WO2017196676A1

WO2017196676A1 - Metal chelation post incorporation detection methods

Info

Publication number: WO2017196676A1
Application number: PCT/US2017/031403
Authority: WO
Inventors: Chiu Tai Andrew Wong; Xiaoling Yang; Collyn SEEGER; Todd Rearick; Steven Menchen; Roman Rozhkov; Barnett Rosenblum; Ronald Graham
Original assignee: Life Technologies Corporation
Priority date: 2016-05-10
Filing date: 2017-05-05
Publication date: 2017-11-16

Abstract

The present disclosure provides methods relating to detecting, identifying and measuring nucleic acids that includes binding metal ions to nucleic acid polymer backbones having negative charges formed as a result of nucleotide incorporation, and detecting or measuring hydrogen ions that are generated as a result of forming metal chelate complexes by addition of chelating agents.

Description

METAL CHELATION POST INCORPORATION DETECTION METHODS

RELATED APPLICATIONS

[0001] This application is Non-provisional application, which claims priority to U.S.

Provisional Application No. 62/333,931, filed May 10, 2016, hereby incorporated by reference in its entirety.

[0002] Throughout this application various publications, patents, and/or patent applications are referenced. The disclosures of the publications, patents and/or patent applications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

[0003] The present disclosure is directed generally to inventive methods relating to identifying and measuring nucleic acids and more specifically to the use of chelating agents for nucleic acid incorporation detection.

BACKGROUND

[0004] Various next generation sequencing platforms employ sequencing-by-synthesis

(SBS) strategies using polymerase or ligase enzymes to detect many nucleic acid strands in parallel. In SBS, template nucleic acid strands or fragments to be sequenced may be first amplified to generate populations of identical template molecules by polymerase chain reaction (PCR). The sequence of these clonally amplified templates is discerned by repeated cycles of primer extension to produce signals that are decoded to obtain nucleotide sequence information for the template nucleic acid molecules. See, e.g., Hert et al., Electrophoresis, 29:4618-4626 (2008); Metzker, Nature Reviews Genetics, 11:31-46 (2010); Droege et al, J. Biotechnology, 136:3-10 (2008).

[0005] Accurate and sensitive sequence determination depends, in part, upon the quality of signals generated as a result of nucleotide incorporation. Various factors can affect the quality of the incorporation signals and may include sampling or reaction time and phasing effects. For example, in sequencing methods where incorporation signals arise by sensing the presence of molecular byproducts arising from a sequencing reaction {e.g., hydrogen ions and/or pyrophosphate molecules) the transient nature of the signals associated with the reactions and the limited amount of byproducts released can significantly affect the overall signal strength. Consequently, there is a need for improved methods for nucleotide incorporation detection that allow for more efficient and accurate sequence determination.

SUMMARY

[0006] Generally disclosed are methods relating to identifying and measuring nucleic acids using chelating agents to improve nucleotide incorporation detection. Such nucleic acid analyses are useful in a variety of applications, including for example, nucleic acid sequencing, detecting the presence of nucleic acids and quantifying nucleic acids.

[0007] In various embodiments, the methods disclosed herein are suitable for use with label-free and/or unterminated sequencing methods. Such methods include ion-based sequencing, where nucleotide incorporation events are detected using chemically sensitive transistors (e.g., field effect transistors (FETs) including chemical field effect transistors (chemFETs) or ion selective field effect transistors (IS FETs). See, e.g., Rothberg et ah, U.S. Patent Application Publication No. 2009/0127589 and Rothberg et al, U.K. Patent Application No. GB24611127.

[0008] In one aspect, provided are methods for nucleic acid sequencing comprising: disposing at least one template nucleic acid in a reaction region communicating with a chemical sensor; introducing a nucleotide into the reaction region along with sequencing reagents to form a synthesized nucleic acid/template nucleic acid complex; introducing one or more metal ions into the reaction region that associates with the synthesized nucleic acid/template nucleic acid complex; introducing one or more chelating agents into the reaction region that disassociates the one or more metal ions from the synthesized nucleic acid/template nucleic acid complex and affects a change in ion concentration for the reaction region; detecting with the chemical sensor the change in ion concentration for the reaction region; and associating the detected change in ion concentration as indicator of nucleotide incorporation whereby the composition of the incorporated nucleotide is used to discern at least a portion of the sequence for the at least one template nucleic acid. In some embodiments, the one or more metal ions reversibly bind to the synthesized nucleic acid/ template nucleic acid complex. [0009] In some embodiments, the template nucleic acid can be attached to or associated with a bead, particle, or other substrate. In some embodiments, the template nucleic acid can be attached to or associated with the bead or particle, and the bead or particle can be disposed (deposited) onto the reaction region that is communicated with a chemical sensor. In some embodiments, the template nucleic acid can be attached to or associated with the reaction region that is communicated with a chemical sensor. The template nucleic acid may further be replicated or clonally amplified to generate one or more copies of the template nucleic acid that are disposed in the same reaction region and sequenced substantially simultaneously. In other embodiments, the metal ion can be one or more of a Group la metal, a Group Ila metal, a transition metal, a post transition metal, a lanthanide or a mixture thereof. In yet other embodiments, the one or more chelating agents can be one or more of ethylenediamine tetraacetic acid (EDTA), diethlyenetriaminepentaacetic acid (DTPA), N-(hydroxyethyl)- ethylenediaminetriacetic acid (HEDTA), ethyleneglycol tetraacetic acid (EGTA), N,N- Ws(carboxymethyl)glycine (NTA), 2,3-dimercaprol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-l-propanesulfonic acid (DMPS), penicillamine, ethylene diamine, terpyridine, 14,7-triazacycononane (TACN), triethylenetetramine (trien), trisil- aminoethyl)amine, ethylenediaminetriacetic acid, 1, 4,7, 10-tetraazacyclododecane- 1,4,7, 10- tetraacetate (DOTA), diethylene triamine pentaacetate (DPTA), deferoxamine (DFOA), deferiprone, tetraethylenetetraamine (TETA), porphine, analogs thereof, salts thereof, or a mixture thereof.

[0010] In some embodiments, the reaction region comprises a well, cavity, or surface. In some embodiments, the at least one template nucleic acid is disposed on or brought in proximity to the reaction region. The reaction region can further include a surface having a nucleic acid primer or other molecular moiety attached thereto for retaining or associating the at least one template nucleic acid with the surface. In other embodiments, the chemical sensor may register the change in ion concentration as an output pulse or signal. The output pulse or signal may further comprise a voltage or current differential generated by the chemical sensor in response to a transient change in ion concentration. In still other embodiments, the nucleic acid template can include an adapter that is at least partially complementary to at least a portion of the primer.

[0011] In some embodiments, nucleotide species introduced into the reaction region comprise nucleotides selected from dATP, dCTP, dGTP, dTTP or dUTP. A single type of nucleotide may be introduced into the reaction region to form the synthesized nucleic acid or template nucleic acid complex when there is complementarity between the nucleotide species and the template nucleic acid. Nucleotides may be serially introduced into the reaction region as singular species forming the synthesized nucleic acid/template nucleic acid complex when there is complementarity between the nucleotide species and the template nucleic acid. In some embodiments, the synthesis of the nucleic acid or template nucleic acid complex can generate nucleotide incorporation byproducts, including pyrophosphate, ions (e.g., hydrogen ions), protons, charge transfer or heat. In other embodiments, the change in ion-concentration can be due at least in part to liberation of ions, including for example hydrogen ions, that result from interactions between the metal ion with the chelating agent.

[0012] In some embodiments, the chemical sensor can comprise an ion- sensitive field- effect transistor (ISFET) including a floating gate coupled (communicated) to the reaction region. Disposing the template nucleic acid in a reaction region can further comprise amplifying the template nucleic acid in the reaction region to form at least one copy of the template nucleic acid. Amplification techniques can include isothermal and polymerase chain reaction amplification to generate additional copies of the template nucleic acid within the reaction region.

[0013] In another aspect, provided are methods for nucleic acid analysis, the method comprising: associating a plurality of template nucleic acids with a plurality of reaction regions, the reaction regions further associated with a plurality of sensors of a sensor array, each sensor comprising a field-effect transistor configured to provide at least one output signal in response to charged atoms or molecules proximate thereto; introducing reagents into the reaction region with the template nucleic acids wherein the reagents may comprise a primer that hybridizes or associates with at least a portion of the template nucleic acids and enzymes including for example polymerases and ligases that may further bind to or associate with the template nucleic/sequencing primer hybrid; introducing a nucleotide species into the reaction region resulting in nucleotide incorporation and extension of the primer when such nucleotide is complementary to a corresponding nucleotide in the template nucleic acid; introducing one or more metal ions into the reaction region wherein the one or more metal ions reversibly binds to the extended template nucleic acid/sequencing primer hybrid; introducing one or more chelating agents into the reaction region that chelates with the one or more metal ions releasing ions, for example hydrogen ions, in proportion to the amount of the template nucleic acid and extended sequencing primer in a respective reaction region; detecting the released hydrogen ions with the field-effect transistor providing at least one output signal indicative of the amount of the template nucleic acid and extended sequencing primer in the reaction region. The nucleic acid analysis method can further The nucleic acid analysis method may further comprise determining a sequence or composition for at least a portion of the template nucleic acid based on the nucleotide species introduced into the reaction region and the at least one output signal provided by the field-effect transistor. In various embodiments, the reaction region may be further washed or flushed removing or substantially reducing the concentration of released ions. In some embodiments, and the steps above can be repeated with the introduction of reagents into the reaction region, where the reagents comprise a primer that hybridizes or associates with at least a portion of the template nucleic acids and enzymes including for example polymerases and ligases that may further bind to or associate with the template nucleic/sequencing primer hybrid. In some embodiments, and the steps above can be repeated with the introduction of another nucleotide species that causes nucleotide incorporation and extension of the primer to further extend the sequencing primer and generating at least one additional output signal that may further be used for template nucleic acid sequence or composition determination.

[0014] In another aspect, provided are methods for nucleic acid analysis without sequencing, the method comprising: disposing a nucleic acid sample in a reaction region communicating with a chemical sensor; introducing one or more metal ions into the reaction region that associates with the nucleic acid sample; introducing one or more chelating agents into the reaction region that disassociates the one or more metal ions from the nucleic acid sample and affects a change in ion concentration in the reaction region; detecting with the chemical sensor the change in ion concentration in the reaction region; and associating the detected change in ion concentration as indicator of the presence of and/or quantity of nucleic acid in the nucleic acid sample. In some embodiments, the one or more metal ions reversibly bind to the nucleic acid sample that is disposed in the reaction region.

[0015] In another aspect, the disclosure relates generally to methods, as well as related systems, compositions, kits, and apparatuses for nucleic acid sequencing comprising: (a) providing a plurality of reaction regions arranged in an array, wherein at least one reaction region in the array is capacitively coupled to a sensor that detects a change in ion concentration; (b) disposing at least one template nucleic acid into the reaction region that is capacitively coupled to the sensor; (c) introducing into the reaction region (i) a primer that hybridizes to a portion of the template nucleic acid, (ii) a nucleotide species, and (iii) an enzyme, under conditions suitable for nucleotide incorporation to generate a primer extension product; (d) introducing into the reaction region one or more metal ions which reversibly binds the primer extension product; (e) introducing into the reaction region one or more chelating agents which binds the metal ions that are reversibly bound to the primer extension product to form a metal ion/chelate complex thereby releasing a plurality of ions which changes the ion concentration in the reaction region; (f) detecting with the sensor the change in the ion concentration in the reaction region; and (g) determining the sequence of the at least one template nucleic acid.

[0016] In some embodiments, the sensor in step (a) comprises an ion-sensitive field- effect transistor (ISFET). In some embodiments, the ion-sensitive field-effect transistor (ISFET) comprises a floating gate which is coupled to the reaction region. In some embodiments, the sensor registers the change in ion concentration as an output pulse or signal.

[0017] In some embodiments, the array in step (a) includes a plurality of reaction regions, each reaction region comprising a well, cavity or surface. In some embodiments, the array in step (a) contains 10 2 - 1010 reaction regions. In some embodiments, at least one of the template nucleic acids in step (b) is disposed into the reaction region that is capacitively coupled to the sensor, where the reaction region comprises a well, cavity or surface. In some embodiments, the template nucleic acid that is disposed into the region is attached to the well, cavity or surface. In some embodiments, the reaction region includes a capture primer attached thereto. In some embodiments, the well, cavity or surface includes a capture primer attached thereto. In some embodiments, the at least one template nucleic acid includes an adapter that hybridizes to at least a portion of the capture primer. In some embodiments, the capture primer comprises an oligonucleotide primer having at least a portion that is complementary to a sequence in the adapter to permit binding between the capture primer and the adaptor. In some embodiments, the at least one template nucleic acid is attached to a bead or particle which is disposed into the reaction region.

[0018] In some embodiments, the nucleotide species that is introduced into the reaction region in step (c) comprises nucleotides selected from the group consisting of dATP, dCTP, dGTP, dTTP and dUTP. [0019] In some embodiments, the enzyme that is introduced into the reaction region in step (c) comprises a DNA or RNA polymerase.

[0020] In some embodiments, the one or more metal ions that are introduced in step (d) are selected from any one or any combination of a Group la metal, a Group Ila metal, a transition metal, a post transition metal, a lanthanide or a mixture thereof.

[0021] In some embodiments, the one or more chelating agents that are introduced in step

(e) are selected from any one or any combination of ethylenediamine tetraacetic acid (EDTA), diethlyenetriaminepentaacetic acid (DTPA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), ethyleneglycol tetraacetic acid (EGTA), N,N-bw(carboxymethyl)glycine (NT A), 2,3- dimercaprol (BAL), meso2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-l- propanesulfonic acid (DMPS), penicillamine, ethylene diamine, terpyridine, 14,7- triazacycononane (TACN), triethylenetetramine(trien), in^'s(2-aminoethyl)amine, ethylenediaminetriacetic acid, l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetate (DOT A), diethylene triamine pentaacetate (DPTA), deferoxamine (DFOA), deferiprone, tetraethylenetetraamine (TETA), porphine, analogs thereof, salts thereof, or a mixture thereof.

[0022] In some embodiments, the plurality of released ions in step (e) are hydrogen ions.

[0023] In some embodiments, the method further comprises disposing at least one template nucleic acid in a reaction region and amplifying the template nucleic acid in the reaction region.

[0024] In some embodiments, the method further comprises at least one washing step performed after any one of steps (b), (c), (d), (e), (f) or (g), or any combination of these steps, before proceeding to the next step. In some embodiments, the reaction region is subjected to a washing step after step (c) to remove or substantially reduce unused primers, nucleotide species and/or enzymes. In some embodiments, the reaction region is subjected to a washing step after step (c) to remove or substantially reduce byproducts of nucleotide incorporation, including pyrophosphate (PPi) and/or hydrogen ions. In some embodiments, the reaction region is subject to a washing after step (d) to remove or substantially reduce unreacted metal ions. In some embodiments, the reaction region is subject to a washing after step (e) to remove or substantially reduce unreacted chelating agents. In some embodiments, the reaction region is subject to a washing after step (f) to remove or substantially reduce the plurality of released ions that are detected by the sensor. In some embodiments, the reaction region is subjected to a washing step after step (b) or after step (c) or after step (d) or after step (e) or after step (f). In some embodiments, the reaction region is subjected to a washing step after steps (c) and (d) and (e) and (f).

[0025] In some embodiments, the nucleotide incorporation of step (c) generates a primer extension product and a plurality of ions which changes the ion concentration in the reaction region. For example, the nucleotide incorporation of step (c) generates hydrogen ions.

[0026] In some embodiments, the sensor detects the change in the ion concentration in the reaction region which is generated from the nucleotide incorporation of step (c).

[0027] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being a part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 depicts a negatively charged nucleic acid polymer backbone and positive ion association.

[0029] Figure 2 depicts an exemplary nucleotide incorporation followed by metal ion binding and metal chelate formation by contact with a chelating agent.

[0030] Figure 3 depicts an exemplary signal generation via formation of hydrogen ion by

DNA counterion exchange (metal ion flow) and signal generation (chelating agent EDTA flow).

[0031] Figure 4 depicts the chemistry of an exemplary nucleotide incorporation and corresponding signal generation. Figure 4a depicts a nucleotide species contacting a template nucleic acid and a primer. Figure 4b depicts incorporation of the nucleotide species for nucleic acid extension and production of byproducts hydrogen ion and PPi.

[0032] Figure 5 depicts the chemistry of an exemplary nucleotide incorporation and corresponding signal generation. Figure 5a depicts a representative negatively charged phosphate linkage between nucleotide units in association with a native potassium counterion. Figure 5b depicts the resulting synthesized nucleic acid/template nucleic acid complex having the nucleotide species incorporated into the top synthesized nucleic acid strand.

[0033] Figure 6 depicts the chemistry of an exemplary nucleotide incorporation and corresponding signal generation. Figure 6a depicts signal generation by a metal chelation reaction and subsequent generation of hydrogen ion. Figure 6b depicts a baseline pre- incorporation signal (graph on the left) and a post-incorporation signal (graph on the right) generated by the incorporation events.

[0034] Figure 7 depicts an exemplary nucleotide non-incorporation and corresponding signal generation. Figure 7a depicts a nucleotide non-incorporation event. Figure 7b shows two graphs showing signal generation before and after a non-incorporation event.

[0035] Figure 8 depicts the chemistry of an exemplary second nucleotide incorporation

(a second dNTP). Figure 8a depicts incorporation of a second nucleotide species contacting a template nucleic acid and primer. Figure 8b depicts incorporation of the second nucleotide species for nucleic extension and production of byproducts hydrogen ion and PPi.

[0036] Figure 9 depicts the chemistry of an exemplary third nucleotide incorporation (a third dNTP). Figure 9a depicts incorporation of a third nucleotide species contacting a template nucleic acid and primer. Figure 9b depicts incorporation of the third nucleotide species for nucleic extension and production of byproducts hydrogen ion and PPi.

[0037] Figure 10 is a graph showing signal generation corresponding to incorporation of a first dNTP, a second dNTP and a third dNTP, compared to a baseline signal.

[0038] Figure 11 depicts an exemplary time-delay metal ion post incorporation detection method. Figure 11a depicts an empty reaction region and a reaction region disposed with nucleic acids a solid substrate (e.g., beads or particles), and nucleotide incorporation and metal ion flow. Figure lib shows a graph showing a time delay in signal generation between the empty reaction region compared to the reaction region containing nucleic acid disposed on beads.

[0039] Figure 12 depicts a proof of principle signal of ladder DNA beads with 20 bp difference in length of primers. Figure 12a is a graph showing a raw trace. Figure 12b is a graph showing an integrated signal histogram. Figure 12c is a graph showing the linearity of signal as a function of primer length.

[0040] Figure 13 is a graph showing signal generation for a sequencing run. [0041] Figure 14 is a graph showing 1-mer signal for two types of beads.

[0042] Figure 15A is a graph showing exemplary template nucleic acid sample detection signals over time.

[0043] Figure 15B is a graph showing an exemplary detection signal distribution over time for samples and controls.

[0044] Figure 16A is a graph showing a normalized sample detection signal distributions over time.

[0045] Figure 16B is a graph showing exemplary peak detection signal linearity properties.

[0046] Figure 17 is a graph showing an exemplary plot of a time delay processed incorporation signal.

[0047] Figure 18 is a schematic of a modified nucleotide having a chelator tag.

[0048] Figure 19A is a schematic of a modified nucleotide carrying a phosphatase chelator tag which can be enzymatically cleaved, as depicted with a circle and hatch.

[0049] Figure 19B is a graph showing signals generated before and after cleavage of the phosphate chelator tag using a phosphatase (CIP).

DETAILED DESCRIPTION

[0050] Following below are more detailed descriptions of various concepts related to, and embodiments of, inventive metal chelation methods for analyzing (e.g. , detecting, identifying and/or quantifying) nucleic acids. Such detection methods are useful in various applications for analyzing nucleic acids, including, for example, identifying and sequencing nucleic acids, quantifying or assaying nucleic acids, single nucleotide polymorphism detection, nucleic acid expression profiling (for example, comparing nucleic acid expression profiles between two or more states or samples, e.g. , comparing between diseased and normal tissue or comparing between untreated tissue and tissue treated with drug, enzymes, radiation or chemical treatment), haplotyping (for example, comparing genes or variations in genes on each of the two alleles present in a human subject), karyotyping (for example, comparing one or more genes from a sample to detect chromosomal differences), genotyping (comparing alleles for one or more genes from a first individual or sample with the same genes from a second individual or sample) and analyzing chemical and/or biological processes (for example, chemical reactions, cell cultures, neural activity, etc.).

[0051] It should be appreciated that various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the disclosed concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. It will be appreciated, that the methods described are not limited to performing nucleic acid analysis and may further be adapted to operate in connection with analyzing other molecular species including for example, biological molecules such as peptides, proteins, antibodies, antigens, enzymes, cells or other biological or chemical moieties.

[0052] In some embodiments, chelation methods for analyzing template molecules, such as nucleic acids, involve synthesis of nucleic acid polymers and/or nucleotide incorporation during template sequencing. Accordingly, certain methods disclosed herein comprise synthesis of nucleic acid polymers (such as a nucleotide incorporation or nucleotide extension reaction) followed by a metal chelation reaction, with byproducts of the metal chelation reaction being measured to provide information useful in nucleic acid analysis.

[0053] Nucleic acid polymer synthesis can involve enzyme-mediated incorporation of individual nucleotides to form and extend a nucleic acid polymer. Nucleotides that are typically used in this process are nucleoside polyphosphates, such as deoxyribonucleotide triphosphates (dNTPs), which can be incorporated in the synthesis of deoxyribonucleic acids (DNA), and ribonucleotide triphosphates (NTPs), which can be incorporated in the synthesis of ribonucleic acids (RNA). Incorporated nucleotides can undergo hydrolysis of one or more of its phosphodiester bonds, which provides the thermodynamic driving force for the overall reaction. Incorporation of nucleotides to form and extend nucleic acid polymers release pyrophosphate (PPi) and hydrogen ions, increasing the overall negative charge of the synthesized nucleic acid polymers.

[0054] Figure 1 shows an exemplary nucleic acid polymer having a negatively charged polymer backbone. Each negative charge of the phosphate groups can be balanced by a native counterion (Ion⁺) commonly occurring in cytoplasm, including for example a Group IA metal ion, such as Na⁺ or K⁺, a Group Ila metal ion, such as Ca²⁺ or Mg²⁺, and the like. The addition of each dNTP at the 5' end of the nucleic acid polymer increases the overall negative charge of the synthesized nucleic acid polymer via the presence of additional negatively-charged phosphate groups. The increase in the overall negative charge of the synthesized nucleic acid polymer can increase the amount of native counterions (Ion+) that balance the negative charge.

[0055] For example, extension of a nucleic acid polymer with a single nucleotide incorporation event can result in the nucleic acid polymer being extended by the single nucleotide with the release of extension byproducts (e.g., PPi and hydrogen ion), increasing the overall negative charge of the synthesized nucleic acid polymer. When the nucleotide incorporation event is conducted at or near a chemical sensor (e.g., ion sensitive field effect transistors), then the release of the extension byproducts from the nucleotide incorporation event can be detected. Optionally, at least one wash step can be performed subsequent to a nucleotide incorporation step. After sufficient washing away of extension byproducts (as an optional step), extended nucleic acid polymers can be contacted with metal ions and the metal ions can bind or associate with the negatively charged backbone of the synthesized nucleic acid polymer while displacing native counterions initially bound thereto. The number of metal ions associated with the negatively charged backbone of the nucleic acid polymer can be quantifiably measured via a metal chelation reaction. Specifically, subsequent contact of the metal-bound/associated nucleic acid backbone with a chelating agent releases a stoichiometric amount of hydrogen ions generally proportional to the amount of nucleic acid present in the sample. Released hydrogen ions can be detected and quantitated by various conventional means including using instruments employing ion sensitive field effect transistors such as Ion Torrent nucleic acid sequencers (ThermoFisher Scientific Inc.). The hydrogen ions released from the chelating agent step can be detected separately from the hydrogen ions released from the nucleotide incorporation step. In stepwise nucleotide incorporation reactions, a difference in the amount of hydrogen ion released before and after introduction of the nucleotide may be used to determine when extension has taken place, by how many nucleotides, and the identity of particular complementary nucleotide of a template nucleic acid molecule. The metal chelation detection methods disclosed herein may be used to measure the corresponding amount of hydrogen ions released as a result of a chelating agent binding with the total amount of metal ions associated with selected nucleic acid polymers, thereby providing an indirect way to measure total amount of and/or identity of nucleic acid present in a sample. [0056] Conventional detection methods measuring nucleotide incorporation byproduct/analyte concentrations, such as PPi or hydrogen ion release, are integrated over time and may be subject to high background and noise. Further, such conventional detection methods are dependent on enzyme kinetics of binding events. The metal chelation post incorporation detection methods disclosed herein address these issues, advantageously facilitating rapid detection of nucleotide incorporation with improved sensitivity and signal-to-noise ratios. In various embodiments, metal ion binding to the negatively charged nucleic acid strands followed by metal ion release with a chelating agent and concomitant liberation of hydrogen ions from the chelating agent provides a mechanism by which to evaluate each incorporation event after completion. Such an approach desirably provides a rapid and consistent ion release that can be readily detected using suitable detection methods, for example using an ion-sensitive field effect transistor (ISFET)-based detector / instrument such as implemented in the Ion Torrent sequencing systems. Desirably, the reaction may further follow first order reaction kinetics thus providing rapid rates of reaction and subsequent signal generation improving sequence detection and or quantitation characteristics.

[0057] Extension of nucleic acid polymers by the incorporation of individual nucleotide species is an important aspect of many processes in molecular biology, both in natural and artificial contexts. Examples of the latter, which entail steps of nucleic acid extension, include nucleic acid amplification, quantitative polymerase chain reaction, and nucleic acid sequencing. Nucleic acid extension involves incorporation of a nucleotide species into a template nucleic acid strand or a complementary nucleic acid strand that is being extended. This incorporation and extension is typically mediated by an enzyme, such as a polymerase or ligase, with a suitable nucleotide species to be incorporated. The nucleotide species typically include one or more high-energy chemical bonds, at least one of which is broken and/or reformed as a lower energy bond, thus providing the free energy to drive the extension reaction.

[0058] One application of nucleic acid extension reactions is in nucleic acid sequencing where the sequence of a target/template nucleic acid is discerned by extending a complementary nucleic acid strand through incorporation of one or more nucleotide species. A number of sequence detection approaches have been devised including methods based on chemical degradation, chain-termination, sequencing-by-synthesis, pyrosequencing, and ion-sensitive detection. [0059] Figure 2 depicts an exemplary metal chelation reaction suitable for nucleic acid analysis by producing a measurable analyte, in particular hydrogen ion. In step 1, a bead, particle or other substrate having a population of template nucleic acid fragments, strands, or samples disposed thereon is contacted with a selected nucleotide species in a reaction region, for example a chamber or well. The template nucleic acid fragments, strands, or samples disposed on the bead or particle or substrate can be duplexed with an oligonucleotide primer. In some embodiments, the nucleotide species can be flowed onto the reaction region and the bead or particle or other substrate that is disposed into the reaction region. The reaction region creates a localized environment facilitating association of the selected nucleotide species with the template nucleic acid and may result in one or more incorporation events where at least a portion of the nucleotide species becomes covalently bound to a growing nucleic acid polymer or strand (e.g. a synthesized nucleic acid or nucleic acid polymer strand) associated with the template nucleic acid. The incorporation event extends the synthesized nucleic acid polymer, increasing the overall negative charge of the nucleic acid backbone, as well as releasing PPi and hydrogen ion byproducts. One or more optional wash flow steps can be conducted to wash away the PPi and hydrogen ion byproducts prior to proceeding to the next step. In step 2, metal ions are contacted and bind with the negatively charged nucleic acid backbone. In some embodiments, the metal ions can be flowed onto the reaction region and the bead or particle or other substrate that is disposed into the reaction region. Such metal ions are selected based on having preferential binding to the negatively charged nucleic acid backbone in comparison to native counterions already bound thereto. In some embodiment, the metal ions can be flowed onto the reaction region and the bead or particle or other substrate that is disposed into the reaction region. Excess reagents, such as unbound metal ion, can optionally be washed away with one or more wash flow steps prior to proceeding to the next step. In step 3, a chelating agent is introduced into the reaction region, where the chelating agent binds or associates with the metal ions to form a metal chelate complex, releasing hydrogen ions that produce a signal proportional to the total amount of nucleic acid, including the nucleic acid of the synthesized nucleic acid strand and the nucleic acid of the template nucleic acid strand. In some embodiment, the chelating agent can be flowed onto the reaction region and the bead or particle or other substrate that is disposed into the reaction region. The release of hydrogen ions and corresponding signal is cumulative and representative of the total amount of metal ions that are bound to the DNA backbone and indirectly to the amount of nucleic acid. The metal chelation reaction shows first order kinetics as reaction proceeds linearly with concentration of reactants. In some embodiments, the reaction region is communicated with a chemical sensor that can detect a change in the concentration of a nucleotide incorporation byproduct, including pyrophosphate, ions (e.g., hydrogen ions), protons, charge transfer or heat. In some embodiments, the chemical sensor comprises an ion selective field effect transistors (ISFET).

[0060] Figure 3 depicts an exemplary metal ion release generated by a metal chelation reaction, namely by generating a hydrogen ion as a result of forming a metal chelate complex when a chelating agent is contacted with a metal ion that is bound to the backbone of nucleic acids. Step 1 depicts a nucleic acid counterion exchange where native counterion potassium ion, which may be associated with the negatively charged backbone of the DNA strands, is displaced in the presence of a metal ion (M^v+) having a higher affinity for the DNA and results in displacement of the potassium ion and preferential association of the M^v+ ion with the DNA. The first step can be followed by at least one wash flow step to wash away displaced ions and excess reagents, e.g. , metal ion M^v+. The second step shows the addition of a chelating agent, for example, EDTA^'H (ethylenediaminetetraacetic acid), where M^v+ preferentially binds with the chelating agent and hydrogen ions are released in proportion to the total amount of metal ions associated with the DNA. Released hydrogen ions can result in a pH change in the reaction environment (e.g., reaction region), which can be detected by an ion sensor, for example an ISFET. Metal chelation detection methods disclosed herein measure additive or cumulative signals of the total amount of hydrogen ions released, advantageously allowing for detection for even small nucleic acid samples. Such hydrogen ion measurements are an indirect method of analyzing nucleic acids of a test sample through quantifying metal ion M^v+ bound to the DNA.

[0061] Figures 4, 5 and 6 depict the chemistry of an exemplary nucleotide incorporation and corresponding signal generation, and in particular the incorporation of a first nucleotide species to a template nucleic acid and primer. In Figure 4a, a template nucleic acid and a primer is contacted with a nucleotide species (in this case, for example, deoxycytidine triphosphosphate ("dCTP")) and in the presence of an enzyme. The sequence of the nucleic acid template is 5'- TAGCTAG-3' and the primer sequence is 5'-CTAG-3' . The cytosine base of dCTP is complementary to the next unbound base of the template nucleic acid (guanine) and results in the elongation of the primer via incorporation of dCTP. The sequence of the elongated primer is 5'- CTAGC-3'. Figure 4b shows the incorporated dCTP to the replicate nucleic acid and extension byproducts hydrogen ion and PPi. This incorporation step can optionally be followed by one or more wash flow steps to wash away hydrogen ion and PPi byproducts, other byproducts and excess reagents.

[0062] Figure 5b depicts the resulting synthesized nucleic acid/template nucleic acid complex having the dCTP incorporated into the top synthesized nucleic acid strand. A more detailed depiction of the synthesized nucleic acid backbone is shown in Figure 5a, showing a representative negatively charged phosphate linkage between nucleotide units in association with a native potassium counterion. Each phosphate linkage of the synthesized nucleic acid/template nucleic acid complex can be negatively charged and in association with a native counterion. Contacting metal ion M^v+ with the synthesized nucleic acid/template nucleic acid complex displaces native counterions and associates M^v+ with the negatively charged phosphate linkages to form a metal bound/associated nucleic acid backbone.

[0063] Figure 6a depicts signal generation by a metal chelation reaction and subsequent generation of hydrogen ion. A chelating agent, for example EDTA, can be contacted with the metal bound/associated nucleic acid backbone, disassociating the metal ion M^v+ from the synthesized nucleic acid/template nucleic acid complex by forming a metal chelate complex while simultaneously affecting a change in ion concentration in the local environment by releasing a hydrogen ion. The hydrogen ion concentration can be measured by a chemical sensor that is in communication with the metal chelation reaction environment. The metal chelation reaction facilitates a cumulative release of hydrogen ion as chelating agent can bind with all metal ion Mv+ in association with the entire synthesized nucleic acid/template nucleic acid complex.

[0064] Figure 6b depicts a baseline pre-incorporation signal (graph on the left) and a post-incorporation signal (graph on the right) generated by the incorporation events of Figures 4- 6. Prior to each attempt to incorporate a nucleotide species, a signal can be measured by contacting a chelating agent with a pre-incorporation nucleic acid sample. The successful incorporation of dCTP extends the synthesized nucleic acid and subsequent metal ion exchange and metal chelation reaction generates a concentration of hydrogen ion that is higher than baseline, as shown by the larger signal generated in the graph on the right. [0065] Figure 7 depicts a non-incorporation event and subsequent signal generation.

Contacting the synthesized nucleic acid/template nucleic acid complex with nucleotide species deoxyguanosine triphosphate ("dGTP") does not result in an incorporation event as dGTP is not complementary to next unbound base (adenosine) on the template nucleic acid, as shown in Figure 7a. As a result, the synthesize nucleic acid is not extended and corresponding signal shows no significant increase compared to a pre-incorporation signal of the synthesized nucleic acid/template nucleic acid complex, as shown in Figure 7b.

[0066] Figures 8 and 9 depict the chemistry of subsequent nucleotide incorporations of the synthesized nucleic acid/template nucleic acid complex of Figure 4. In particular, Figures 8 a and 8b depict the incorporation of a second nucleotide species (deoxythymidine triphosphate ("dTTP")), where dTTP is complementary to the next unbound base (adenosine) on the template nucleic acid. The sequence of the nucleic acid template is 5'-TAGCTAG-3' and the primer sequence is 5'-CTAG-3', and the sequence of the elongated primer is 5'-CTAGCT-3'. Figures 9a and 9b depict the incorporation of a third nucleotide species (deoxyadenosine triphosphate ("dATP")), where dATP is complementary to the next unbound base (thymine) on the template nucleic acid. The sequence of the elongated primer is 5'-CTAGCTA-3' .

[0067] Figure 10 depicts an overlay of signal generation corresponding to incorporation of a first nucleotide species, a second nucleotide species, and a third nucleotide species of Figures 4, 8 and 9. Successive incorporations of nucleotide species can increase signal generation that correspond with the amount hydrogen ion released during their respective metal chelation reaction.

[0068] In some embodiments, the methods provided herein are post-incorporation detection (PID) methods, where nucleic acid analysis is conducted after all incorporation events have occurred, providing a cumulative detection of analyte, such as hydrogen ion. Such PID methods are advantageous in that they utilize natural nucleotides while allowing for low reagent cost and avoiding typical enzymatic cleavage chemistry, provide proton-like run times where signals are generated by fast first order chemical kinetics instead of enzyme kinetic rates, allows for multiple measurements that improve signal to noise ratios, and measures total nucleic acids, which always produces signals even for low nucleic acid copies in test samples.

[0069] In some embodiments, nucleic acids can be analyzed using chelation methods that do not involve synthesis of nucleic acid polymers. Nucleic acid polymer samples can be directly subject to metal chelation reaction, with byproducts of the metal chelation reaction being measured to provide information useful in nucleic acid analysis. In particular, a nucleic acid polymer sample can be contacted with a metal ion, facilitating an exchange of native counterions associated with the nucleic acid polymer sample with the metal ion and resulting in the metal ion being associated with the negatively charged nucleic acid polymer sample.

[0070] Suitable metal ions (M^v+) can be selected from metal ions capable of associating with the negative charges of a nucleic acid, including for example, alkali (Group IA) metals, alkaline earth (Group IIA) metals, transition metals, post transition metals, or lanthanides. In some embodiments, metal ions are selected from yttrium (Y³⁺), manganese (Mn²⁺), lanthanum (La³⁺), manganese (Mn³⁺), iron (Fe²⁺, Fe³⁺), nickel (Ni²⁺), copper (Cu²⁺), chromium (Cr³⁺, Cr⁶⁺), cobalt (Co²⁺, Co³⁺), titanium (Ti³⁺, Ti⁴⁺), vanadium (V²⁺, V³⁺), zirconium (Zr⁴⁺), silver (Ag⁺), zinc (Zn²⁺), aluminum (Al³⁺), tin (Sn²⁺, Sn⁴⁺), cerium (Ce³⁺), neodynium (Nd³⁺), lithium (Li+), sodium (Na+), potassium (K+), magnesium (Mg²⁺), calcium (Ca²⁺), strontium (Sr²⁺), barium (Ba²⁺) or mixtures thereof. Such metal ions are selected for having a higher affinity with the nucleic acid compared to affinity of native counterions initially associated with the nucleic acid prior to metal ion exchange. For example, the metal ions (M^v+) can be selected from yttrium (Y³⁺), manganese (Mn²⁺), lanthanum (La³⁺), manganese (Mn³⁺), iron (Fe²⁺, Fe³⁺), nickel (Ni²⁺), copper (Cu²⁺), chromium (Cr³⁺, Cr⁶⁺), cobalt (Co²⁺, Co³⁺), titanium (Ti³⁺, Ti⁴⁺), vanadium (V²⁺, V³⁺), zirconium (Zr⁴⁺), silver (Ag⁺), zinc (Zn²⁺), aluminum (Al³⁺), tin (Sn²⁺, Sn⁴⁺), cerium (Ce³⁺), neodynium (Nd³⁺), lithium (Li+), magnesium (Mg²⁺), calcium (Ca²⁺), strontium (Sr²⁺), barium (Ba²⁺) or mixtures thereof. In a particular example, the metal ions are selected from yttrium (Y³⁺), manganese (Mn²⁺), lanthanum (La³⁺), manganese (Mn³⁺), iron (Fe²⁺, Fe³⁺), nickel (Ni²⁺), copper (Cu²⁺) chromium (Cr³⁺, Cr⁶⁺), cobalt (Co²⁺, Co³⁺), titanium (Ti³⁺, Ti⁴⁺), vanadium (V²⁺, V³⁺), zirconium (Zr⁴⁺), silver (Ag⁺), zinc (Zn²⁺), aluminum (Al³⁺), tin (Sn²⁺, Sn⁴⁺), cerium (Ce³⁺), neodynium (Nd³⁺), lithium (Li+), strontium (Sr²⁺), barium (Ba²⁺) or mixtures thereof. For example, the metal ions can include yttrium (Y³⁺), manganese (Mn²⁺), lanthanum (La³⁺), manganese (Mn³⁺), cobalt (Co²⁺, Co³⁺), vanadium (V²⁺, V³⁺), cerium (Ce³⁺), neodynium (Nd³⁺), or mixtures thereof. In particular, the metal ions can include manganese (Mn²⁺), lanthanum (La³⁺), manganese (Mn³⁺), or mixtures thereof. In some embodiments, metal ions are introduced in the presence of one or more salts, for example a monovalent, divalent salt or a mixture thereof. In other embodiments, metal ions are introduced in the presence of one or more divalent salts. [0071] Chelating agents include organic or inorganic compounds capable of associating with metal ions. Such chelating agents typically comprise "ligand" binding atoms that form two or more separate coordinate bonds between a polydentate ligand and a single central atom, i.e., metal ion. Suitable chelating agents have a higher affinity of chelating with a metal ion compared to the affinity of a nucleic acid sample for the same metal ion. Suitable chelating agents include, but are not limited to, ethylenediamine tetraacetic acid (EDTA), diethlyenetriaminepentaacetic acid (DTPA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), ethyleneglycol tetraacetic acid (EGTA), N,N-bw(carboxymethyl)glycine (NT A), 2,3- dimercaprol (BAL), meso2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-l- propanesulfonic acid (DMPS), penicillamine, ethylene diamine, terpyridine, 14,7- triazacycononane (TACN), triethylenetetramine(trien), ira(2-aminoethyl)amine, ethylenediaminetriacetic acid, l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetate (DOT A), diethylene triamine pentaacetate (DPTA), deferoxamine (DFOA), deferiprone, tetraethylenetetraamine (TETA), porphine, analogs thereof, salts thereof, or a mixture thereof. In some embodiments, chelating agents are introduced in the presence of one or more salts, for example a monovalent, divalent salt or a mixture thereof. In other embodiments, chelating agents are introduced in the presence of one or more divalent salts.

[0072] In some embodiments, the metal chelation reaction is conducted in the presence of a buffer. Buffer systems can be selected to provide suitable pH ranges that facilitate first order reaction kinetics for metal chelation. Suitable pH ranges facilitate sufficient protonation of the chelator so that hydrogen ions are released after binding with metal ions. Further, suitable pH ranges also provide sufficiently low buffering capacity of the chelating environment so that pH changes caused by hydrogen ion release is maximized. In some embodiments, a reagent or buffer can include a source of ions, for example, one or more monovalent salts, such as KC1, K- acetate, NH₄-acetate, K-glutamate, NH₄C1, or ammonium sulfate. In some embodiments, a reagent or buffer can include a source of divalent ions, such as Mg²⁺ or Mn²⁺, MgCl₂, MnCl₂, or Mg-acetate. In some embodiments, a reagent or buffer can include magnesium, manganese and/or calcium. In some embodiments, a buffer can include trisaminomethane (Tris), N-(2- hydroxy-l,l-bis(hydroxymethyl)ethyl)glyince (Tricine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid ) (HEPES), 3-(N- morpholino)propanesulfonic acid (MOPS), N-2-aminoethanesulfonic acid (ACES), 2- ethanesulfonic acid (MES), citric acid, acetic acid, or inorganic buffers such as phosphate, borate or acetate-based buffers, which can provide a pH range of about 4 to about 12. In some embodiments, a buffer can include dithiothreitol (DTT), glycerol, spermidine, and/or BSA (bovine serum albumin). In some embodiments, a buffer can include adenosine triphosphate (ATP). Alternatively, the metal chelation reaction is performed absent a buffer or absent competing native counter ions.

[0073] In some embodiments, the metal chelation reaction is conducted under other suitable conditions that include various parameters, such as time, temperature, reagents, salts, and co-factors.

[0074] Nucleotide incorporations and metal chelation reactions can be performed in the same reaction region, to which a chemical sensor is coupled. Advantages of performing the metal chelation reaction and nucleotide incorporations in a defined reaction region, such as a chamber or a well, include the ability to control the influx and efflux of reagents and byproducts. Another advantage is the ability to perform a plurality of metal chelation reactions and nucleotide incorporation equivalents in the same defined reaction region, which can produce a corresponding increase in equivalents of reaction products. Particularly if multiple nucleotide incorporations are extensions of a homogenous population of template nucleic acids, such as would be performed in a sequencing reaction, multiple parallel metal chelation reaction and nucleotide incorporations will additively produce a larger signal to be detected and measured.

[0075] In some embodiments, provided are methods for identifying a base at a position in a target nucleic acid, including incorporating a nucleotide species at a terminus of an extension primer that is hybridized to the target nucleic acid using a polymerase enzyme and at least one nucleotide species, and identifying the position in a target nucleic acid based on the incorporation of the nucleotide species using metal chelation methods described herein, wherein the nucleotide species is incorporated when the nucleotide includes a base that is complementary to the corresponding position in the target nucleic acid.

[0076] A nucleotide comprises any compound that can bind selectively to, or can be polymerized by, a polymerase. Typically, but not necessarily, selective binding of the nucleotide to the polymerase is followed by polymerization of the nucleotide into a nucleic acid strand by the polymerase. Occasionally, however, the nucleotide may dissociate from the polymerase without becoming incorporated into the nucleic acid strand, an event referred to herein as a "non- productive" event. A nucleotide incorporation reaction (also called a "nucleotide polymerization" reaction) can include primer extension reactions, nucleic acid amplification reactions, or sequence-by-synthesis reactions. Nucleotides include not only naturally occurring nucleotides, but also any analogs, regardless of their structure, that can bind selectively to, or can be polymerized by, a polymerase. While naturally occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides of the present disclosure can include compounds lacking any one, some or all of such moieties. In some embodiments, the nucleotide can optionally include a chain of phosphorus atoms comprising three, four, five, six, seven, eight, nine, ten or more phosphorus atoms. In some embodiments, the phosphorus chain can be attached to any carbon of a sugar ring, such as the 5' carbon. The phosphorus chain can be linked to the sugar with an intervening O or S. In some embodiments, one or more phosphorus atoms in the chain can be part of a phosphate group having P and O. In some embodiments, the phosphorus atoms in the chain can be linked together with intervening O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or 1-imidazole). In some embodiments, the phosphorus atoms in the chain can have at least one side group including O, B¾, or S. In the phosphorus chain, a phosphorus atom with a side group other than O can be a substituted phosphate group. In the phosphorus chain, phosphorus atoms with an intervening atom other than O can be a substituted phosphate group.

[0077] Some examples of nucleotides that can be used in the disclosed methods include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, modified peptide nucleotides, metallonucleosides, phosphonate nucleosides, and modified phosphate-sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing compounds. In some embodiments, the nucleotide can comprise non-oxygen moieties such as, for example, thio- or borano- moieties, in place of the oxygen moiety bridging the alpha phosphate and the sugar of the nucleotide, or the alpha and beta phosphates of the nucleotide, or the beta and gamma phosphates of the nucleotide, or between any other two phosphates of the nucleotide, or any combination thereof. [0078] In some embodiments, a nucleotide can include a purine or pyrimidine base, including adenine, guanine, cytosine, thymine, or uracil. In some embodiments, a nucleotide includes dexoy adenosine triphosphate ("dATP"), deoxyguanosine triphosphate ("dGTP"), deoxycytidine triphosphate ("dCTP"), deoxythymidine triphosphate ("dTTP"), or deoxyuridine triphosphate ("dUTP").

[0079] In some embodiments, the nucleotide is natural or unlabeled. The methods described herein are advantageous in that nucleic acid analyses can be conducted using natural or unlabeled nucleotides, which help avoid unwanted cleavage chemistry or high reagent costs. In some embodiments, the nucleotide comprises a label and referred to herein as a "labeled nucleotide." In some embodiments, the label can be attached to any portion of a nucleotide including a base, sugar or any intervening phosphate group or a terminal phosphate group, i.e., the phosphate group most distal from the sugar.

[0080] In some embodiments, a nucleotide (or analog thereof) can be attached to a label.

In some embodiments, a label comprises a detectable moiety. In some embodiments, a label can generate, or cause to generate, a detectable signal. A detectable signal can be generated from a chemical or physical change (e.g. , heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). For example, a proximity event can include two reporter moieties approaching each other, or associating with each other, or binding each other. A detectable signal can be detected optically, electrically, chemically, enzymatically, thermally, or via mass spectroscopy or Raman spectroscopy. A label can include compounds that are luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, or electrochemical. A label can include compounds that are fluorophores, chromophores, radioisotopes, haptens, affinity tags, atoms, or enzymes. In some embodiments, the label comprises a moiety not typically present in naturally occurring nucleotides. For example, the label can include fluorescent, luminescent, or radioactive moieties.

[0081] In some embodiments, a nucleotide can be labeled with a chelator tag. Chelator tags can be directly attached to a nucleotide (i.e. , nucleotide-chelator tag) or attached to a nucleotide by a linker group (i.e., nucleotide-linker-chelator tag). In some embodiments, the chelator tag is attached to any portion of a nucleotide, including attached to the base, sugar or phosphate tail (Figure 18). Chelator tags can include moieties that can generate a hydrogen ion when metal ions replace hydrogen ions associated with the chelator tag, producing a detectable H change. Non-limiting examples of chelator tags include phosphates (e.g. , monophosphates, diphosphates, triphosphates etc.), carboxylic acids and other moieties with a readily-ionizable hydrogen. Thus in some embodiments, 1) a nucleotide labeled or modified with a chelator tag is incorporated into a template nucleic acid molecule (e.g. , DNA or RNA), 2) metal ions are flowed and hydrogen ions associated with the chelator tag is replaced with metal ions and a pH spike is produced, 3) chelating agent is flowed to remove metal ions, and 4) a linker, if present, is cleaved from the nucleotide to remove the chelator tag. In some embodiments, the linker can be cleaved chemically or enzymatically. The enzymatic cleaving can be achieved with a phosphatase if the chelator tag is a phosphate group or chain (Figure 19A). An example of signals generated before and after cleavage of a chelator tag is shown in Figure 19B. The process steps 1) to 4 can be repeated, allowing for multiple measurements that can improve signal to noise ratios. This method also provides a signal generated by fast chemical kinetics, reducing specific errors due to polymerase kinetics associated with more conventional methods. In addition, a reversible terminator is utilized, measuring one base at a time, which can provide higher homopolymer accuracy.

[0082] By way of a non-limiting example of nucleotide incorporation (e.g. , DNA polymerization), the steps or events of nucleotide incorporation are well known and generally comprise: (1) complementary base-pairing a template nucleic acid with a primer having a terminal 3' OH (the terminal 3' OH provides the polymerization initiation site for polymerase); (2) binding the base-paired template nucleic acid/primer duplex with a polymerase to form a complex; (3) a nucleotide species binds with the polymerase, which interrogates the nucleotide species for complementarity with the template nucleotide on the template nucleic acid molecule; (4) the polymerase may undergo a conformational change (e.g. , from an open to a closed complex if the candidate nucleotide is complementary); (5) the polymerase catalyzes nucleotide incorporation.

[0083] In one embodiment, the polymerase catalyzes nucleotide incorporation by forming a bond between the nucleotide species and the nucleotide at the terminal end of the polymerization initiation site. The polymerase can catalyze the terminal 3' OH of the primer exerting a nucleophilic attack on the bond between the a and β phosphates of the candidate nucleotide to mediate a nucleotidyl transferase reaction resulting in phosphodiester bond formation between the terminal 3' end of the primer and the nucleotide species (i.e. , nucleotide incorporation in a template-dependent manner), and concomitant cleavage to form a cleavage product. The polymerase can liberate the cleavage product. In some embodiments, where the polymerase incorporates a nucleotide having phosphate groups, the cleavage product includes one or more phosphate groups. In some embodiments, where the polymerase incorporates a nucleotide having substituted phosphate groups, the cleavage product may include one or more substituted phosphate groups. In some embodiments, nucleotide incorporation reactions produce one or more cleavage products (e.g. , byproducts) including polyphosphate compounds (pyrophosphates), hydrogen ions, or protons.

[0084] The nucleotide species may or may not be complementary to the template nucleotide on the template nucleic acid molecule. The nucleotide species can bind the polymerase and then dissociate from the polymerase. If the nucleotide dissociates from the polymerase (e.g. , it is not incorporated), it can be liberated and typically carries intact polyphosphate groups.

[0085] In some embodiments, nucleotide incorporation can be a reverse transcriptase reaction, which includes a nucleic acid template (RNA or DNA), primers, nucleotide species (or analogs thereof) and reverse transcriptase enzyme. In some embodiments, nucleotide incorporation can be a transcription reaction which includes an RNA template, nucleotide species (or analogs thereof) and a DNA-dependent RNA polymerase enzyme. Nucleotide incorporation events involving reverse transcriptase or DNA-dependent RNA polymerase are well known in the art.

[0086] In some embodiments, a nucleotide incorporation reaction can include natural nucleotides, nucleotide analogs, or a combination of both.

[0087] A polymerase comprises any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent fashion. Such polymerases can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase can be a mutant polymerase comprising one or more mutations involving the replacement of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids from the polymerase, or the linkage of parts of two or more polymerases. The term "polymerase" and its variants, as used herein, also refers to fusion proteins comprising at least two portions linked to each other, where the first portion comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand and is linked to a second portion that comprises a second polypeptide, such as, for example, a reporter enzyme or a processivity-enhancing domain. In some embodiments, a polymerase can be a high fidelity polymerase. Typically, the polymerase comprises one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes or lacks other enzymatic activities, such as for example, 3' to 5' exonuclease activity, 5' to 3' exonuclease activity, or strand displacement activity. In some embodiments, a polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. In some embodiments, a polymerase can be post-translationally modified proteins or fragments thereof.

[0088] In some embodiments, the polymerase can include any one or more polymerases, or biologically active fragment of a polymerase, which are described in any of: U.S. published application No. 2011/0262903, published 10/27/2011; International PCT Publication No. WO 2013/023176, published 2/14/2013; International PCT Publication No. WO 2013/023176, published February 14, 2013; U.S. 61/884,921, filed September 30, 2013; U.S. published application No. 2011/0262903, published October 27, 2011; U.S. published application No. 2011/0301041, published December 8, 2011; U.S. published application No. 2012/0202276; U.S. 13/035177, filed February 25, 2011, and published as U.S. published application No. 2011/0318748 on December 29, 2011; U.S. 13/572,488, filed August 10, 2012; and U.S. 61/884,921, filed September 30, 2013.

[0089] In some embodiments, a polymerase can be a DNA polymerase and include without limitation bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases and phage DNA polymerases.

[0090] In some embodiments, a polymerase can be a replicase, DNA-dependent polymerase, primases, RNA-dependent polymerase (including RNA-dependent DNA polymerases such as, for example, reverse transcriptases), a thermo-labile polymerase, or a thermo-stable polymerase. In some embodiments, a polymerase can be any Family A or B type polymerase. Many types of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C (e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., human Pol beta), and Y (e.g., E. coli UmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum variants) polymerases are described in Rothwell and Watsman 2005 Advances in Protein Chemistry 71:401-440. In some embodiments, a polymerase can be a T3, T5, T7, or SP6 RNA polymerase.

[0091] In some embodiments, nucleotide incorporation reactions can be conducted with one type or a mixture of different types of polymerases. In some embodiments, nucleotide incorporation reactions can be conducted with a low fidelity or high fidelity polymerase.

[0092] In some embodiments, nucleic acid amplification reactions can be catalyzed by heat- stable or heat-labile polymerases.

[0093] In some embodiments, an archaeal DNA polymerase can be, without limitation, a thermostable or thermophilic DNA polymerase such as, for example: a Bacillus subtilis (Bsu) DNA polymerase I large fragment; a Thermus aquaticus (Taq) DNA polymerase; a Thermus filiformis (Tfi) DNA polymerase; a Phi29 DNA polymerase; a Bacillus stearothermophilus (Bst) DNA polymerase; a Thermococcus sp. 9° N-7 DNA polymerase; a Bacillus smithii (Bsm) DNA polymerase large fragment; a Thermococcus litoralis (Tli) DNA polymerase or Vent™ (exo-) DNA polymerase (from New England Biolabs); or "Deep Vent" (exo-) DNA polymerase (New England Biolabs).

[0094] A nucleic acid comprises single- stranded or double- stranded polynucleotides, or a mixture of both. In some embodiments, a plurality of different polynucleotides comprises polynucleotides having the same or different sequences. In some embodiments, a plurality of different polynucleotides comprises polynucleotides having the same or different lengths. In some embodiments, a plurality of different polynucleotides comprises about 2 to about 10, or about 10 to about 50, or about 50 to about 100, or about 100 to about 500, or about 500 to about 1,000, or about 1,000 to about 5,000, or about 10³ to about 10⁶, or about 10⁶ to about 10¹⁰, or more different polynucleotides. In some embodiments, a plurality of different polynucleotides comprises polymers of deoxyribonucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, a plurality of different polynucleotides comprises naturally-occurring, synthetic, recombinant, cloned, amplified, unamplified or archived (e.g., preserved) forms. In some embodiments, a plurality of different polynucleotides comprises DNA, cDNA RNA or chimeric RNA/DNA, and nucleic acid analogs. [0095] In some embodiments, a template nucleic acid molecule comprises any nucleic acid, including DNA, mitochondrial DNA, cDNA, RNA, mRNA, miRNA, or RNA/DNA hybrids. A template nucleic acid molecule can be single- stranded or double- stranded nucleic acids, and can have any length. A template nucleic acid molecule can be chromosomal, genomic, transcriptomic, organellar, methylated, chromatin-linked, cloned, unamplified, amplified, natural or synthetic, and can be isolated from any source, for example, from an organism, normal or diseased cells or tissues, body fluids, archived tissue (e.g., tissue archived in formalin and/or in paraffin). In some embodiments, the template nucleic acid molecule can originate from any organism including human, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, simian, ape, plant, insect, bacteria, virus or fungus. In some embodiments, the template nucleic acid molecule can originate from water, soil or food. In some embodiments, the template nucleic acid molecule can be PCR products, cosmids, plasmids, naturally occurring or synthetic libraries, and the like. In some embodiments, a template nucleic acid molecule can be isolated from any source including prokaryotes, eukaryotes (e.g. , humans, plants and animals), fungus, and viruses; cells; tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen; environmental samples; culture samples; or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. In some embodiments, template nucleic acid molecules can be isolated from a formalin-fixed tissue, or from a paraffin-embedded tissue, or from a formalin-fix paraffin-embedded (FFPE) tissue. In some embodiments, a template nucleic acid molecule can be about 100 bp to about 1000 bp, or about 1 kb to about 50 kb, or about 50 kb to about 100 kb, or longer.

[0096] In some embodiments, the template nucleic acid molecule is attached to a spacer which is used to distance the template nucleic acid molecule (and in particular the target nucleic acid sequence comprised therein) from the bead. Examples of suitable linkers are known in the art (see Diehl et ah , Nature Methods, 2006, 3(7):551-559) and include, but are not limited to, carbon-carbon linkers, such as, but not limited to, iSpl8 which are commercially available from GeneLink (e.g., spacer 18 (hexaethyleneglycol) catalog No. 26-6447).

[0097] In some embodiments, a template nucleic acid molecule includes at least one universal sequence, such as a primer binding site, an amplification primer sequence, a sequencing primer sequence, a capture primer sequence and/or a cleavable site. A template nucleic acid molecule can be generated by joining together an initial polynucleotide (from any source) to a nucleic acid adaptor having a primer binding sequence. For example, the initial polynucleotide and adaptor can be joined by ligation, hybridization or primer extension methods. An adapter can be joined to at least one end of a linear template, or within the body of a linear or circular initial polynucleotide. Optionally, the template can be circularized after the adapter is joined.

[0098] In some embodiments, primers comprise polymers of deoxyribonucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, primers comprise naturally- occurring, synthetic, recombinant, cloned, amplified, or unamplified forms. In some embodiments, primers comprise DNA, cDNA RNA, chimeric RNA/DNA, or nucleic acid analogs. In some embodiments, primers comprise single-stranded or double-stranded forms.

[0099] In some embodiments, at least a portion of a primer can hybridize with a portion of at least one strand of a template nucleic acid molecule in the nucleotide incorporation reaction mixture. In some embodiments, at least a portion of a primer can be partially or fully complementary to a portion of the template nucleic acid molecule. A template nucleic acid molecule can include a polynucleotide sequence of interest, or a nucleic acid adaptor sequence joined to the polynucleotide sequence of interest.

[00100] In some embodiments, a primer can include or lack a terminal 3' OH which can serve as an initiation site for nucleotide incorporation. In some embodiments, a primer can include a terminal 3' blocking group that does not serve as an initiation site for nucleotide incorporation.

[00101] In some embodiments, primers can be any length, including about 5 to about 20 nucleotides, or about 20 to about 40 nucleotides, or about 40 to about 60 nucleotides, or about 60 to about 80 nucleotides, or longer.

[00102] In some embodiments, a primer can have a 5' or 3' overhang tail (tailed primer) that does not hybridize with a portion of at least one strand of a template nucleic acid molecule. In some embodiments, a non-complementary portion of a tailed primer can be any length, including about 1 to about 50 or more nucleotides in length.

[00103] In some embodiments, a plurality of primers includes individual primers that are essentially the same or are different. For example, primers in the plurality can have essentially the same sequences or different sequences, or can have essentially the same length or different lengths, or can include natural or synthetic forms or a mixture of both.

[00104] In some embodiments, a nucleotide incorporation reaction mixture can contain at least one reagent for conducting a nucleotide incorporation reaction. For example, a nucleotide incorporation reaction mixture can include any one or any combination of reagents: at least one nucleotide, one or more polymerases, at least one template nucleic acid molecule, at least one primer, at least one divalent cation. Optionally, a nucleotide incorporation reaction mixture can include other enzymes, including at least one phosphatase. Optionally, a nucleotide incorporation reaction mixture can include at least one accessory protein that can: bind single- stranded or double- stranded nucleic acids; mediate loading other protein onto a nucleic acid; unwind nucleic acid substrates; relax nucleic acids; resolve nucleic acid structures; disassemble complexes of nucleic acids and proteins, or disassemble nucleic acid structures; or hydrolyze nucleic acids. In some embodiments, an accessory protein comprises a sliding clamp protein. In some embodiments, an accessory protein comprises a multimeric protein complex. In some embodiments, a multimeric protein complex comprises 2, 3, 4, 5, 6, 7, 8, or more subunits. In some embodiments, a multimeric accessory protein complex comprises a homo-meric or hetero- meric protein complex.

[00105] Optionally, a nucleotide incorporation reaction mixture includes one or more additives for enhancing nucleotide incorporation, including betaine, DMSO, proline, trehalose, MMNO (4-methylmorpholine N-oxide) or a PEG-like compound.

[00106] In some embodiments, methods for nucleic acid analysis can be conducted under conditions that are suitable for: binding a nucleotide to a polymerase (where the polymerase is bound to a duplex that includes a template molecule and primer); incorporating the nucleotide into the primer; associating a metal ion to an incorporation product, chelating the metal ion with a chelating agent and generating hydrogen ions, detecting the hydrogen ions or one or any combination of these steps.

[00107] In other embodiments, methods for nucleic acid analysis can be conducted under conditions that are suitable for: associating a metal ion to a nucleic acid sample, chelating the metal ion with a chelating agent and generating hydrogen ions, detecting the hydrogen ions or one or any combination of these steps. [00108] In some embodiments, suitable nucleic acid analysis conditions include parameters, such as: time, temperature, pH, buffers, reagents, cations, salts, co-factors, nucleotides, nucleic acids, and enzymes.

[00109] In some embodiments, suitable conditions include conducting metal chelation reactions and/or nucleotide incorporation reactions in a liquid phase, including an aqueous fluid or immiscible fluid. In some embodiments, metal chelation reactions and/or nucleotide incorporation reactions can be conducted in a continuous aqueous phase, or in a hydrophilic phase of an emulsion having a discontinuous hydrophilic phase and a continuous hydrophobic phase. In some embodiments, an aqueous fluid can be water-based. In some embodiments, a hydrophobic phase can be oil-based. In some embodiments, different metal chelation reactions and/or nucleotide incorporation reactions can be conducted in separate compartments (e.g. , droplets) forming part of a hydrophilic phase of an emulsion having a discontinuous hydrophilic phase and a continuous hydrophobic phase.

[00110] In some embodiments, suitable nucleotide incorporation reaction conditions include conducting a nucleotide incorporation reaction with a polymerase enzyme and one or more nucleotides.

[00111] In some embodiments, suitable metal chelation reaction conditions and nucleotide incorporation reaction conditions include cyclical temperature changes, or essentially isothermal temperature conditions, or a combination of both. In some embodiments, a reaction can be conducted at a temperature lower than the melting point temperature of double stranded DNA. In some embodiments, a reaction can be conducted at a temperature range of about 0 °C to about 10 °C, or about 10 °C to about 20 °C, or about 20 °C to about 30 °C, or about 30 °C to about 40 °C, or about 40 °C to about 50 °C, or about 50 °C to about 60 °C, or about 60 °C to about 70 °C, or about 70 °C to about 80 °C, or about 80 °C to about 90 °C, or about 90 °C to about 100 °C, or higher temperatures.

[00112] In some embodiments, suitable metal chelation reaction conditions and nucleotide incorporation reaction conditions include conducting a reaction for a time, such as about 10 seconds to about 30 seconds, or about 30 seconds to about 60 seconds, or about 1 minutes to about 3 minutes, or about 3 minutes to about 5 minutes, or about 5 minutes to about 6 minutes, or about 6 minutes to about 7 minutes, or about 7 minutes to about 8 minutes, or about 8 minutes to about 9 minutes, or about 9 minutes to about 10 minutes, or about 10 minutes to about 11 minutes, or about 11 minutes to about 12 minutes, or about 12 minutes to about 13 minutes, or about 13 minutes to about 14 minutes, or about 14 minutes to about 15 minutes, or about 15 minutes to about 20 minutes, or about 20 minutes to about 30 minutes, or about 30 minutes to about 45 minutes, or about 45 minutes to about 60 minutes, or about 1 hour to about 3 hours, or about 3 hours to about 6 hours, or about 6 hours to about 10 hours, or longer.

[00113] In some embodiments, suitable metal chelation reaction conditions and nucleotide incorporation reaction conditions include conducting such reactions in a volume of about 1 μL· to about 10 μί, or about 10 μΐ_^ to about 25 μί, or about 25 μΐ_^ to about 50 μί, or about 50 μΐ_^ to about 75 μί, or about 75 μΐ_^ to about 100 μί, or about 100 μΐ_^ to about 125 μί, or about 125 μΐ_^ to about 150 μί, or about 150 μΐ_^ to about 200 μί, or more.

[00114] In some embodiments, suitable metal chelation reaction conditions and nucleotide incorporation reaction conditions include conducting a reaction in a tube or well. In some embodiments, the well can be a part of a 96-well plate.

[00115] In some embodiments, methods for metal chelation and nucleotide incorporation comprise one or more surfaces. In some embodiments, a surface can be attached with a plurality of first primers, the first primers of the plurality sharing a common first primer sequence. In some embodiments, a surface can be attached with a plurality of first and second primers, where the first and second primers have different sequences.

[00116] In some embodiments, a surface can be an outer or top-most layer or boundary of an object. In some embodiments, a surface can be interior to the boundary of an object.

[00117] In some embodiments, a surface can be porous, semi-porous or non-porous. In some embodiments, a surface can be a planar surface, as well as concave, convex, or any combination thereof. In some embodiments, a surface can be a bead, particle, microparticle, sphere, filter, flowcell, well, groove, channel reservoir, gel or inner wall of a capillary. In some embodiments, a surface includes the inner walls of a capillary, a channel, a well, groove, channel, reservoir. In some embodiments, a surface can include texture (e.g., etched, cavitated, pores, three-dimensional scaffolds or bumps).

[00118] In some embodiments, particles can have a shape that is spherical, hemispherical, cylindrical, barrel- shaped, toroidal, rod-like, disc-like, conical, triangular, cubical, polygonal, tubular, wire-like or irregular. [00119] In some embodiments, a surface can be made from any material, including glass, polymers, borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g. , films of gold, silver, aluminum, or diamond).

[00120] In some embodiments, a surface can be magnetic or paramagnetic bead (e.g. , magnetic or paramagnetic nanoparticles or microparticles). In some embodiments, paramagnetic microparticles can be paramagnetic beads attached with streptavidin (e.g. , Dynabeads™ M-270 from Invitrogen, Carlsbad, CA). Particles can have an iron core, or comprise a hydrogel or agarose (e.g. , Sepharose™).

[00121] In some embodiments, the surface can be attached with a plurality of a first primer. A surface can be coated with an acrylamide, carboxylic or amine compound for attaching a nucleic acid (e.g. , a first primer). In some embodiments, an amino-modified nucleic acid (e.g. , primer) can be attached to a surface that is coated with a carboxylic acid. In some embodiments, an amino-modified nucleic acid can be reacted with l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC or EDAC) for attachment to a carboxylic acid coated surface (with or without succinimidyl ester (NHS)). A first primer can be immobilized to an acrylamide compound coating on a surface. Particles can be coated with an avidin-like compound (e.g., streptavidin) for binding biotinylated nucleic acids.

[00122] In some embodiments, the surface comprises the surface of a bead, particle or other substrate. In some embodiments, a bead, particle or other substrate comprises a polymer material. For example, a bead, particle or other substrate comprises a gel, hydrogel or acrylamide polymers. A bead, particle or other substrate can be porous. A bead, particle or other substrate can have cavitation or pores, or can include three-dimensional scaffolds. In some embodiments, a bead, particle or other substrate can be Ion Sphere™ particles.

[00123] In some embodiments, the disclosed methods include immobilizing one or more templates nucleic acid molecules onto one or more supports. Template nucleic acid molecules may be immobilized on the solid support by any method including but not limited to physical adsorption, by ionic or covalent bond formation, or combinations thereof. A solid support may include a polymeric, a glass, or a metallic material. Examples of solid supports include a membrane, a planar surface, a microtiter plate, a bead, a filter, a test strip, a slide, a cover slip, and a test tube. A support includes any solid phase material upon which an oligomer is synthesized, attached, ligated or otherwise immobilized. A support can optionally comprise a "resin," "phase," "surface," and "support." A support may be composed of organic polymers, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A support may also be inorganic, such as glass, silica, controlled-pore-glass (CPG), or reverse-phase silica. The configuration of a support may be in the form of beads, spheres, particles, granules, a gel, or a surface. Surfaces may be planar, substantially planar, or non-planar. Supports may be porous or non-porous, and may have swelling or non- swelling characteristics. A support can be shaped to comprise one or more wells, depressions or other containers, vessels, features or locations. A plurality of supports can be configured in an array at various locations. A support is optionally addressable (e.g. , for robotic delivery of reagents), or by detection means including scanning by laser illumination and confocal or deflective light gathering. An amplification support (e.g. , a bead) can be placed within or on another support (e.g. , within a well of a second support).

[00124] In some embodiments the solid support is a "microparticle," "bead," "microbead," or other micro-sized substrate, (optionally but not necessarily spherical in shape) having a smallest cross-sectional length (e.g. , diameter) of about 50 μιη or less, preferably about 10 μιη or less, about 3 μιη or less, about 1 μιη or less, about 0.5 μιη or less, e.g. , about 0.1 μιη, about 0.2 μηι, about 0.3 μιη, or about 0.4 μιη, or smaller (e.g. , less than about 1 nm, about 1 nm to about 10 nm, about 10 nm to about 100 nm, or about 100 nm to about 500 nm). Microparticles, microbeads or other micro-sized substrates (e.g. , Dynabeads from Dynal, Oslo, Norway) may 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, polymethyacrylate, titanium dioxide, latex, polystyrene, etc. Magnetization can facilitate collection and concentration of the attached reagents (e.g. , polynucleotides or ligases) after amplification, and can also facilitate additional steps (e.g. , washes, reagent removal, etc.). In certain embodiments of the invention, a population of microparticles, microbeads or other micro-sized substrate having different shapes sizes and/or colors can be used. Microparticles, microbeads or other micro-sized substrates can optionally be encoded, e.g. , with quantum dots such that each microparticle can be individually or uniquely identified. [00125] In some embodiments, a bead, particle or other substrate surface can be functionalized for attaching a plurality of a first primer. In some embodiments, a bead, particle or other substrate can be any size that can fit into a reaction region, e.g. , chamber or well. For example, one bead, particle or other substrate can fit in a reaction region, e.g. , chamber or well. In some embodiments, more than one bead, particle or other substrate can fit in a reaction region, e.g. , chamber or well. In some embodiments, the smallest cross-sectional length of a bead, particle or other substrate (e.g. , diameter) can be about 50 μιη or less, or about 10 μιη or less, or about 3 μηι or less, about 1 μιη or less, about 0.5 μιη or less, e.g. , about 0.1 μιη, about 0.2 μιη, about 0.3 μηι, or about 0.4 μιη, or smaller (e.g., less than about 1 nm, about 1 nm to about 10 nm, about 10 nm to about 100 nm, or about 100 nm to about 500 nm).

[00126] In some embodiments, a bead, particle or other substrate can be attached with a plurality of one or more different primer sequences. In some embodiments, a bead, particle or other substrate can be attached with a plurality of one primer sequence, or can be attached a plurality of two or more different primer sequences. In some embodiments, a bead, particle or other substrate can be attached with a plurality of at least about 1,000 primers, or about 1,000 primers to about 10,000 primers, or about 10,000 primers to about 50,000 primers, or about 50,000 primers to about 75,000 primers, or about 75,000 primers to about 100,000 primers, or more.

[00127] In some embodiments, nucleotides can be used in any nucleic acid sequencing workflow, including sequencing by oligonucleotide probe ligation and detection (e.g. , SOLiD™ from Life Technologies, WO 2006/084131), probe-anchor ligation sequencing (e.g. , Complete Genomics™ or Polonator™), sequencing- by-synthesis (e.g. , Genetic Analyzer and HiSeq™, from Illumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454 Life Sciences), ion-sensitive sequencing (e.g. , Personal Genome Machine (PGM™) and Ion Proton™ Sequencer, both from Ion Torrent Systems, Inc.), and single molecule sequencing platforms (e.g. , HeliScope™ from Helicos™).

[00128] In some embodiments, template nucleic acids produced using the methods can be used as a substrate for a biological or chemical reaction that is analyzed by a sensor including a field-effect transistor (FET). In various embodiments, the FET is 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 and it is the structural analog of a MOSFET transistor, where the charge on the gate electrode is applied by a chemical process. An "ISFET" or ion-sensitive field-effect transistor, is used for measuring ion concentrations in solution; when the ion concentration (such as H⁺) changes, the current through the transistor will change accordingly. A detailed theory of operation of an ISFET is given in "Thirty years of ISFETOLOGY: what happened in the past 30 years and what may happen in the next 30 years," P. Bergveld, Sens. Actuators, 88 (2003), pp. 1- 20.

[00129] In some embodiments, the FET may be a FET array. As used herein, an "array" is a planar arrangement of elements such as sensors or wells. The array may be one or two dimensional. A one dimensional array can be an array having one column (or row) of elements in the first dimension and a plurality of columns (or rows) in the second dimension. The number of columns (or rows) in the first and second dimensions may or may not be the same. The FET or array can comprise about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10^s or more FETs.

[00130] In some embodiments, one or more microfluidic structures can be fabricated above the FET sensor array to provide for containment and/or confinement of biological or chemical reactions, including metal chelation reactions. For example, in one implementation, the microfluidic structure(s) can be configured as one or more regions (e.g. , microwells, or reaction chambers, or reaction wells, as the terms are used interchangeably herein) disposed above one or more sensors of the array, such that the one or more sensors over which a given well is disposed detect and measure analyte presence, level, and/or concentration in the given well. In some embodiments, there can be a 1 : 1 correspondence of FET sensors and reaction wells.

[00131] Reaction regions comprising microwells or reaction chambers are typically hollows or wells having well-defined shapes and volumes, which can be manufactured into a substrate and can be fabricated using conventional microfabrication techniques, e.g. , as disclosed in the following references: Doering and Nishi, Editors, Handbook of Semiconductor Manufacturing Technology, Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMS and Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al, Silicon Micromachining (Cambridge University Press, 2004); and the like. Examples of configurations (e.g. , spacing, shape and volumes) of microwells or reaction chambers are disclosed in Rothberg et ah , U.S. patent publication 2009/0127589; Rothberg et ah , U.K. patent application GB24611127. [00132] In some embodiments, biological or chemical reactions and metal chelation reactions can be performed in a solution or a reaction region that is in contact with, operatively coupled, or capacitively coupled to a FET, such as a chemFET or an ISFET. The FET (or chemFET or ISFET) and/or reaction region can be an array of FETs or reaction chambers, respectively. In some embodiments, chemFETs and ISFETs are configured as large scale arrays to allow for multiple and simultaneous analysis.

[00133] In some embodiments, biological or chemical reactions and metal chelation reactions can be carried out in a two-dimensional array of reaction regions, wherein each reaction region can be coupled to a FET, and each reaction region is no greater than about 10 μηι (i.e., 1 pL) in volume. In some embodiments each reaction region is no greater than about 0.34 pL, about 0.096 pL or even about 0.012 pL in volume. A reaction region can optionally be no greater than about 2 μηι 2 , about 5 μπι 2 , about 10 μιη 2 , about 15 μιη 2 , about 22 μιη 2 , about 32 μηι 2 , about 42 μιη 2 , about 52 μιη 2 , about 62 μιη 2 , about 72 μιη 2 , about 82 μιη 2 , about 92 μιη 2 , or about 102 μηι 2 in cross-sectional area at the top. Preferably, the array has at least about 102 , about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10^s, about 10⁹, or more reaction regions. In some embodiments, at least one of the reaction regions is operatively coupled to at least one of the FETs.

[00134] FET arrays as used in various embodiments according to the disclosure can be fabricated according to conventional CMOS fabrications techniques, as well as modified CMOS fabrication techniques and other semiconductor fabrication techniques beyond those conventionally employed in CMOS fabrication. Additionally, various lithography techniques can be employed as part of an array fabrication process.

[00135] Exemplary FET arrays suitable for use in the disclosed methods, as well as microwells and attendant fluidics, and methods for manufacturing them, are disclosed, for example, in U.S. Patent Publication No. 2010/0301398; U.S. Patent Publication No. 2010/0300895; U.S. Patent Publication No. 2010/0300559 (now U.S. Patent No. 8,546,128); U.S. Patent Publication No. 2010/0197507 (now U.S. Patent No. 8,306,757); U.S. Patent Publication No. 2010/0137143; U.S. Patent Publication No. 2009/0127589 (now U.S. Patent No. 7,948,015); and U.S. Patent Publication No. 2009/0026082 (now U.S. Patent No. 8,262,900), which are incorporated by reference in their entireties. [00136] In one aspect, the disclosed methods can be used for carrying out label-free nucleic acid sequencing, and in particular, ion-based nucleic acid sequencing. The concept of label-free detection of nucleotide incorporation has been described in the literature, including the following references that are incorporated by reference: Rothberg et al, U.S. Patent Publication 2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86 (2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006). Briefly, in nucleic acid sequencing applications, nucleotide incorporations are determined by measuring natural byproducts of polymerase-catalyzed extension reactions, including hydrogen ions, polyphosphates, PPi, and Pi {e.g., in the presence of phosphatase or pyrophosphatase). Examples of such ion-based nucleic acid sequencing methods and platforms include the Ion Torrent PGM™ or Proton™ sequencer (Ion Torrent™ Systems, Life Technologies Corporation).

[00137] In some embodiments, the present disclosure provides nucleotides employed in a nucleic acid sequencing method. In one exemplary embodiment, the disclosure relates generally to a method for obtaining sequence information from template polynucleotides, comprising: performing template-dependent nucleic acid synthesis using any one, or a combination of any, of the nucleotides described herein.

[00138] In some embodiments, the template-dependent synthesis includes incorporating one or more nucleotide species in a template-dependent fashion into a newly synthesized nucleic acid strand.

[00139] In some embodiments, the methods can include producing one or more ionic byproducts, including for example, hydrogen ions, resulting from metal chelation reactions.

[00140] In some embodiments, the disclosure relates generally to a method for sequencing a template nucleic acid, comprising: (a) disposing a template nucleic acid into a plurality of reaction regions, wherein one or more of the reaction regions are in contact with at least one field effect transistor (FET). Optionally, the method further includes contacting a template nucleic acid, which are disposed into one of the reaction regions, with a polymerase thereby synthesizing a new nucleic acid strand by sequentially incorporating one or more nucleotides into the new nucleic acid strand. Optionally, the method further includes associating one or more metal ions with the nucleic acid. Optionally, the method further includes adding a chelating agent to form a metal chelate with the one or more metal ions and generating one or more hydrogen ions as a byproduct of such metal chelation. Optionally, the method further includes detecting the incorporation of the one or more nucleotides by detecting the generation of the one or more hydrogen ions as a byproduct of metal chelation using the FET.

[00141] In some embodiments, the detecting includes detecting a change in voltage and/or current at the at least one FET within the array in response to the generation of the one or more hydrogen ions as a byproduct of metal chelation.

[00142] In some embodiments, the FET can be selected from the group consisting of: ion- sensitive FET (IS FET) and chemically- sensitive FET (chemFET).

[00143] One exemplary system involving nucleic acid molecule sequencing via detection of ionic byproducts of metal chelation is the Ion Torrent PGM™, Proton™, or S5™ sequencer (Thermo Fisher Scientific), which is an ion-based sequencing system that can sequence template nucleic acid molecules by detecting hydrogen ions produced as a byproduct of metal chelation reactions. Typically, hydrogen ions are released as byproducts of metal chelation reactions occurring when a chelating agent is contacted with a metal ion bound to a nucleic acid molecule, which is formed after a metal ion is contacted with a nucleic acid molecule that has been subject to a nucleotide incorporation event. The Ion Torrent PGM™, Proton™, or S5™ sequencer indirectly detects nucleotide incorporations by detecting the hydrogen ion byproducts of metal chelation. The Ion Torrent PGM™, Proton™, or S5™ sequencer can include a plurality of nucleic acid templates to be sequenced, each template disposed within a respective sequencing reaction well in an array. The wells of the array can each be coupled to at least one ion sensor that can detect the release of hydrogen ions or changes in solution pH produced as a byproduct of metal chelation. The ion sensor comprises a field effect transistor (FET) coupled to an ion- sensitive detection layer that can sense the presence of hydrogen ions or changes in solution pH. The ion sensor can provide output signals indicative of nucleotide incorporation, which can be represented as voltage changes whose magnitude correlates with the hydrogen ion concentration in a respective well or reaction chamber. Different nucleotide species can be flowed serially into the reaction region, and can be incorporated by the polymerase into an extending primer (or polymerization site) in an order determined by the sequence of the template nucleic acid. Metal ions can be introduced and associate with the negatively charged nucleic acid molecule, followed by introducing a chelating agent that can preferentially associate with the bound metal ions and generate hydrogen ions. Each metal chelation can be accompanied by the release of hydrogen ions in the reaction well, along with a concomitant change in the localized pH. The release of hydrogen ions can be analyzed by the FET of the sensor, which produces signals indicating the occurrence of nucleotide incorporation. Nucleotides that are not incorporated during a particular nucleotide flow may not produce signals or may not produce a signal change compared to a baseline signal taken prior to an attempted incorporation event. The amplitude of the signals from the FET can also be correlated with the number of nucleotides of a particular type incorporated into the extending nucleic acid molecule thereby permitting homopolymer regions to be resolved. Thus, during a run of the sequencer multiple nucleotide flows into the reaction region along with incorporation monitoring across a multiplicity of reaction regions can permit the instrument to resolve the sequence of many nucleic acid templates simultaneously. Further details regarding the compositions, design and operation of the Ion Torrent PGM™ or Proton™ sequencer can be found, for example, in U.S. Patent Publication No. 2009/0026082 (now U.S. Patent No. 8,262,900); U.S. Patent Publication No. 2010/0137143; and U.S. Patent Publication No. 2010/0282617 (now U.S. Patent No. 8,349, 167), all of which applications are incorporated by reference herein in their entireties.

[00144] In some exemplary embodiments, the methods, systems, and computer readable media described herein may advantageously be used to process and/or analyze data and signals obtained from electronic or charged -based nucleic acid sequencing. In electronic or charged- based sequencing (such as, pH-based sequencing), a nucleotide incorporation event may be determined by detecting ions (e.g. , hydrogen ions) that are generated as byproducts of metal chelation reactions. This may be used to sequence a template nucleic acid molecule, which may be a fragment of a nucleic acid sequence of interest, for example, and which may be directly or indirectly attached as a clonal population to a solid support, such as a particle, microparticle, bead, or other substrate. The sample or template nucleic acid may be operably associated to a primer and polymerase and may be subjected to repeated cycles or "flows" of nucleotide addition (which may be referred to herein as "nucleotide flows" from which nucleotide incorporations may result) and washing. The primer may be annealed to the sample or template so that the primer's 3' end can be extended by a polymerase whenever nucleotides complementary to the next base in the template are added. Then, based on the known sequence of nucleotide flows followed by metal ion flows, chelating agent flows, and on measured output signals of the chemical sensors indicative of ion concentration during each chelating agent flow, the identity of the type, sequence and number of nucleotide(s) associated with a sample nucleic acid present in a reaction region coupled to a chemical sensor can be determined.

[00145] In a typical embodiment of ion-based nucleic acid sequencing, nucleotide incorporations can be detected by detecting the presence and/or concentration of hydrogen ions generated by metal chelation reactions. In one embodiment, templates, optionally pre-bound to a sequencing primer and/or a polymerase, can be loaded into reaction chambers (such as the microwells disclosed in Rothberg et ah), after which repeated cycles of nucleotide addition, metal ion addition, chelating agent addition and washings can be carried out. In some embodiments, such templates can be attached as clonal populations to a solid support, such as particles, bead, or other substrate, and clonal populations can be loaded into reaction regions.

[00146] In other embodiments, the templates, optionally bound to a polymerase, are distributed, deposited or positioned to different sites of the array. The sites of the array include primers and the methods can include hybridizing different templates to the primers within different sites.

[00147] In each addition step of the cycle, the polymerase can extend the primer by incorporating added nucleotide species only if the next base in the template is the complement of the added nucleotide species. If there is one complementary base, there is one incorporation, if two complementary bases, there are two incorporations, if three complementary bases, there are three incorporations, and so on. With each such incorporation the overall negative charge is increased on the resulting nucleic acid molecule. Subsequent introduction of a metal ion will associate the metal ion to the extended nucleic acid in accordance with the valence of the metal ion and the number of negative charges on the resulting nucleic acid molecule. Introduction of a chelating agent will bind the metal ions to the chelating agent to form a metal chelate, and as a result a corresponding amount of hydrogen ions are released. Collectively releasing hydrogen ions changes the local pH of the reaction region. The production of hydrogen ions is monotonically related to the number of contiguous complementary bases in the template (as well as the total number of template molecules with primer and polymerase that participate in an extension reaction). Thus, when there are a number of contiguous identical complementary bases in the template nucleic acid molecule {i.e. , a homopolymer region), the number of hydrogen ions generated as a result of metal chelation, and therefore the magnitude of the local pH change, can be proportional to the number of contiguous identical complementary bases. If the next base in the template is not complementary to the added nucleotide, then no incorporation occurs and no hydrogen ion is released after addition of metal ion and chelating agent. In some embodiments, after each step of adding a nucleotide species, followed by adding a metal ion followed by adding a chelating agent, additional steps can be performed, in which an unbuffered wash solution at a predetermined pH is used to remove the nucleotide, metal ion and/or chelating agent of the previous step in order to prevent misincorporations, mis-metal binding and mis- metal chelations in later cycles. In some embodiments, after each step of adding a nucleotide, an additional step can be performed wherein the reaction regions are treated with a nucleotide- destroying agent, such as apyrase, to eliminate any residual nucleotides remaining in the reaction region, which may result in spurious extensions in subsequent cycles.

[00148] In one exemplary embodiment, different kinds of nucleotide species are added sequentially to the reaction regions, so that each reaction can be exposed to the different nucleotides one at a time. For example, nucleotide species can be added in the following sequence: dATP, dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed by a wash step, metal ion addition step, chelating agent addition step and other appropriate wash steps. The cycles may be repeated for about 50 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 750 times, or more, depending on the length of sequence information desired.

[00149] In some embodiments, the disclosure relates generally to methods for sequencing a population of template nucleic acids, comprising: (a) generating a plurality of amplicons by clonally amplifying a plurality of template nucleic acids onto a plurality of surfaces, wherein the amplifying is performed within a single continuous phase of a reaction mixture and wherein at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the resulting amplicons are substantially monoclonal in nature. In some embodiments, a sufficient number of substantially monoclonal amplicons are produced in a single amplification reaction to generate at least about 100 MB, about 200MB, about 300 MB, about 400 MB, about 500MB, about 750 MB, about 1GB or about 2 GB of AQ20 sequencing reads on an Ion Torrent PGM™ 314, 316 or 318 sequencer. The term "AQ20 and its variants," as used herein, refers to a particular method of measuring sequencing accuracy in the Ion Torrent PGM™ sequencer. Accuracy can be measured in terms of the Phred-like Q score, which measures accuracy on logarithmic scale that: Q10 = 90%, Q20= 99%, Q30 = 99.9%, Q40 = 99.99%, and Q50 = 99.999%. For example, in a particular sequencing reaction, accuracy metrics can be calculated either through prediction algorithms or through actual alignment to a known reference genome. Predicted quality scores ("Q scores") can be derived from algorithms that look at the inherent properties of the input signal and make fairly accurate estimates regarding if a given single base included in the sequencing "read" will align. In some embodiments, such predicted quality scores can be useful to filter and remove lower quality reads prior to downstream alignment. In some embodiments, the accuracy can be reported in terms of a Phred-like Q score that measures accuracy on logarithmic scale such that: Q10 = 90%, Q17 = 98%, Q20= 99%, Q30 = 99.9%, Q40 = 99.99%, and Q50 = 99.999%. In some embodiments, the data obtained from a given polymerase reaction can be filtered to measure only polymerase reads measuring "N" nucleotides or longer and having a Q score that passes a certain threshold, e.g. , Q10, Q17, Q100 (referred to herein as the "NQ17" score). For example, the 100Q20 score can indicate the number of reads obtained from a given reaction that are at least 100 nucleotides in length and have Q scores of Q20 (99%) or greater. Similarly, the 200Q20 score can indicate the number of reads that are at least 200 nucleotides in length and have Q scores of Q20 (99%) or greater.

[00150] In some embodiments, accuracy can also be calculated based on proper alignment using a reference genomic sequence, referred to herein as the "raw" accuracy. This is single pass accuracy, involving measurement of the "true" per base error associated with a single read, as opposed to consensus accuracy, which measures the error rate from the consensus sequence which is the result of multiple reads. Raw accuracy measurements can be reported in terms of "AQ" scores (for aligned quality). In some embodiments, the data obtained from a given polymerase reaction can be filtered to measure only polymerase reads measuring "N" nucleotides or longer having a AQ score that passes a certain threshold, e.g. , AQ10, AQ17, AQ100 (referred to herein as the "NAQ17" score). For example, the 100AQ20 score can indicate the number of reads obtained from a given polymerase reaction that are at least 100 nucleotides in length and have AQ scores of AQ20 (99%) or greater. Similarly, the 200AQ20 score can indicate the number of reads that are at least 200 nucleotides in length and have AQ scores of AQ20 (99%) or greater.

[00151] Another aspect of the invention includes time-delay cation post incorporation detection. Figure 11 shows an exemplary example of time-delay cation post incorporation detection. In Figure 11a, template nucleic acid, e.g. , DNA, disposed on a solid substrate (e.g. , beads or particles) can be subject to nucleotide incorporation in a reaction region, such as a chamber or well. The bottom of the reaction region can be coated with a coating comprising one or more chelating agent. Nucleic acid analysis can be conducted by a metal ion flow where metal ions bind to negative charges on the nucleic acid. Once a saturation concentration of metal ion is reached (i.e. , metal ions bind with all negative charges on the nucleic acid), excess metal ion diffuses towards and contacts the chelating agent coating on the bottom of the reaction region, forming a metal chelate complex and generating hydrogen ion that can be detected by a FET sensor. As metal ions also bind with the nucleic acid, there is a time delay in detecting the hydrogen ions created by the metal ions diffusing to and contacting the chelating agent coating on the bottom of the reaction region. As a control, metal ions are flowed into an empty reaction region having a chelating agent coating on the bottom of the well. Figure l ib shows a time delay in signal generation between the empty reaction region compared to the reaction region containing nucleic acid disposed on beads. Such time delay can be correlated to and is a measure of nucleic acid negative charges formed by nucleotide incorporation. Example 3 below provides additional details and experimental results demonstrating mechanisms for time delay post- incorporation detection according to the present disclosure.

[00152] The post incorporation detection methods described herein may be applied to a variety of analytical methods for nucleic acid analysis and are not limited to detection of single nucleotide incorporation events or associated sequence determination methods. For example, the metal chelator-based detection methods of the present disclosure may be adapted for use in connection with other applications in which ions, such as hydrogen or pyrophosphate (PPi), are generated or released as a product or byproduct of an enzymatic or biochemical reaction. The methods of the present disclosure may therefore be useful for improving detection and / or quantitation of polymerase and/or ligase-mediated extension of template nucleic acids including those for primer extension, amplicon rescue, probe-based hybridization or fragment-based detection methods. Additionally, the methods may be adapted to detect nucleic acid products associated with digital amplification by polymerase chain reaction (dPCR) and quantitative PCR (qPCR) and is particularly useful in accurate quantification of clonally amplified nucleic acids. EXAMPLES

[00153] Embodiments of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

Example 1. Proof of principle for metal chelation post incorporation detection.

[00154] Ladder DNA beads with 20 bp length of primers were generated by typical sequencing extension. Ladder DNA beads include beads having 26 bp, 49 bp, 77 bp, 105 bp and 126 bp. Each of the ladder DNA beads were loaded and subjected to 40 sequencing flows. The results are shown in Figure 12.

[00155] Figure 12a (left figure) shows a raw trace (frame = 1/60 s time), the trace being taken for the entire population of beads and the trace of those beads were averaged. The higher the bp count showed lower pH as a result of a proportionate amount of hydrogen ions generated. Figure 12b (middle figure) shows an integrated signal histogram for each individual ladder DNA beads. Count signal is released and different depending on number of bp and length of primers. Figure 12c (right figure) shows the linearity of signal as a function of primer length.

[00156] Figure 12 in general shows that primers with about 20 bp difference have differentiated signal and the signal in general is linear.

[00157] Figure 13 shows proof of principle of signal for a sequencing run. The bottom control signal is a result of no primer to facilitate any primer extension resulting in low signals and no dramatic change in observed signal. The top control signal shows fully extended double stranded DNA with a high signal. The two control signals show the two boundaries of the upper and lower range of signals. The middle sequencing signal shows an expected linear increase in signal throughout a sequencing run as a result of additional primer being hybridized onto DNA.

Example 2. Modified Nucleotide Detection.

[00158] Figure 14 shows 1-mer signal for two types of beads, namely a first bead type having incorporated natural nucleotide and a second bead type having incorporated modified nucleotide. The modified nucleotide contains a chelator tag (Figure 18) that can facilitate binding of more metal ions that the first beads type with natural nucleotide. The first bead type and the second bead type are loaded and subject to flow of 200 μιη La³⁺, metal ions displaced from the chelator tag in the second bead type will produce a signal as shown in flow 1. In comparison, the first bead type with natural nucleotide produces no signal as shown in flow 2, but signal is then generated upon addition of chelating agent EDTA.

Example 3. Time Delay Post-Incorporation Detection

[00159] Figures 15 and 16 show time delay nucleotide detection for beads containing template nucleic acids having 37 base pairs, 66 base pairs, 92 base pairs and 126 base pairs. Each bead sample was subject to a nucleotide incorporation, followed by metal cation flow in their respective reaction region. Hydrogen ion signals were detected by a FET sensor, where hydrogen ion was generated after reaching a metal ion saturation concentration and excess metal ion diffused and contacted a chelating agent coating on the bottom of the reaction region. Figure 15a shows the detection signals over time for each template nucleic acid sample. Figure 15b shows the detection signal distribution over time for each sample, as well as for a control sample having an empty reaction region. Figure 16a shows a normalized detection signal distribution over time for each sample, having a signal-to-noise ratio of 7.8493. Figure 16b shows that the time to peak detection signal increases linearly with the number of base pairs in each template nucleotide sample.

[00160] In various embodiments, time delay nucleotide detection can be modeled using selected sample containment region and diffusion rate characteristics. In one approach, the change in detection signal over time is determined by assessing the convergence of pH related to sample nucleotide incorporation at or in proximity to the sensor surface relative to the bulk pH for the liquid or environment in proximity the sample containment region or well and takes into consideration the coating response associated with the sensor that binds the metal ions.

Equation 1: —

dt

[00161] In Equation 1, the change in signal over time— can be determined for time (t) between signal s_T— s, where s_T is the bulk pH signal associated with the liquid or environment above or in proximity to the sample containment region or well (e.g. well top bulk pH signal) and s is the signal at or in proximity to the sensor surface (e.g. well signal). An estimated or calculated reagent exchange or diffusion rate factor a applied to s_T— s is summed with a coating response characteristic φ where φ (τ, t) may be representative of the flux produced when metal ions bind or associate with the sensor coating according to a time delay constant τ.

[00162] In various embodiments, detected incorporation signal time delays may be modeled by scaling versus flux associated with a sensor well or containment region lacking sample. Detected signal well flux time delays may further be due to metal ion binding to DNA. An exemplary signal modeling approach taking into consideration wells or sample containment regions with and without sample may be addressed by the following equations.

Equation 2: ^{dS f} = a(s_T— s_re^) + <p(T_ref) for a well lacking sample ds

Equation 3:— = a(s_T— s) + φ(τ_νβ^ + δτ) for a well containing sample

ds

Equation 4:— = as + δφ combines the equations above and eliminates s_Twhere s = s— s_ref is the background- subtracted signal and δφ = φ{τ_Τβ^ + δτ)— φ{τ_γβ^) is differential flux

[00163] Solving for the close form solution s provides a fitting equation that may be used to model signal incorporations according to:

Equation 5: s(t) = e^~at f* δφβ^~αί' dt'

[00164] Figure 17 illustrates an exemplary plot of an incorporation signal processed according to the time delay methods described above. In various embodiments, a quadratic fitting operation may be applied to the signal to improve single to noise determinations for peak evaluation and associated identification of a maximal time frame for which the signal is observed.

[00165] While the principles of the disclosure have been illustrated in relation to the exemplary embodiments shown herein, the principles of the disclosure are not limited thereto and include any modification, variation or permutation thereof. Further, the examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the devices, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Modifications of the above-described modes for carrying out the disclosure that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.

[00166] The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "plurality" includes two or more referents unless the content clearly dictates otherwise. A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED:

1. A method for nucleic acid sequencing comprising:

a) providing a plurality of reaction regions arranged in an array, wherein at least one reaction region in the array is capacitively coupled to a sensor that detects a change in ion concentration;

b) disposing at least one template nucleic acid into the reaction region that is capacitively coupled to the sensor;

c) introducing into the reaction region (i) a primer that hybridizes to a portion of the template nucleic acid, (ii) a nucleotide species, and (iii) an enzyme, under conditions suitable for nucleotide incorporation to generate a primer extension product;

d) introducing into the reaction region one or more metal ions which

reversibly binds the primer extension product;

e) introducing into the reaction region one or more chelating agents which binds the metal ions that are reversibly bound to the primer extension product to form a metal ion/chelate complex thereby releasing a plurality of ions which changes the ion concentration in the reaction region;

f) detecting with the sensor the change in the ion concentration in the

reaction region; and

g) determining the sequence of the at least one template nucleic acid.

2. The method of claim 1, wherein the sensor comprises an ion-sensitive field-effect

transistor (ISFET).

3. The method of claim 2, wherein the ion-sensitive field-effect transistor (ISFET)

comprises a floating gate coupled to the reaction region.

4. The method of claim 1, wherein the sensor registers the change in ion concentration as an output pulse or signal.

5. The method of claim 1, wherein the reaction region comprises a well, cavity or surface.

6. The method of claim 5, wherein the at least one template nucleic acid is disposed into the well, cavity or surface.

7. The method of claim 6, wherein the at least one template nucleic acid is attached to the well, cavity or surface.

8. The method of claim 5, wherein the well, cavity or surface includes a capture primer attached thereto.

9. The method of claim 8, wherein the at least one template nucleic acid includes an adapter that hybridizes to at least a portion of the capture primer.

10. The method of claim 1, wherein the at least one template nucleic acid is attached to a bead or particle which is disposed into the reaction region.

11. The method of claim 1, wherein the nucleotide species introduced into the reaction region comprise nucleotides selected from the group consisting of dATP, dCTP, dGTP, dTTP and dUTP.

12. The method of claim 1, wherein the enzyme comprises a DNA or RNA polymerase.

13. The method of claim 1, wherein the one or more metal ions is selected from a Group la metal, a Group Ila metal, a transition metal, a post transition metal, a lanthanide or a mixture thereof.

14. The method of claim 1, wherein the one or more chelating agents is selected from

ethylenediamine tetraacetic acid (EDTA), diethlyenetriaminepentaacetic acid (DTPA), N- (hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), ethyleneglycol tetraacetic acid (EGTA), N,N-Ws(carboxymethyl)glycine (NT A), 2,3-dimercaprol (BAL), meso2,3- dimercaptosuccinic acid (DMSA), 2,3-dimercapto-l-propanesulfonic acid (DMPS), penicillamine, ethylene diamine, terpyridine, 14,7-triazacycononane (TACN), triethylenetetramine(trien), ira(2-aminoethyl)amine, ethylenediaminetriacetic acid, l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetate (DOT A), diethylene triamine pentaacetate (DPTA), deferoxamine (DFOA), deferiprone, tetraethylenetetraamine (TETA), porphine, analogs thereof, salts thereof, or a mixture thereof.

15. The method of claim 1, wherein the plurality of released ions in step (e) are hydrogen ions.

16. The method of claim 1, wherein the disposing of at least one template nucleic acid in a reaction region further comprises amplifying the template nucleic acid in the reaction region.

17. The method of claim 1, further comprising:

(i) washing the reaction region after step (b) or after step (c) or after step (d) or after step (e) or after step (f); or

(ii) washing the reaction region after steps (c) and (d) and (e) and (f).

18. The method of claim 1, wherein the nucleotide incorporation of step (c) generates a

primer extension product and a plurality of ions which changes the ion concentration in the reaction region.

19. The method of claim 18, wherein the sensor detects the change in the ion concentration in the reaction region which is generated from the nucleotide incorporation of step (c).

20. The method of claim 1, wherein the array contains 10 2 - 1010 reaction regions.