions on a single double-stranded nucleic acid molecule and a single bead in a single reaction mixture, the same single reaction mixture can contain multiple double-stranded nucleic acid molecules and multiple beads that undergo the same series of reactions to produce at least two beads, each having one template nucleic acid or population of monoclonal or substantially monoclonal templates attached thereto.

[0256] In some embodiments, a preseeding (or seeding) method can be performed as illustrated in Figure 56. In this example, the method is designed to generate a desired support-attached nucleic acid molecule from a series of amplification cycles in which only one of the amplification products, the desired target nucleic acid, will be attached to the support. The desired target contains a linker moiety, such as biotin (labeled with the letter L in Figure 56), attached to the 5' end of the nucleic acid and an adapter nucleotide sequence (labeled with the letter B' in Figure 56) at the 3' end that is complementary to a primer (labeled with the letter B in Figure 56) immobilized on a support (e.g., a bead). In contrast, the method shown in Figure 55 generates two nucleic acid amplification products that hybridize to the support, only one of which has the desired linker moiety. In generating only one amplification product that will be attached to the support, the method depicted in Figure 56 avoids producing supports that do not contain the desired target nucleic acid, e.g., one lacking a linker moiety, which would not be used for downstream analysis. This method therefore avoids overutilization of supports and nucleic acids and ensures that only a single nucleic acid target molecule hybridizes to the support, which is desirable for maintaining a high level of monoclonality in subsequent templated amplifications using supports bound to only a single nucleic acid. As illustrated in Figure 56, double-stranded nucleic acids (e.g., library nucleic acids) contain different adapter sequences at each end, shown as an A adapter sequence at the 5' end and a P1 adapter sequence at the 3' end (e.g., standard Ion Torrent A and P1 library adapters, Thermo Fisher Scientific). To initiate the seeding process, the library nucleic acids are subjected to one cycle of amplification (i.e., denaturation, primer annealing, and primer extension) in the presence of primers as depicted in Figure 56. Exemplary primers used for amplification are biotinylated primer A (forward primer) and reverse fusion primer.A fusion primer (e.g., the primer labeled trP1 in Figure 56) is a fusion of a sequence complementary to a portion of the adapter sequence at the 3' end of the target nucleic acid and a B primer sequence identical to the sequence of the B primer immobilized on the support. In the example shown in Figure 56, trP1 is a 23-mer segment of the IonP1 adapter, having the sequence of SEQ ID NO:1. The fusion primer hybridizes and primes with an internal portion of the 3' adapter sequence of the library nucleic acid molecule near the library insert sequence and does not hybridize to the remainder of the adapter sequence at the 3'-most end of the library nucleic acid. This creates a mismatched end between the fusion primer sequence and the 3' extreme portion of the adapter on the library nucleic acid. As shown in Figure 56, after two cycles of amplification (e.g., PCR), four amplification products are generated, but only one product is capable of seeding (or hybridizing) to a support (e.g., an Ionsphere particle). Thus, upon subsequent denaturation of the amplification product, only one single strand of the product will hybridize to the B primer on the support. This primer can be extended to form a double-stranded template nucleic acid, in which the single strand contains a linker moiety that can be used to attach the support-bound nucleic acid to a magnetic bead, for example, for use in enriching or loading the template beads into a sequencing device. While Figure 56 depicts a single double-stranded nucleic acid molecule and a series of reactions on a single support in a single reaction mixture, the same single reaction mixture can contain multiple double-stranded nucleic acid molecules and multiple supports that undergo the same series of reactions to produce at least two supports, each with an attached template nucleic acid.

[0257] Certain embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, include methods for generating nucleic acid templates having specific nucleotide sequences. Such methods are particularly useful for preseeding one or more, two or more, or multiple supports or surfaces with a single nucleic acid.In one embodiment, a method for generating a nucleic acid template having a specific nucleotide sequence includes: (a) obtaining a nucleic acid, or an initial plurality of nucleic acids, or a population of nucleic acids, containing a nucleic acid strand having a first sequence of contiguous nucleotides at the 5' end of the nucleic acid strand, a second sequence of contiguous nucleotides at the 3' end of the nucleic acid strand, and a third nucleotide sequence positioned between the first and second sequence of contiguous nucleotides, wherein the first sequence of contiguous nucleotides and the second sequence of contiguous nucleotides are different from each other, and the first sequence of contiguous nucleotides is substantially identical among the plurality of nucleic acids or population of nucleic acids, and the second sequence of contiguous nucleotides is substantially identical among the plurality of nucleic acids or population of nucleic acids; and (b) subjecting the nucleic acid, or the initial plurality of nucleic acids, or population of nucleic acids to cycles of nucleic acid amplification in the presence of a first primer and a second primer, wherein the first primer amplifies the first sequence of contiguous nucleotides. and (c) subjecting the product of the nucleic acid amplification cycles of (b) to nucleic acid amplification cycles in the presence of the first and second primers to nucleic acid amplification cycles in the presence of the first and second primers to generate a plurality of distinct nucleic acid products, wherein only one of the plurality of distinct nucleic acid products of the nucleic acid amplification of the nucleic acid, or each distinct nucleic acid of the initial plurality or population of nucleic acids in step (a), comprises a sequence of nucleotides complementary to the fourth nucleotide sequence. When step (a) includes an initial plurality or population of nucleic acids, the method generates a population of distinct nucleic acids, each nucleic acid comprising a sequence of nucleotides complementary to the fourth nucleotide sequence.In some embodiments, the method further comprises subjecting the products of the nucleic acid amplification cycles of (c) to one or more cycles of nucleic acid amplification in the presence of a first and a second primer to generate additional nucleic acid products containing a sequence of nucleotides complementary to the fourth nucleotide sequence. In some embodiments, the population of nucleic acids described in step (a) is subjected to two or more cycles of nucleic acid amplification in the presence of one or more forward primers comprising an oligonucleotide sequence substantially identical to the first sequence of contiguous nucleotides and a reverse primer comprising an oligonucleotide sequence complementary to the second sequence of contiguous nucleotides, blocked at the 3' end and linked at the 5' end of the oligonucleotide sequence to a fourth nucleotide sequence that is not complementary to the second sequence, to generate nucleic acid products in which substantially all or all of the products comprise a sequence of nucleotides complementary to the fourth nucleotide sequence. In some embodiments of any of the methods for generating nucleic acid templates having specific nucleotide sequences, the forward or first primer comprises a modified nucleotide-containing attachment. In some embodiments, the attachment to the modified nucleotide comprises a linker moiety, e.g., biotin. In some embodiments, the sequence of the fourth nucleotide sequence is substantially identical to the sequence of a primer immobilized to one or more supports or surfaces, or sites on the surface, and the method can further include attaching a product comprising a sequence of nucleotides complementary to the fourth nucleotide sequence to one or more supports, surfaces, or sites on the surface by hybridization with the immobilized primer. In some embodiments, the method further includes isolating the nucleic acid strand attached to the support by removing the support from any other nucleic acid not bound to the support.

[0258] In some embodiments of the method for generating a nucleic acid template having a specific nucleotide sequence or a population of two or more nucleic acid templates having a specific nucleotide sequence, one or more primers comprise a modified nucleotide containing an attachment. For example, in some embodiments, the forward primer or first primer, or the reverse primer or second primer, comprises a modified nucleotide containing an attachment. In some embodiments, the attachment to the modified nucleotide comprises a linker moiety, such as biotin. In some embodiments, one or more primers (e.g., the forward or first primer) comprise a 3'-terminal nucleotide sequence substantially identical to the first nucleotide sequence, a 5'-terminal nucleotide sequence, and a non-replicable portion positioned between the 3'-terminal nucleotide sequence and the 5'-terminal nucleotide sequence. In embodiments in which the forward or first primer comprises a non-replicable portion between the 3'-terminal nucleotide sequence and the 5'-terminal nucleotide sequence substantially identical to the first nucleotide sequence, the only product from the final nucleic acid amplification that comprises a sequence of nucleotides complementary to the fourth nucleotide sequence also comprises a 5'-terminal single-stranded region comprising the non-replicable portion and 5'-terminal nucleotide sequence of the first primer. In some embodiments, the method further comprises combining or contacting the single-stranded nucleic acid of the product of the final cycle of amplification with a single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence under annealing conditions, thereby hybridizing the product containing a sequence of nucleotides complementary to the fourth nucleotide sequence to the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence to generate a partially double-stranded nucleic acid. In some embodiments, the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence is attached to one or more supports or surfaces or sites on a surface. In some embodiments, the support is a solid support. In certain embodiments, the support is a particle or bead.In some embodiments, the method further comprises generating an extended double-stranded nucleic acid by extending the 3' end of the oligonucleotide portion of the partially double-stranded nucleic acid, thereby synthesizing a nucleic acid strand comprising a single-stranded oligonucleotide having a nucleotide sequence substantially identical to the fourth nucleotide sequence and complementary to the product to which the single-stranded oligonucleotide is hybridized. In some embodiments, the nucleic acid strand comprising the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence is attached to the support at the 5' end of the strand through the portion of the strand that is the oligonucleotide sequence. In some embodiments, the method further comprises isolating the nucleic acid strand attached to the support by collecting the support or removing the nucleic acid strand from other nucleic acids or reaction components that are not bound to the support.

[0259] In one embodiment, a method for preseeding or seeding a support includes: (a) obtaining a nucleic acid comprising a nucleic acid strand containing a first sequence of contiguous nucleotides at the 5'-end of the nucleic acid strand, a second sequence of contiguous nucleotides at the 3'-end of the nucleic acid strand, and a third nucleotide sequence positioned between the first and second sequences of contiguous nucleotides; (b) subjecting the nucleic acid to cycles of nucleic acid amplification in the presence of a capture primer, wherein the capture primer comprises a nucleotide sequence complementary to the second sequence of contiguous nucleotides and is attached to a support; and (c) subjecting the product of the nucleic acid amplification of (b) to cycles of nucleic acid amplification in the presence of a capture primer not attached to a support and a first primer comprising a nucleotide sequence substantially identical to the first sequence of contiguous nucleotides. In some embodiments, the first primer is attached to a linker moiety.

[0260] In some embodiments, the preseeding (or seeding) method is performed essentially as illustrated in Figures 54-56, with the addition of one or more amplification (e.g., PCR) cycles of the method. Thus, in any such preseeding method that uses multiple cycles of amplification (e.g., PCR), two or more cycles of amplification, e.g., three, four, five, or more, can be included in the preseeding method. For example, if the maximum possible nucleic acid library input falls below the optimal range, additional amplification cycles can be included in the seeding process to generate a sufficient amount of template-seeded supports that can be used in further methods, including, for example, templated amplification and downstream sequencing processes to generate a substantially monoclonal population of nucleic acid templates. Such situations may include, for example, lower-than-expected library concentrations resulting from the library preparation method. In such situations, it may be possible to increase the copy number of library templates to a more optimal level by scaling up seeding to accommodate a larger library input volume, but this may not be an available option due to volume constraints of the reaction vessel or process. An increased number of amplification cycles can be included at any point in the preseeding method, for example, before or after introducing the support with attached oligonucleotide primers into the amplification scheme. For example, with respect to the method depicted in Figure 56, if an additional amplification cycle is included before the point at which the support is introduced into the reaction mixture, a total of four amplification products will be generated that will hybridize to the B primer on the support, compared to only one product that will hybridize in the scheme shown without adding another cycle of amplification. Furthermore, each of the four product strands will include a linker moiety attached to the 5' end of the nucleic acid.Thus, if supports with immobilized B primers are added to the reaction mixture after an additional amplification cycle, the next amplification cycle of denaturation, primer hybridization, and primer extension will result in all four of the amplification products containing sequences complementary to the B primers on the supports, and these amplification products will hybridize to the supports and extend, thereby seeding four supports compared to one support. If an additional amplification cycle is included after the final amplification cycle shown in Figure 56 and supports with attached B primers are added to the reaction mixture, a total of four amplification products will result in nucleic acid strands that hybridize to the B primers on the supports, compared to only one product strand hybridizing in the scheme shown without adding another cycle of amplification. Furthermore, each of the four product strands will include a linker moiety attached to the 5' end of the nucleic acid. Thus, assuming a sufficient number of supports with immobilized B primers are added to the reaction mixture after an additional amplification cycle, a total of four supports will be seeded with nucleic acid template, compared to one seeded support when no additional amplification is included. Thus, by increasing the number of amplification cycles in preseeding methods such as these, a greater number of seeded supports is obtained from the same number of input library nucleic acid molecules.

[0261] In some embodiments, the preseeding (or seeding) methods provided herein involve a combined process of attaching nucleic acids to a support or surface by hybridization while simultaneously amplifying the attached nucleic acids to a low level, e.g., about 10 or more, 20 or more, 50 or more, 100 or more, 250 or more, 500 or more, or 1000 or more copies. In some embodiments, the preseeding reaction generates a plurality of preseeded supports, surfaces, or sites on a surface, where each preseeded support, or site in the plurality of preseeded supports, or site on the surface, comprises a plurality of first primers attached to the support or site, the plurality of first primers having substantially identical sequences, and some of the plurality of first primers are conjugated to template nucleic acid molecules and some of the plurality of first primers are not conjugated to template nucleic acid molecules. In these embodiments, the preseeding reaction mixture typically contains some or all of the following: a population of nucleic acid molecules, a polymerase, nucleotides, a population of first primers, a population or plurality of supports or surfaces, or cofactors such as divalent cations. A variety of methods can be used to preseed the support with substantially monoclonal template nucleic acid molecules. As non-limiting examples, the preseeding reaction can be performed using a recombinase-polymerase amplification (RPA) reaction, a template walking reaction, PCR, emulsion PCR, or bridge PCR. The preseeding reaction or templated reaction can be performed in bulk in solution. Furthermore, the preseeding reaction mixture or templated reaction mixture can include a first universal primer attached to one or more supports, a second universal primer in solution (a soluble second universal primer), and multiple nucleic acid molecules, each of which is attached to at least one universal primer binding sequence that can be added during library preparation, and the universal primer binding sequence binds the first and optional second universal primers. In some embodiments, a pre-seeding or templated reaction is carried out in the well.In some embodiments, the pre-seeding and templated reactions are performed using sequential RPA reactions, and the template nucleic acid molecule is washed away after the pre-seeding reaction before performing the templated reaction.

[0262] In some embodiments, different nucleic acid molecules are preseeded onto one or more distinct individual surfaces or supports (e.g., beads or particles) without the need for compartmentalization prior to amplification. In other embodiments, the nucleic acid molecules are partitioned or distributed into an emulsion prior to amplification. In some embodiments, the preseeding reaction can be carried out in parallel in multiple compartmentalized reaction volumes, as opposed to amplification within a single continuous liquid phase. Each reaction volume can contain a preseeding reaction mixture. For example, the nucleic acid molecules can be distributed or deposited into an array of reaction chambers or reaction volumes, such that at least two such chambers or volumes of the array receive a single nucleic acid molecule. In some embodiments, multiple separate reaction volumes are formed. The reaction chambers (or reaction volumes) can optionally be sealed prior to amplification. A preseeding reaction can be carried out in each of the reaction chambers to generate a substantially monoclonal population of template nucleic acid molecules. In another embodiment, the reaction mixture is compartmentalized or separated into multiple microreactors dispersed within the continuous phase of an emulsion. Each compartment or microreactor functions as an independent amplification reactor; thus, the entire emulsion can support many separate amplification reactions in separate (discontinuous) liquid phases in a single reaction vessel (e.g., an Eppendorf tube or well). As used herein, the term "emulsion" includes any composition comprising a mixture of a first liquid and a second liquid, where the first and second liquids are substantially immiscible with each other. The compartmentalized or separate reaction volumes optionally mix, do not communicate with each other, or cannot mix or communicate with each other. In such embodiments, the pre-seeding reaction mixture in the microreactor can be any of the pre-seeding reaction mixtures described herein. In some embodiments in which the reaction mixture is dispersed within an emulsion, the method further includes recovering from the emulsion at least some of the supports attached to the substantially monoclonal population of template nucleic acid molecules.In some embodiments, the method further comprises depositing on a surface at least some of the supports having attached thereto the substantially monoclonal population of template nucleic acid molecules, hi some embodiments, the method further comprises forming an array by depositing on a surface at least some of the supports having attached thereto the substantially monoclonal population of template nucleic acid molecules.

[0263] In some embodiments, the preseeding reaction may produce supports with no template nucleic acid molecules attached (empty supports), other preseeded supports with one type of template nucleic acid molecule attached, and other preseeded supports with two or more types of template nucleic acid molecules attached. The number of template nucleic acid molecules attached to one or more preseeded supports is the preseeding number. In some embodiments of the preseeding method, the preseeding number is 1 or about 1 to 150,000 template nucleic acid molecules, e.g., about 1 to 100,000, 1 to 75,000, 1 to 50,000, 1 to 25,000, 1 to 10,000, 1 to 5,000, 1 to 2,500, 10 to 100,000, 10 to 75,000, 10 to 50,000, 10 to 25,000, 10 to 10,000, 10 to 5,000, or 10 to 2,500 template nucleic acid molecules. In some embodiments, after the preseeding reaction, the majority of any primers attached to the support are not bound to the template nucleic acid molecules. These unbound primers can be used in a subsequent template-based reaction to further amplify the template nucleic acid molecules. For example, after the pre-seeding reaction, at least 90%, 95%, 96%, 97%, 98%, or 99% of the primers attached to the support are typically not bound to a template nucleic acid molecule, or all but one of the primers attached to the support are not bound to a template nucleic acid molecule.

[0264] In some embodiments, the preseeding or templated reaction involves using a PCR amplification method. In some embodiments, the preseeding or templated reaction is carried out in a single round or cycle of PCR. In other embodiments, the preseeding or templated reaction is carried out in multiple rounds or cycles of PCR. For example, in some methods using amplification (e.g., PCR) cycles, one or more, or two or more cycles of PCR can be performed in the absence (or presence) of a support to generate the desired template or amount thereof, followed by one, one or more, or two or more cycles of PCR in the presence of a support to seed the desired template nucleic acid onto the support (see, e.g., Figure 56). These methods can include diluting the amount of nucleic acid molecules reacting with the support to reduce the percentage of supports reacting with two or more nucleic acid molecules. In some embodiments, the nucleic acid molecules are diluted so that the preseeding reaction has a support-to-template nucleic acid molecule ratio selected to optimize the percentage of supports attached with one template nucleic acid molecule or a substantially monoclonal population of template nucleic acid molecules. For example, preseeding can be performed at support to nucleic acid molecule ratios of at least about 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, and 100:1. In some embodiments, PCR is performed in bulk in solution in a single reaction mixture. In some embodiments, PCR is performed in wells or reaction chambers in a continuous solution. In some embodiments, PCR is performed in an emulsion where PCR is performed in multiple microreactors in an emulsion, as described elsewhere herein.

[0265] In some embodiments, the preseeding or templated reaction involves template walking, in which a portion of a double-stranded nucleic acid molecule dissociates so that a primer binds to one of the strands to initiate a new round of replication (see, e.g., U.S. Patent Publication No. 2012 / 0156728, published June 21, 2012, which is incorporated herein by reference in its entirety). Template walking reactions are typically performed at isothermal temperatures. In some embodiments, template walking is performed in an emulsion.

[0266] In some embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, the preseeding or templated reaction mixture includes components for partially denaturing the template nucleic acid molecule. In some embodiments, the partially denaturing conditions include treating or contacting the template nucleic acid molecule to be amplified with one or more enzymes that can partially denature the nucleic acid template, optionally in a sequence-specific or sequence-directed manner, as in an RPA reaction. In some embodiments, at least one enzyme catalyzes strand invasion or unwinding, optionally in a sequence-specific manner. Optionally, the one or more enzymes include one or more enzymes selected from the following: a recombinase, a topoisomerase, and a helicase. In some embodiments, partially denaturing the template includes contacting the template with a recombinase and forming a nucleoprotein complex containing the recombinase. Optionally, the template nucleic acid molecule is contacted with the recombinase in the presence of a first and, optionally, a second primer. Partially denaturing can include catalyzing strand exchange using a recombinase and hybridizing a first primer to the first primer binding sequence (or hybridizing a second primer to the second primer binding sequence). In some embodiments, partially denaturing includes performing strand exchange and using a recombinase to hybridize a first primer to the first primer binding sequence and a second primer to the second primer binding sequence.

[0267] In some embodiments, partially denaturing the template nucleic acid molecule comprises contacting the template with one or more recombinases or nucleoprotein complexes, at least one of which may comprise a recombinase. Without being limited by theory, it is believed that recombinases coat single-stranded DNA (ssDNA) to form nucleoprotein filament strands that invade double-stranded regions of homology on the template nucleic acid molecule. This creates a short hybrid and displaced strand bubble known as a D-loop (see, e.g., U.S. Patent No. 5,223,414 to Zarling, U.S. Patent Nos. 5,273,881 and 5,670,316 to Sena, and U.S. Patent Nos. 7,270,981, 7,399,590, 7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and 8,071,308, which are incorporated herein by reference in their entireties). The free 3' end of the hybridized primer is extended by DNA polymerase to synthesize a new complementary strand. The complementary strand displaces the originally paired partner strand as the template nucleic acid molecule is extended. In one embodiment, one or more of the paired primers are contacted with one or more recombinases before contacting them with a template nucleic acid molecule, which is optionally double-stranded. At least one of the nucleoprotein complexes can include a primer (e.g., a first primer or a second primer, or a primer including a sequence complementary to a corresponding primer binding sequence in the template). In some embodiments, partially denaturing the template includes contacting the template with a nucleoprotein complex including the primer. Partial denaturing can include hybridizing a primer of the nucleoprotein complex to a corresponding primer binding sequence in the template, thereby forming a primer-template duplex. In some embodiments, partially denaturing the template nucleic acid molecule includes contacting the template with a first nucleoprotein complex including a first primer.Partially denaturing can include hybridizing a first primer of a first nucleoprotein complex to a first primer binding sequence of the forward strand, thereby forming a first primer-template duplex. In some embodiments, partially denaturing the template includes contacting the template with a second nucleoprotein complex comprising a second primer. Partially denaturing can include hybridizing a second primer of the second nucleoprotein complex to a second primer binding sequence of the reverse strand, thereby forming a second primer-template duplex.

[0268] In some embodiments of the preseeding or templated methods provided herein, including a population of immobilized first primers and a population of second primers in solution during the preseeding or templated reaction, the template nucleic acid is at least partially denatured, and the first primer binding site on the template binds to the first primer attached to the solid support. The first primer is used by the polymerase to generate a strand complementary to one strand of the template nucleic acid. That complementary strand is then covalently attached to the solid support through the primer. The second primer in solution is complexed with a recombinase and binds to the primer binding site on the complementary strand, thus partially denaturing the bound template nucleic acid molecule. The polymerase uses the primer to synthesize a new strand identical to the original template nucleic acid strand. In some such embodiments, this strand is then thought to be partially denatured by the binding of the recombinase to the nearby first primer attached to the solid support, and the polymerase synthesizes another complementary strand. Through the repeated steps of this process, a substantially monoclonal population of template nucleic acid molecules is generated during some embodiments of amplification in the pre-seeding or templated reaction.

[0269] In some embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, preseeding or templating involves partial denaturation or amplification, including any one or more steps or methods described herein, using a recombinase and, optionally, a recombinase accessory protein. Recombinases can include any agent capable of inducing or increasing the frequency of occurrence of recombination events, including any event in which two different polynucleotide strands recombine with each other. Recombinases can be enzymes that catalyze homologous recombination. Suitable recombinases include RecA and its prokaryotic or eukaryotic homologs, or functional fragments or variants thereof, optionally in combination with one or more single-stranded binding proteins (SSBs). In some embodiments, homologous recombination enzymes include enzymes from any organism, including Myoviridae (e.g., uvsX from bacteriophage T4, RB69, etc.), Escherichia coli (e.g., recA), or humans (e.g., RAD51). In embodiments, the reaction mixture includes one or more recombinases selected from uvsX, RecA, RadA, RadB, Rad51, homologs thereof, functional analogs thereof, or combinations thereof. In an exemplary embodiment, the recombinase is uvsX. The uvsX protein may be present, for example, at 50-1000 ng / μl, 100-750 ng / μl, 200-600 ng / μl, or 250-500 ng / μl. In some embodiments of the methods provided herein, the preseeding or templating reaction mixture includes one or more recombinase accessory proteins. For example, the accessory protein can improve the activity of the recombinase enzyme. In some embodiments, the accessory protein can bind to a single strand of the template nucleic acid molecule or can load the recombinase onto the template nucleic acid molecule. The accessory protein can be derived, for example, from any bacteriophage, including myoviridae phages, or bacterial species.In some embodiments, methods for nucleic acid amplification can include a single-stranded binding protein, such as a myovirus gp32 (e.g., T4 or RB69). In some embodiments, the reaction mixture includes a protein that improves recombinase loading onto nucleic acids. For example, UvsY protein is a recombinase loading protein. In some embodiments, UvsY can be present at about 20-500 ng / μL, e.g., about 20-250 ng / μL, 20-125 ng / μL, or 75-125 ng / μL.

[0270] In some embodiments, the preseeding or templated reaction mixture can further include other components. For example, the composition can include nucleotides, a population of first primers, optionally a second primer, cofactors, and a buffer. The population of first primers and optionally a population of second primers can be attached to one or more supports. As a non-limiting example, the composition can include one or more supports, a recombinase such as uvsX, a polymerase such as Sau DNA polymerase, a recombinase loading protein such as uvsY, a single-stranded binding protein such as gp32 protein, nucleotides, ATP, phosphocreatine, and creatine kinase. The reaction component composition can be in liquid form or in solid form, such as a dried pellet that can be rehydrated. The dehydrated pellet can include, for example, a recombinase, optional recombinase accessory proteins, optionally gp32, DNA polymerase, dNTPs, ATP, optionally phosphocreatine, optional crowding agent, and optionally creatine kinase. The rehydration buffer can include, for example, Tris buffer, potassium acetate, and optionally, a crowding agent such as PEG. The DNA polymerase can be, for example, T4 or T7 DNA polymerase, and if the polymerase is T7 DNA polymerase, thioredoxin can be further included. In some embodiments, when a dehydrated pellet containing the reaction mixture components is used, the pellet is rehydrated with the rehydration buffer, and the template nucleic acid molecule, primers, and additional nuclease-free water are added to the final volume. Furthermore, the components of the composition can be divided so that any combination of components can be in pellet or liquid form, and one or more remaining combinations of components can be in one or more separate pellets or liquid forms. Such combinations can form kits or vials containing at least two of such combinations. For example, the kit or vial can include a pellet containing all reaction mixture components except the polymerase enzyme, which can be provided in a separate pellet or liquid form in a vial, for example, of the kit.In one non-limiting example, the composition comprises a population of nucleic acid molecules, a polymerase, a recombinase, a forward primer, a reverse primer, nucleotides, and a buffer. In some embodiments, the composition comprises nucleic acid molecules, a forward primer, a reverse primer, uvsX recombinase, a uvsY recombinase loading protein, a gp32 protein, a Sau DNA polymerase, dNTPs, ATP, phosphocreatine, and creatine kinase. In some embodiments, the composition comprises at least two different nucleic acid molecules having both a first primer binding sequence and a second primer binding sequence, a recombinase, a recombinase accessory protein, a polymerase, a first universal primer, a second universal primer, dNTPs, and a buffer. In some embodiments, the composition further comprises one or more supports. In an exemplary embodiment, the composition includes at least two different template nucleic acid molecules having both a first primer binding sequence and a second primer binding sequence, a uvsX recombinase, a uvsY recombinase loading protein, a gp32 protein, a Sau DNA polymerase, ATP, phosphocreatine, creatine kinase, a first universal primer attached to a beaded support, a second universal primer, and a buffer.

[0271] In some embodiments of the methods provided herein, as well as instruments, devices, systems, compositions, and kits for carrying out these methods, templated reactions (e.g., one, more than one, two, or more than two templated reactions) are performed after the preseeding reaction, and the template nucleic acid molecules on the preseeded surfaces or supports are amplified or further amplified (referred to herein as templated reactions). The preseeded solid supports are typically generated in a preseeding reaction that is separate from the templated reaction. Generally, in such embodiments, the templated reaction mixture does not contain additional template nucleic acid molecules in solution, such that the template nucleic acid molecules attached to the one or more preseeded supports are the predominant or sole source of template nucleic acid molecules in the templated reaction mixture before the templated reaction is initiated. In some embodiments, the template nucleic acid molecules are present in solution in the reaction mixture when the templated reaction is initiated. In some embodiments, one or more washes are performed on the one or more preseeded supports before introducing them into the templated reaction mixture. In some embodiments, two or more reactions are carried out in a templated manner.

[0272] In some embodiments, one or more templated reactions are performed (or a templated reaction is performed in two steps of two separate amplifications, e.g., two separate RPA reactions). In some embodiments, two or more separate reactions are performed in a templated method. For example, in some methods provided herein, a first, or initial, templated reaction is performed, followed by a second, or subsequent, templated reaction. The initial templated reaction includes one or more supports to which one or more template polynucleotides are attached, in some cases generated in a separate pre-seeding process prior to the initiation of the templated reaction. The initial templated reaction includes amplification of one or more template polynucleotides on the pre-seeded supports, e.g., using a recombinase and a polymerase (i.e., RPA) under substantially isothermal conditions. In such embodiments, the separate templated or RPA reactions can be performed for the same or different periods of time. The initial first templated reaction, and the subsequent second or subsequent templated reaction, are typically carried out for a period of time that is shorter than the duration of the subsequent second templated reaction. For example, the initial first templating reaction, and subsequent second or subsequent templating reactions, can be carried out over about 1 to 10 minutes, about 1 to 9 minutes, about 1 to 8 minutes, about 1 to 7 minutes, about 1 to 6 minutes, about 1 to 5 minutes, about 1 to 4 minutes, about 1 to 3 minutes, about 1 to 2.5 minutes, about 1 to 2 minutes, about 1 to 1.5 minutes, about 2 to 10 minutes, about 2 to 9 minutes, about 2 to 8 minutes, about 2 to 7 minutes, about 2 to 6 minutes, about 2 to 5 minutes, about 2 to 4 minutes, about 2 to 3 minutes, about 2 to 2.5 minutes, about 2.5 to 10 minutes, about 2.5 to 9 minutes, about 2.5 to 8 minutes, about 2.5 to 7 minutes, about 2.5 to 6 minutes, about 2.5 to 5 minutes, about 2.5 to 4 minutes, or about 2.5 to 3 minutes. In some examples, the initial, first, and subsequent second or subsequent templated reactions can be carried out for less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2.5 minutes, less than about 2 minutes, less than about 1.5 minutes, or less than about 1 minute. In one non-limiting example, the initial templated reaction is carried out for about 2.5 minutes at about 40° C. using an RPA. In some embodiments, the initial, or first, templated reaction is terminated or limited before the initiation of the second or subsequent templated reaction.For example, a termination composition, such as a templated reaction inhibitor, may be added to the initial or first templated reaction to inhibit or stop the reaction or prevent it from continuing. Examples of templated reaction inhibitors include, but are not limited to, components that inhibit one or more components of a nucleic acid amplification reaction, such as a polymerase inhibitor or a recombinase inhibitor, or compositions that limit components of the reaction required for the reaction to proceed. For example, in an initial templated reaction involving RPA, the templated reaction inhibitor may be a chelator, such as EDTA, that binds to cations such as magnesium, which may be required for recombinase-mediated reactions in recombinase-polymerase amplification. In another example, the templated reaction can be limited or stopped by removing reaction components, such as by washing the templated reaction site (e.g., reaction chamber or well) with a solution that does not contain one or more components required for the templated reaction. For example, the templated reaction site may be washed or rinsed with a solution lacking the recombinase, polymerase, cations, nucleotides, or other components of the RPA reaction used in the templated reaction. In some embodiments, a second or subsequent templated reaction following an initial or first templated reaction is initiated or facilitated by adding or contacting one or more templated reaction components to the template-bound support that was subjected to the initial or first templated reaction. For example, in a second or subsequent templated reaction involving recombinase-polymerase nucleic acid amplification, one or more of the following components may be added to or contacted with the template-bound support: recombinase, polymerase, nucleotides, or cations. A second or subsequent templated reaction following a first or initial templated reaction is typically carried out for a period longer than the duration of the first templated reaction.For example, a second or subsequent templated reaction following a first templated reaction may take about 5 to 60 minutes, about 5 to 50 minutes, about 5 to 45 minutes, about 5 to 40 minutes, about 5 to 35 minutes, about 5 to 30 minutes, about 5 to 25 minutes, about 5 to 20 minutes, about 5 to 15 minutes, about 5 to 10 minutes, about 10 to 60 minutes, about 10 to 50 minutes, about 10 to 45 minutes, about 10 to 40 minutes, about 10 to 35 minutes, about 10 to 30 minutes, about 10 to 25 minutes, about 10 ... The reaction may be carried out for 5 minutes, about 10-20 minutes, about 10-15 minutes, about 15-60 minutes, about 15-50 minutes, about 15-45 minutes, about 15-40 minutes, about 15-35 minutes, about 15-30 minutes, about 15-25 minutes, about 15-20 minutes, about 20-60 minutes, about 20-50 minutes, about 20-45 minutes, about 20-40 minutes, about 20-35 minutes, about 20-30 minutes, or about 20-25 minutes. In some examples, a second or subsequent templated reaction following a first or initial templated reaction may be carried out for at least about 60 minutes, at least about 55 minutes, at least about 50 minutes, at least about 45 minutes, at least about 40 minutes, at least about 35 minutes, at least about 30 minutes, at least about 25 minutes, at least about 20 minutes, less than 2 minutes, less than 1.5 minutes, or less than 1 minute. In one non-limiting example, the second or subsequent templated reaction is carried out using RPA for about 20 minutes at about 40° C. In some embodiments, the second or subsequent templated reaction is terminated, stopped, or limited at a set time, e.g., as described for the initial or first templated reaction.

[0273] One advantage of including two or more reactions in a templated process to generate a substantially monoclonal population of nucleic acids is that it facilitates control of template amplification, which can reduce or prevent polyclonality of the nucleic acid population. For example, an initial or first amplification of template polynucleotides on a preseeded support, which is limited in amount or duration, can limit the amount of free template nucleic acid available for migration to another templated reaction site. The initial or first templated reaction provides for binding of the replicated template nucleic acid to additional immobilized primers on the preseeded support or surface or surface site, thus providing an environment that is less open to the diffusion of template nucleic acid that occurs in the second or subsequent templated reaction amplification.

[0274] For example, in some embodiments of the preseeding or templated methods provided herein, including those in which two or more reactions are performed in the templated method, one or more of the reactions contain one or more diffusion limiting agents. When amplifying two or more template nucleic acid molecules within a single, continuous liquid phase of the reaction mixture, including a diffusion limiting agent can be advantageous. The diffusion limiting agent can further prevent or retard the diffusion of template nucleic acid molecules or amplified polynucleotides produced through replication of at least some of the template nucleic acid molecules within the templated or preseeding reaction mixture, thereby converting nucleic acids in one templated reaction to generate a monoclonal template population into another templated reaction to generate a different monoclonal template population, thereby reducing the formation of polyclonal contaminants during nucleic acid amplification in the templated or preseeding reaction. Thus, the diffusion limiting agent can prevent or reduce the formation of polyclonal contaminants during amplification without requiring compartmentalization of the preseeding or templated reaction mixture by physical or encapsulation means (e.g., emulsion). In some embodiments, a diffusion limiting agent is included in the first or initial templated reaction. In some embodiments, a diffusion limiting agent is included in the first or initial templated reaction but not in the second or subsequent templated reaction. In some embodiments, a diffusion limiting agent is included in both the first or initial templated reaction and one or more subsequent templated reactions. In some embodiments, the diffusion limiting agent included in one or more reactions is one or more sieving agents, e.g., a polymer such as methylcellulose, or the diffusion limiting agent provides a matrix with multiple pores. A sieving agent can be any agent that is effective in sieving, restricting or retarding the movement of one or more template nucleic acid molecules or polynucleotides present in a pre-seeding reaction mixture or templated reaction mixture, such as amplification reaction products or template nucleic acid molecules. Thus, the sieving agent can reduce the Brownian motion of polynucleotides. In some embodiments, the sieving agent is a polymeric compound.By way of non-limiting example, the sieving agent can include a polysaccharide, a polypeptide, an organic polymer, or any other suitable polymer. In some embodiments, the sieving agent is one or more of the following polymers: cellulose, dextran, starch, glycogen, agar, chitin, pectin, or agarose. In some embodiments, the sieving agent comprises a cellulose derivative, such as sodium carboxymethylcellulose, sodium carboxymethyl 2-hydroxyethylcellulose, methylcellulose, hydroxylethylcellulose, 2-hydroxypropylcellulose, carboxymethylcellulose, hydroxylpropylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethylcellulose, (hydroxypropyl)methylcellulose, or hydroxyethylethylcellulose, or a mixture comprising any one or more of such polymers. In some embodiments, the pre-seeding reaction mixture comprises a crowding agent. For example, a crowding agent can increase the concentration of one or more components in a nucleic acid amplification reaction by creating a crowded reaction environment. In some embodiments, the pre-seeding reaction mixture or the templated reaction mixture comprises both a sieving agent and a diffusion-limiting reagent or a crowding agent. The diffusion-limiting agent comprises a diffusion-reducing agent. Diffusion reducers include any compound that reduces the migration of template nucleic acid molecules or polynucleotides from regions of higher concentration to regions having lower concentration. In some embodiments, diffusion reducers include any compound that reduces the migration of any component of a nucleic acid amplification reaction, regardless of size.

[0275] In some embodiments, the diffusion-limiting agent included in one or more reactions is a drag compound. The term "drag compound" and variations thereof refer to any compound, e.g., a chemical compound, that can attach to nucleic acids and retard the diffusion of these nucleic acids through a reaction mixture while allowing diffusion synthesis to proceed using such polynucleotides, primers, templates, or amplification products in a nucleic acid synthesis reaction. For example, a drag compound can provide hydrodynamic drag when attached to a nucleic acid by altering the overall size, length, radius, shape, or charge of the modified nucleic acid compared to a nucleic acid lacking the attached compound. In some embodiments, a drag compound attached to a nucleic acid can alter the interaction between the nucleic acid and an aqueous medium compared to the interaction between the aqueous medium and a nucleic acid lacking the attached compound. Attachment of such a drag compound to nucleic acids within a synthesis reaction typically reduces the mobility of such nucleic acids in the reaction mixture, which can help prevent cross-contamination of amplification products or templates between different synthesis reactions occurring within the same reaction mixture. For example, a drag compound can be a compound that binds to or attaches to a template nucleic acid during a templated reaction, e.g., during template nucleic acid amplification, reducing the mobility of the template nucleic acid in the templated reaction mixture. In some embodiments, the template nucleic acid is modified to include a moiety (referred to as a "drag tag") that binds to the drag compound. For example, an affinity moiety, such as a linker moiety, can be attached to the nucleic acid, and the drag compound is a binding partner moiety, e.g., a receptor-type moiety, that binds to the affinity moiety. In one non-limiting example, the template nucleic acid is attached to a biotin moiety that can bind an avidin-like moiety (e.g., streptavidin or a derivative thereof, e.g., neutravidin), which functions as a drag compound included in one or more templated or pre-seeding reactions. In some embodiments, the drag compound is included in the first or initial templated reaction. In some embodiments, the drag compound is included in the first or initial templated reaction but not in second or subsequent templated reactions. In some embodiments, the drug compound is included in the first, or initial, templated reaction and in one or more subsequent templated reactions.In some embodiments, the diffusion reducer or sieving agent comprises polyacrylamide, agar, agarose, or a cellulose polymer such as hydroxyethyl cellulose (HEC), methyl cellulose (MC), or carboxymethyl cellulose (CMC).

[0276] In some embodiments of the templated methods provided herein, the templated reaction comprises an RPA reaction. The templated reaction mixture typically comprises all or some of the following: one or more preseeded solid supports containing a population of substantially identical attached first primers and having attached thereto a substantially monoclonal template nucleic acid molecule, e.g., one or more template molecules (template molecule), a polymerase, a recombinase, an optional single-stranded binding protein, an optional recombinase-loading protein, an optional second or reverse primer that may be attached to a solid support or in solution, dNTPs, ATP, a buffer, and optionally one or both of phosphocreatine and creatine kinase. The reaction can be initiated by the addition of a divalent cation, such as MgCl2 or Mg(OAc)2. In some embodiments, the buffer comprises a crowding agent or diffusion-limiting agent, such as a sieving agent or anti-bacterial compound, such as PEG, Tris buffer, or potassium acetate salt. In some embodiments, the forward primer binding sequence on the template nucleic acid molecule is complementary to or identical to at least a portion of the forward primer, and the reverse primer binding sequence on the template nucleic acid molecule is complementary to or identical to at least a portion of the reverse primer. In some embodiments, the templated reaction comprises bulk amplification (e.g., bulk isothermal amplification) or is performed in a reaction chamber, e.g., the well of a multi-well solid support or surface.

[0277] In some embodiments, the compositions, as well as related systems, methods, kits, and devices, include a templated reaction mixture comprising a population of preseeded supports, nucleotides, a recombinase, and a polymerase, each of which has one or more, e.g., 10 to 50,000, substantially monoclonal template nucleic acid molecules with attached first primers, and further comprising attached first primers that are attached to the preseeded supports and are not bound to template nucleic acid molecules. In some embodiments, the reaction mixture does not contain a cation capable of initiating a recombinase-polymerase amplification reaction, or at least 95% of the template nucleic acid molecules in the reaction mixture are attached to one or more supports. In some embodiments, the templated reaction mixture further comprises a cation capable of initiating a recombinase-polymerase amplification reaction. In some embodiments, the template nucleic acid molecules comprise two or more template nucleic acid molecules with different sequences. In some embodiments, the templated reaction mixture comprises a recombinase accessory protein. In further embodiments, the recombinase accessory protein is a single-stranded binding protein or a recombinase loading protein. In some embodiments, the preseeded support is generated in a preseeding reaction comprising PCR cycles or a recombinase-polymerase amplification (RPA) reaction. In some embodiments, the RPA reaction is carried out by incubating the RPA reaction mixture at a temperature of 35°C to 45°C for 2 to 5 minutes.

[0278] In some embodiments of the methods provided herein, the pre-seeding method can include an RPA reaction, or the templated process can include two or more RPA reactions, so these methods can include sequential RPA reactions. For example, the first RPA reaction can be a pre-seeding reaction followed by a second RPA-templated reaction. In another example, the pre-seeding reaction can be one or more PCR cycles or non-isothermal amplification cycles, followed by one or more templated reactions with an RPA reaction. The RPA reactions can be performed under the same conditions. However, in some embodiments, the pre-seeding RPA reaction, or the first templated RPA reaction followed by one or more subsequent templated RPA reactions, is performed so that fewer amplification cycles occur than the templated RPA reaction or templated reaction occurring after the first templated RPA reaction. For example, the pre-seeding RPA reaction can be performed for a shorter time than the templated RPA reaction that amplifies the template nucleic acid molecule attached to the pre-seeded solid support produced by the pre-seeded RPA reaction. As non-limiting illustrative examples, a pre-seeding RPA reaction, or an initial templated RPA reaction followed by one or more templated RPA reactions, may be run, for example, for about 2-5 minutes to generate one or more pre-seeded or templated supports, e.g., a population, which are then subjected to a templated RPA reaction run for about 10-60 minutes. In some of these non-limiting illustrative examples, reaction components, including the template nucleic acid, are washed off the pre-seeded solid support prior to the templated RPA reaction.

[0279] In some embodiments, the pre-seeding or templated reaction mixture is pre-incubated under conditions that inhibit premature initiation. For example, one or more components in the pre-seeding reaction mixture can be retained outside the reaction vessel to prevent premature initiation. In some embodiments, a divalent cation (e.g., magnesium or manganese) is added to initiate the reaction. In another example, the reaction mixture is pre-incubated at a temperature that inhibits enzymatic activity, such as about 0-15°C or about 15-25°C. The reaction is then incubated at a higher temperature to increase enzymatic activity. In an exemplary embodiment, the pre-seeding or templated reaction mixture is not exposed to temperatures above 42°C during the reaction. In some embodiments, the pre-seeding or templated reaction is carried out under isothermal conditions. In some embodiments, isothermal conditions include reactions that are exposed to temperature changes constrained within a limited range during at least a portion of the amplification (or the entire amplification process), including, for example, a temperature change of about 10°C or less, or about 5°C, or about 1-5°C, or about 0.1-1°C, or about 0.1°C or less. The temperature of an isothermal reaction can typically be about 15°C to 65°C, e.g., about 15°C to 55°C, about 15°C to 45°C, about 15°C to 37°C, about 30°C to 60°C, about 40°C to 60°C, about 55°C to 60°C, about 35°C to 45°C, or about 37°C to 42°C. In other embodiments, the preseeding reaction is not exposed to temperatures above 40°C, 41°C, 42°C, 43°C, 45°C, or 50°C. Thus, in certain embodiments, the reaction mixture is not exposed to hot start conditions. In some embodiments, these methods are carried out without subjecting the double-stranded template nucleic acid molecule to extreme denaturing conditions during amplification. For example, these methods involve subjecting the nucleic acid template to denaturing conditions during amplification. m In some embodiments, these methods are performed without exposing the template to chemical denaturants, such as NaOH, urea, or guanidinium, during amplification.

[0280] In some embodiments, after performing the templating method, the templated surfaces or supports or sites have at least 50,000, 75,000, 100,000, 125,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 10 6 In some embodiments, after performing the templating method, the templated surface or support or site has about 50,000 to 500,000 substantially monoclonal template nucleic acid molecules attached to each templated support, e.g., about 50,000 to 400,000, about 50,000 to 300,000, about 50,000 to 200,000, about 50,000 to 100,000, about 100,000 to 400,000, about 100,000 to 300,000, about 100,000 to 200,000, or about 150,000 to 300,000 substantially monoclonal template nucleic acid molecules attached to each template support.

[0281] In some embodiments, a method for generating one or more templated supports or surfaces, e.g., solid supports, includes, for example, a) forming a templated reaction mixture by combining one or more pre-seeded supports, nucleotides, a recombinase, and a polymerase, wherein the one or more pre-seeded supports comprise a population of substantially identical first primers attached thereto, and have one template molecule attached thereto, or have substantially monoclonal template nucleic acid molecules attached thereto, and the one or more pre-seeded supports comprise, in some embodiments, 100 template nucleic acid molecules attached thereto. a) attaching a template nucleic acid segment to a pre-seeded solid support, the pre-seeded support further comprising an attached first primer that is not bound to the template nucleic acid molecule; a templating reaction mixture further comprising a population of substantially identical second primers, which may be soluble or in solution, the template nucleic acid molecule comprising a primer binding site for the second primer at or near the opposite end of the proximal segment; and b) performing one or more templated reactions. In some embodiments, when performing the templated reactions, the templated reaction mixture is incubated under isothermal conditions to amplify the template nucleic acid molecule and generate one or more templated surfaces or supports. Some templated reactions include the addition of a cation to the templated reaction mixture. In some embodiments, the template nucleic acid molecule is not in solution in the reaction mixture when the templated reaction is initiated. In some embodiments, the template nucleic acid molecule comprises two or more template nucleic acid molecules with different sequences. In some embodiments of these methods, at least 100-fold more substantially monoclonal template nucleic acid molecules are present on the templated support than were present on the preseeded support.

[0282] The templated reaction can be preceded by a preseeding method, in which one or more preseeded supports or a population of preseeded supports are generated for use in forming the templated reaction mixture. The preseeding method involves using the preseeding reaction mixture under preseeding conditions to generate preseeding supports bearing one template molecule or a plurality, e.g., 10 to 100,000, substantially monoclonal template nucleic acid molecules, each having an attached first primer and some attached first primers that are not bound to the template nucleic acid molecule. Typically, the preseeding reaction involves incubating a preseeding reaction mixture containing a population of nucleic acid molecules with a population of supports or surfaces containing a population of attached, substantially identical first primers. In some embodiments, the population of nucleic acid molecules comprises a plurality of nucleic acid molecules comprising a target sequence and one or more universal adaptor sequences. For example, each nucleic acid molecule of the plurality contains, with respect to one of the strands of the molecule, a first sequence of contiguous nucleotides at the 5'-end of the molecule (e.g., a first adaptor), a second sequence of contiguous nucleotides at the 3'-end of the molecule (e.g., a second adaptor), and a third nucleotide sequence, e.g., a target sequence, positioned between the first and second sequences of contiguous nucleotides, wherein the first and second sequences of contiguous nucleotides of the nucleic acid molecule are different, the first sequence of contiguous nucleotides of the nucleic acid molecule are substantially identical, and the second sequence of contiguous nucleotides of the nucleic acid molecule are substantially identical among the population of nucleic acid molecules. One of the sequences of contiguous nucleotides at the end of the nucleic acid molecule (e.g., the adaptor sequence) is typically complementary to at least a portion of a first primer attached to a support, or at least a portion of a first primer attached to a support is added to the end of the nucleic acid molecule in a pre-seeding reaction. In some embodiments, incubation in the pre-seeding reaction comprises cycles of amplification (e.g., PCR) under conditions in which the nucleic acid anneals to the first primer attached to the support. The pre-seeding reaction mixture, in some embodiments, comprises components (e.g., one or more polymerases, dNTPs) for extension of the first primer hybridized to the nucleic acid.In some embodiments, the preseeding reaction comprises one or more cycles of nucleic acid amplification (e.g., including denaturation, primer annealing, and primer extension), in which a contiguous nucleotide sequence complementary to at least a portion of a first primer attached to a support is added t...

Claims

1. 1. A sequencing system comprising: an automated sequencing device adapted to determine variant calls for one or more extracted polynucleotide samples with a performance of at least 98.5% raw read accuracy and a runtime ranging from 5 hours to 14 hours when sequencing four extracted polynucleotide samples using a targeted assay having one DNA pool per sample and an average amplicon size ranging from 100 to 120 bases.

2. 1. A sequencing system comprising: an automated sequencing device adapted to determine variant calls for one or more extracted polynucleotide samples with a performance of at least 98.5% raw read accuracy and a runtime ranging from 15 hours to 24 hours for determining variant calls upon sequencing of 32 extracted polynucleotide samples using a targeted assay having one DNA pool per sample and an average amplicon size ranging from 100 to 120 bases.

3. 1. A sequencing system comprising: an automated sequencing device adapted to determine variant calls upon sequencing of six extracted polynucleotide samples using a targeted assay having two DNA and two RNA pools per sample and an average amplicon size in the range of 100-120 bases, with a performance of at least 98.5% raw read accuracy and a runtime in the range of 20 hours to 30 hours to determine variant calls of one or more extracted polynucleotide samples.

4. 1. A sequencing system comprising: an automated sequencing device adapted to determine variant calls for one or more extracted polynucleotide samples with a performance of at least 98.5% raw read accuracy and a runtime in the range of 20 hours to 28 hours for determining variant calls upon sequencing of 12 extracted polynucleotide samples using a targeted assay having one DNA pool per sample and an average amplicon size in the range of 190-220 bases.

5. 10. The sequencing system of claim 1, wherein the runtime ranges from 5 hours to 12 hours.

6. 6. The sequencing system of claim 1, wherein the raw read accuracy is at least 99.0% raw read accuracy.

7. 7. The sequencing system of claim 6, wherein the raw read accuracy is at least 99.1%.

8. 8. The sequencing system of any one of claims 1-7, wherein the performance is further characterized by a Q20 median read length of at least 100 bases and no more than 450 bases.

9. 9. The sequencing system of claim 8, wherein the Q20 median read length is at least 102 bases and no more than 300 bases.

10. 10. The sequencing system of claim 9, wherein the Q20 median read length is at least 104 bases and no more than 300 bases.

11. 11. The sequencing system of any one of claims 1 to 10, wherein the performance is further characterized by a uniformity of at least 97%.

12. 12. The sequencing system of claim 11, wherein the uniformity is at least 98%.

13. 13. The sequencing system of claim 12, wherein the uniformity is at least 98.5%.

14. 14. The sequencing system of any one of claims 1 to 13, wherein the performance is further characterized by an average percent reads on target of at least 97%.

15. 15. The sequencing system of claim 14, wherein the average percent reads on target is at least 98%.

16. 16. The sequencing system of claim 15, wherein the average percent reads on target is at least 98.1%.

17. 17. The sequencing system of any one of claims 1 to 16, wherein the performance is further characterized by total reads in the range of 13 million bases to 100 million bases.

18. 18. The sequencing system of claim 17, wherein the total reads are in the range of 13 million bases to 60 million bases.

19. 19. The sequencing system of claim 18, wherein the total reads are in the range of 14.5 million bases to 25 million bases.

20. 1. A method for sequencing comprising: Providing a sequencing device with four extracted polynucleotide samples, a sequencing substrate, one pool of DNA for each sample, and reagents for an assay having an average amplicon size in the range of 100-120 bases; generating a plurality of polynucleotides from the assay reagents with the sequencing device; sequencing the plurality of polynucleotides with the sequencing device with a raw read accuracy of at least 98.5%; generating, at the sequencing device, a variant call report based on the sequencing; The method, wherein said generating, said sequencing, and said creating are performed with a runtime within a range of 5 hours to 14 hours.

21. 21. The method of claim 20, wherein the runtime ranges from 5 hours to 12 hours.

22. 22. The method of claim 20 or 21, wherein the raw read accuracy is at least 99.1%.

23. 23. The method of claim 22, wherein the raw read accuracy is at least 99.2%.

24. 24. The method of any one of claims 20-23, wherein the raw read accuracy performance is further characterized by a Q20 median read length of at least 100 bases and no more than 300 bases.

25. 25. The method of claim 24, wherein the Q20 median read length is at least 102 bases and no more than 300 bases.

26. 26. The method of claim 25, wherein the Q20 median read length is at least 104 bases and no more than 300 bases.

27. The method of any one of claims 20 to 26, wherein the raw read precision performance is further characterized by a uniformity of at least 97%.

28. 28. The method of claim 27, wherein the uniformity is at least 98%.

29. 29. The method of claim 28, wherein the uniformity is at least 98.5%.

30. 30. The method of any one of claims 20-29, wherein the raw read accuracy performance is further characterized by an average percent reads on target of at least 97%.

31. 31. The method of claim 30, wherein the average percent reads on target is at least 98%.

32. 32. The method of claim 31 , wherein the average percent reads on target is at least 98.1%.

33. 33. The method of any one of claims 20-32, wherein the raw read accuracy performance is further characterized by total reads in the range of 13 million bases to 25 million bases.

34. 34. The method of claim 33, wherein the total reads are in the range of 14 million bases to 25 million bases.

35. 35. The method of claim 34, wherein the total reads range from 14.5 million bases to 25 million bases.

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