WO2021163621A2

WO2021163621A2 - Methods and systems for nucleic acid amplification and sequencing

Info

Publication number: WO2021163621A2
Application number: PCT/US2021/018032
Authority: WO
Inventors: Hesaam Esfandyarpour; Seth Stern; Maryam Jouzi; Meysam R. Barmi; Shankar Shastry; Seyyed Mir Abolhassan VAEZI
Original assignee: Genapsys, Inc.
Priority date: 2020-02-14
Filing date: 2021-02-12
Publication date: 2021-08-19
Also published as: EP4103746A2; WO2021163621A3; EP4103746A4

Abstract

Disclosed herein are methods and systems for nucleic acid amplification, processing nucleic acid molecules, and sequencing nucleic acid molecule. The systems and methods described herein provide for blocking subsets of probes used for nucleic acid amplification and nucleic acid amplification at a subset of probes used for nucleic acid amplification. The systems and methods described herein provide for multiplexed sequencing of nucleic acid molecules. The systems and methods described herein thereby provide for sequencing one or more nucleic acid molecules substantially simultaneously. The systems and methods described herein provide for efficient incorporation of nucleotide bases. The systems and methods described herein thereby provide for sequencing one or more nucleic acid molecules with reduced dephasing.

Description

METHODS AND SYSTEMS FOR NUCLEIC ACID AMPLIFICATION AND

SEQUENCING

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/976,982, filed February 14, 2020, U.S. Provisional Patent Application No. 62/978,744, filed February 19, 2020, and U.S. Provisional Patent Application No. 62/987,818, filed March 10, 2020, each of which is entirely incorporated here by reference.

BACKGROUND

[0002] Genetic analysis has emerged as one of the most important processes in the scientific enterprise and has given rise to several new fields and processes. Practical applications from this broad ability include numerous medical breakthroughs, computational advancements, and fundamental insight into the world around us. Deoxyribonucleic acid (DNA) amplification is an indispensable tool in a variety of genetic analysis. Amplification of DNA from small quantities is critical in collection of DNA from a crime scene, archeological analysis, identification of genes of interest, and in medical diagnostics. The preparation of the genetic material for testing, for example multiplexed testing, and subsequent analysis in such a manner to capture an entire genome and maintain its integrity is a challenging and critical limitation in any genetic methodology.

SUMMARY

[0003] In another aspect, the present disclosure provides a method for nucleic acid amplification, comprising: bringing a template nucleic acid molecule in contact with an array of oligonucleotides, wherein said template nucleic acid molecule binds to an oligonucleotide of said array of oligonucleotides; using said template nucleic acid molecule to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other oligonucleotides of said array of oligonucleotides; binding nucleic acid molecules of said plurality of nucleic acid molecules to said other oligonucleotides of said array of oligonucleotides, thereby generating occupied oligonucleotides; removing at least a portion of said nucleic acid molecules of said plurality of nucleic acid molecules from said occupied oligonucleotides, thereby generating active oligonucleotides; and using said template nucleic acid molecule and said active oligonucleotides to amplify said template nucleic acid molecule, thereby generating amplicons coupled to said active oligonucleotides. In some embodiments, (b) and (c) occur contemporaneously. In some embodiments, (b) and (c) occur consecutively. In some embodiments, said other oligonucleotides of said array of oligonucleotides comprise a common sequence. In some embodiments, said plurality of nucleic acid molecules are at least partially complementary to said common sequence. In some embodiments, oligonucleotides of said array of oligonucleotides are identical. In some embodiments, said plurality of nucleic acid molecules is a plurality of ribonucleic acid (RNA) molecules. In some embodiments, (b) is performed with the aid of an RNA polymerase. In some embodiments, said RNA polymerase is T7 RNA polymerase. In some embodiments, (d) comprises removing at least a portion of said nucleic acid molecules of said plurality of nucleic acid molecules from said occupied oligonucleotides with use of an enzyme. In some embodiments, said enzyme is an RNase. In some embodiments, said RNase is RNase H. In some embodiments, (b) is performed when said template nucleic acid molecule is bound to said oligonucleotide. In some embodiments, after (b), said nucleic acid molecules of said plurality of nucleic acid molecules are transported from said oligonucleotide to said other oligonucleotides of said array of oligonucleotides. In some embodiments, after (b), said nucleic acid molecules of said plurality of nucleic acid molecules are transported from said oligonucleotide to said other oligonucleotides of said array of oligonucleotides via diffusion. In some embodiments, said template nucleic acid molecule comprises a promoter sequence. In some embodiments, said oligonucleotides comprises a complementary promoter sequence complementary to said promoter sequence. In some embodiments, said promoter sequence is a

T7 ribonucleic acid (RNA) polymerase promoter sequence. In some embodiments, (b) is performed by binding of said template nucleic acid molecule to at least two oligonucleotides of said array of oligonucleotides. In some embodiments, said array of oligonucleotides is attached to a solid support. In some embodiments, said solid support is a bead. In some embodiments, said solid support is planar. In some embodiments, said solid support is a surface of a well. In some embodiments, said array of oligonucleotides is in sensory communication with a sensor. In some embodiments, said sensor comprises an electrode. In some embodiments, said sensor comprises a plurality of electrodes. In some embodiments, said sensor is among an array of sensors. In some embodiments, at least one sensor of said array of sensors is individually addressable. In some embodiments, said array of oligonucleotides is among a plurality of arrays of oligonucleotides. In some embodiments, the method, after (b), further comprises excluding said plurality of nucleic acid molecules from other arrays of said plurality of arrays of oligonucleotides. In some embodiments, said excluding comprises applying an electric field to said plurality of nucleic acid molecules. In some embodiments, at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said electric field. In some embodiments, said label is a particle. In some embodiments, said excluding comprises applying a magnetic field to said plurality of nucleic acid molecules. In some embodiments, at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said magnetic field. In some embodiments, said label is a particle. In some embodiments, said excluding is performed with the aid of a diffusion barrier. In some embodiments, said excluding is performed by degrading a subset of said plurality of nucleic acid molecules. In some embodiments, said degrading is performed with an enzyme. In some embodiments, said enzyme is an RNase. In some embodiments, said enzyme is coupled to a support. In some embodiments, said support is a particle. In some embodiments, the method further comprises applying an electric field to said support. In some embodiments, said array of oligonucleotides comprises oligonucleotides having sequences different from oligonucleotides of at least one other array of said plurality of arrays of oligonucleotides. In some embodiments, the method further comprises repeating (a) - (e) at least one other array of said plurality of arrays of oligonucleotides. In some embodiments, (e) comprises conducting a reaction with aid of a recombinase. In some embodiments, (e) comprises conducting a reaction with aid of a polymerase. In some embodiments, said amplicons coupled to said active oligonucleotides are a clonal population of nucleic acids. In some embodiments, the method further comprises sequencing at least a subset of said amplicons coupled to said active oligonucleotides or derivatives thereof. In some embodiments, said sequencing is completed via sequencing-by-synthesis. In some embodiments, said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

[0004] In another aspect, the present disclosure provides a method for processing a template nucleic acid molecule, comprising: providing a template nucleic acid molecule coupled to an oligonucleotide of an array of oligonucleotides, wherein other oligonucleotides of said array of oligonucleotides are blocked such that other template nucleic acid molecules are incapable of stably coupling to said other oligonucleotides; deblocking at least a subset of said other oligonucleotides; and using said template nucleic acid molecule and said active oligonucleotides to amplify said template nucleic acid molecule, thereby generating amplicons coupled to said active oligonucleotides. In some embodiments, said other oligonucleotides of said array of oligonucleotides are blocked with nucleic acid molecules bound to said other oligonucleotides of said array of oligonucleotides. In some embodiments, said nucleic acid molecules are ribonucleic acid (RNA) molecules. In some embodiments, oligonucleotides of said array of oligonucleotides are coupled to a support. In some embodiments, said support is a bead. In some embodiments, said support is planar. In some embodiments, in (b), said deblocking is performed with the aid of an enzyme. In some embodiments, said enzyme is an RNase. In some embodiments, (b),(c), or both occurs in a well. In some embodiments, the method further comprises applying an electric field to said array of oligonucleotides. In some embodiments, the method further comprises applying a magnetic field to said array of oligonucleotides. In some embodiments, said amplicons coupled to said active oligonucleotides are a clonal population of nucleic acids. In some embodiments, the method further comprises sequencing at least a subset of said amplicons coupled to said active oligonucleotides or derivatives thereof. In some embodiments, said sequencing is completed via sequencing-by-synthesis. In some embodiments, said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

[0005] In another aspect, the present disclosure provides a method for nucleic acid amplification, comprising: bringing a plurality of target nucleic acid molecules in contact with an array of oligonucleotides, wherein said plurality of target nucleic molecules is present at a concentration such that most a target nucleic acid molecule of said plurality of target nucleic acid molecules hybridizes to an oligonucleotide of said array of oligonucleotides; subjecting said array of oligonucleotides to conditions sufficient to synthesize a first plurality of nucleic acid molecules from said target nucleic acid molecule hybridized to said oligonucleotide, wherein said first plurality of nucleic acid molecules is hybridized to other oligonucleotides of said array of oligonucleotides; subjecting said array of oligonucleotides to conditions sufficient to remove or degrade at least a subset of said first plurality of nucleic acid molecules; and subsequent to (c), subjecting said array of oligonucleotides to conditions sufficient to amplify said target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to said array of oligonucleotides. In some embodiments, said oligonucleotides of said array of oligonucleotides comprise a common sequence. In some embodiments, said plurality of nucleic acid molecules are at least partially complementary to said common sequence. In some embodiments, said oligonucleotides of said array of oligonucleotides are identical. In some embodiments, said first plurality of nucleic acid molecules is a plurality of ribonucleic acid (RNA) molecules. In some embodiments, (b) is performed with the aid of an RNA polymerase. In some embodiments, said

RNA polymerase is T7 RNA polymerase. In some embodiments, (c) comprises removing or degrading said subset of said nucleic acid molecules with an enzyme. In some embodiments, said enzyme is an RNase. In some embodiments, said RNase is RNase H. In some embodiments, (b) further comprises transporting a subset of said first plurality of nucleic acid molecules to said other oligonucleotides of said array of oligonucleotides. In some embodiments, (b) further comprises transporting a subset of said first plurality of nucleic acid molecules to said other oligonucleotides of said array of oligonucleotides via diffusion. In some embodiments, said target nucleic acid molecule hybridized to said oligonucleotide comprises a promoter sequence. In some embodiments, said oligonucleotides of said array of oligonucleotides comprise a complementary promoter sequence complementary to said promoter sequence. In some embodiments, said promoter sequence is a T7 ribonucleic acid (RNA) polymerase promoter sequence. In some embodiments, said array of oligonucleotides is attached to a solid support. In some embodiments, said solid support is a bead. In some embodiments, said solid support is planar. In some embodiments, said solid support is a surface of a well. In some embodiments, said array of oligonucleotides is in sensory communication with a sensor. In some embodiments, said sensor comprises an electrode. In some embodiments, said sensor comprises a plurality of electrodes. In some embodiments, said sensor is among an array of sensors. In some embodiments, at least one sensor of said array of sensors is individually addressable. In some embodiments, said array of oligonucleotides is among a plurality of arrays of oligonucleotides. In some embodiments, (b) further comprises excluding said plurality of nucleic acid molecules from other arrays of said plurality of arrays of oligonucleotides. In some embodiments, said excluding comprises applying an electric field to said plurality of nucleic acid molecules. In some embodiments, at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said electric field. In some embodiments, said label is a particle. In some embodiments, said excluding comprises applying a magnetic field to said plurality of nucleic acid molecules. In some embodiments, at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said magnetic field.

In some embodiments, said label is a particle. In some embodiments, said excluding is performed with the aid of a diffusion barrier. In some embodiments, said excluding is performed by degrading a subset of said plurality of nucleic acid molecules. In some embodiments, said degrading is performed with an enzyme. In some embodiments, said enzyme is an RNase. In some embodiments, said enzyme is coupled to a support. In some embodiments, said support is a particle. In some embodiments, the method further comprises applying an electric field to said support. In some embodiments, said array of oligonucleotides comprises oligonucleotides having sequences different from oligonucleotides of at least one other array of said plurality of arrays of oligonucleotides. In some embodiments, the method further comprises repeating (a) - (d) at another array of said plurality of arrays of oligonucleotides. In some embodiments, (d) comprises conducting a reaction with aid of a recombinase. In some embodiments, (d) comprises conducting a reaction with aid of a polymerase. In some embodiments, the method further comprises (e) sequencing at least a subset of said second plurality of nucleic acid molecules hybridized to said array of oligonucleotides. In some embodiments, the method further comprises sequencing at least a subset of said substantially clonal populations at said another array of said plurality of arrays of oligonucleotides. In some embodiments, said sequencing is completed via sequencing-by-synthesis. In some embodiments, said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge. [0006] In another aspect, the present disclosure provides a method for sequencing a nucleic acid molecule, comprising: providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence, wherein said nucleic acid molecule comprises a first primer hybridized to said third sequence; subjecting said third sequence to sequencing to generate a first sequencing read comprising at least a portion of said third sequence; bringing a second primer having a sequence complementarity with said second sequence in contact with said nucleic acid molecule under conditions sufficient for said second primer to hybridize to said second sequence, and subjecting said second sequence to sequencing to generate a second sequencing read comprising at least a portion of said second sequence; and bringing a third primer having a sequence complementarity with said first sequence in contact with said nucleic acid molecule under conditions sufficient for said third primer to hybridize to said first sequence, and subjecting said first sequence to sequencing to generate a third sequencing read comprising at least a portion of said first sequence. In some embodiments, said sequencing of said first sequence, second sequence, third sequence, or any combination thereof comprises use of a polymerizing enzyme. In some embodiments, said polymerizing enzyme comprises strand displacement activity. In some embodiments, said second sequencing read displaces said first sequencing read. In some embodiments, said third sequencing read displaces said second sequencing read. In some embodiments, said sequencing of said first sequence, third sequence, or both, generates an identification tag for said second sequence. In some embodiments, said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof. In some embodiments, the method further comprises, prior to providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, coupling said first sequence, said third sequence, or both to said second sequence. In some embodiments, said first sequence or said third sequence is coupled to said second sequence via ligation. In some embodiments, said first sequence or said third sequence is coupled to said second sequence via hybridization. In some embodiments, said nucleic acid molecule is coupled to said support via a probe coupled to said support. In some embodiments, said probe comprises an oligonucleotide. In some embodiments, said support is a bead. In some embodiments, said support is planar. In some embodiments, said support is a surface of a well. In some embodiments, said probe is in sensory communication with a sensor.

In some embodiments, said sensor comprises an electrode. In some embodiments, said sensor comprises a plurality of electrodes. In some embodiments, said sequencing is completed via sequencing-by-synthesis. In some embodiments, said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

[0007] In another aspect, the present disclosure provides a method for processing a nucleic acid molecule, comprising: providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence and a second sequence; subjecting said nucleic acid molecule to a first extension reaction to generate a first strand complementary to said first sequence, wherein a 5’ end of said first strand comprises a blocking group; and subjecting said nucleic acid molecule to a second extension reaction to generate a second strand complementary to said second sequence, wherein a 5’ end of said second strand comprises an additional blocking group. In some embodiments, subjecting said nucleic acid molecule to a second extension reaction to generate a second strand complementary to said second sequence is performed subsequent to subjecting said nucleic acid molecule to a first extension reaction to generate a first strand complementary to said first sequence. In some embodiments, said nucleic acid molecule further comprises a third sequence. In some embodiments, the method further comprises subjecting said nucleic acid molecule to a third extension reaction to generate a third strand complementary to said third sequence. In some embodiments, said sequencing of said first sequence generates an identification tag for said second sequence. In some embodiments, said sequencing of said first sequence, third sequence, or both, generates an identification tag for said second sequence. In some embodiments, said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof. In some embodiments, the method further comprises, prior to providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, coupling said first sequence, said third sequence, or both to said second sequence. In some embodiments, said first sequence or third sequence is coupled to said second sequence via ligation. In some embodiments, said first sequence or third sequence is coupled to said second sequence via hybridization. In some embodiments, said nucleic acid molecule is coupled to said support via a probe coupled to said support. In some embodiments, said probe comprises an oligonucleotide. In some embodiments, said support is a bead. In some embodiments, said support is planar. In some embodiments, said support is a surface of a well. In some embodiments, said probe is in sensory communication with a sensor. In some embodiments, said sensor comprises an electrode. In some embodiments, said sensor comprises a plurality of electrodes. In some embodiments, said sequencing is completed via sequencing-by-synthesis. In some embodiments, said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge. In some embodiments, said blocking group comprises one or more biologic molecules. In some embodiments, said one or more biologic molecules comprise one or more nucleotides, one or more enzymes, or both. In some embodiments, said blocking group comprises one or more metals.

[0008] In another aspect, the present disclosure provides a method for processing a nucleic acid molecule, comprising: providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence, wherein said nucleic acid molecule comprises a first primer hybridized to said third sequence; subjecting said third sequence to sequencing to generate a first non-optical sequencing read comprising at least a portion of said third sequence; bringing a second primer having sequence complementarity with said second sequence in contact with said nucleic acid molecule under conditions sufficient for said second primer to hybridize to said second sequence, and subjecting said second sequence to non-optical sequencing to generate a second sequencing read comprising at least a portion of said second sequence; and bringing a third primer having sequence complementarity with said first sequence in contact with said nucleic acid molecule under conditions sufficient for said third primer to hybridize to said first sequence, and subjecting said first sequence to non-optical sequencing to generate a third sequencing read comprising at least a portion of said first sequence. In some embodiments, subjecting said third sequence to sequencing to generate a first non-optical sequencing read comprising at least a portion of said third sequence, bringing a second primer having sequence complementarity with said second sequence in contact with said nucleic acid molecule under conditions sufficient for said second primer to hybridize to said second sequence, and subjecting said second sequence to non-optical sequencing to generate a second sequencing read comprising at least a portion of said second sequence, and bringing a third primer having sequence complementarity with said first sequence in contact with said nucleic acid molecule under conditions sufficient for said third primer to hybridize to said first sequence, and subjecting said first sequence to non-optical sequencing to generate a third sequencing read comprising at least a portion of said first sequence are performed in any order of sequence. In some embodiments, said sequencing of said first sequence, third sequence, or both, generates an identification tag for said second sequence. In some embodiments, said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof. In some embodiments, the method further comprises, prior to providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, coupling said first sequence, said third sequence, or both to said second sequence. In some embodiments, said first sequence or third sequence is coupled to said second sequence via ligation. In some embodiments, said first sequence or third sequence is coupled to said second sequence via hybridization. In some embodiments, said nucleic acid molecule is coupled to said support via a probe coupled to said support. In some embodiments, said probe comprises an oligonucleotide.

In some embodiments, said support is a bead. In some embodiments, said support is planar. In some embodiments, said support is a surface of a well. In some embodiments, said probe is in sensory communication with a sensor. In some embodiments, said sensor comprises an electrode. In some embodiments, said sensor comprises a plurality of electrodes. In some embodiments, said sequencing is completed via sequencing-by-synthesis. In some embodiments, said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge. In some embodiments, the method further comprises, between any combination of subjecting said third sequence to sequencing to generate a first non-optical sequencing read comprising at least a portion of said third sequence, bringing a second primer having sequence complementarity with said second sequence in contact with said nucleic acid molecule under conditions sufficient for said second primer to hybridize to said second sequence, and subjecting said second sequence to non-optical sequencing to generate a second sequencing read comprising at least a portion of said second sequence, and bringing a third primer having sequence complementarity with said first sequence in contact with said nucleic acid molecule under conditions sufficient for said third primer to hybridize to said first sequence, and subjecting said first sequence to non-optical sequencing to generate a third sequencing read comprising at least a portion of said first sequence, performing an annealing operation. In some embodiments, said annealing operation is a thermal annealing operation.

[0009] In another aspect, the present disclosure provides a method for sequencing a template nucleic acid molecule, comprising: (a) providing a plurality of nucleic acid molecules immobilized adjacent to a support, wherein each of the plurality of nucleic acid molecules comprises a sequence of the template nucleic acid molecule; (b) in a first phase, sequentially bringing the plurality of nucleic acid molecules in contact with nucleotides of one or more types that are fewer than four types of nucleotides and detecting a first set of signals from the plurality of nucleic acid molecules; and (c) in a second phase subsequent to the first phase, sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides and detecting a second set of signals from the plurality of nucleic acid molecules, to obtain sequences of the plurality of nucleic acid molecules, wherein a sequential order of nucleotides in the first phase is different than a sequential order of nucleotides in the second phase, wherein a sequence of the plurality of nucleic acid molecules has a phase lag or phase lead of at most 5 bases with respect to another sequence of the plurality of nucleic acid molecules. [0010] In some embodiments, the method further comprises (d) in a third phase, sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides, wherein a sequential order of nucleotides in the third phase is different than a sequential order of nucleotides in the first phase and the second phase. In some embodiments, the method further comprises (e) in a fourth phase, sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides, wherein a sequential order of nucleotides in the fourth phase is different than a sequential order of nucleotides in the first phase, the second phase, and the third phase. In some embodiments, the method further comprises (f) repeating (b), (c), (d), (e), or any combination thereof. In some embodiments, the phase lag or phase lead is at most 4 bases. In some embodiments, the phase lag or phase lead is at most 3 bases. In some embodiments, the phase lag or phase lead is at most 2 bases. In some embodiments, the phase lag or phase lead is at most 1 base. In some embodiments, the first set of signals or the second set of signals are associated with an impedance, conductivity, charge, or change thereof, associated with the plurality of nucleic acid molecules.

[0011] In another aspect, the present disclosure provides a method of performing a stepwise extension of a plurality of primers hybridized to a plurality of nucleic acid molecules as part of a clonal population, comprising: (a) contacting, in a first phase, the clonal population with each of four types of nucleotides under conditions sufficient to extend the primers in a template directed synthesis; and (b) contacting, in a second phase, the clonal population with fewer than each of four types of nucleotides.

[0012] In some embodiments, the method further comprises (c) contacting, in a third phase, the clonal population with fewer than each of four types of nucleotides wherein a sequential order of nucleotides in the third phase is different than a sequential order of nucleotides in the first phase and the second phase. In some embodiments, the method further comprises (d) contacting, in a fourth phase, the clonal population with fewer than each of four types of nucleotides wherein a sequential order of nucleotides in the fourth phase is different than a sequential order of nucleotides in the first phase, the second phase, and the third phase. In some embodiments, the method further comprises (e) repeating (b), (c), (d), or any combination thereof. In some embodiments, the method further comprises detecting signals from the plurality of nucleic acid molecules to generate a plurality of sequences of the plurality of nucleic acid molecules. In some embodiments, a sequence of the plurality of nucleic acid molecules has a phase lag or phase lead of at most 5 bases with respect to another sequence of the plurality of nucleic acid molecules. In some embodiments, the phase lag or phase lead is at most 4 bases. In some embodiments, the phase lag or phase lead is at most 3 bases. In some embodiments, the phase lag or phase lead is at most 2 bases. In some embodiments, the phase lag or phase lead is at most 1 base. In some embodiments, a sequential order of nucleotides in the first phase is different than a sequential order of nucleotides in the second phase. In some embodiments, the sequencing comprises sequencing via sequencing-by-synthesis. In some embodiments, the sequencing comprises measuring one or more signals associated with sequencing-by-synthesis. In some embodiments, the signals associated with an impedance, conductivity, charge, or change thereof, associated with the plurality of nucleic acid molecules.

[0013] In another aspect, the present disclosure provides a method for sequencing a template nucleic acid molecule, comprising: (a) providing a plurality of nucleic acid molecules immobilized adjacent to a support, wherein each of the plurality of nucleic acid molecules comprises a sequence of the template nucleic acid molecule; (b) in a first phase, bringing the plurality of nucleic acid molecules in contact with fewer than each of four types of nucleotides; and (c) in a second phase, bringing the plurality of nucleic acid molecules in contact with the four types of nucleotides, to obtain sequences of the plurality of nucleic acid molecules, wherein a sequence of the plurality of nucleic acid molecules has a phase lag or phase lead of at most 5 bases with respect to another sequence of the plurality of nucleic acid molecules.

[0014] In some embodiments, the second phase is subsequent to the first phase. In some embodiments, the second phase is prior to the first phase. In some embodiments, the method further comprises (d) in a third phase, bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides, wherein a sequential order of nucleic acid molecules in the third phase is different than a sequential order of nucleic acid molecules in the first phase and the second phase. In some embodiments, the method further comprises (e) in a fourth phase, bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides, wherein a sequential order of nucleic acid molecules in the fourth phase is different than a sequential order of nucleic acid molecules in the first phase, the second phase, and the third phase. In some embodiments, the method further comprises (f) repeating (b), (c), (d), (e), or any combination thereof. In some embodiments, the phase lag or phase lead is at most 4 bases. In some embodiments, the phase lag or phase lead is at most 3 bases. In some embodiments, the phase lag or phase lead is at most 2 bases. In some embodiments, the phase lag or phase lead is at most 1 base. In some embodiments, the obtaining sequences comprises sequencing via sequencing-by-synthesis. In some embodiments, the obtaining sequences comprises measuring one or more signals associated with sequencing-by-synthesis. In some embodiments, the signals are associated with an impedance, conductivity, charge, or change thereof, associated with the plurality of nucleic acid molecules. [0015] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0016] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0017] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS [0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0020] FIG. l is a flow chart of an example process for nucleic acid amplification.

[0021] FIG. 2 is an example overview of a ribonucleic acid (RNA) blocking process.

[0022] FIG. 3 is an example of sensor selective RNA blocking.

[0023] FIG. 4 is a flow chart of an example process for processing a template nucleic acid molecule.

[0024] FIGS. 5A - 5D are example schematics of confining the nucleic acid molecules generated in the nucleic acid molecule blocking process. [0025] FIG. 6A - 6B are an example of confining the nucleic acid molecules using the electrophoretic force and an example electrode material for the electrodes of FIGs. 5A, 5C, and

6A.

[0026] FIG. 7 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0027] FIG. 8 shows a flowchart for an example method for sequencing a nucleic acid molecule.

[0028] FIG. 9 shows a flowchart for an example method for processing a nucleic acid molecule.

[0029] FIG. 10 shows a flowchart for an example method for processing a nucleic acid molecule.

[0030] FIG. 11 shows an example of a nucleic acid molecule comprising multiple sequences.

[0031] FIG. 12 shows an example overview of a run-off sequencing process.

[0032] FIG. 13 shows an example overview of a blocking sequencing process.

[0033] FIG. 14 shows an example overview of a meltoff sequencing process.

[0034] FIG. 15 shows a flowchart for an example method for sequencing a template nucleic acid molecule.

[0035] FIG. 16 shows a flowchart for an example method of performing a stepwise extension of a plurality of primers hybridized to a plurality of nucleic acid molecules as part of a clonal population.

[0036] FIG. 17 shows a flowchart for an example method for sequencing a template nucleic acid molecule.

DETAILED DESCRIPTION

[0037] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0038] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3

[0039] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0040] The term “nucleotide,” as used herein, generally refers to a nucleotide or a nucleotide analog. A nucleotide may be a naturally occurring nucleotide. The nucleotide may be a non- naturally occurring or synthetic (or engineered) nucleotide.

[0041] The term “oligonucleotide,” as used herein, generally refers to a nucleic acid molecule comprising at least two nucleotides. An oligonucleotide may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000, 100,000 or more nucleotides. In some examples, an oligonucleotide comprises at most about 100,000,

50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 175, 150, 125, 100,

90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 9 R fi 4 3 or 2 nucleotides. An oligonucleotide may be unbound (e.g., in solution) or bound (e.g., chemically bonded to a substrate).

[0042] The term “probe,” as used herein, generally refers to a first moiety configured to bind to a second moiety. In some embodiments the probe is an antibody. In some embodiments the probe is a ligand (e.g., a small molecule ligand). In some embodiments the probe is an oligonucleotide at least partially complimentary to an oligonucleotide of the second moiety. In some embodiments the probe is any combination thereof.

[0043] The phrase “active probes,” as used herein, generally refers to probes available for binding to nucleic acids. For example, a probe that had a ribonucleic acid (RNA) molecule or another species of molecule that blocks probe binding removed from it can be an active probe, as it is configured to accept a copy of a target nucleic acid molecule.

[0044] The phrase “diffusion barrier,” as used herein, generally refers to a material configured to inhibit or slow diffusion. The material may be a viscous liquid (e.g., l-Butyl-3- methylimidazolium hexafluorophosphate or glycerol) or a solid (e.g., a polymer).

[0045] The phrase “promoter sequence,” as used herein, generally refers to a region of a nucleic acid that initiates a polymerase. In some embodiments, the promoter sequence may correspond to a polymerase used in a nucleic acid extension reaction. In some embodiments, the promoter sequence may be a promoter sequence for a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), a phage T3 RNA polymerase, and the like.

[0046] The phrase “sensory communication,” as used herein, generally refers to an event being detectable by a sensor. For example, a binding of a nucleic acid molecule to a probe is in sensory communication with an impedance sensor if the sensor can detect the change in impedance caused by the binding of the nucleic acid to the probe. In some embodiments, the sensory communication may be electrical communication (e.g., detecting electrical signals). In some embodiments, the sensory communication may be optical communication (e.g., detecting fluoresce events). In some embodiments, the sensory communication may be chemical communication (e.g., detecting a change in pH).

[0047] The phrase “clonal population,” as used herein, generally refers to nucleic acids that comprise an identical or substantially identical sequence to a template nucleic acid molecule. [0048] The term “degrade,” as used herein, generally refers to an at least partial removal of a material. For example, degrading an RNA strand hybridized to a probe can involve dehybridizing the RNA strand from a probe with addition of a high ionic strength buffer. Degrading may be partial or full. Degrading may remove at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more of a material. Degrading may remove most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of a material. In some embodiments, degrading may be performed by an enzyme (e.g., an RNase). In some embodiments, degrading may be performed by a chemical agent (e.g., sodium hydroxide, formamide, guanidine, sodium salicylate, dimethyl sulfoxide, propylene glycol, urea). In some embodiments, degrading may be performed by heating. In some embodiments, degrading may be performed by increasing the ionic strength around the material (e.g., adding sodium chloride). In some embodiments, degrading may be performed with any combination of aforementioned processes (e.g., heating and adding a salt).

[0049] The phrase “conditions sufficient to synthesize,” as used herein, generally refers to a presence of materials configured to amplify a nucleic acid. The materials configured to amplify a nucleic acid may comprise a polymerase and a nucleotide or nucleoside. In some embodiments the nucleotide or nucleoside further comprises at least one, two, three, or more phosphate groups (e.g., the nucleoside is a nucleotide, a nucleoside diphosphate, a nucleoside triphosphate, etc.) The nucleotide or nucleoside may comprise a base. In some embodiments the base is adenine, guanine, cytosine, uracil, or thymine. A plurality of different bases may be added to generate conditions sufficient to synthesize. The conditions sufficient to synthesize may further comprise additional materials (e.g., cellular energy sources, etc.).

[0050] The term “amplicons,” as used herein, generally refers to one or more copies of a nucleic acid molecule, such as, for example, an amplification product or nucleic acid extension product. The amplification products may be clonal copies of a starting or template nucleic acid molecule. In some embodiments, the amplicons can be ribonucleic acid (RNA) molecules. In some embodiments the amplicons can be deoxyribonucleic acid (DNA) molecules.

[0051] The term “barcode,” “index,” “identification tag,” or “barcode sequence,” as used herein, generally refers to a known sequence of nucleic acid bases coupled to a nucleic acid molecule of interest.

[0052] The phrase “incapable of stably coupling to said other probes,” as used herein, generally refers to an inability of a nucleic acid to hybridize to one or more other probes under a given set of conditions. For example, a nucleic acid may partially bind to a probe such that at high temperatures the nucleic acid is free floating while at lower temperatures the nucleic acid is able to stay partially bound to the probe.

[0053] In an aspect, the present disclosure provides methods for nucleic acid amplification. A method for nucleic acid amplification may comprise bringing a template nucleic molecule in contact with an array of probes. The template nucleic acid molecule may bind to a probe of the array of probes. The template nucleic acid molecule may be used to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other probes of the array of probes. The nucleic acid molecules of the plurality of nucleic acid molecules may bind to the other probes of the array of probes, thereby generating occupied probes. At least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules may be removed from the occupied probes, thereby generating active probes. The template nucleic acid molecule and the active probes may be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active probes.

[0054] The present disclosure provides methods and systems for sample processing.

Methods and systems of the present disclosure may be used to process a nucleic acid molecule for subsequent analysis, such as, for example, sequencing.

[0055] FIG. l is a flow chart of an example process nucleic acid amplification. The process 100 can be implemented on an appropriately configured system as described elsewhere herein. The appropriately configured system may be a system configured to perform a nucleic acid sequencing. The system may be configured to amplify one or more nucleic acids. The system can bring a template nucleic molecule in contact with an array of nucleotides (110). The template nucleic acid molecule may bind to a probe of the array of probes. The template nucleic molecule may comprise a nucleic molecule of interest (e.g., a DNA molecule to be sequenced). The template nucleic molecule may further comprise one or more moieties configured to bind to a probe. For example, the template nucleic molecule can be a fragment of a DNA sample with a probe attached to the 3’ end. In another example, the template nucleic molecule can be a fragment of a DNA sample with a probe attached to the 5’ end. The moiety configured to bind to the probe may be configured to bind with a portion of the probe. For example, the probe can be a

36-base probe, and the moiety can be 15 bases complimentary to the free end of the probe. The binding of the template nucleic acid molecule with the probe may be a hybridization of complimentary bases. The template nucleic acid molecule may have a concentration of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550

600, 700, 800, 900, 1,000 or more nanograms per microliter. The template nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350,

300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70,

65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,

0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less nanograms per microliter. The template nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 to 4 nanograms per microliter.

[0056] The system can use the template nucleic acid molecule to synthesize a plurality of nucleic acid molecules that are at least partially complementary to sequences of other probes of the array of probes (120). The synthesizing a plurality of nucleic acid molecules may be a polymerase chain reaction. The plurality of nucleic acid molecules may be RNA molecules,

DNA molecules, or probes. The RNA molecules may be synthesized from the template nucleic acid molecule with the aid of a reagent. The reagent may be an enzyme. The enzyme may be an RNA polymerase. The RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase. The plurality of nucleic acid molecules may be at least partially complimentary to sequences of other probes of the array of nucleotides. The plurality of nucleic acids may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more complimentary to sequences of other probes of the array of nucleotides. The plurality of nucleic acid molecules may be at most about 100 %, 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less complimentary to sequences of other probes of the array of nucleotides.

[0057] The other probes of the array of probes may comprise a common sequence. The probes of the array of probes may be identical. The plurality of nucleic acid molecules may be at least partially complementary to the common sequence. For example, an RNA molecule can be complimentary to all probes within a 15 micrometer (micron) square area (15 pm²), but not to probes outside that area. The other probes of the array of probes may have a common sequence with other probes in an area of at least about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 50, 75, 100,

150, 200, 250, 500, 750, 1,000, 5,000, 10,000, 50,000, 100,000, or more square microns ( pm²).

The other probes of the array of probes may have a common sequence with other probes in an area of at most about 100,000, 50,000, 10,000, 5,000, 1,000, 750, 500, 250, 200, 150, 100, 75,

50, 25, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or less square microns. Operation 120 may be performed with binding of the template nucleic acid molecule to at least two probes of the array of probes. The binding of the template nucleic acid molecule to at least two probes may impart a bridge geometry to the template nucleic acid molecule. The array of probes may be among a plurality of arrays of probes. The array of probes may comprise probes having sequences different from probes of at least one other array of the plurality of arrays of probes. For example, the probes coupled to the sensors of a 3x3 grid of sensors can each have a different sequence, leading to 9 different probe sequences. The different probe sequences may result in less cross contamination of nucleic acids between sensors. The lack of cross contamination may be particularly relevant in sensing arrays that do not comprise wells. For example, each bead of an array of beads having different probe sequences can prevent the RNA produced at each bead from diffusing to and binding onto another bead.

[0058] In some embodiments, the arrays of the plurality of arrays of probes can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. In some embodiments, select areas of the arrays of probes can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. In some embodiments, a subset of the arrays of probes are blocked from binding to nucleic acid molecules. In some embodiments, the subset of the arrays of probes can be blocked by a nucleic acid molecule of a first plurality of nucleic acid molecules. In some embodiments, the first plurality of nucleic acid molecules comprises RNA molecules. In some embodiments, the template nucleic acid molecule can be among a plurality of template nucleic acid molecules. In some embodiments, individual template nucleic acid molecules can comprise different sequences. In some embodiments, distinct template nucleic acid molecules can be bound to distinct select areas of the arrays of probes. In some embodiments, the distinct template nucleic acid molecules can be selectively amplified or sequenced at corresponding, distinct, or select areas of the arrays of probes. In some embodiments, selective amplification of the distinct template nucleic acid molecules generates a second plurality of nucleic acid molecules. In some embodiments, the second plurality of nucleic acid molecules comprise DNA molecules.

[0059] Operation 130 may be performed when the template nucleic acid is bound to the probe. The nucleic acid molecules of the plurality of nucleic acid molecules may, after operation 130, be transported from the probe to the other probes of the array of probes. The transportation may be via diffusion. The transportation may be assisted diffusion. The transportation may be an active transportation. The active transportation may comprise cellular transportation methods

(e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow (e.g., flowing a liquid carrier in the direction of transport), or any combination thereof. The transportation may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of diffusion.

[0060] The system can bind nucleic acid molecules of the plurality of nucleic acid molecules to the other probes of the array of probes, thereby generating occupied probes (130). The binding of the nucleic acid to the probe may be configured to prevent additional nucleic acids or other template nucleic acid molecules from binding to the probe. The binding of the nucleic acid to the probe may allow for one template nucleic acid to bind to a given area. For example, a target nucleic acid binds to a probe and produces a plurality of nucleic acids that block the surrounding probes from other target nucleic acids binding.

[0061] The operations 120 and 130 may occur contemporaneously. For example, an RNA molecule generated by the template nucleic acid molecule can bind to a nearby probe immediately after being generated. The operations 120 and 130 may occur consecutively. For example, an RNA molecule generated by the template nucleic acid molecule can float in solution for a time before binding to a nearby probe. The time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other probe may be at least about 0.1 s, 1 s, 2 s, 3 s, 4 s, 5 s, 10 s, 30 s, 60 s, 120 s, 180 s, 240 s, 300 s, 360 s, 600 s, 1200 s, 2400 s, 3600 s, or more. The time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other probe may be at most about 3600 s, 2400 s, 1200 s, 600 s, 360 s, 300 s, 240 s, 180 s, 120 s, 60 s, 30 s, 10 s, 5 s, 4 s, 3 s, 2 s, 1 s, 0.1 s, or less. [0062] The system can remove at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied probes, thereby generating active probes (140). Operation 140 may comprise removing at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied probes with a reagent. The removing at least a portion of the nucleic acid molecules may be removing substantially all nucleic acid molecules within an area. For example, all of the probes in a well of a sensing array can have the bound nucleic acid molecules removed. In another example, the nucleic acids bound to probes on the surface of a bead can be removed. The removing at least a portion of the nucleic acid molecules may be removing nucleotides of a given sequence. For example, nucleotides with the sequence ATACG can be removed, but nucleotides with the sequence TTAAG can remain. The reagent may be an enzyme. The enzyme may be an RNase. The RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V. The reagent may be a chemical compound. The chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea.

[0063] The system can use the template nucleic acid molecule and the active probes to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active probes (150). Operation 150 may comprise conducting a reaction with aid of at least one recombinase, polymerase, or a combination thereof. The recombinase may be a Tre recombinase, a Cre recombinase, a Hin recombinase, a Dmcl recombinase, a Rad51 recombinase, or a FLP recombinase. The polymerase may be a DNA polymerase or an RNA polymerase. The RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase. The DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y. The amplicons coupled to the active probes may be a clonal population of nucleic acids. The clonal population of nucleic acids may be clones of the template nucleic acid. The amplicons may be a partially clonal population. The amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal. The amplicons may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal. Operation 150 may further comprise sequencing at least a subset of the amplicons coupled to the active probes or derivatives thereof. The derivatives may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid. The derivatives may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less similar in sequence to the template nucleic acid. The subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons. The subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the amplicons. The sequencing may be sequencing-by synthesis, sequencing-by-ligation, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof. The nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof. The sequencing may be performed by methods and systems as described elsewhere herein. [0064] The array of probes may be among a plurality of arrays of probes. The plurality of arrays of probes may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000,

10,000, 50,000, 100,000, 500,000, 1,000,000, or more arrays of probes. The plurality of arrays of probes may be at most about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500,

250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less arrays of probes. The operations 110 - 150 may be repeated at another array of the plurality of arrays of probes. The operations may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000,

500,000, 1,000,000, or more other arrays. The operations may be repeated for at least about

1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4,

3, 2, or less other arrays. The operations may be repeated at each other array of the plurality of arrays of probes.

[0065] In an aspect, the present disclosure provides methods for processing a template nucleic acid molecule. A method for processing a template nucleic acid molecule may comprise providing a template nucleic molecule coupled to a probe of an array of probes. The other probes of the array of probes may be blocked such that other template nucleic acid molecules are incapable of stably coupling to the other probes. At least a subset of the other probes may be blocked. The template nucleic acid molecule and the deblocked or active probes may be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the deblocked or active probes.

[0066] FIG. 2 is an example overview of an RNA blocking process. A template nucleic acid molecule 201 may comprise a promoter sequence 202. The template nucleic acid molecule may further comprise a sequence 203 that is at least partially complimentary to probe 204. Probe 204 may comprise a complimentary promoter sequence 205. Complimentary promoter sequence 205 may be complimentary to promoter sequence 202. Promoter sequence 202 may be a T7 RNA polymerase promoter sequence. The promoter sequence 202 may be configured to initiation production of one or more RNA strands 206. The one or more RNA strands 206 may be at least partially complimentary to probe 204. The one or more RNA strands may block the other probes such that another template nucleic acid molecule 207 may not stably bind to the other probes. [0067] Other blocking and techniques can be utilized instead of or in combination with the RNA blocking process. Nucleic acid molecules can be loaded onto the arrays of probes described herein to achieve a Poisson or super Poisson distribution of probes bound to nucleic acid molecules. Kinetic exclusion can be used to achieve a Poisson or super Poisson distribution of probes bound to nucleic acid molecules. Non-template biologic molecules can be provided with the template nucleic acid molecules. The non-template biologic molecules can occupy a subset of probes of the arrays of probes. Template nucleic acid molecules can be loaded onto unoccupied probes to achieve a Poisson or super Poisson distribution of probes bound to template nucleic acid molecules. In some embodiments, the non-template biologic molecules comprise nucleic acid molecules. In some embodiments, the nucleic acid molecules comprise DNA. In some embodiments, the nucleic acid molecules comprise degraded DNA. In some embodiments, the nucleic acid molecules comprise RNA. In some embodiments, the nucleic acid molecules comprise degraded RNA. In some embodiments, the non-template biologic molecules comprise proteins. In some embodiments, the non-template biologic molecules comprise enzymes. The concentration of template nucleic acid molecules in a sample solution is controlled such that the rate of probe binding of a template nucleic acid molecule by any probe of the arrays of probes is much lower than the rate of clonal amplification and sufficient exhaustion of the bound probe’s capacity to capture another template nucleic acid molecule.

[0068] The initial sample solution can comprise target nucleic acid molecules and non-target biologic molecules. The target nucleic acid molecules and non-target biologic molecules may be present in the solution at a ratio of target nucleic acid molecules to non-target biologic molecules of at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100. The target nucleic acid molecules and non-target biologic molecules may be immobilized at a reaction site at a ratio of at least 1:1, 1:2, 1:3, 1:4, 1:5, 1 :6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100. The ratio may be greater than, less than, or some value in between those stated above. The amplifying may occur at a rate sufficient to generate the amplicons of the target nucleic acid molecules and/or non-target biologic molecules without amplification of other target nucleic acid molecules and/or non-target biologic molecules of the plurality of target nucleic acid molecules and/or non-target biologic molecules at a reaction site using kinetic exclusion. This amplification may occur at a rate sufficient to generate amplicons of target nucleic acid molecules and/or non target biologic molecules of a plurality of target nucleic acid molecules and/or non-target biologic molecules at a reaction site. A target nucleic acid molecule(s) and/or non-target biologic molecule(s) may be amplified to generate a copy or complement, or a plurality of copies or complements, of a target nucleic acid molecule(s) and/or non-target biologic molecule(s) immobilized at a reaction site. Amplicons of either the target nucleic acid molecules and/or non target biologic molecules may be a clonal population, complementary, or partially complementary.

[0069] The target nucleic acid molecule may have a concentration of at least about 0.001 nanograms (ng)/microliter (pL), 0.005 ng/pL, 0.01 ng/pL, 0.05 ng/pL, 0.1 ng/pL, 0.2 ng/pL, 0.3 ng/pL, 0.4 ng/pL, 0.5 ng/pL, 0.6 ng/pL, 0.7 ng/pL, 0.8 ng/pL, 0.9 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 4 ng/pL, 5 ng/pL, 6 ng/pL, 7 ng/pL, 8 ng/pL, 9(ng/pL), 10(ng/pL), 11 (ng/pL), 12(ng/gL), 13 (ng/gL), 14(n g/gL), 15 (ng/gL), 16 ng/gL, 17 ng/gL, 18 ng/gL, 19 ng/gL, 20 ng/mL, 25 ng/gL, 30 ng/gL, 35 ng/gL, 40 ng/gL, 45 ng/gL, 50 ng/gL, 55 ng/gL, 60 ng/gL, 65 ng/gL, 70 ng/gL, 80 ng/gL, 85 ng/gL, 90 ng/gL, 95 ng/gL, 100 ng/gL, 110 ng/gL, 120 ng/gL,

130 ng/gL, 140 ng/gL, 150 ng/gL, 160ng/gL, 170ng/gL, 180 ng/gL, 190 ng/gL, 200 ng/gL, 225 ng/gL, 250 ng/gL, 275 ng/gL, 300 ng/gL, 350 ng/gL, 400 ng/gL, 450 ng/gL, 500 ng/gL, 550 ng/gL, 600 ng/gL, 700 ng/gL, 800 ng/gL, 900 ng/gL, 1,000 ng/gL or more nanograms per microliter. The target nucleic acid molecule may have a concentration of at most about 1,000 ng/gL, 900 ng/gL, 800 ng/gL, 700 ng/gL, 600 ng/gL, 550 ng/gL, 500 ng/gL, 450 ng/gL, 400 ng/gL, 350 ng/gL, 300 ng/gL, 275 ng/gL, 250 ng/gL, 225 ng/gL, 200 ng/gL, 190 ng/gL, 180 ng/gL, 170 ng/gL, 160 ng/gL, 150 ng/gL, 140 ng/gL, 130 ng/gL, 120 ng/gL, 110 (ng/gL, 100 ng/gL, 95 ng/gL, 90 ng/gL, 85 ng/gL, 80 ng/gL, 75 ng/gL, 70 ng/gL, 65 ng/gL, 60 ng/gL, 55 ng/gL, 50 ng/gL, 45 ng/gL, 40 ng/gL, 35 ng/gL, 30 ng/gL, 25 ng/gL, 20 ng/gL, 19 ng/gL, 18 ng/gL, 17 ng/gL, 16 ng/gL, 15 ng/gL, 14 ng/gL, 13 (ng/gL, 12 ng/gL, 11 ng/gL, 10 ng/gL, 9 ng/gL, 8 ng/gL, 7 ng/gL, 6 ng/gL, 5 ng/gL, 4 ng/gL, 3 ng/gL, 2 ng/gL, 1 ng/gL, 0.9 ng/gL, 0.8 ng/gL, 0.7 ng/gL, 0.6 ng/gL, 0.5 ng/gL, 0.4 ng/gL, 0.3 ng/gL, 0.2 ng/gL, 0.1 ng/gL, 0.05 ng/gL,

0.01 ng/gL, 0.005 ng/gL, 0.001 ng/gL, or less nanograms per microliter. The target nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 nanograms per microliter to 4 nanograms per microliter.

[0070] The sample solution comprising the target nucleic acid molecules may be flowed at a flow rate about 1 microliter (gL)/minute (min) to about 12 gL/min. The solution comprising the target nucleic acid molecules may be flowed at a flow rate about 1 gL/min to about 2 gL/min, about 1 gL/min to about 3 gL/min, about 1 gL/min to about 4 gL/min, about 1 gL/min to about 5 gL/min, about 1 gL/min to about 6 gL/min, about 1 gL/min to about 7 gL/min, about 1 gL/min to about 8 gL/min, about 1 gL/min to about 9 gL/min, about 1 gL/min to about 10 gL/min, about

1 gL/min to about 11 gL/min, about 1 gL/min to about 12 gL/min, about 2 gL/min to about 3 gL/min, about 2 gL/min to about 4 gL/min, about 2 gL/min to about 5 gL/min, about 2 gL/min to about 6 gL/min, about 2 gL/min to about 7 gL/min, about 2 gL/min to about 8 gL/min, about

2 gL/min to about 9 gL/min, about 2 gL/min to about 10 gL/min, about 2 gL/min to about 11 gL/min, about 2 gL/min to about 12 gL/min, about 3 gL/min to about 4 gL/min, about 3 gL/min to about 5 gL/min, about 3 gL/min to about 6 gL/min, about 3 gL/min to about 7 gL/min, about

3 gL/min to about 8 gL/min, about 3 gL/min to about 9 gL/min, about 3 gL/min to about 10 gL/min, about 3 gL/min to about 11 gL/min, about 3 gL/min to about 12 gL/min, about 4 gL/min to about 5 gL/min, about 4 gL/min to about 6 gL/min, about 4 gL/min to about 7 gL/min, about 4 gL/min to about 8 gL/min, about 4 gL/min to about 9 gL/min, about 4 gL/min to about 10 gL/min, about 4 gL/min to about 11 gL/min, about 4 gL/min to about 12 gL/min, about 5 mL/min to about 6 gL/min, about 5 gL/min to about 7 gL/min, about 5 mL/min to about 8 gL/min, about 5 gL/min to about 9 gL/min, about 5 gL/min to about 10 gL/min, about 5 gL/min to about 11 gL/min, about 5 gL/min to about 12 gL/min, about 6 gL/min to about 7 gL/min, about 6 gL/min to about 8 gL/min, about 6 mL/min to about 9 mL/min, about 6 gL/min to about

10 gL/min, about 6 gL/min to about 11 gL/min, about 6 gL/min to about 12 gL/min, about 7 gL/min to about 8 gL/min, about 7 gL/min to about 9 gL/min, about 7 gL/min to about 10 gL/min, about 7 gL/min to about 11 gL/min, about 7 gL/min to about 12 gL/min, about 8 gL/min to about 9 gL/min, about 8 gL/min to about 10 gL/min, about 8 gL/min to about 11 gL/min, about 8 gL/min to about 12 gL/min, about 9 gL/min to about 10 gL/min, about 9 gL/min to about 11 gL/min, about 9 gL/min to about 12 gL/min, about 10 gL/min to about 11 gL/min, about 10 gL/min to about 12 gL/min, or about 11 gL/min to about 12 gL/min. The solution comprising the target nucleic acid molecules may be flowed at about 1 gL/min, about 2 gL/min, about 3 gL/min, about 4 gL/min, about 5 gL/min, about 6 gL/min, about 7 gL/min, about 8 gL/min, about 9 gL/min, about 10 gL/min, about 11 gL/min, or about 12 gL/min. The solution comprising the target nucleic acid molecules may be flowed at least about 1 gL/min, about 2 gL/min, about 3 gL/min, about 4 gL/min, about 5 gL/min, about 6 gL/min, about 7 gL/min, about 8 gL/min, about 9 gL/min, about 10 gL/min, or about 11 gL/min. The solution comprising the target nucleic acid molecules may be flowed at most about 2 gL/min, about 3 gL/min, about 4 gL/min, about 5 gL/min, about 6 gL/min, about 7 gL/min, about 8 gL/min, about 9 gL/min, about 10 gL/min, about 11 gL/min, or about 12 gL/min.

[0071] The sample solution comprising the target nucleic acid molecules may be flowed at about 13 microliters (gL)/minute (min) to about 24 gL/min. The solution comprising the target nucleic acid molecules may be flowed at about 13 gL/min to about 14 gL/min, about 13 gL/min to about 15 gL/min, about 13 gL/min to about 16 gL/min, about 13 gL/min to about 17 gL/min, about 13 gL/min to about 18 gL/min, about 13 gL/min to about 19 gL/min, about 13 gL/min to about 20 gL/min, about 13 gL/min to about 21 gL/min, about 13 gL/min to about 22 gL/min, about 13 gL/min to about 23 gL/min, about 13 gL/min to about 24 gL/min, about 14 gL/min to about 15 gL/min, about 14 gL/min to about 16 gL/min, about 14 gL/min to about 17 gL/min, about 14 gL/min to about 18 gL/min, about 14 gL/min to about 19 gL/min, about 14 gL/min to about 20 gL/min, about 14 gL/min to about 21 gL/min, about 14 gL/min to about 22 gL/min, about 14 gL/min to about 23 gL/min, about 14 gL/min to about 24 gL/min, about 15 gL/min to about 16 gL/min, about 15 gL/min to about 17 gL/min, about 15 gL/min to about 18 gL/min, about 15 gL/min to about 19 gL/min, about 15 gL/min to about 20 gL/min, about 15 gL/min to about 21 gL/min, about 15 gL/min to about 22 gL/min, about 15 gL/min to about 23 gL/min, about 15 gL/min to about 24 gL/min, about 16 gL/min to about 17 gL/min, about 16 gL/min to about 18 gL/min, about 16 gL/min to about 19 gL/min, about 16 gL/min to about 20 gL/min, about 16 gL/min to about 21 gL/min, about 16 gL/min to about 22 gL/min, about 16 gL/min to about 23 gL/min, about 16 gL/min to about 24 gL/min, about 17 gL/min to about 18 gL/min, about 17 gL/min to about 19 gL/min, about 17 gL/min to about 20 gL/min, about 17 gL/min to about 21 gL/min, about 17 gL/min to about 22 gL/min, about 17 gL/min to about 23 gL/min, about 17 gL/min to about 24 gL/min, about 18 gL/min to about 19 gL/min, about 18 gL/min to about 20 gL/min, about 18 gL/min to about 21 gL/min, about 18 gL/min to about 22 gL/min, about 18 gL/min to about 23 gL/min, about 18 gL/min to about 24 gL/min, about 19 gL/min to about 20 gL/min, about 19 gL/min to about 21 gL/min, about 19 gL/min to about 22 gL/min, about 19 gL/min to about 23 gL/min, about 19 gL/min to about 24 gL/min, about 20 gL/min to about 21 gL/min, about 20 gL/min to about 22 gL/min, about 20 gL/min to about 23 gL/min, about 20 gL/min to about 24 gL/min, about 21 gL/min to about 22 gL/min, about 21 gL/min to about 23 gL/min, about 21 gL/min to about 24 gL/min, about 22 gL/min to about 23 gL/min, about 22 gL/min to about 24 gL/min, or about 23 gL/min to about 24 gL/min. The solution comprising the target nucleic acid molecules may be flowed at about 13 gL/min, about 14 gL/min, about 15 gL/min, about 16 gL/min, about 17 gL/min, about 18 gL/min, about 19 gL/min, about 20 gL/min, about 21 gL/min, about 22 gL/min, about 23 gL/min, or about 24 gL/min. The solution comprising the target nucleic acid molecules may be flowed at least about

13 gL/min, about 14 gL/min, about 15 gL/min, about 16 gL/min, about 17 gL/min, about 18 gL/min, about 19 gL/min, about 20 gL/min, about 21 gL/min, about 22 gL/min, or about 23 gL/min. The solution comprising the target nucleic acid molecules may be flowed at most about

14 gL/min, about 15 gL/min, about 16 gL/min, about 17 gL/min, about 18 gL/min, about 19 gL/min, about 20 gL/min, about 21 gL/min, about 22 gL/min, about 23 gL/min, or about 24 gL/min.

[0072] The sample solution comprising the target nucleic acid molecules may be flowed at about 25 microliters (gL)/minute (min) to about 36 gL/min. The solution comprising the target nucleic acid molecules may be flowed at about 25 gL/min to about 26 gL/min, about 25 gL/min to about 27 gL/min, about 25 gL/min to about 28 gL/min, about 25 gL/min to about 29 gL/min, about 25 gL/min to about 30 gL/min, about 25 gL/min to about 31 gL/min, about 25 gL/min to about 32 gL/min, about 25 gL/min to about 33 gL/min, about 25 gL/min to about 34 gL/min, about 25 gL/min to about 35 gL/min, about 25 gL/min to about 36 gL/min, about 26 gL/min to about 27 gL/min, about 26 gL/min to about 28 gL/min, about 26 gL/min to about 29 gL/min, about 26 gL/min to about 30 gL/min, about 26 gL/min to about 31 gL/min, about 26 gL/min to about 32 gL/min, about 26 gL/min to about 33 gL/min, about 26 gL/min to about 34 gL/min, about 26 gL/min to about 35 gL/min, about 26 gL/min to about 36 gL/min, about 27 gL/min to about 28 gL/min, about 27 gL/min to about 29 gL/min, about 27 gL/min to about 30 gL/min, about 27 gL/min to about 31 gL/min, about 27 gL/min to about 32 gL/min, about 27 gL/min to about 33 gL/min, about 27 gL/min to about 34 gL/min, about 27 gL/min to about 35 gL/min, about 27 gL/min to about 36 gL/min, about 28 gL/min to about 29 gL/min, about 28 gL/min to about 30 gL/min, about 28 gL/min to about 31 gL/min, about 28 gL/min to about 32 gL/min, about 28 gL/min to about 33 gL/min, about 28 gL/min to about 34 gL/min, about 28 gL/min to about 35 gL/min, about 28 gL/min to about 36 gL/min, about 29 gL/min to about 30 gL/min, about 29 gL/min to about 31 gL/min, about 29 gL/min to about 32 gL/min, about 29 gL/min to about 33 gL/min, about 29 gL/min to about 34 gL/min, about 29 gL/min to about 35 gL/min, about 29 gL/min to about 36 gL/min, about 30 gL/min to about 31 gL/min, about 30 gL/min to about 32 gL/min, about 30 gL/min to about 33 gL/min, about 30 gL/min to about 34 gL/min, about 30 gL/min to about 35 gL/min, about 30 gL/min to about 36 gL/min, about 31 gL/min to about 32 gL/min, about 31 gL/min to about 33 gL/min, about 31 gL/min to about 34 gL/min, about 31 gL/min to about 35 gL/min, about 31 gL/min to about 36 gL/min, about 32 gL/min to about 33 gL/min, about 32 gL/min to about 34 gL/min, about 32 gL/min to about 35 gL/min, about 32 gL/min to about 36 gL/min, about 33 gL/min to about 34 gL/min, about 33 gL/min to about 35 gL/min, about 33 gL/min to about 36 gL/min, about 34 gL/min to about 35 gL/min, about 34 gL/min to about 36 gL/min, or about 35 gL/min to about 36 gL/min The solution comprising the target nucleic acid molecules may be flowed at about 25 gL/min, about 26 gL/min, about 27 gL/min, about 28 gL/min, about 29 gL/min, about 30 gL/min, about 31 gL/min, about 32 gL/min, about 33 gL/min, about 34 gL/min, about 35 gL/min, or about 36 gL/min. The solution comprising the target nucleic acid molecules may be flowed at least about

25 gL/min, about 26 gL/min, about 27 gL/min, about 28 gL/min, about 29 gL/min, about 30 gL/min, about 31 gL/min, about 32 gL/min, about 33 gL/min, about 34 gL/min, or about 35 gL/min. The solution comprising the target nucleic acid molecules may be flowed at most about

26 gL/min, about 27 gL/min, about 28 gL/min, about 29 gL/min, about 30 gL/min, about 31 gL/min, about 32 gL/min, about 33 gL/min, about 34 gL/min, about 35 gL/min, or about 36 gL/min.

[0073] A diffusion barrier may be used to contain template nucleic acid molecules in a probe. The high viscosity buffer may be a hydrocarbon (e.g., an oil, squalene), a chemical compound (e.g., 1 -Butyl-3 -methylimidazolium hexafluorophosphate or glycerol), a gel buffer, a viscoelastic polymer, or the like. The buffer may have a viscosity of at least about 1 x 10 ³ Pascal-seconds (IE-3 Pa s), 5E-3 Pa s, IE-2 Pa s, 5E-2 Pa s, IE-1 Pa s, 5E-1 Pa s, 1 Pa s, 5 Pa s, 10 Pa s, 50 Pa s, 100 Pa s, 500 Pa s, 1,000 Pa s, or more. The buffer may have a viscosity of at most about 1,000 Pa s, 500 Pa s, 100 Pa s, 50 Pa s, 10 Pa s, 5 Pa s, 1 Pa s, 5E-1 Pa s, IE-1 Pa s, 5E-2 Pa s, lE-2 Pa s, 5E-3 Pa s, IE-3 Pa s, or less.

[0074] A subset of nucleic acid molecules may be degraded to exclude the nucleic acid molecules for other arrays. The nucleic acid molecules may be contained within well by the use of degrading elements. Degrading elements may comprise enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof. The enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof. The chemical degrading elements may be an acid (e.g., / - toluene sulfonic acid, nitric acid, ascorbic acid), a base (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof. The light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule). For example, a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA. The degrading element 506 may be coupled to a support. The support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof. An electric field may be applied to the support. A generator may generate the electric field. The electric field may have a potential of at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V,

9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may have a potential of at most about 10,000V, 5,000V, 1,000V, 240V, 120V, 50V, 20V, 15V, 12V, 10V, 9V, 8V, 7V, 6V, 5V, 4V, 3V, 2V, IV, 0.9V, 0.8V, 0.7V, 0.6V, 0.5V, 0.4V, 0.3 V, 0.2V, 0.1V, 0.05V, 0.01V, 0.005V, 0.001V or less volts. The electric field may be applied via electrodes that are electronically coupled to a generator. The support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode. The electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof. [0075] The array of probes may be attached to a solid support 208. The solid support 208 may be a bead, planar, a surface of a well, or any combination thereof. For example, a bead functionalized with probes can rest on a planar surface. The bead may be a functionalized bead comprising a tosylated surface. The bead may have a diameter of at least about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1,000, or more micrometers. The bead may have a diameter of at most about 1,000, 750, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25, 10, 5, 1, or less micrometers. The bead may be a component of a well-less sensing array. The bead may be a polymer bead (e.g., latex, polystyrene), a glass bead, a metal bead, or the like. The planar solid support may be a well-less sensing array. The planar solid support may comprise one or more electrodes. The electrodes may be dielectric stacks, metals, or a combination thereof. The electrodes may be nanoneedles. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,

75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500,

600, 700, 800, 900, 1,000, or more micrometers. The well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50

45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, the well can have an x dimension of 434 micrometers, a y dimension of 30 micrometers, and a z dimension of 510 micrometers. In another example, the well can have an x and y dimension of 16 micrometers and a z dimension of 1 micron. The system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors. The mechanisms may be mechanisms as described in FIGs. 5A-5D.

[0076] The array of probes may be in sensory communication with a sensor. The sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof. The sensor may comprise an electrode. The electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof. The sensor may comprise a plurality of electrodes. The plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes. The plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes. The sensor may be among an array of sensors. The array of sensors may comprise sensors of one or more types. For example, an array of sensor may comprise an optical sensor and an electrical sensor. The sensors of the array of sensors may be individually addressable. For example, each electrode of an array of 1,000,000 electrodes can be measured independently of each other electrode.

[0077] FIG. 4 is a flow chart of an example process for processing a template nucleic acid molecule. The process 400 can be implemented on an appropriately configured system as described elsewhere herein. The system may provide a template nucleic acid molecule coupled to a probe of an array of probes (410). The template nucleic acid molecule may have a concentration of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65

70, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300

350, 400, 450, 500, 550, 600, 700, 800, 900, 1,000 or more nanograms per microliter. The template nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less nanograms per microliter. The template nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 to 4 nanograms per microliter. The other probes of the array of probes may be blocked such that other template nucleic acid molecules may be incapable of stably coupling to the other probes. The other probes of the array of probes may be blocked with nucleic acid molecules bound to the other probes of the array of probes. The nucleic acid molecules may be DNA molecules or RNA molecules. For example, the other probes can be blocked with RNA molecules that bind to enough of the probe to prevent stable binding. In this example, the amount the RNA molecules are configured to be bound to prevent stable binding can be a function of temperature and the ionic strength of the buffer solution around the probes. The stability of the binding can be modulated by factors such as the length of the blocking nucleic acid, the sequence of the probe, the ionic strength of the solution (e.g., the salt concentration), the temperature, the presence of solvents (e.g., formamide, DMSO), the presence of ligands, the presence of metal ions, the pH of the solution, or any combination thereof. The nucleic acids blocking the other probes may isolate the template nucleic acid.

[0078] The probes of the array of probes may be coupled to a support. The support may be planer. The support may be a bead. The bead may be a component of a welldess sensing array. The probes may be coupled to a functional unit on the surface of the bead. The support may be the interior of a well. The support may be an electrode. The probes of the array of probes may be coupled to the support by a linking unit. The linking unit may be a polymer, a thiol group, a silane group, or the like. An electric field may be applied to the array of probes. The electric field may be at least about 0.001 Volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V,

50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may be at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,

0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 or less volts. The electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4- ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof. The electric field may be applied over a distance of at least about 0.1, 1, 5,

10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150

160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers.

The electric field may be applied over a distance of at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65,

60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a pair of gold electrodes 100 micrometers apart can be used to apply a 0.5 V potential to the array of probes. A magnetic field may be applied to the array of probes. The magnetic field may be at least about 1 x 10⁶ Tesla (IE-6 T), IE-5 T, IE-4 T, IE-3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more. The magnetic field may be at most about 1E1 T, 1E0 T, IE-1 T, IE-2 T, IE-3 T, IE-4, 1E- 5 T, IE-6 T, or less. The magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet) or an electromagnet (e.g., a solenoid). The magnetic field may be applied over a distance of at least about 0.1, 1, 5, 10, 15,

20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,

170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers. The magnetic field may be applied over a distance of at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65

60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a solenoid coil can be placed 500 micrometers behind the array of probes and used to apply a 0.3 Tesla magnetic field.

[0079] The system may deblock at least a subset of the other probes (420). The deblocking may be performed with the aid of a reagent. The reagent may be a chemical reagent, a physical process, an enzyme, or any combination thereof. The chemical reagent may be a solvent (e.g., methanol, formamide), a ligand, a metal ion source, a proton source (e.g., an acid), a base (e.g., sodium hydroxide), a radical source, or any combination thereof. The physical process may be applying energy (e.g., heating, sonication), applying light (e.g., an ultraviolet laser), or a combination thereof. The enzyme may be an RNase or a DNase. The RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V. The DNase may be DNase I, II, or micrococcal nuclease.

[0080] The system may use the template nucleic acid molecule and the deblocked or active probes to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the deblocked or active probes (430). Operation 420 and/or 430 may occur in a well. The well may be a well of a plurality of wells of a sensing array. The well may comprise one or more beads. For example, a single bead may be at least partially contained by the well. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,

The well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, the well can have a width of 150 micrometers, a depth of 105 micrometers, and a height of 437 micrometers. In another example the well can have a length and width of 15 microns and a depth of 3 microns.

[0081] The amplicons coupled to the active probes may be a clonal population of nucleic acids. For example, a template nucleic acid molecule can be coupled to a probe surrounded by an array of nucleotides that were recently deblocked. In this example, the template nucleic acid molecule can be amplified such that clones of the template nucleic acid molecule occupy the recently deblocked or active probes. The amplicons may be a partially clonal population. The amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal. The amplicons may be at most about 100%, 99.9%,

99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal. For example, a template nucleic acid can be coupled to a probe in a well, where all of the other probes in the well are blocked. In this example, after deblocking the other probes and generating amplicons of the template nucleotide, the other probes can have a 100% clonal population, as all of the amplicons are derived from the template nucleic acid. Operation 430 may further comprise sequencing at least a subset of the amplicons coupled to the active probes or derivatives thereof. The derivatives may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid. The derivatives may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less similar in sequence to the template nucleic acid. The subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons. The subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the amplicons. The sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof. The nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof. The sequencing may be performed by methods and systems as described elsewhere herein.

[0082] FIGs. 5A-5D are example schematics of confining the nucleic acid molecules generated in the nucleic acid molecule blocking process. The nucleic acid molecules may be RNA. The methods and systems as described elsewhere herein may comprise methods and mechanisms configured to exclude a plurality of nucleic acid molecules from other arrays of a plurality of arrays of probes. The excluding may generate arrays with less than about 99.9%,

99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,

6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less contamination from other arrays.

The excluding may generate arrays with more than about 0.01%, 0.05 %, 0.1%, 0.5%, 1%, 2%,

3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

95%, 98%, 99%, 99.9%, or more contamination from other arrays. The confining the RNA may be for a time. For example, the RNA can be confined while the RNA is being generated, and then the excess RNA can be washed away.

[0083] FIG. 5A shows an example use of a high viscosity buffer 501 as a diffusion barrier to contain nucleic acid molecules 502 in well 503. The high viscosity buffer may be a hydrocarbon (e.g., an oil, squalene), a chemical compound (e.g., 1 -Butyl-3 -methylimidazolium hexafluorophosphate or glycerol), a gel buffer, a viscoelastic polymer, or the like. The buffer may have a viscosity of at least about 1 x 10³ Pascal-seconds (IE-3 Pa s), 5E-3 Pa s, IE-2 Pa s, 5E-2 Pa s, IE-1 Pa s, 5E-1 Pa s, 1 Pa s, 5 Pa s, 10 Pa s, 50 Pa s, 100 Pa s, 500 Pa s, 1,000 Pa s, or more. The buffer may have a viscosity of at most about 1,000 Pa s, 500 Pa s, 100 Pa s, 50 Pa s, 10 Pa s, 5 Pa s, 1 Pa s, 5E-1 Pa s, IE-1 Pa s, 5E-2 Pa s, IE-2 Pa s, 5E-3 Pa s, IE-3 Pa s, or less.

[0084] FIG. 5B shows an example of degrading at least a subset of the nucleic acid molecules to exclude the nucleic acid molecules for other arrays. The nucleic acid molecules 504 may be contained within well 505 by the use of degrading elements 506. Degrading elements 506 may comprise enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof. The enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof. The chemical degrading elements may be an acid (e.g.,/>-toluene sulfonic acid, nitric acid, ascorbic acid), abase (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof. The light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule). For example, a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA. The degrading element 506 may be coupled to a support.

The support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof. For example, a plurality of RNase enzymes can be coupled to a plurality of support beads, and the support beads can be placed above the wells. An electric field may be applied to the support. Generator 508 may generate the electric field. The electric field may have a potential of at least about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2

V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10

V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may have a potential of at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9,

8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 or less volts.

The electric field may be applied via electrodes 507 that are electronically coupled to generator

508. The support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode. The electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof.

[0085] FIG. 5C shows an example method for confining the nucleic acids comprising applying an electric field. The nucleic acid molecules 509 may be within well 510. Generator 513 may be electronically coupled to electrodes 512, which may apply an electric field between the electrodes. The electric field may interact with labels 511 attached to one or more of nucleic acid molecules 509. The interacting may draw the nucleic acid molecules away from the top of the well and thus contain the nucleic acid molecules. The labels may be a particle. The particle may be a di electrophoretic particle. The particle may be a metal particle (e.g., gold, aluminum, silver, platinum), a semiconductor particle (e.g., silicon, carbon, zinc sulfide), or a molecular unit (e.g., Ru(bpy)₃ ²⁺, ferrocene). The particle may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different particle may be attached to each end of the nucleic acid molecule.

[0086] FIG. 5D shows an example method for confining the nucleic acids comprising applying a magnetic field. The magnetic field may be applied to the plurality of nucleic acid molecules 514 in well 515 using magnet 516. The magnet may be a permanent magnet (e.g., a rare-earth magnet, an iron-based magnet) or an electromagnet (e.g., a solenoid, a superconducting magnet). At least one nucleic acid molecule of the nucleic acid molecules 514 may comprise a label 517 that interacts with the magnetic field. Label 517 may be a particle (e.g., an iron nanoparticle), a molecular species (e.g., a single molecule magnet, an iron containing molecule), or a combination thereof. For example, a nucleic acid can be attached to the surface of an iron nanoparticle cluster. The label may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different label may be attached to each end of the nucleic acid molecule.

[0087] FIG. 6A shows an example of confining the nucleic acid molecules using the electrophoretic force. Nucleic acid molecules 601 may be generated in well 602. To prevent the nucleic acid molecules from leaving the well, an electric field can be applied between electrodes 603 and 604. The electric field may generate an electrophoretic force that attracts the nucleic acid molecules down into the well 602. A generator 605 may generate the electric field. The generator may generate a potential of at least about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V,

0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V,

15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The generator may generate a potential of at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or less volts. The electrodes may be separated by at least about 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1,000 or more micrometers. The electrodes may be separated by at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20,

15, 10, 5, 1, 0.1, or less micrometers.

[0088] FIG. 6B shows an example electrode material for the electrodes of FIGs. 5A, 5C, and 6A. Other examples of electrode materials may be metals, semiconductors, or conductive polymers. The metals may be gold, silver, platinum, nickel, copper, iron, other transition metals, or alloys thereof. The semiconductors may be organic semiconductors (e.g., O_όo, phenyl-C61- butyric acid methyl ester), inorganic semiconductors (e.g., silicon, cadmium telluride, indium tin oxide, gallium arsenide), or a combination thereof. The conductive polymers may be polyfluroenes, polyacetylenes, poly(p-phenylene vinylene)s, polypyrroles, polyanilines, polythiophenes, poly(3,4-ethylenedioxythiophene), poly(p-phenylene sulfide), or any combination thereof.

[0089] In an aspect, the present disclosure provides methods for nucleic acid amplification. A method for nucleic acid amplification may comprise brining a plurality of target nucleic acid molecules in contact with an array of probes. The plurality of target nucleic molecules may be present at a concentration such that at most a target nucleic acid molecule of the plurality of target nucleic acid molecules hybridizes to a probe of the array of probes. The array of probes may be subject to conditions sufficient to synthesize a first plurality of nucleic acid molecules from the target nucleic acid molecule hybridized to the probe. The first plurality of nucleic acid molecules may be hybridized to other probes of the array of probes. The array of probes may be subject to conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules. The array of probes may be subject to conditions sufficient to amplify the target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to the array of probes.

[0090] The target nucleic acid molecule may have a concentration of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1,000 or more nanograms per microliter. The target nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275, 250,

225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7,

0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less nanograms per microliter. The target nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the target nucleic acid molecule may have a concentration from 0.4 to 4 nanograms per microliter.

[0091] The probes of the array of nucleotides may comprise a common sequence. The probes of the array of probes may be identical. The plurality of nucleic acid molecules may be at least partially complementary to the common sequence. The plurality of nucleic acids may be at least about 1 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 97 %, 98 %, 99 %, or more complimentary to sequences of other probes of the array of nucleotides. The plurality of nucleic acid molecules may be at most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less complimentary to sequences of other probes of the array of nucleotides. The first plurality of nucleic acid molecules may be a plurality of RNA molecules. The synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may be performed with the aid of an enzyme. The enzyme may be an RNA polymerase. The RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase. The synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may involve transporting a subset of the first plurality of nucleic acid molecules to the other probes of the array of probes. The transporting may be via diffusion. The transporting may be assisted diffusion. The transporting may be an active transporting. The active transporting may comprise cellular transportation methods (e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow (e.g., flowing a liquid carrier in the direction of transport), or any combination thereof. The transporting may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of simple diffusion.

[0092] The conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules may comprise removing or degrading the subset of the nucleic acid molecules with a reagent. The reagent may be an enzyme. The enzyme may be an RNase. The RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V. The reagent may be a chemical compound. The chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea. The target nucleic acid molecule hybridized to the probe may comprise a promoter sequence. The promoter sequence may be a T7 RNA polymerase promoter sequence. The probes of the array of probes may comprise a complementary promoter sequence. The complementary promoter sequence may be complimentary to the promoter sequence of the target nucleic acid molecule. For example, the target nucleic acid molecule may be able to hybridize with a probe via interaction of the promoter sequence with the complementary promoter sequence.

[0093] The array of probes may be attached to a solid support. The array of probes may be attached to a solid support. The solid support may be a bead, planar, a surface of a well, or any combination thereof. For example, a bead functionalized with probes can rest on a planar surface. The bead may be a functionalized bead comprising a tosylated surface. The bead may have a diameter of at least about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1,000, or more microns. The bead may have a diameter of at most about 1,000, 750, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25, 10, 5, 1, or less microns. The bead may be a component of a well-less sensing array. The bead may be a polymer bead (e.g., latex, polystyrene), a glass bead, a metal bead, or the like. The planar solid support may be a well-less sensing array. The planar solid support may comprise one or more electrodes. The electrodes may be dielectric stacks, metals, or a combination thereof. The electrodes may be nanoneedles. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35,

40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190

200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers. The well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000, 900,

800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90

85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, the well can have an x dimension of 434 micrometers, a y dimension of 30 micrometers, and a z dimension of 510 micrometers. In another example, the well can have an x and w dimension of 15 micrometers and a z dimension of 1 micron. The system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors. The mechanisms may be mechanisms as described in FIGs. 5A-5D.

[0094] The array of probes may be in sensory communication with a sensor. The sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof. The sensor may comprise an electrode. The electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof. The sensor may comprise a plurality of electrodes. The plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000,

50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes. The plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000,

10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes. The sensor may be among an array of sensors. The array of sensor may comprise sensors of one or more types. For example, an array of sensor may comprise an optical sensor and an electrical sensor. The sensors of the array of sensors may be individually addressable. For example, each electrode of an array of

1,000,000 can be measured independently of each other electrode.

[0095] The array of probes may be among a plurality of arrays of probes. The plurality of arrays of probes may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more arrays of probes. The plurality of arrays of probes may be at most about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500,

250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less arrays of probes.

[0096] The excluding may comprise applying an electric field to the plurality of nucleic acid molecules. The electric field may be at least about 0.001 Volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1

V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V,

9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may be at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or less volts. The electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4- ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof. The electric field may be applied over a distance of at least about 0.1, 1, 5,

60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a pair of gold electrodes 100 micrometers apart can be used to apply a 0.5 V potential to the array of probes. A nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the electric field. At least one nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the electric field. The label may be a particle. The particle may be a di electrophoretic particle. The particle may be a metal particle (e.g., gold, aluminum, silver, platinum), a semiconductor particle (e.g., silicon, carbon, zinc sulfide), or a molecular unit (e.g., Ru(bpy)32+, ferrocene). The particle may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different particle may be attached to each end of the nucleic acid molecule.

[0097] The excluding may comprise applying a magnetic field to the plurality of nucleic acid molecules. The magnetic field may be at least about 1 x 10-6 Tesla (IE-6 T), IE-5 T, IE-4 T, 1E- 3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more. The magnetic field may be at most about 1E1 T,

1E0 T, IE-1 T, IE-2 T, IE-3 T, IE-4, IE-5 T, IE-6 T, or less. The magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet) or an electromagnet (e.g., a solenoid). The magnetic field may be applied over a distance of at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers. The magnetic field may be applied over a distance of at most about 1,000,

900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,

95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a solenoid coil can be placed 500 micrometers behind the array of probes and used to apply a 0.3 Tesla magnetic field. A nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the magnetic field. At least one nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the magnetic field. The label may be a particle (e.g., an iron nanoparticle), a molecular species (e.g., a single molecule magnet, an iron containing molecule), or a combination thereof. For example, a nucleic acid can be attached to the surface of a 3 nm iron nanoparticle. The label may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different label may be attached to each end of the nucleic acid molecule.

[0098] The excluding may be performed with the aid of a diffusion barrier. The diffusion barrier may be a high viscosity buffer. The high viscosity buffer may be a hydrocarbon (e.g., an oil, squalene), a chemical compound (e.g., 1 -Butyl-3 -methylimidazolium hexafluorophosphate or glycerol), a gel buffer, a viscoelastic polymer, or the like. The buffer may have a viscosity of at least about 1 x 10-3 Pascal-seconds (IE-3 Pa s), 5E-3 Pa s, IE-2 Pa s, 5E-2 Pa s, IE-1 Pa s, 5E- 1 Pa s, 1 Pa s, 5 Pa s, 10 Pa s, 50 Pa s, 100 Pa s, 500 Pa s, 1,000 Pa s, or more. The buffer may have a viscosity of at most about 1,000 Pa s, 500 Pa s, 100 Pa s, 50 Pa s, 10 Pa s, 5 Pa s, 1 Pa s, 5E-1 Pa s, IE-1 Pa s, 5E-2 Pa s, IE-2 Pa s, 5E-3 Pa s, IE-3 Pa s, or less.

[0099] The excluding may be performed by degrading a subset of the plurality of nucleic acid molecules. The degrading may be performed with degrading elements. The degrading elements may be enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof. The enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof. The chemical degrading elements may be an acid (e.g., p-toluene sulfonic acid, nitric acid, ascorbic acid), abase (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof. The light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule). For example, a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA. The degrading elements may be coupled to a support. The support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof. For example, a plurality of RNase enzymes can be coupled to a plurality of support beads, and the support beads can be placed above the wells. An electric field may be applied to the support. A generator may generate the electric field. The electric field may have a potential of at least about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V,

0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V,

20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may be at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7,

0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or less volts. The electric field may be applied via electrodes that are electronically coupled to the generator. The support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode. The electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof. [00100] The array of probes may comprise probes having sequences different from probes of at least one other array of the plurality of arrays of probes. The array of probes may comprise probes having sequences different from probes of at least one other array of the plurality of arrays of probes. For example, the probes coupled to the sensors of a 3x3 grid of sensors can each have a different sequence, leading to 9 different probe sequences. The different probe sequences may result in less cross contamination of nucleic acids between sensors. The lack of cross contamination may be particularly relevant in well-less sensing arrays. For example, each bead of an array of beads having a different probe sequence can prevent the RNA produced at each bead from diffusing to and binding onto another bead. The synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may include excluding the plurality of nucleic acid molecules from other arrays of the plurality of arrays of probes. For examples, the nucleotides can be contained to a sensor in a well and not leak to other wells containing other sensors.

[00101] The method may be repeated at another array of the plurality of arrays of probes. The method may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays. The method may be repeated for at least about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250,

100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays. The method may be repeated at each other array of the plurality of arrays of probes. The repeating the method may further comprise sequencing at least a subset of the substantially clonal populations at the another array of the plurality of arrays of probes.

[00102] The subjecting the array of probes to conditions sufficient to amplify the target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to the array of probes may comprise conducing a reaction with aid of a recombinase, a polymerase, or any combination thereof. The recombinase may be a Tre recombinase, a Cre recombinase, a Hin recombinase, aDmcl recombinase, aRad51 recombinase, or a FLP recombinase. The polymerase may be a DNA polymerase or an RNA polymerase. The RNA polymerase may be an RNA polymerase as described elsewhere herein. The DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y.

[00103] The method may further comprise sequencing at least a subset of the second plurality of nucleic acid molecules hybridized to the array of probes. The subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the second plurality of nucleic acid molecules. The subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the second plurality of nucleic acid molecules. The sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof. The sequencing may be performed with measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof. The sequencing may be performed by methods and systems as described elsewhere herein.

OLIGONUCLEOTIDE PROBES

[00104] The phrase “active oligonucleotides,” as used herein, generally refers to oligonucleotides available for binding to nucleic acids. For example, an oligonucleotide that recently had an RNA molecule removed from it can be an active oligonucleotide, as it is configured to accept a copy of a target nucleic acid molecule.

[00105] An array of probes may be used instead of the array of oligonucleotides in the methods and systems described herein. For example, antibodies can be used instead of oligonucleotides. An array of probes may be intermixed with the array of oligonucleotides. [00106] The nucleic acid amplification process can be implemented on an appropriately configured system as described elsewhere herein. The system can bring a template nucleic molecule in contact with an array of nucleotides. The template nucleic acid molecule may bind to an oligonucleotide of the array of oligonucleotides. The template nucleic molecule may comprise a nucleic molecule of interest (e.g., a DNA molecule to be sequenced). The template nucleic molecule may further comprise one or more moieties configured to bind to a probe. For example, the template nucleic molecule can be a fragment of a DNA sample with an oligonucleotide attached to the 3’ end. In another example, the template nucleic molecule can be a fragment of a

DNA sample with an oligonucleotide attached to the 5’ end. The moiety configured to bind to the probe may be configured to bind with a portion of the probe. For example, the probe can be a 36- base oligonucleotide, and the moiety can be 15 bases complimentary to the free end of the oligonucleotide. The binding of the template nucleic acid molecule with the oligonucleotide may be a hybridization of complimentary bases. The template nucleic acid molecule may have a concentration of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,

70, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300,

350, 400, 450, 500, 550, 600, 700, 800, 900, 1,000 or more nanograms per microliter. The template nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less nanograms per microliter. The template nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 to 4 nanograms per microliter.

[00107] The system can use the template nucleic acid molecule to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other oligonucleotides of the array of oligonucleotides. The synthesizing a plurality of nucleic acid molecules may be a polymerase chain reaction. The plurality of nucleic acid molecules may be RNA molecules,

DNA molecules, or oligonucleotides. The RNA molecules may be synthesized from the template nucleic acid molecule with the aid of a reagent. The reagent may be an enzyme. The enzyme may be a RNA polymerase. The RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase. The plurality of nucleic acid molecules may be at least partially complimentary to sequences of other oligonucleotides of the array of nucleotides. The plurality of nucleic acids may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,

99%, or more complimentary to sequences of other oligonucleotides of the array of nucleotides. The plurality of nucleic acid molecules may be at most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less complimentary to sequences of other oligonucleotides of the array of nucleotides.

[00108] The other oligonucleotides of the array of oligonucleotides may comprise a common sequence. The oligonucleotides of the array of oligonucleotides may be identical. The plurality of nucleic acid molecules may be at least partially complementary to the common sequence. For example, an RNA molecule can be complimentary to all oligonucleotides within a 15 micrometer square area, but not to oligonucleotides outside that area. The other oligonucleotides of the array of oligonucleotides may have a common sequence with other oligonucleotides in an area of at least about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 750, 1,000, 5,000, 10,000, 50,000, 100,000, or more square microns. The other oligonucleotides of the array of oligonucleotides may have a common sequence with other oligonucleotides in an area of at most about 100,000, 50,000, 10,000, 5,000, 1,000, 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or less square microns. The synthesizing may be performed with binding of the template nucleic acid molecule to at least two oligonucleotides of the array of oligonucleotides. The binding of the template nucleic acid molecule to at least two oligonucleotides may impart a bridge geometry to the template nucleic acid molecule. The array of oligonucleotides may be among a plurality of arrays of oligonucleotides. The array of oligonucleotides may comprise oligonucleotides having sequences different from oligonucleotides of at least one other array of the plurality of arrays of oligonucleotides. For example, the oligonucleotides coupled to the sensors of a 3x3 grid of sensors can each have a different sequence, leading to 9 different oligonucleotide sequences. The different oligonucleotide sequences may result in less cross contamination of nucleic acids between sensors. The lack of cross contamination may be particularly relevant in sensing arrays that do not comprise wells. For example, each bead of an array of beads having different oligonucleotide sequences can prevent the RNA produced at each bead from diffusing to and binding onto another bead.

[00109] In some embodiments, the arrays of the plurality of arrays of oligonucleotides can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. In some embodiments, select areas of the arrays of oligonucleotides can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. In some embodiments, a subset of the arrays of oligonucleotides are blocked from binding to nucleic acid molecules. In some embodiments, the template nucleic acid molecule can be among a plurality of template nucleic acid molecules. In some embodiments, individual template nucleic acid molecules can comprise different sequences. In some embodiments, distinct template nucleic acid molecules can be bound to distinct select areas of the arrays of oligonucleotides. In some embodiments, the distinct template nucleic acid molecules can be selectively amplified or sequenced at corresponding, distinct, or select areas of the arrays of oligonucleotides.

[00110] Transporting the plurality of nucleic acid molecules produced from the template nucleic acid molecule may be performed when the template nucleic acid is bound to the oligonucleotide. The nucleic acid molecules of the plurality of nucleic acid molecules may be transported from the oligonucleotide to the other oligonucleotides of the array of oligonucleotides. The transportation may be via diffusion. The transportation may be assisted diffusion. The transportation may be an active transportation. The active transportation may comprise cellular transportation methods (e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow

(e.g., flowing a liquid carrier in the direction of transport), or any combination thereof. The transportation may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of diffusion.

[00111] The system can bind nucleic acid molecules of the plurality of nucleic acid molecules to the other oligonucleotides of the array of oligonucleotides, thereby generating occupied oligonucleotides. The binding of the nucleic acid to the oligonucleotide may be configured to prevent additional nucleic acids or other template nucleic acid molecules from binding to the oligonucleotide. The binding of the nucleic acid to the oligonucleotide may allow for one template nucleic acid to bind to a given area. For example, a target nucleic acid binds to an oligonucleotide and produces a plurality of nucleic acids that block the surrounding oligonucleotides from other target nucleic acids binding.

[00112] The synthesis of the plurality of nucleic acid molecules and the transport of the plurality of nucleic acid molecules may occur contemporaneously. For example, an RNA molecule generated by the template nucleic acid molecule can bind to a nearby oligonucleotide immediately after being generated. The synthesis of the plurality of nucleic acid molecules and the transport of the plurality of nucleic acid molecules may occur consecutively. For example, an RNA molecule generated by the template nucleic acid molecule can float in solution for a time before binding to a nearby oligonucleotide. The time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other oligonucleotide may be at least about 0.1 s, 1 s, 2 s, 3 s, 4 s, 5 s, 10 s, 30 s, 60 s, 120 s, 180 s, 240 s, 300 s, 360 s, 600 s, 1200 s, 2400 s, 3600 s, or more. The time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other oligonucleotide may be at most about 3600 s, 2400 s, 1200 s, 600 s, 360 s, 300 s, 240 s, 180 s, 120 s, 60 s, 30 s, 10 s, 5 s, 4 s, 3 s,

2 s, 1 s, 0.1 s, or less.

[00113] The system can remove at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied oligonucleotides, thereby generating active oligonucleotides. Removal of the nucleic acid molecules may comprise removing at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied oligonucleotides with a reagent. The removing at least a portion of the nucleic acid molecules may be removing substantially all nucleic acid molecules within an area. For example, all of the oligonucleotides in a well of a sensing array can have the bound nucleic acid molecules removed. In another example, the nucleic acids bound to oligonucleotides on the surface of a bead can be removed. The removing at least a portion of the nucleic acid molecules may be removing nucleotides of a given sequence. For example, nucleotides with the sequence ATACG can be removed, but nucleotides with the sequence TTAAG can remain. The reagent may be an enzyme. The enzyme may be an RNase. The RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl,

T2, U2, or V. The reagent may be a chemical compound. The chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea.

[00114] The system can use the template nucleic acid molecule and the active oligonucleotides to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active oligonucleotides. The amplification may comprise conducting a reaction with aid of at least one recombinase, polymerase, or a combination thereof. The recombinase may be a Tre recombinase, a Cre recombinase, a Hin recombinase, a Dmcl recombinase, a Rad51 recombinase, or a FLP recombinase. The polymerase may be a DNA polymerase or an RNA polymerase. The RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase. The DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y. The amplicons coupled to the active oligonucleotides may be a clonal population of nucleic acids. The clonal population of nucleic acids may be clones of the template nucleic acid. The amplicons may be a partially clonal population. The amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal. The amplicons may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal. The amplification may further comprise sequencing at least a subset of the amplicons coupled to the active oligonucleotides or derivatives thereof. The derivatives may be at least about 1%, 5%,

10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid. The derivatives may be at least about 99.9%, 99%, 98%,

95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less similar in sequence to the template nucleic acid. The subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%,

60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons. The subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%,

1%, or less of the amplicons. The sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof. The nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof. The sequencing may be performed by methods and systems as described elsewhere herein.

[00115] The array of oligonucleotides may be among a plurality of arrays of oligonucleotides. The plurality of arrays of oligonucleotides may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more arrays of oligonucleotides. The plurality of arrays of oligonucleotides may be at most about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less arrays of oligonucleotides. The operations 110 - 150 may be repeated at another array of the plurality of arrays of oligonucleotides. The operations may be repeated for at least about 2, 3, 4,

5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays. The operations may be repeated for at least about 1,000,000, 500,000,

100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays. The operations may be repeated at each other array of the plurality of arrays of oligonucleotides.

[00116] In an aspect, the present disclosure provides methods for processing a template nucleic acid molecule. A method for processing a template nucleic acid molecule may comprise providing a template nucleic molecule coupled to an oligonucleotide of an array of oligonucleotides. The other oligonucleotides of the array of oligonucleotides may be blocked such that other template nucleic acid molecules are incapable of stably coupling to the other oligonucleotides. At least a subset of the other oligonucleotides may be blocked. The template nucleic acid molecule and the deblocked oligonucleotides may be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active oligonucleotides. [00117] In some embodiments, a template nucleic acid molecule may comprise a promoter sequence. The template nucleic acid molecule may further comprise a sequence that is at least partially complimentary to oligonucleotide. The oligonucleotide may comprise a complimentary promoter sequence. The complimentary promoter sequence may be complimentary to promoter sequence. The promoter sequence may be a T7 RNA polymerase promoter sequence. The promoter sequence may be configured to initiation production of one or more RNA strands. The one or more RNA strands may be at least partially complimentary to oligonucleotide. The one or more RNA strands may block the other oligonucleotides such that another template nucleic acid molecule may not stably bind to the other oligonucleotides.

[00118] The array of oligonucleotides may be attached to a solid support. The solid support 208 may be a bead, planar, a surface of a well, or any combination thereof. For example, a bead functionalized with oligonucleotides can rest on a planar surface. The bead may be a functionalized bead comprising a tosylated surface. The bead may have a diameter of at least about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1,000, or more micrometers. The bead may have a diameter of at most about 1,000, 750, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25, 10, 5, 1, or less micrometers. The bead may be a component of a welldess sensing array. The bead may be a polymer bead (e.g., latex, polystyrene), a glass bead, a metal bead, or the like. The planar solid support may be a well-less sensing array. The planar solid support may comprise one or more electrodes. The electrodes may be dielectric stacks, metals, or a combination thereof. The electrodes may be nanoneedles. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35,

85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, the well can have an x dimension of 434 micrometers, a y dimension of 30 micrometers, and a z dimension of 510 micrometers. In another example, the well can have an x and y dimension of 16 micrometers and a z dimension of 1 micron. The system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors. The mechanisms may be mechanisms as described in FIGs. 5A-5D.

[00119] The array of oligonucleotides may be in sensory communication with a sensor. The sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof. The sensor may comprise an electrode. The electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof. The sensor may comprise a plurality of electrodes. The plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000,

5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes. The plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000,

100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes. The sensor may be among an array of sensors. The array of sensors may comprise sensors of one or more types.

For example, an array of sensor may comprise an optical sensor and an electrical sensor. The sensors of the array of sensors may be individually addressable. For example, each electrode of an array of 1,000,000 electrodes can be measured independently of each other electrode.

[00120] In some embodiments, processing a template nucleic acid molecule can be implemented on an appropriately configured system as described elsewhere herein. The system may provide a template nucleic acid molecule coupled to an oligonucleotide of an array of oligonucleotides. The template nucleic acid molecule may have a concentration of at least about

0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700,

800, 900, 1,000 or more nanograms per microliter. The template nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275,

250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9,

0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less nanograms per microliter. The template nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 to 4 nanograms per microliter. The other oligonucleotides of the array of oligonucleotides may be blocked such that other template nucleic acid molecules may be incapable of stably coupling to the other oligonucleotides. The other oligonucleotides of the array of oligonucleotides may be blocked with nucleic acid molecules bound to the other oligonucleotides of the array of oligonucleotides. The nucleic acid molecules may be DNA molecules or RNA molecules. For example, the other oligonucleotides can be blocked with RNA molecules that bind to enough of the oligonucleotide to prevent stable binding. In this example, the amount the RNA molecules are configured to be bound to prevent stable binding can be a function of temperature and the ionic strength of the buffer solution around the oligonucleotides. The stability of the binding can be modulated by factors such as the length of the blocking nucleic acid, the sequence of the oligonucleotide, the ionic strength of the solution (e.g., the salt concentration), the temperature, the presence of solvents (e.g., formamide, DMSO), the presence of ligands, the presence of metal ions, the pH of the solution, or any combination thereof. The nucleic acids blocking the other oligonucleotides may isolate the template nucleic acid.

[00121] The oligonucleotides of the array of oligonucleotides may be coupled to a support. The support may be planer. The support may be a bead. The bead may be a component of a well less sensing array. The oligonucleotides may be coupled to a functional unit on the surface of the bead. The support may be the interior of a well. The support may be an electrode. The oligonucleotides of the array of oligonucleotides may be coupled to the support by a linking unit. The linking unit may be a polymer, a thiol group, a silane group, or the like. An electric field may be applied to the array of oligonucleotides. The electric field may be at least about 0.001 Volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may be at most about 10,000, 5,000, 1,000,

240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 or less volts. The electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof. The electric field may be applied over a distance of at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500,

600, 700, 800, 900, 1,000, or more micrometers. The electric field may be applied over a distance of at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150,

140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a pair of gold electrodes 100 micrometers apart can be used to apply a 0.5 V potential to the array of oligonucleotides. A magnetic field may be applied to the array of oligonucleotides. The magnetic field may be at least about 1 x 10⁶ Tesla (IE-6 T), IE-5 T, IE-4 T, IE-3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more. The magnetic field may be at most about 1E1 T, 1E0 T, IE-1 T, IE-2 T, IE-3 T, IE-4, IE-5 T, IE-6 T, or less. The magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet) or an electromagnet (e.g., a solenoid). The magnetic field may be applied over a distance of at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers. The magnetic field may be applied over a distance of at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140,

130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a solenoid coil can be placed 500 micrometers behind the array of oligonucleotides and used to apply a 0.3 Tesla magnetic field.

[00122] The system may deblock at least a subset of the other oligonucleotides, thereby generating active oligonucleotides. The deblocking may be performed with the aid of a reagent.

The reagent may be a chemical reagent, a physical process, an enzyme, or any combination thereof. The chemical reagent may be a solvent (e.g., methanol, formamide), a ligand, a metal ion source, a proton source (e.g., an acid), a base (e.g., sodium hydroxide), a radical source, or any combination thereof. The physical process may be applying energy (e.g., heating, sonication), applying light (e.g., an ultraviolet laser), or a combination thereof. The enzyme may be an RNase or a DNase. The RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V. The

DNase may be DNase I, II, or micrococcal nuclease.

[00123] The system may use the template nucleic acid molecule and the deblocked or active oligonucleotides to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the deblocked or active oligonucleotides. The deblocking of oligonucleotides and the amplification of the template nucleic acid molecule may occur in a well. The well may be a well of a plurality of wells of a sensing array. The well may comprise one or more beads. For example, a single bead may be at least partially contained by the well. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15,

170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers. The well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120,

110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, the well can have a width of 150 micrometers, a depth of 105 micrometers, and a height of 437 micrometers. In another example the well can have a length and width of 15 microns and a depth of 3 microns.

[00124] The amplicons coupled to the active oligonucleotides may be a clonal population of nucleic acids. For example, a template nucleic acid molecule can be coupled to an oligonucleotide surrounded by an array of nucleotides that were recently deblocked. In this example, the template nucleic acid molecule can be amplified such that clones of the template nucleic acid molecule occupy the recently unblocked oligonucleotides. The amplicons may be a partially clonal population. The amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal. The amplicons may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%,

1%, or less clonal. For example, a template nucleic acid can be coupled to an oligonucleotide in a well, where all of the other oligonucleotides in the well are blocked. In this example, after deblocking the other oligonucleotides and generating amplicons of the template nucleotide, the other oligonucleotides can have a 100% clonal population, as all of the amplicons are derived from the template nucleic acid. The amplification of the template nucleic acid molecule may further comprise sequencing at least a subset of the amplicons coupled to the active oligonucleotides or derivatives thereof. The derivatives may be at least about 1%, 5%, 10%,

20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid. The derivatives may be at least about 99.9%, 99%, 98%,

[00125] In some embodiments, the nucleic acid molecules used for blocking the oligonucleotides comprise RNA molecules. The methods and systems as described elsewhere herein may comprise methods and mechanisms configured to exclude a plurality of nucleic acid molecules from other arrays of a plurality of arrays of oligonucleotides. The excluding may generate arrays with less than about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less contamination from other arrays. The excluding may generate arrays with more than about 0.01%, 0.05 %, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more contamination from other arrays. The confining the RNA may be for a time. For example, the RNA can be confined while the RNA is being generated, and then the excess RNA can be washed away.

[00126] In some embodiments, a high viscosity buffer may be used as a diffusion barrier to contain nucleic acid molecules in well. The high viscosity buffer may be a hydrocarbon (e.g., an oil, squalene), a chemical compound (e.g., 1 -Butyl-3 -methylimidazolium hexafluorophosphate or glycerol), a gel buffer, a viscoelastic polymer, or the like. The buffer may have a viscosity of at least about 1 x 10³ Pascal-seconds (IE-3 Pa s), 5E-3 Pa s, IE-2 Pa s, 5E-2 Pa s, IE-1 Pa s, 5E-1

Pa s, 1 Pa s, 5 Pa s, 10 Pa s, 50 Pa s, 100 Pa s, 500 Pa s, 1,000 Pa s, or more. The buffer may have a viscosity of at most about 1,000 Pa s, 500 Pa s, 100 Pa s, 50 Pa s, 10 Pa s, 5 Pa s, 1 Pa s,

5E-1 Pa s, IE-1 Pa s, 5E-2 Pa s, IE-2 Pa s, 5E-3 Pa s, IE-3 Pa s, or less.

[00127] In some embodiments, degrading at least a subset of the nucleic acid molecules can be used to exclude the nucleic acid molecules for other arrays. The nucleic acid molecules may be contained within well by the use of degrading elements. The degrading elements may comprise enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof. The enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof. The chemical degrading elements may be an acid (e.g.,/>-toluene sulfonic acid, nitric acid, ascorbic acid), a base (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof. The light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule). For example, a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA. The degrading element 506 may be coupled to a support. The support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof. For example, a plurality of RNase enzymes can be coupled to a plurality of support beads, and the support beads can be placed above the wells. An electric field may be applied to the support. The generator may generate the electric field. The electric field may have a potential of at least about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may have a potential of at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2,

1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 or less volts. The electric field may be applied via electrodes 507 that are electronically coupled to generator 508. The support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode.

The electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof.

[00128] In some embodiments, the confining of the nucleic acids comprises applying an electric field. The nucleic acid molecules may be within well. The generator may be electronically coupled to electrodes, which may apply an electric field between the electrodes.

The electric field may interact with labels attached to one or more of nucleic acid molecules. The interacting may draw the nucleic acid molecules away from the top of the well and thus contain the nucleic acid molecules. The labels may be a particle. The particle may be a dielectrophoretic particle. The particle may be a metal particle (e.g., gold, aluminum, silver, platinum), a semiconductor particle (e.g., silicon, carbon, zinc sulfide), or a molecular unit (e.g., Ru(bpy)₃ ²⁺, ferrocene). The particle may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different particle may be attached to each end of the nucleic acid molecule.

[00129] In some embodiments, the confining of the nucleic acids comprises applying a magnetic field. The magnetic field may be applied to the plurality of nucleic acid molecules in well using magnet. The magnet may be a permanent magnet (e.g., a rare-earth magnet, an iron- based magnet) or an electromagnet (e.g., a solenoid, a superconducting magnet). At least one nucleic acid molecule of the nucleic acid molecules may comprise a label that interacts with the magnetic field. The label may be a particle (e.g., an iron nanoparticle), a molecular species (e.g., a single molecule magnet, an iron containing molecule), or a combination thereof. For example, a nucleic acid can be attached to the surface of an iron nanoparticle cluster. The label may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different label may be attached to each end of the nucleic acid molecule.

[00130] In some embodiments, the confining of the nucleic acids comprises applying electrophoretic force. Nucleic acid molecules may be generated in well. To prevent the nucleic acid molecules from leaving the well, an electric field can be applied between electrodes. The electric field may generate an electrophoretic force that attracts the nucleic acid molecules down into the well. A generator may generate the electric field. The generator may generate a potential of at least about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7

V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The generator may generate a potential of at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or less volts. The electrodes may be separated by at least about 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1,000 or more micrometers. The electrodes may be separated by at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110,

100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers

[00131] Examples of electrode material for the electrodes are shown in FIG. 6B. Other examples of electrode materials may be metals, semiconductors, or conductive polymers. The metals may be gold, silver, platinum, nickel, copper, iron, other transition metals, or alloys thereof. The semiconductors may be organic semiconductors (e.g., O_όo, phenyl-C61 -butyric acid methyl ester), inorganic semiconductors (e.g., silicon, cadmium telluride, indium tin oxide, gallium arsenide), or a combination thereof. The conductive polymers may be polyfluroenes, polyacetylenes, poly(p-phenylene vinylene)s, polypyrroles, polyanilines, polythiophenes, poly(3,4-ethylenedioxythiophene), poly(p-phenylene sulfide), or any combination thereof.

[00132] In an aspect, the present disclosure provides methods for nucleic acid amplification. A method for nucleic acid amplification may comprise brining a plurality of target nucleic acid molecules in contact with an array of oligonucleotides. The plurality of target nucleic molecules may be present at a concentration such that at most a target nucleic acid molecule of the plurality of target nucleic acid molecules hybridizes to an oligonucleotide of the array of oligonucleotides.

The array of oligonucleotides may be subject to conditions sufficient to synthesize a first plurality of nucleic acid molecules from the target nucleic acid molecule hybridized to the oligonucleotide. The first plurality of nucleic acid molecules may be hybridized to other oligonucleotides of the array of oligonucleotides. The array of oligonucleotides may be subject to conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules. The array of oligonucleotides may be subject to conditions sufficient to amplify the target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to the array of oligonucleotides.

[00133] The target nucleic acid molecule may have a concentration of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 110, 120,

130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1,000 or more nanograms per microliter. The target nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275, 250,

225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7,

[00134] The oligonucleotides of the array of nucleotides may comprise a common sequence. The oligonucleotides of the array of oligonucleotides may be identical. The plurality of nucleic acid molecules may be at least partially complementary to the common sequence. The plurality of nucleic acids may be at least about 1 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 97 %, 98 %, 99 %, or more complimentary to sequences of other oligonucleotides of the array of nucleotides. The plurality of nucleic acid molecules may be at most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%,

70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less complimentary to sequences of other oligonucleotides of the array of nucleotides. The first plurality of nucleic acid molecules may be a plurality of RNA molecules. The synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may be performed with the aid of an enzyme. The enzyme may be an RNA polymerase. The RNA polymerase may be a

T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase. The synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may involve transporting a subset of the first plurality of nucleic acid molecules to the other oligonucleotides of the array of oligonucleotides. The transporting may be via diffusion.

The transporting may be assisted diffusion. The transporting may be an active transporting. The active transporting may comprise cellular transportation methods (e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow (e.g., flowing a liquid carrier in the direction of transport), or any combination thereof. The transporting may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of simple diffusion.

[00135] The conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules may comprise removing or degrading the subset of the nucleic acid molecules with a reagent. The reagent may be an enzyme. The enzyme may be an RNase. The

RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V. The reagent may be a chemical compound. The chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea. The target nucleic acid molecule hybridized to the oligonucleotide may comprise a promoter sequence. The promoter sequence may be a T7 RNA polymerase promoter sequence. The oligonucleotides of the array of oligonucleotides may comprise a complementary promoter sequence. The complementary promoter sequence may be complimentary to the promoter sequence of the target nucleic acid molecule. For example, the target nucleic acid molecule may be able to hybridize with an oligonucleotide via interaction of the promoter sequence with the complementary promoter sequence.

[00136] The array of oligonucleotides may be attached to a solid support. The array of oligonucleotides may be attached to a solid support. The solid support may be a bead, planar, a surface of a well, or any combination thereof. For example, a bead functionalized with oligonucleotides can rest on a planar surface. The bead may be a functionalized bead comprising a tosylated surface. The bead may have a diameter of at least about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1,000, or more microns. The bead may have a diameter of at most about 1,000, 750, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25, 10, 5, 1, or less microns. The bead may be a component of a well-less sensing array. The bead may be a polymer bead (e.g., latex, polystyrene), a glass bead, a metal bead, or the like. The planar solid support may be a well -less sensing array. The planar solid support may comprise one or more electrodes. The electrodes may be dielectric stacks, metals, or a combination thereof. The electrodes may be nanoneedles. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,

90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800,

900, 1,000, or more micrometers. The well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190,

180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30,

25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, the well can have an x dimension of 434 micrometers, a y dimension of 30 micrometers, and a z dimension of 510 micrometers. In another example, the well can have an x and w dimension of 15 micrometers and a z dimension of 1 micron. The system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors. The mechanisms may be mechanisms as described in FIGs. 5A-5D.

[00137] The array of oligonucleotides may be in sensory communication with a sensor. The sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof. The sensor may comprise an electrode. The electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof. The sensor may comprise a plurality of electrodes. The plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes. The plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes. The sensor may be among an array of sensors. The array of sensor may comprise sensors of one or more types. For example, an array of sensor may comprise an optical sensor and an electrical sensor. The sensors of the array of sensors may be individually addressable. For example, each electrode of an array of 1,000,000 can be measured independently of each other electrode.

[00138] The array of oligonucleotides may be among a plurality of arrays of oligonucleotides. The plurality of arrays of oligonucleotides may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more arrays of oligonucleotides. The plurality of arrays of oligonucleotides may be at most about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less arrays of oligonucleotides.

[00139] The excluding may comprise applying an electric field to the plurality of nucleic acid molecules. The electric field may be at least about 0.001 Volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1

9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may be at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or less volts. The electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4- ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof. The electric field may be applied over a distance of at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers.

60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a pair of gold electrodes 100 micrometers apart can be used to apply a 0.5 V potential to the array of oligonucleotides. A nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the electric field. At least one nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the electric field. The label may be a particle. The particle may be a di electrophoretic particle. The particle may be a metal particle (e.g., gold, aluminum, silver, platinum), a semiconductor particle (e.g., silicon, carbon, zinc sulfide), or a molecular unit (e.g., Ru(bpy)₃ ²⁺, ferrocene). The particle may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different particle may be attached to each end of the nucleic acid molecule.

[00140] The excluding may comprise applying a magnetic field to the plurality of nucleic acid molecules. The magnetic field may be at least about 1 x 10⁶ Tesla (IE-6 T), IE-5 T, IE-4 T, 1E- 3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more. The magnetic field may be at most about 1E1 T,

1E0 T, IE-1 T, IE-2 T, IE-3 T, IE-4, IE-5 T, IE-6 T, or less. The magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet) or an electromagnet (e.g., a solenoid). The magnetic field may be applied over a distance of at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers. The magnetic field may be applied over a distance of at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,

95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers. For example, a solenoid coil can be placed 500 micrometers behind the array of oligonucleotides and used to apply a 0.3 Tesla magnetic field. A nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the magnetic field. At least one nucleic acid molecule of the plurality of nucleic acid molecules may comprise a label that interacts with the magnetic field. The label may be a particle (e.g., an iron nanoparticle), a molecular species (e.g., a single molecule magnet, an iron containing molecule), or a combination thereof. For example, a nucleic acid can be attached to the surface of a 3 nm iron nanoparticle. The label may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule. A different label may be attached to each end of the nucleic acid molecule.

[00141] The excluding may be performed with the aid of a diffusion barrier. The diffusion barrier may be a high viscosity buffer. The high viscosity buffer may be a hydrocarbon (e.g., an oil, squalene), a chemical compound (e.g., 1 -Butyl-3 -methylimidazolium hexafluorophosphate or glycerol), a gel buffer, a viscoelastic polymer, or the like. The buffer may have a viscosity of at least about 1 x 10³ Pascal-seconds (IE-3 Pa s), 5E-3 Pa s, IE-2 Pa s, 5E-2 Pa s, IE-1 Pa s, 5E-1 Pa s, 1 Pa s, 5 Pa s, 10 Pa s, 50 Pa s, 100 Pa s, 500 Pa s, 1,000 Pa s, or more. The buffer may have a viscosity of at most about 1,000 Pa s, 500 Pa s, 100 Pa s, 50 Pa s, 10 Pa s, 5 Pa s, 1 Pa s, 5E-1 Pa s, IE-1 Pa s, 5E-2 Pa s, IE-2 Pa s, 5E-3 Pa s, IE-3 Pa s, or less.

[00142] The excluding may be performed by degrading a subset of the plurality of nucleic acid molecules. The degrading may be performed with degrading elements. The degrading elements may be enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof. The enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof. The chemical degrading elements may be an acid (e.g.,/>-toluene sulfonic acid, nitric acid, ascorbic acid), abase (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof. The light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule). For example, a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA. The degrading elements may be coupled to a support. The support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof. For example, a plurality of RNase enzymes can be coupled to a plurality of support beads, and the support beads can be placed above the wells. An electric field may be applied to the support. A generator may generate the electric field. The electric field may have a potential of at least about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more. The electric field may be at most about 10,000, 5,000, 1,000, 240, 120, 50, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7,

0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or less volts. The electric field may be applied via electrodes that are electronically coupled to the generator. The support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode. The electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof. [00143] The array of oligonucleotides may comprise oligonucleotides having sequences different from oligonucleotides of at least one other array of the plurality of arrays of oligonucleotides. The array of oligonucleotides may comprise oligonucleotides having sequences different from oligonucleotides of at least one other array of the plurality of arrays of oligonucleotides. For example, the oligonucleotides coupled to the sensors of a 3x3 grid of sensors can each have a different sequence, leading to 9 different oligonucleotide sequences. The different oligonucleotide sequences may result in less cross contamination of nucleic acids between sensors. The lack of cross contamination may be particularly relevant in well -less sensing arrays. For example, each bead of an array of beads having a different oligonucleotide sequence can prevent the RNA produced at each bead from diffusing to and binding onto another bead. The synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may include excluding the plurality of nucleic acid molecules from other arrays of the plurality of arrays of oligonucleotides. For examples, the nucleotides can be contained to a sensor in a well and not leak to other wells containing other sensors.

[00144] The method may be repeated at another array of the plurality of arrays of oligonucleotides. The method may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays. The method may be repeated for at least about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays. The method may be repeated at each other array of the plurality of arrays of oligonucleotides. The repeating the method may further comprise sequencing at least a subset of the substantially clonal populations at the another array of the plurality of arrays of oligonucleotides.

[00145] The subjecting the array of oligonucleotides to conditions sufficient to amplify the target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to the array of oligonucleotides may comprise conducing a reaction with aid of a recombinase, a polymerase, or any combination thereof. The recombinase may be a Tre recombinase, a Cre recombinase, aHin recombinase, aDmcl recombinase, aRad51 recombinase, or a FLP recombinase. The polymerase may be a DNA polymerase or an RNA polymerase. The RNA polymerase may be an RNA polymerase as described elsewhere herein. The DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y.

[00146] The method may further comprise sequencing at least a subset of the second plurality of nucleic acid molecules hybridized to the array of oligonucleotides. The subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the second plurality of nucleic acid molecules. The subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the second plurality of nucleic acid molecules. The sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof. The sequencing may be performed with measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof. The sequencing may be performed by methods and systems as described elsewhere herein. Multiplexing Sequencing

[00147] The present disclosure provides methods and systems for sequencing a nucleic acid molecule. A method for sequencing a nucleic acid molecule may comprise providing the nucleic acid molecule coupled to a support at a 3’ end of the nucleic molecule. The nucleic acid molecule may comprise, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence. The nucleic acid molecule may comprise a first primer hybridized to the third sequence. The third sequence may be subjected to sequencing to generate a first sequencing read comprising at least a portion of the third sequence. A second primer having a sequence complementarity with the second sequence may be brought in contact with the nucleic acid molecule under conditions sufficient for the second primer to hybridize to the second sequence. The second sequence may be subjected to sequencing to generate a second sequencing read comprising at least a portion of the second sequence. A third primer having a sequence complementarity with the first sequence may be brought in contact with the nucleic acid molecule under conditions sufficient for the third primer to hybridize to the first sequence. The first sequence may be subjected to sequencing to generate a third sequencing read comprising at least a portion of the first sequence.

[00148] FIG. 8 shows a flowchart for an example method 800 for sequencing a nucleic acid molecule. In an operation 810, the method 800 may comprise providing a nucleic acid molecule coupled to a support at a 3’ end of the nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence, wherein the nucleic acid molecule comprises a first primer hybridized to the third sequence. The nucleic acid molecule may be an oligonucleotide. The first sequence may comprise a primer hybridization location. The primer hybridization location may be placed on the 5’ end of a first barcode sequence. The second sequence may comprise a second primer hybridization location.

The second primer hybridization location may be placed on the 5’ end of a target nucleic acid molecule (e.g., a nucleic acid molecule of interest to be sequenced). The third sequence may comprise a third primer hybridization location. The third primer hybridization location may be placed on the 5’ end of a second barcode sequence. Though referred to herein with reference to a first and third sequence, the method 800 may be performed with one or both of a first or a third sequence coupled to the second sequence. For example, the nucleic acid molecule can be a first sequence, a first barcode, a second sequence, and a target sequence. In this example, the method

800 does not comprise operation 820.

[00149] The nucleic acid molecule may be a deoxyribose nucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof. The method 800 may further comprise, prior to operation 810, coupling the first sequence, the third sequence, or both to the second sequence. For example, an RNA molecule can be prepared for sequencing by coupling a first primer and a first barcode to the 3’ end of the RNA and a second primer and a second barcode to the 5’ end of the RNA. The first sequence and/or the third sequence may be coupled to the second sequence via ligation. The ligation may be a sticky end ligation or a blunt end ligation.

The ligation may be performed with the aid of one or more enzymes. The first sequence and/or the third sequence may be coupled to the second sequence via hybridization. The hybridization may involve hybridizing at least a portion of the first and/or third sequence to at least a portion of the second sequence. A new strand of DNA may be generated complimentary to the hybridized strand. For example, a first sequence can be partially hybridized onto a second sequence and a DNA polymerase can generate two complimentary strands comprising the first sequence and the second sequence.

[00150] The nucleic acid molecule may be coupled to the support via a probe coupled to the support. The probe may be a probe as described elsewhere herein. The probe may be coupled to the support as described elsewhere herein. The probe may comprise an oligonucleotide. The nucleic acid molecule may hybridize to the probe. The support may be a support as described elsewhere herein (e.g., a bead, planar, a surface of a well).

[00151] In another operation 820, the method 800 may further comprise subjecting the third sequence to sequencing to generate a first sequencing read comprising at least a portion of the third sequence. The subjecting the third sequence to sequencing may comprise applying reagents and conditions sufficient to sequence the third sequence as described elsewhere herein. The at least a portion of the third sequence may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%,

60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the third sequence. The at least a portion of the third sequence may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%,

60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the third sequence. The first sequencing read may comprise at least a portion of the nucleic acid molecule adjacent to the third sequence.

For example, the first sequencing read can also sequence a first barcode region of the nucleic acid molecule. The sequencing may comprise use of a polymerizing enzyme as described elsewhere herein. The first sequencing read may comprise identifying the sequence of the third sequence and/or a portion of the nucleic acid molecule adjacent to the third sequence. The sequencing the third sequence and/or a portion of the nucleic acid molecule adjacent to the third sequence may generate an identification tag for the target nucleic acid molecule. The sequencing may be a sequencing-by-synthesis. The sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge. The sequencing may be performed with aid of a sensor. The probe and/or the nucleic acid molecule may be in sensory communication with one or more sensors.

The sensors may be sensors as described elsewhere herein (e.g., one or more electrodes, one or more optical sensors). The conditions sufficient for a primer to hybridize may be an appropriately configured temperature, ionic strength, presence or absence of a denaturant, or the like.

[00152] In another operation 830, the method 800 may further comprise brining a second primer having a sequence complementarity with the second sequence in contact with the nucleic acid molecule under conditions sufficient for the second primer to hybridize to the second sequence, and subjecting the second sequence to sequencing to generate a second sequencing read comprising at least a portion of the second sequence. The sequencing may be sequencing as described elsewhere herein. The sequencing of operation 830 may be sequencing of the same type as operation 820. The sequencing may comprise use of a polymerizing enzyme. The polymerizing enzyme may comprise strand displacement activity. The second sequencing read may displace the first sequencing read. For example, a first sequencing read can generate a complimentary strand hybridized to the nucleic acid molecule from the location of the third sequence on. In this example, an enzyme with strand displacement activity can be used in the sequencing of the second sequence and the enzyme can displace the complimentary strand at the location of the third sequence. The displacing the first sequencing read product may enable another sequencing read of the first sequence on. For example, a first barcode can be resequenced by the same enzyme that was used to sequence the second sequence. Resequencing the third sequence on may increase the accuracy of the sequencing. For example, displacing the first read product of the third sequence and resequencing it can reduce the chance that an error results in the misidentification of a barcode. The at least a portion of the second sequence may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the second sequence. The at least a portion of the second sequence may be at most about

99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the second sequence. The displacing of the first read product may increase the speed at which the sequencing of the nucleic acid molecule may be performed. For example, running off the first sequencing product can remove the process of otherwise removing the product, thus removing an operation in the sequencing process.

[00153] In another operation 840, the method 800 may further comprise bringing a third primer having a sequence complementarity with the first sequence in contact with the nucleic acid molecule under conditions sufficient for the third primer to hybridize to the first sequence, and subjecting the first sequence to sequencing to generate a third sequencing read comprising at least a portion of the first sequence. The at least a portion of the first sequence may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the first sequence. The at least a portion of the first sequence may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the first sequence. The sequencing may be sequencing of the same type as operations 820 and/or 830. The sequencing may comprise displacing the second sequencing read. For example, after sequencing the first sequence, the strand displacing polymerase used can continue down the nucleic acid molecule and displace the second sequencing read. In this example, the sequencing can continue in order to provide an additional read of the second and third sequences on to reduce the error associated with those sequences. The sequencing of the first and/or the third sequences on may generate an identification tag for the second sequence.

[00154] The present disclosure provides methods and systems for processing a nucleic acid molecule. A method for processing a nucleic acid molecule may comprise providing the nucleic acid molecule coupled to a support at a 3’ end of the nucleic acid molecule. The nucleic acid molecule may comprise, from a 5’ end to a 3’ end, a first sequence and a second sequence. The nucleic acid molecule may be subjected to a first extension reaction to generate a first strand complementary to the first sequence. A 5’ end of the first strand may comprise a blocking group. The nucleic acid molecule may be subjected to a second extension reaction to generate a second strand complementary to the second sequence. A 5’ end of the second strand may comprise an additional blocking group.

[00155] FIG. 9 shows a flowchart for an example method 900 for processing a nucleic acid molecule. In an operation 910, the method 900 may comprise providing a nucleic acid molecule coupled to a support at a 3’ end of the nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence and a second sequence. The nucleic acid molecule may be a DNA molecule, an RNA molecule, or a derivative thereof. Prior to operation

910, the method 900 may further comprise coupling the first sequence, a third sequence, or both to the second sequence. The coupling may be via ligation or hybridization, as described elsewhere herein. The first, second, and/or third sequences may be primer hybridization locations. For example, the first location can be complimentary to a primer configured as an initiator for an RNA polymerase. The first, second, and/or third sequences may be different from one another. The first, second, and/or third sequences may be coupled to a first, second, and/or third target nucleic acid molecule, respectively. The second target nucleic acid molecule may be a nucleic acid molecule of interest for sequencing (e.g., a nucleic acid molecule derived from a biological sample). The first and/or third target nucleic acid molecules may be barcode nucleic acid molecules (e.g., of a known sequence for use in identifying the second nucleic acid molecule). For example, a known sequence of 8 nucleic acids can be coupled to the first sequence and a target nucleic acid molecule can be coupled to the second sequence. In this example, the two resulting coupled nucleic acid molecules can be coupled together and used as the nucleic acid molecule of method 900. The nucleic acid molecule may be coupled to the support via a probe coupled to the support. The probe may be a probe as described elsewhere herein (e.g., an oligonucleotide, an antibody). The support may be a support as described elsewhere herein (e.g., a bead, planar, a surface of a well).

[00156] In another operation 920, the method 900 may further comprise subjecting the nucleic acid molecule to a first extension reaction to generate a first strand complementary to the first sequence, wherein a 5’ end of the first strand comprises a blocking group. The sequencing of the first sequence may generate an identification tag for the second sequence (e.g., the first sequence is a barcode for the second sequence). The first extension reaction may further comprise sequencing the first sequence. The probe and/or the nucleic acid may be in sensory communication with a sensor (e.g., in optical communication, in electrical communication). The sensor may comprise an electrode. The sensor may comprise a plurality of electrodes. The sensor may be used to aid in the sequencing of the first sequence. The sequencing may be completed via sequencing-by-synthesis. The sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge. For example, an electrical sensor can be used to measure a change in impedance as nucleotides are incorporated into the first strand, and thus identify which nucleotides are present in the first sequence.

[00157] The blocking group may be configured to inhibit an activity of a polymerase used in the first extension reaction. The blocking group may comprise a dideoxy group, an at least partially complimentary RNA strand, a chemical blocking group, and enzymatic block, a photonic block, one or more biologic molecules, one or more metals, one or more ions, or any combination thereof. The dideoxy group may prevent a further elongation of the first strand by removing the hydroxy group that is used for further extension. The dideoxy group may be attached to a nucleotide base, forming a dideoxy nucleotide. The at least partially complimentary

RNA strand may be an RNA strand that blocks further growth of the first strand by hybridizing to the nucleic acid molecule and blocking further progress of a polymerase. For example, a nucleic acid molecule comprising a first and second sequence can have an RNA strand bind to the second sequence such that a polymerase generating a complementary strand of the first sequence stops when it reaches the blocking RNA strand. A polymerase without strand displacement characteristics may be used when the blocking group is an at least partially complimentary RNA strand. The chemical blocking group may be an azido group (e.g., forming a 3’ -azi do-nucleotide), a fluorescent label, or the like. The one or more biologic molecules may comprise one or more nucleotides, enzymes, or both. The one or more metals may be one or more metal ions. The one or more metals may be introduced into solution (e.g., freely solvated ions) or associated with the strand (e.g., attached to the strand via a chelating moiety). The one or more metals may be one or more different metals (e.g., iron and nickel). The one or more ions may be alkali ions (e.g., sodium, potassium), alkali earth ions (e.g., calcium), non-metal ions

(e.g., phosphorous), chalcogen ions, halide ions, or the like.

[00158] In another operation 930, the method 900 may further comprise subjecting the nucleic acid molecule to a second extension reaction to generate a second strand complementary to the second sequence, wherein a 5’ end of the second strand comprises an additional blocking group. Operation 930 may be performed before or subsequent to operation 920. The second extension reaction may be using the same reagents, conditions, and/or sensors as the first extension reaction. The additional blocking group may be a blocking group as described above. The additional blocking group may be a same blocking group or a different blocking group as the blocking group of operation 920.

[00159] The nucleic acid molecule of method 900 may further comprise a third sequence. The method 900 may further comprise subjecting the nucleic acid molecule to a third extension reaction to generate a third strand complimentary to the third sequence. The third extension reaction may further comprise sequencing the third strand using sequencing methods described elsewhere herein. The sequencing the third strand may generate an identification tag for the second sequence. For example, known sequences can be attached to the second strand as the first and third strand, and the extension reactions and related sequencing of those known sequences can identify the second strand as a particular sample. The presence of the identification tag may enable a plurality of target nucleic acid molecules to be sequenced at substantially the same time.

For example, five sequences of interest can be identified with five different identification tags, sequenced simultaneously, and the sequences generated by the sequencing can be matched to the sequences of interest using the different identification tags. The presence of the identification tag may enable at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,

40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,

225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1,000 or more target nucleic acid sequences to be sequenced substantially simultaneously. The presence of the identification tag may enable at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275, 250,

225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50,

45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less target nucleic acid sequences to be sequenced substantially simultaneously.

[00160] The present disclosure provides methods and systems for processing a nucleic acid molecule. A method for processing a nucleic acid molecule may comprise providing the nucleic acid molecule coupled to a support at a 3’ end of the nucleic acid molecule. The nucleic acid molecule may comprise, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence. The third sequence may be subjected to sequencing to generate a first non-optical sequencing read comprising at least a portion of the third sequence. The nucleic acid molecule may comprise a first primer hybridized to the third sequence A second primer having sequence complementarity with the second sequence may be brought in contact with the nucleic acid molecule under conditions sufficient for the second primer to hybridize to the second sequence. The second sequence may be subject to non-optical sequencing to generate a second sequencing read comprising at least a portion of the second sequence. A third primer having sequence complementarity with the first sequence may be brought in contact with the nucleic acid molecule under conditions sufficient for the third primer to hybridize to the first sequence. The first sequence may be subjected to non-optical sequencing to generate a third sequencing read comprising at least a portion of the first sequence.

[00161] FIG. 10 shows a flowchart for an example method 1000 for processing a nucleic acid molecule. In an operation 1010, the method 1000 may comprise providing a nucleic acid molecule coupled to a support at a 3’ end of the nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence, wherein said nucleic acid molecule comprises a first primer hybridized to the third sequence. The nucleic acid molecule may be a DNA molecule, an RNA molecule, or a derivative thereof. Prior to operation 1010, the method 1000 may further comprise coupling the first sequence, the third sequence, or both to the second sequence. The coupling may be via ligation or hybridization, as described elsewhere herein. The nucleic acid molecule may be coupled to the support via a probe coupled to the support. The probe may be a probe as described elsewhere herein (e.g., an oligonucleotide, an antibody). The support may be a support as described elsewhere herein (e.g., a bead, planar, a surface of a well).

[00162] In another operation 1020, the method 1000 may further comprise subjecting the third sequence to sequencing to generate a first non-optical sequencing read comprising at least a portion of the third sequence. The probe may be in sensory communication with a sensor. The sensor may be used in generating the first non-optical sequencing read. The sensor may comprise an electrode. The sensor may comprise a plurality of electrodes. The sequencing may be completed via sequencing-by-synthesis. The sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge. The sequencing may be performed using a sequencing platform as described elsewhere herein. An annealing operation may be performed subsequent to operation 1020. The annealing operation may remove the primer and/or the sequencing product generated in operation 1020. The annealing operation may be a thermal annealing operation (e.g., heating), a chemical annealing operation (e.g., increasing the ionic strength of a solution around the nucleic acid molecule to anneal), an optical annealing operation (e.g., applying energy via one or more light sources), or any combination thereof.

[00163] In another operation 1030, the method 1000 may further comprise brining a second primer having sequence complementarity with the second sequence in contact with the nucleic acid molecule under conditions sufficient for the second primer to hybridize to the second sequence, and subjecting the second sequence to non-optical sequencing to generate a second sequencing read comprising at least a portion of the second sequence. The conditions sufficient for the second primer to hybridize may be conditions as described elsewhere herein. The non- optical sequencing may be the same sequencing process as in operation 1020 or it may be a different sequencing process. The second sequencing read may further comprise a read of a target nucleic acid molecule coupled to the second sequence. For example, the second sequencing read can read a target nucleic acid molecule of an unknown sequence, thus sequencing the target nucleic acid molecule. An annealing operation may be performed subsequent to operation 1030. The annealing operation may remove the primer and/or the sequencing product generated in operation 1030. The annealing operation may be a thermal annealing operation (e.g., heating), a chemical annealing operation (e.g., increasing the ionic strength of a solution around the nucleic acid molecule to anneal), an optical annealing operation (e.g., inducing energy via one or more light sources), or any combination thereof. [00164] In another operation 1040, the method 1000 may further comprise brining a third primer having sequence complementarity with the first sequence in contact with the nucleic acid molecule under conditions sufficient for the third primer to hybridize to the first sequence, and subjecting the first sequence to non-optical sequencing to generate a third sequencing read comprising at least a portion of the first sequence. The conditions sufficient for the second primer to hybridize may be conditions as described elsewhere herein. The non-optical sequencing may be the same sequencing process as in operations 1020 and/or 1030. The second sequencing read may further comprise a read of a target nucleic acid molecule coupled to the second sequence.

For example, the second sequencing read can read a target nucleic acid molecule of an unknown sequence, thus sequencing the target nucleic acid molecule. An annealing operation may be performed subsequent to operation 1040. The annealing operation may remove the sequencing product and/or primer generated in operation 1040. The annealing operation may be a thermal annealing operation (e.g., heating), a chemical annealing operation (e.g., increasing the ionic strength of a solution around the nucleic acid molecule to anneal), an optical annealing operation

(e.g., inducing energy via one or more light sources), or any combination thereof. The operations

1020, 1030, and 1040 may be performed in any order. In other words, the precise order the operations are performed in does not impact the net result of the method 1000. Operations 1020 and/or 1030 may generate an identification tag for the second sequence (e.g., be used as barcodes for the second sequence).

[00165] FIG. 11 shows an example of a nucleic acid molecule 1110 comprising multiple sequences. Target nucleic acid molecule 1101 may be a nucleic acid molecule derived from a subject (e.g., a human, an animal, a bacteria). The purpose of the sequencing may be to determine the sequence of target nucleic acid molecule 1101. Promoter sequences 1102, 1104, and 1109 may be coupled to target nucleic acid molecule 1101. The promoter sequences may be placed adjacent to target nucleic acid molecule 1101, as well as barcode sequences 1103 and 1105. The barcode sequences may be known sequences (e.g., the sequences are artificially generated). The barcode sequences may be associated with a source of nucleic acid molecule 1101. For example, two known sequences can be coupled to a target nucleic acid molecule derived from a patient in order to identify that target nucleic acid molecule as being from that patient. The barcode sequences may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more nucleotides long. The barcode sequences may be at most about 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer nucleotides long. The nucleic acid molecule 1110 may comprise at least about 1, 2, 3, 4, or more barcode sequences. The nucleic acid molecule may comprise at most about 4, 3, 2, 1, or fewer barcode sequences. Promoter sequence 1109 may be complimentary to promoter strand 1106. The promoter strand may initialize a sequencing-by-synthesis reading of target nucleic acid molecule 1101. Similar promoter strands may be hybridized to promoter sequences 1102 or 1104 to read indexes 1103 and 1105. The nucleic acid molecule 1110 may be bound to support 1108 via probe 1107. The probe may be a probe as described elsewhere herein, and the support may be a support as described elsewhere herein.

Efficient Incorporation of Nucleotide Bases

[00166] The present disclosure provides methods and systems for sequencing a template nucleic acid molecule. A method for sequencing a template nucleic acid molecule may comprise providing a plurality of nucleic acid molecules immobilized adjacent to a support. Each of the plurality of nucleic acid molecules may comprise a sequence of the template nucleic acid molecule. In a first phase, the plurality of nucleic acid molecules may be sequentially brought in contact with nucleotides of one or more types that are fewer than four types of nucleotides and a first set of signals from the plurality of nucleic acid molecules is detected. In some embodiments, the first set of signals can be indicative of nucleic acid molecule synthesis or a nucleic acid extension reaction. In a second phase, which may be subsequent to the first phase, the plurality of nucleic acid molecules may be sequentially brought in contact with up to the four types of nucleotides and a second set of signals from the plurality of nucleic acid molecules is detected, to obtain sequences of the plurality of nucleic acid molecules. A sequential order of nucleotides in the first phase may be different than a sequential order of nucleotides in the second phase. A nucleic acid molecule undergoing a synthesis or extension reaction may have a phase lag or phase lead of at most 5 bases with respect to another nucleic acid molecule also undergoing a synthesis or extension reaction. The methods and systems for sequencing a template nucleic acid molecule described herein reduce phase lag or phase lead to at most 5 bases.

[00167] FIG. 15 shows a flowchart for an example method 1500 for sequencing a template nucleic acid molecule. In an operation 1510, the method 1500 may comprise providing a plurality of nucleic acid molecules immobilized adjacent to a support. The plurality of nucleic acid molecules may be nucleic acid molecules as described elsewhere herein. The plurality of nucleic acid molecules may be one or more clonal or substantially clonal nucleic acid populations. The plurality of nucleic acid molecules may be target nucleic acid molecules. The immobilization may comprise binding the nucleic acid molecules to a probe (e.g., an oligonucleotide, an antibody). The support may be a support as described elsewhere herein. The support may be a surface of a bead, a planar surface, a well, or the like. The support may position the nucleic acid molecules in sensory communication with one or more sensors. For example, the support can be a bead adjacent to two electrodes. The nucleic acid molecules may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,

100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, or more nucleotides. The nucleic acid molecules may comprise at most about 5,000, 1,000, 900, 800, 700,

600, 500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less nucleotides.

[00168] In another operation 1520, the method 1500 may further comprise sequentially bringing the plurality of nucleic acid molecules in contact with nucleotides of one or more types that are fewer than four types of nucleotides and detecting a first set of signals from the plurality of nucleic acid molecules. The bringing of the plurality of nucleic acid molecules in contact with nucleotides may be via flowing the nucleotides into a chamber where the nucleic acid molecules are located. The plurality of nucleic acid molecules may be suspended in a fluid. The fluid may comprise a buffer. The nucleotides of one or more types may be monophosphate nucleotides, nucleoside diphosphates, or nucleoside triphosphates. The nucleotide bases may be adenine, thymine, guanine, cytosine, uracil, or any combination thereof. The one or more types may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types. The one or more types may be at most about

10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less types. The detecting may be detecting using electrical sensors, optical sensors, or the like. The signals may be electrical signals (e.g., ionic potentials, potentials, impedance measurements), optical signals (e.g., fluorescence intensity, fluorescence lifetime, fluorescence wavelength), chemical potentials, or any combination thereof. The electrical signals may be electrical signals as described elsewhere herein. The signals may be indicative of one or more nucleotide bases incorporating into one or more strands complimentary to the nucleic acid molecules. The sequentially bringing of nucleotides in contact may comprise introducing the nucleotide(s) and/or other reagents (e.g., polymerases, buffers). The sequentially bringing nucleotides in contact may further comprise a washing operation subsequent to introducing the nucleotide and/or other reagents. A phase may comprise one or more operations of sequentially bringing the plurality of nucleic acid molecules into contact with the nucleotides.

[00169] In another operation 1530, the method 1500 may further comprise sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides and detecting a second set of signals from the plurality of nucleic acid molecules, to obtain sequences of the plurality of nucleic acid molecules. The sequential order of nucleotides in operation 1520 may be different than a sequential order of nucleotides in operation 1530. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more bases with respect to another sequence of said plurality of nucleic acid molecules. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at most about 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,

6, 5, 4, 3, 2, 1, or fewer bases with respect to another sequence of said plurality of nucleic acid molecules. The second set of signals may be the same type of signals a in operation 1520, or the second set of signals may be a different type of signals. The obtaining the sequences may be completed within at least about 0.5, 1, 5, 10, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132,

144, 156, 168, 180, 192, 204, 226, 238, 250, or more hours of initiating the sequencing run. The obtaining the sequence may be completed within at most about 250, 238, 226, 204, 192, 180,

168, 156, 144, 132, 120, 108, 96, 84, 72, 60, 48, 36, 24, 12, 10, 5, 1, 0.5, or less hours of initiating the sequencing run.

[00170] In an additional operation, the method 1500 may further comprise operation 1540. Operation 1540 may comprise sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides. A sequential order of nucleotides in the third phase may be different than a sequential order of nucleotides in operation 1520 and/or operation 1530.

[00171] In an additional operation, the method 1500 may further comprise operation 1550. Operation 1550 may comprise sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides. A sequential order of nucleotides in the operation 1550 may be of a different than a sequential order of nucleotides in any of operations 1520, 1530, and/or 1540. The method 1500 may further comprise repeating operation 1520, operation 1530, operation 1540, operation 1550, or any combination thereof.

[00172] The first set of signals and/or the second set of signals may be associated with an impedance, conductivity, charge, or change thereof, associated with said plurality of nucleic acid molecules. The first and/or second set of signals may be optical signals (e.g., fluorescence intensity signals, fluorescence lifetime signals, wavelength measurements). The sequences may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,

98%, 99%, 99.9%, or more accurate. The sequences may be at most about 99.9%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less accurate. [00173] The present disclosure provides methods and systems of performing a nucleic acid molecule extension reaction of a plurality of primers hybridized to a plurality of nucleic acid molecules as part of a clonal population. A method of performing a nucleic acid molecule extension reaction of a plurality of primers hybridized to a plurality of nucleic acid molecules as part of a clonal population may comprise contacting, in a first phase, the clonal population with each of four types of nucleotides under conditions sufficient to extend the primers in a template directed synthesis. In a second phase, the clonal population may be contacted with fewer than each of four types of nucleotides.

[00174] FIG. 16 shows a flowchart for an example method 1600 of performing a stepwise extension of a plurality of primers hybridized to a plurality of nucleic acid molecules as part of a clonal population. In an operation 1610, the method 1600 may comprise contacting the clonal population with each of four types of nucleotides under conditions sufficient to extend the primers in a template directed synthesis. The plurality of nucleic acid molecules may be nucleic acid molecules as described elsewhere herein. The plurality of nucleic acid molecules may be target nucleic acid molecules. The plurality of nucleic acid molecules may be immobilized to a support, which immobilization may comprise binding the nucleic acid molecules to a probe (e.g., an oligonucleotide, an antibody). The support may be a support as described elsewhere herein.

The support may be a surface of a bead, a planar surface, a well, or the like. The support may position the nucleic acid molecules in sensory communication with one or more sensors. For example, the support can be a bead adjacent to two electrodes. The primers may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,

70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, or more nucleotides. The primers may comprise at most about 5,000, 1,000, 900, 800, 700, 600,

500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17

16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less nucleotides. The contacting the clonal population may comprise introducing the nucleotide and/or other reagents (e.g., polymerases, buffers). The contacting the clonal population may further comprise a washing operation subsequent to introducing the nucleotide and/or other reagents. A phase may comprise one or more operations of sequentially brining the plurality of nucleic acid molecules into contact with the nucleotides.

[00175] In another operation 1620, the method 1600 may comprise contacting the clonal population with fewer than each of four types of nucleotides. The contacting may be contacting with at least about 1, 2, 3, or more types of nucleotides. The contacting may be contacting with at most about 3, 2, 1, or fewer types of nucleotides. A sequential order of nucleotides in operation 1610 may be different than a sequential order of nucleotides in operation 1620. The contacting may be under conditions similar to the conditions of operation 1610. The conditions may be conditions sufficient to extend the primers in a template directed synthesis.

[00176] In an additional operation, the method 1600 may further comprise operation 1630. Operation 1630 may comprise sequentially brining the plurality of nucleic acid molecules in contact with up to the four types of nucleotides. A sequential order of nucleotides in operation 1630 may be different than a sequential order of nucleotides in operation 1610 and/or operation

1620.

[00177] In an additional operation, the method 1600 may further comprise operation 1640. Operation 1640 may comprise sequentially brining the plurality of nucleic acid molecules in contact with up to the four types of nucleotides. A sequential order of nucleotides in operation 1630 may be different than a sequential order of nucleotides in operation 1610, operation 1620, and/or operation 1630. The method 1600 may further comprise repeating operation 1620, operation 1630, operation 1640, or any combination thereof.

[00178] The method 1600 may further comprise detecting signals from the plurality of nucleic acid molecules to generate a plurality of sequences of the plurality of nucleic acid molecules. The generating of a plurality of sequences may completed within at least about 0.5, 1, 5, 10, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 226, 238, 250, or more hours of initiating the sequencing run. The generating of a plurality of sequences may completed within at most about 250, 238, 226, 204, 192, 180, 168, 156, 144, 132, 120, 108, 96, 84, 72, 60, 48, 36, 24, 12, 10, 5, 1, 0.5, or less hours of initiating the sequencing run. The signals may be signals as described elsewhere herein. The plurality of sequences may be at least about 10%,

20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more accurate. The plurality of sequences may be at most about 99.9%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less accurate. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more bases with respect to another sequence of said plurality of nucleic acid molecules. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at most about 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer bases with respect to another sequence of said plurality of nucleic acid molecules. The sequencing may comprise sequencing via sequencing-by-synthesis, sequencing-by-ligation, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The sequencing may comprise measuring one or more signals associated with the sequencing (e.g., the sequencing by synthesis). The signals may be signals as described elsewhere herein (e.g., associated with an impedance, conductivity, charge, or change thereof). The signals may be associated with the plurality of nucleic acid molecules.

[00179] The present disclosure provides methods and systems for sequencing a template nucleic acid molecule. A method for sequencing a template nucleic acid molecule may comprise providing a plurality of nucleic acid molecules immobilized adjacent to a support. Each of the plurality of nucleic acid molecules may comprise a sequence of the template nucleic acid molecule. In a first phase, the plurality of nucleic acid molecules may be brought in contact with fewer than each of four types of nucleotides. In a second phase, the plurality of nucleic acid molecules may be brought in contact with the four types of nucleotides, to obtain sequences of the plurality of nucleic acid molecules. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at most 5 bases with respect to another sequence of the plurality of nucleic acid molecules. [00180] FIG. 17 shows a flowchart for an example method 1700 for sequencing a template nucleic acid molecule. In an operation 1710, the method 1700 may comprise providing a plurality of nucleic acid molecules immobilized adjacent to a support. Each of the plurality of nucleic acid molecules may comprise a sequence of the template nucleic acid molecule. The plurality of nucleic acid molecules may be nucleic acid molecules as described elsewhere herein. The plurality of nucleic acid molecules may be one or more clonal or substantially clonal nucleic acid populations. The plurality of nucleic acid molecules may be target nucleic acid molecules. The immobilization may comprise binding the nucleic acid molecules to a probe (e.g., an oligonucleotide, an antibody). The support may be a support as described elsewhere herein. The support may be a surface of a bead, a planar surface, a well, or the like. The support may position the nucleic acid molecules in sensory communication with one or more sensors. For example, the support can be a bead adjacent to two electrodes. The nucleic acid molecules may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,

60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, or more nucleotides. The nucleic acid molecules may comprise at most about 5,000, 1,000, 900,

800, 700, 600, 500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25,

20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less nucleotides.

[00181] In another operation 1720, the method 1700 may further comprise brining the plurality of nucleic acid molecules in contact with fewer than each of four types of nucleotides.

The contacting may be under conditions sufficient to extend the primers in a template directed synthesis. For example, the contacting may be in the presence of a polymerase. The contacting may comprise introducing the nucleotide and/or other reagents (e.g., polymerases, buffers). The contacting may further comprise a washing operation subsequent to introducing the nucleotide and/or other reagents. A phase may comprise one or more operations of sequentially brining the plurality of nucleic acid molecules into contact with the nucleotides.

[00182] In another operation 1730, the method 1700 may further comprise bringing the plurality of nucleic acid molecules in contact with the four types of nucleotides, to obtain sequences of the plurality of nucleic acid molecules. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, or more bases with respect to another sequence of said plurality of nucleic acid molecules. A sequence of the plurality of nucleic acid molecules may have a phase lag or phase lead of at most about 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer bases with respect to another sequence of the plurality of nucleic acid molecules. Operation 1730 may be subsequent to operation 1720. Alternatively, operation 1730 may be prior to operation 1720. The contacting may be under conditions similar to those of operation 1720. [00183] In an additional operation, the method 1700 may further comprise operation 1740.

Operation 1740 may comprise sequentially bringing the plurality of nucleic acid molecules in contact with up to the four types of nucleotides. A sequential order of nucleic acid molecules in operation 1740 may be different than a sequential order of nucleic acid molecules in operation

1720 and/or operation 1730. The contacting may be under conditions similar to operations 1720 and/or 1730.

[00184] In an additional operation, the method 1700 may further comprise operation 1750. Operation 1750 may comprise sequentially brining the plurality of nucleic acid molecules in contact with up to the four types of nucleotides. A sequential order of nucleic acid molecules in operation 1750 may be different than a sequence of nucleic acid molecules in operation 1720, operation 1730, and/or operation 1740. The bringing into contact may be under conditions similar to operation 1720, 1730, and/or 1740. The method 1700 may further comprise repeating operation 1720, operation 1730, operation 1740, operation 1750, or any combination thereof. The sequencing may comprise sequencing via sequencing-by-synthesis, sequencing-by-ligation, Sanger sequencing, hydrogen ion detection sequencing, polony sequencing, nanopore sequencing, rolling circle sequencing, or the like. The sequencing may comprise measuring one or more signals associated with the sequencing (e.g., the sequencing by synthesis). The signals may be signals as described elsewhere herein (e.g., associated with an impedance, conductivity, charge, or change thereof). The signals may be associated with the plurality of nucleic acid molecules.

Computer systems

[00185] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 7 shows a computer system 701 that is programmed or otherwise configured to implement methods for nucleic acid amplification. The computer system 701 can regulate various aspects of the methods of the present disclosure, such as, for example, implementing synthesis procedures to generate nucleotides that bind to probes. The computer system 701 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. [00186] The computer system 701 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 705, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 701 also includes memory or memory location 710 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 715 (e.g., hard disk), communication interface 720 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 725, such as cache, other memory, data storage and/or electronic display adapters. The memory 710, storage unit 715, interface 720 and peripheral devices 725 are in communication with the CPU 705 through a communication bus (solid lines), such as a motherboard. The storage unit 715 can be a data storage unit (or data repository) for storing data. The computer system 701 can be operatively coupled to a computer network (“network”) 730 with the aid of the communication interface 720.

The network 730 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 730 in some cases is a telecommunication and/or data network. The network 730 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 730, in some cases with the aid of the computer system 701, can implement a peer-to-peer network, which may enable devices coupled to the computer system 701 to behave as a client or a server.

[00187] The CPU 705 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 710. The instructions can be directed to the CPU 705, which can subsequently program or otherwise configure the CPU 705 to implement methods of the present disclosure.

Examples of operations performed by the CPU 705 can include fetch, decode, execute, and writeback.

[00188] The CPU 705 can be part of a circuit, such as an integrated circuit. One or more other components of the system 701 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[00189] The storage unit 715 can store files, such as drivers, libraries and saved programs.

The storage unit 715 can store user data, e.g., user preferences and user programs. The computer system 701 in some cases can include one or more additional data storage units that are external to the computer system 701, such as located on a remote server that is in communication with the computer system 701 through an intranet or the Internet.

[00190] The computer system 701 can communicate with one or more remote computer systems through the network 730. For instance, the computer system 701 can communicate with a remote computer system of a user (e.g., a server system). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 701 via the network 730.

[00191] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 701, such as, for example, on the memory 710 or electronic storage unit 715. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 705. In some cases, the code can be retrieved from the storage unit

715 and stored on the memory 710 for ready access by the processor 705. In some situations, the electronic storage unit 715 can be precluded, and machine-executable instructions are stored on memory 710.

[00192] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre compiled or as-compiled fashion.

[00193] Aspects of the systems and methods provided herein, such as the computer system 701, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[00194] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a

PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00195] The computer system 701 can include or be in communication with an electronic display 735 that comprises a user interface (EΊ) 740 for providing, for example, an interface showing the progress of an amplification operation. Examples of LT’s include, without limitation, a graphical user interface (GET) and web-based user interface.

[00196] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 705. The algorithm can, for example, determine optimal conditions to use for sequencing.

EXAMPLES

[00197] The following examples are illustrative of certain systems and methods described herein and are not intended to be limiting.

EXAMPLE 1 Selective RNA Blocking

[00198] FIG. 3 is an example of sensor selective RNA blocking. An array of sensors is represented by sensors 301, 302, and 303. Sensor 302 is decorated with an array of oligonucleotides 304. The oligonucleotides of array 304 comprise a T7 RNA polymerase promoter sequence 305 and an adhesion sequence 306. Sensors 301 and 303, however, have oligonucleotide arrays 307, where the oligonucleotides comprise the T7 RNA polymerase promoter sequence and a different adhesion sequence 308. Adhesion sequences 306 and 308 are not the same sequence, so RNA that binds to 306 will not bind to 308. A template nucleic acid molecule 309, having the compliment to the T7 RNA polymerase promoter sequence 305 and an adhesion sequence 310 complimentary to sequence 306, is added to the array of sensors. Because adhesion sequence 310 is complimentary to adhesion sequence 306, nucleic acid molecule 309 binds to an oligonucleotide on sensor 302. Lacking an appropriate adhesion layer for sensors 301 and 303, the target nucleic acid molecule 309 does not adhere to those sensors. After the target nucleic acid molecule 309 binds to the oligonucleotide of array 304, T7 RNA polymerase begins producing RNA 311 based on the adhesion sequence 310, which then diffuse out and bind to the other oligonucleotides of array 304. This process is sufficiently fast that other target nucleic acid molecules are unable to bind to the oligonucleotides of array 304, leaving target nucleic acid molecule isolated on sensor 302. RNA 311 does not have a fully complimentary sequence to the oligonucleotides of arrays 307, so no binding occurs.

[00199] In this example, another target nucleic acid molecule 312 is introduced into the sensor array. Because all of the oligonucleotides of array 304 on sensor 302 are bound with RNA 311, the new target nucleic acid molecule binds to the oligonucleotides of array 307 on sensors 301 and 303. The same RNA production process then initiates on sensors 301 and 303, generating RNA 313. RNA 313 is complimentary to the oligonucleotides of array 307, so it binds and excludes other target nucleic acid molecules from binding to sensors 301 or 303. After washing out the excess RNA, the system is left with sensors 301 and 303 having a single bound strand of target nucleic acid molecule 312 surrounded by oligonucleotides blocked with RNA 313. Similarly, sensor 302 has a single molecule of target nucleic acid molecule 309 surrounded by oligonucleotides blocked by RNA 311.

[00200] Adding in RNase H in operation 314, RNA 311 and 313 is digested and removed from the arrays of oligonucleotides, leaving sensors 301 and 303 with target nucleic acid molecule 312 surrounded by open oligonucleotides of arrays 307 and similarly sensor 302 with target nucleic acid molecule 309 surrounded by the open oligonucleotides of array 304. Since the target nucleic acid molecules are isolated, performing an amplification operation 315 generates clonal populations of nucleic acids 316 and 317. Clonal population 316 contains clones of target nucleic acid molecule 312 while clonal population 317 contains clones of target nucleic acid molecule 309. These clonal populations can then be sequenced with greater accuracy due to the lack of contamination of each clonal population by other nucleic acid molecules.

EXAMPLE 2 Run-Off Sequencing

[00201] FIG. 12 shows an example overview of a run-off sequencing process. Nucleic acid molecule 1201 is bound to the support via a probe, which in this case is an oligonucleotide. A first primer 1203 is hybridized to a complementary region of the nucleic acid molecule, which itself is attached to the 5’ end of a first barcode index. The primer serves as an initiation point for a polymerase-based extension reaction, and the extension is combined with a detection of the incorporation of the nucleotides during the reaction to sequence the first barcode index. The extension reaction results in a complimentary strand 1204 hybridized to the nucleic acid molecule. [00202] After the first extension reaction, another primer 1205 is bound to a different sequence of the nucleic acid molecule 1208. In this example, the primer 1205 was flowed in and the solution was slightly heated to anneal the primer to the nucleic acid molecule 1201. Another sequencing-by-synthesis reaction is performed using primer 1205 as the initiation point for the synthesis. This sequencing by synthesis generates a sequence of the insert, which in this case is a nucleic acid molecule with an unknown sequence. The polymerase used to generate the complimentary strand 1206 possesses strand displacement properties, so when the extension reaction comes to primer 1203 and complimentary strand 1204, the polymerase displaces those strands and continues producing complimentary strand 1206. The displacement of the previous primer and complementary strand removes the operation of de-hybridizing those products which in turn decreases the length of time used to perform the sequencing. Additionally, the sequence of the index can be read again, which decreases the uncertainty of the identification of the index.

[00203] The process can then be repeated by annealing primer 1207 to the complimentary portion 1209 of nucleic acid 1201. Performing a sequencing-by-synthesis extension reaction can read the identity of a second index, permitting a greater number of inserts to be read on a given chip, as the number of possible indexes increases significantly with the number of bases contained within the indexes. Adding a second index can enable in excess of 350 different inserts to be sequenced on the same chip, significantly improving the throughput of the process. The sequencing-by-synthesis of the second index can continue in order to provide a second read of the insert, again improving performance by decreasing error in the read.

EXAMPLE 3 Blocking Sequencing

[00204] FIG. 13 shows an example overview of a blocking sequencing process. The nucleic acid 1301 comprises a sequence 1302 that is complimentary to primer 1303. The primer is configured to be the initiation point for a polymerase to initiate a chain elongation reaction. A first elongation reaction is performed, which generates a complementary strand 1304, which is complimentary to a known sequence of nucleotides within nucleic acid 1301. The generation of the complementary strand is accompanied by a sequencing-by-synthesis read of the bases that incorporate into the complimentary strand. For example, optical measurements of fluorescent labels attached to the bases can be taken as they incorporate. In another example, measurements of the local electronic environment can be taken to determine when an incorporation event occurs. After the index is sequenced, dideoxy nucleotides are added to the reaction mixture, which terminates the complimentary strand 1304 and makes it unable to be further extended.

This is done in order to prevent future extension reactions from further elongating the strand and inducing error into later measurements. Further, by allowing the complimentary strand to stay bound, it removes removal operations that in turn decrease the time and reagents used to perform the sequencing.

[00205] After the first extension reaction is complete and the complimentary strand 1304 has been blocked, another primer 1306 is hybridized to a complimentary region 1305 of nucleic acid molecule 1301. Similarly to how complimentary strand 1304 was generated, primer 1306 serves as the initiation location of the generation of complimentary strand 1307. The sequencing-by synthesis reaction that generates complimentary strand 1307 provides a sequence for the unknown insert sequence. After the strand complimentary to the insert has been generated, it too is terminated with a dideoxy nucleotide to prevent further extension reactions from occurring. Then, a third primer 1309 is bound to a third complimentary region 1308, and the second index of the nucleic acid molecule 1301 is sequenced. Though shown in this order, the sequencing of the first index, the second index, and the insert can be done in any order.

EXAMPLE 4 Melt off Sequencing

[00206] FIG. 14 shows an example overview of a melt off sequencing process. Nucleic acid 1401 comprises a first sequence 1402 that is complimentary to a primer 1403. The primer is hybridized to the sequence, and a chain elongation reaction starting at the primer generates complimentary strand 1404. The generation of the complimentary strand is part of a sequencing- by-synthesis process that identifies index 1. After the sequencing by synthesis is performed, the entire complimentary strand comprising primer 1403 and complimentary strand 1404 is melted off (dehybridized) from nucleic acid molecule 1401. This melt off can be performed using conditions including ionic strength changes, adding denaturants, and the like. After the first melt off operation, a second primer 1405 is introduced to the area around nucleic acid molecule 1401, and it is annealed to hybridize to a complimentary sequence 1409. Another sequencing by synthesis is performed to sequence a second index, which results in a second complimentary strand 1406. Upon completion, primer 1405 and complimentary strand 1406 are melted off of nucleic acid molecule 1401 and removed. Subsequently, a third primer is introduced and hybridized to a complementary portion 1410 of nucleic acid molecule 1401. Another sequencing by synthesis reaction is performed to sequence the insert, which generates complimentary strand 1408. Though shown in a particular order in this example, the sequencing of the first index, the second index, and the insert can be performed in any order.

EXAMPLE 5 Efficient Incorporation of Nucleotide Bases

[00207] During the sequencing-by-synthesis methods as described elsewhere herein, nucleotide bases are flowed in to provide nucleotides for the synthesis of one or more nucleic acid molecules. A target nucleic acid sequence is clonally amplified to produce a clonal population of target nucleic acid molecules that all share the target nucleic acid sequence

(“amplicons”). Nucleic acid molecules complementary to the amplicons are synthesized to sequence the amplicons and identify the target nucleic acid sequence. The nucleotides are flowed into contact with the amplicons in a particular nucleotide flow order as to starve the amplicons of a specific nucleotide type. An example of a nucleotide flow order that starve the amplicons of specific nucleotide types is (1) ACGAGCACG (2) TCTGCGT (3) AGATGTA and (4) CTCAT.

In phase 1, the nucleotide base thymine (T) is excluded from being introduced, and instead the other three bases (A, C, and G) are repeatedly introduced. As such, the extension reaction occurring at each of the target nucleic acid molecules that are being sequenced stops at the next thymine in the sequence. Because of this, any amplicon that is out of phase, with respect to other amplicons, has the opportunity to catch up to the common phase. For example, for a sequence

GACT, all of the synthesis reactions occurring at the amplicons halt at the C before the T. In this example, an amplicon that was lagging by one base has the opportunity to catch up to the rest of the nucleic acid molecules by incorporating a C during one of the nucleotide flows before T is introduced.

[00208] In phase 2, instead of thymine being removed from the nucleotides that are introduced, adenine is not flowed into contact with the nucleic acid molecules. As such, all of the extension reactions that were previously stopped at thymine continue until an adenine in the amplicon is reached. Once the adenine is reached, the synthesis reactions stop again, and the amplicons will catch up to the next occurrence of adenine in the sequence. Similarly, phases 3 and 4 remove C and G, respectively, from the nucleotides that are brought into contact with the amplicons. In doing so, the phase lag or phase lead is reduced to zero after each of phases 1, 2, 3, and 4. By removing the phase lag or lead, the number of in phase, signal generating nucleic acid molecules increase, which increases signal and decreases noise. The flow order can then be repeated, going from phase 4 back to phase 1, until the sequencing-by-synthesis is completed. [00209] The order of nucleotides to be excluded does not impact the efficacy of the nucleotide incorporation. For example, the first nucleotide to be removed from the flow can be A instead of T. Other non-limiting examples of flow orders include ACGAGCAACG,TTCTGCGT, AGATGTA, CTCAT; ACGGAAGCACG, TTCCTGCGT, AAGGTAGTA, CCTTCAT; ACGGAGCACG, TTCTGCGT, AAGTAGTA, CCTCAT; and ACGGAGCAACG, TTCTGCCGT, AAGTAGGTA, CCTCATT. Each different flow order can have a different balance of effectiveness and speed. For example, a flow order with more repeats without a particular base can increase the likelihood that all of the phasing errors have been removed, but this flow order can also increase the time taken to perform the sequencing. [00210] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for nucleic acid amplification, comprising:

(a) bringing a template nucleic acid molecule in contact with an array of oligonucleotides, wherein said template nucleic acid molecule binds to an oligonucleotide of said array of oligonucleotides;

(b) using said template nucleic acid molecule to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other oligonucleotides of said array of oligonucleotides;

(c) binding nucleic acid molecules of said plurality of nucleic acid molecules to said other oligonucleotides of said array of oligonucleotides, thereby generating occupied oligonucleotides;

(d) removing at least a portion of said nucleic acid molecules of said plurality of nucleic acid molecules from said occupied oligonucleotides, thereby generating active oligonucleotides; and

(e) using said template nucleic acid molecule and said active oligonucleotides to amplify said template nucleic acid molecule, thereby generating amplicons coupled to said active oligonucleotides.

2. The method of claim 1, wherein (b) and (c) occur contemporaneously.

3. The method of claim 1, wherein (b) and (c) occur consecutively.

4. The method of claim 1, wherein said other oligonucleotides of said array of oligonucleotides comprise a common sequence.

5. The method of claim 4, wherein said plurality of nucleic acid molecules are at least partially complementary to said common sequence.

6. The method of claim 4, wherein oligonucleotides of said array of oligonucleotides are identical.

7. The method of claim 1, wherein said plurality of nucleic acid molecules is a plurality of ribonucleic acid (RNA) molecules.

8. The method of claim 7, wherein (b) is performed with the aid of an RNA polymerase.

9. The method of claim 8, wherein said RNA polymerase is T7 RNA polymerase.

10. The method of claim 1, wherein (d) comprises removing at least a portion of said nucleic acid molecules of said plurality of nucleic acid molecules from said occupied oligonucleotides with use of an enzyme.

11. The method of claim 10, wherein said enzyme is an RNase.

12. The method of claim 11, wherein said RNase is RNase H.

13. The method of claim 1, wherein (b) is performed when said template nucleic acid molecule is bound to said oligonucleotide.

14. The method of claim 13, wherein, after (b), said nucleic acid molecules of said plurality of nucleic acid molecules are transported from said oligonucleotide to said other oligonucleotides of said array of oligonucleotides.

15. The method of claim 14, wherein, after (b), said nucleic acid molecules of said plurality of nucleic acid molecules are transported from said oligonucleotide to said other oligonucleotides of said array of oligonucleotides via diffusion.

16. The method of claim 1, wherein said template nucleic acid molecule comprises a promoter sequence.

17. The method of claim 16, wherein said oligonucleotides comprises a complementary promoter sequence complementary to said promoter sequence.

18. The method of claim 16, wherein said promoter sequence is a T7 ribonucleic acid (RNA) polymerase promoter sequence.

19. The method of claim 1, wherein (b) is performed by binding of said template nucleic acid molecule to at least two oligonucleotides of said array of oligonucleotides.

20. The method of claim 1, wherein said array of oligonucleotides is attached to a solid support.

21. The method of claim 20, wherein said solid support is a bead.

22. The method of claim 20, wherein said solid support is planar.

23. The method of claim 20, wherein said solid support is a surface of a well.

24. The method of claim 1, wherein said array of oligonucleotides is in sensory communication with a sensor.

25. The method of claim 24, wherein said sensor comprises an electrode.

26. The method of claim 25, wherein said sensor comprises a plurality of electrodes.

27. The method of claim 24, wherein said sensor is among an array of sensors.

28. The method of claim 27, wherein at least one sensor of said array of sensors is individually addressable.

29. The method of claim 1, wherein said array of oligonucleotides is among a plurality of arrays of oligonucleotides.

30. The method of claim 29, further comprising, after (b), excluding said plurality of nucleic acid molecules from other arrays of said plurality of arrays of oligonucleotides.

31. The method of claim 30, wherein said excluding comprises applying an electric field to said plurality of nucleic acid molecules.

32. The method of claim 31, wherein at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said electric field.

33. The method of claim 32, wherein said label is a particle.

34. The method of claim 30, wherein said excluding comprises applying a magnetic field to said plurality of nucleic acid molecules.

35. The method of claim 34, wherein at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said magnetic field.

36. The method of claim 35, wherein said label is a particle.

37. The method of claim 30, wherein said excluding is performed with the aid of a diffusion barrier.

38. The method of claim 30, wherein said excluding is performed by degrading a subset of said plurality of nucleic acid molecules.

39. The method of claim 38, wherein said degrading is performed with an enzyme.

40. The method of claim 39, wherein said enzyme is an RNase.

41. The method of claim 39, wherein said enzyme is coupled to a support.

42. The method of claim 41, wherein said support is a particle.

43. The method of claim 41, further comprising apply an electric field to said support.

44. The method of claim 29, wherein said array of oligonucleotides comprises oligonucleotides having sequences different from oligonucleotides of at least one other array of said plurality of arrays of oligonucleotides.

45. The method of claim 29, further comprising repeating (a) - (e) at least one other array of said plurality of arrays of oligonucleotides.

46. The method of claim 1, wherein (e) comprises conducting a reaction with aid of a recombinase.

47. The method of claim 1, wherein (e) comprises conducting a reaction with aid of a polymerase.

48. The method of claim 1, wherein said amplicons coupled to said active oligonucleotides are a clonal population of nucleic acids.

49. The method of claim 1, further comprising sequencing at least a subset of said amplicons coupled to said active oligonucleotides or derivatives thereof.

50. The method of claim 49, wherein said sequencing is completed via sequencing-by synthesis.

51. The method of claim 49, wherein said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

52. A method for processing a template nucleic acid molecule, comprising:

(a) providing a template nucleic acid molecule coupled to an oligonucleotide of an array of oligonucleotides, wherein other oligonucleotides of said array of oligonucleotides are blocked such that other template nucleic acid molecules are incapable of stably coupling to said other oligonucleotides;

(b) deblocking at least a subset of said other oligonucleotides; and

(c) using said template nucleic acid molecule and said active oligonucleotides to amplify said template nucleic acid molecule, thereby generating amplicons coupled to said active oligonucleotides.

53. The method of claim 52, wherein said other oligonucleotides of said array of oligonucleotides are blocked with nucleic acid molecules bound to said other oligonucleotides of said array of oligonucleotides.

54. The method of claim 53, wherein said nucleic acid molecules are ribonucleic acid (RNA) molecules.

55. The method of claim 52, wherein oligonucleotides of said array of oligonucleotides are coupled to a support.

56. The method of claim 55, wherein said support is a bead.

57. The method of claim 55, wherein said support is planar.

58. The method of claim 52, wherein, in (b), said deblocking is performed with the aid of an enzyme.

59. The method of claim 58, wherein said enzyme is an RNase.

60. The method of claim 52, wherein (b),(c), or both occurs in a well.

61. The method of claim 52, further comprising applying an electric field to said array of oligonucleotides.

62. The method of claim 52, further comprising applying a magnetic field to said array of oligonucleotides.

63. The method of claim 52, wherein said amplicons coupled to said active oligonucleotides are a clonal population of nucleic acids.

64. The method of claim 52, further comprising sequencing at least a subset of said amplicons coupled to said active oligonucleotides or derivatives thereof.

65. The method of claim 64, wherein said sequencing is completed via sequencing-by synthesis.

66. The method of claim 64, wherein said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

67. A method for nucleic acid amplification, comprising:

(a) bringing a plurality of target nucleic acid molecules in contact with an array of oligonucleotides, wherein said plurality of target nucleic molecules is present at a concentration such that most a target nucleic acid molecule of said plurality of target nucleic acid molecules hybridizes to an oligonucleotide of said array of oligonucleotides;

(b) subjecting said array of oligonucleotides to conditions sufficient to synthesize a first plurality of nucleic acid molecules from said target nucleic acid molecule hybridized to said oligonucleotide, wherein said first plurality of nucleic acid molecules is hybridized to other oligonucleotides of said array of oligonucleotides;

(c) subjecting said array of oligonucleotides to conditions sufficient to remove or degrade at least a subset of said first plurality of nucleic acid molecules; and

(d) subsequent to (c), subjecting said array of oligonucleotides to conditions sufficient to amplify said target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to said array of oligonucleotides.

68. The method of claim 67, wherein said oligonucleotides of said array of oligonucleotides comprise a common sequence.

69. The method of claim 68, wherein said plurality of nucleic acid molecules are at least partially complementary to said common sequence.

70. The method of claim 68, wherein said oligonucleotides of said array of oligonucleotides are identical.

71. The method of claim 67, wherein said first plurality of nucleic acid molecules is a plurality of ribonucleic acid (RNA) molecules.

72. The method of claim 71, wherein (b) is performed with the aid of an RNA polymerase.

73. The method of claim 72, wherein said RNA polymerase is T7 RNA polymerase.

74. The method of claim 67, wherein (c) comprises removing or degrading said subset of said nucleic acid molecules with an enzyme.

75. The method of claim 74, wherein said enzyme is an RNase.

76. The method of claim 75, wherein said RNase is RNase H.

77. The method of claim 67, wherein (b) further comprises transporting a subset of said first plurality of nucleic acid molecules to said other oligonucleotides of said array of oligonucleotides.

78. The method of claim 77, wherein (b) further comprises transporting a subset of said first plurality of nucleic acid molecules to said other oligonucleotides of said array of oligonucleotides via diffusion.

79. The method of claim 67, wherein said target nucleic acid molecule hybridized to said oligonucleotide comprises a promoter sequence.

80. The method of claim 79, wherein said oligonucleotides of said array of oligonucleotides comprise a complementary promoter sequence complementary to said promoter sequence.

81. The method of claim 79, wherein said promoter sequence is a T7 ribonucleic acid (RNA) polymerase promoter sequence.

82. The method of claim 67, wherein said array of oligonucleotides is attached to a solid support.

83. The method of claim 82, wherein said solid support is a bead.

84. The method of claim 82, wherein said solid support is planar.

85. The method of claim 82, wherein said solid support is a surface of a well.

86. The method of claim 67, wherein said array of oligonucleotides is in sensory communication with a sensor.

87. The method of claim 86, wherein said sensor comprises an electrode.

88. The method of claim 87, wherein said sensor comprises a plurality of electrodes.

89. The method of claim 86, wherein said sensor is among an array of sensors.

90. The method of claim 89, wherein at least one sensor of said array of sensors is individually addressable.

91. The method of claim 67, wherein said array of oligonucleotides is among a plurality of arrays of oligonucleotides.

92. The method of claim 91, wherein (b) further comprises excluding said plurality of nucleic acid molecules from other arrays of said plurality of arrays of oligonucleotides.

93. The method of claim 92, wherein said excluding comprises applying an electric field to said plurality of nucleic acid molecules.

94. The method of claim 93, wherein at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said electric field.

95. The method of claim 94, wherein said label is a particle.

96. The method of claim 92, wherein said excluding comprises applying a magnetic field to said plurality of nucleic acid molecules.

97. The method of claim 96, wherein at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a label that interacts with said magnetic field.

98. The method of claim 97, wherein said label is a particle.

99. The method of claim 92, wherein said excluding is performed with the aid of a diffusion barrier.

100. The method of claim 92, wherein said excluding is performed by degrading a subset of said plurality of nucleic acid molecules.

101. The method of claim 100, wherein said degrading is performed with an enzyme.

102. The method of claim 101, wherein said enzyme is an RNase.

103. The method of claim 101, wherein said enzyme is coupled to a support.

104. The method of claim 103, wherein said support is a particle.

105. The method of claim 103, further comprising apply an electric field to said support.

106. The method of claim 91, wherein said array of oligonucleotides comprises oligonucleotides having sequences different from oligonucleotides of at least one other array of said plurality of arrays of oligonucleotides.

107. The method of claim 91, further comprising repeating (a) - (d) at another array of said plurality of arrays of oligonucleotides.

108. The method of claim 67, wherein (d) comprises conducting a reaction with aid of a recombinase.

109. The method of claim 67, wherein (d) comprises conducting a reaction with aid of a polymerase.

110. The method of claim 67, wherein the method further comprises (e) sequencing at least a subset of said second plurality of nucleic acid molecules hybridized to said array of oligonucleotides.

111. The method of claim 107, further comprising sequencing at least a subset of said substantially clonal populations at said another array of said plurality of arrays of oligonucleotides.

112. The method of claim 110, wherein said sequencing is completed via sequencing-by synthesis.

113. The method of claim 110, wherein said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

114. A method for sequencing a nucleic acid molecule, comprising:

(a) providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence, a second sequence and a third sequence, wherein said nucleic acid molecule comprises a first primer hybridized to said third sequence;

(b) subjecting said third sequence to sequencing to generate a first sequencing read comprising at least a portion of said third sequence; (c) bringing a second primer having a sequence complementarity with said second sequence in contact with said nucleic acid molecule under conditions sufficient for said second primer to hybridize to said second sequence, and subjecting said second sequence to sequencing to generate a second sequencing read comprising at least a portion of said second sequence; and

(d) bringing a third primer having a sequence complementarity with said first sequence in contact with said nucleic acid molecule under conditions sufficient for said third primer to hybridize to said first sequence, and subjecting said first sequence to sequencing to generate a third sequencing read comprising at least a portion of said first sequence.

115. The method of claim 114, wherein said sequencing of said first sequence, second sequence, third sequence, or any combination thereof comprises use of a polymerizing enzyme.

116. The method of claim 115, wherein said polymerizing enzyme comprises strand displacement activity.

117. The method of claim 116, wherein said second sequencing read displaces said first sequencing read.

118. The method of claim 117, wherein said third sequencing read displaces said second sequencing read.

119. The method of claim 114, wherein said sequencing of said first sequence, third sequence, or both, generates an identification tag for said second sequence.

120. The method of claim 114, wherein said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof.

121. The method of claim 114, further comprising, prior to (a), coupling said first sequence, said third sequence, or both to said second sequence.

122. The method of claim 121, wherein said first sequence or said third sequence is coupled to said second sequence via ligation.

123. The method of claim 121, wherein said first sequence or said third sequence is coupled to said second sequence via hybridization.

124. The method of claim 114, wherein said nucleic acid molecule is coupled to said support via a probe coupled to said support.

125. The method of claim 124, wherein said probe comprises an oligonucleotide.

126. The method of claim 114, wherein said support is a bead.

127. The method of claim 114, wherein said support is planar.

128. The method of claim 114, wherein said support is a surface of a well.

129. The method of claim 114, wherein said probe is in sensory communication with a sensor.

130. The method of claim 129, wherein said sensor comprises an electrode.

131. The method of claim 129, wherein said sensor comprises a plurality of electrodes.

132. The method of claim 114, wherein said sequencing is completed via sequencing-by synthesis.

133. The method of claim 132, wherein said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

134. A method for processing a nucleic acid molecule, comprising:

(a) providing said nucleic acid molecule coupled to a support at a 3’ end of said nucleic acid molecule, which nucleic acid molecule comprises, from a 5’ end to a 3’ end, a first sequence and a second sequence;

(b) subjecting said nucleic acid molecule to a first extension reaction to generate a first strand complementary to said first sequence, wherein a 5’ end of said first strand comprises a blocking group; and

(c) subjecting said nucleic acid molecule to a second extension reaction to generate a second strand complementary to said second sequence, wherein a 5’ end of said second strand comprises an additional blocking group.

135. The method of claim 134, wherein (c) is performed subsequent to (b).

136. The method of claim 134, wherein said nucleic acid molecule further comprises a third sequence.

137. The method of claim 136, further comprising subjecting said nucleic acid molecule to a third extension reaction to generate a third strand complementary to said third sequence.

138. The method of claim 134, wherein said sequencing of said first sequence generates an identification tag for said second sequence.

139. The method of claim 136, wherein said sequencing of said first sequence, third sequence, or both, generates an identification tag for said second sequence.

140. The method of claim 134, wherein said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof.

141. The method of claim 136, further comprising, prior to (a), coupling said first sequence, said third sequence, or both to said second sequence.

142. The method of claim 141, wherein said first sequence or third sequence is coupled to said second sequence via ligation.

143. The method of claim 141, wherein said first sequence or third sequence is coupled to said second sequence via hybridization.

144. The method of claim 134, wherein said nucleic acid molecule is coupled to said support via a probe coupled to said support.

145. The method of claim 144, wherein said probe comprises an oligonucleotide.

146. The method of claim 134, wherein said support is a bead.

147. The method of claim 134, wherein said support is planar.

148. The method of claim 134, wherein said support is a surface of a well.

149. The method of claim 144, wherein said probe is in sensory communication with a sensor.

150. The method of claim 149, wherein said sensor comprises an electrode.

151. The method of claim 149, wherein said sensor comprises a plurality of electrodes.

152. The method of claim 134, wherein said sequencing is completed via sequencing-by synthesis.

153. The method of claim 151, wherein said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

154. The method of claim 134, wherein said blocking group comprises one or more biologic molecules.

155. The method of claim 154, wherein said one or more biologic molecules comprise one or more nucleotides, one or more enzymes, or both.

156. The method of claim 134, wherein said blocking group comprises one or more metals.

157. A method for processing a nucleic acid molecule, comprising:

(b) subjecting said third sequence to sequencing to generate a first non-optical sequencing read comprising at least a portion of said third sequence;

(c) bringing a second primer having sequence complementarity with said second sequence in contact with said nucleic acid molecule under conditions sufficient for said second primer to hybridize to said second sequence, and subjecting said second sequence to non-optical sequencing to generate a second sequencing read comprising at least a portion of said second sequence; and

(d) bringing a third primer having sequence complementarity with said first sequence in contact with said nucleic acid molecule under conditions sufficient for said third primer to hybridize to said first sequence, and subjecting said first sequence to non-optical sequencing to generate a third sequencing read comprising at least a portion of said first sequence.

158. The method of claim 157, wherein (b), (c), and (d) are performed in any order of sequence.

159. The method of claim 157, wherein said sequencing of said first sequence, third sequence, or both, generates an identification tag for said second sequence.

160. The method of claim 157, wherein said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a derivative thereof.

161. The method of claim 157, further comprising, prior to (a), coupling said first sequence, said third sequence, or both to said second sequence.

162. The method of claim 161, wherein said first sequence or third sequence is coupled to said second sequence via ligation.

163. The method of claim 162, wherein said first sequence or third sequence is coupled to said second sequence via hybridization.

164. The method of claim 157, wherein said nucleic acid molecule is coupled to said support via a probe coupled to said support.

165. The method of claim 164, wherein said probe comprises an oligonucleotide.

166. The method of claim 157, wherein said support is a bead.

167. The method of claim 157, wherein said support is planar.

168. The method of claim 157, wherein said support is a surface of a well.

169. The method of claim 164, wherein said probe is in sensory communication with a sensor.

170. The method of claim 169, wherein said sensor comprises an electrode.

171. The method of claim 169, wherein said sensor comprises a plurality of electrodes.

172. The method of claim 157, wherein said sequencing is completed via sequencing-by synthesis.

173. The method of claim 172, wherein said sequencing is performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, or change in charge.

174. The method of claim 157, further comprising, between any combination of (b), (c), and (d), performing an annealing operation.

175. The method of claim 174, wherein said annealing operation is a thermal annealing operation.

176. A method for sequencing a template nucleic acid molecule, comprising:

(a) providing a plurality of nucleic acid molecules immobilized adjacent to a support, wherein each of said plurality of nucleic acid molecules comprises a sequence of said template nucleic acid molecule;

(b) in a first phase, sequentially bringing said plurality of nucleic acid molecules in contact with nucleotides of one or more types that are fewer than four types of nucleotides and detecting a first set of signals from said plurality of nucleic acid molecules; and (c) in a second phase subsequent to said first phase, sequentially bringing said plurality of nucleic acid molecules in contact with up to said four types of nucleotides and detecting a second set of signals from said plurality of nucleic acid molecules, to obtain sequences of said plurality of nucleic acid molecules, wherein a sequential order of nucleotides in said first phase is different than a sequential order of nucleotides in said second phase, wherein a sequence of said plurality of nucleic acid molecules has a phase lag or phase lead of at most 5 bases with respect to another sequence of said plurality of nucleic acid molecules.

177. The method of claim 176, further comprising (d) in a third phase, sequentially bringing said plurality of nucleic acid molecules in contact with up to said four types of nucleotides, wherein a sequential order of nucleotides in said third phase is different than a sequential order of nucleotides in said first phase and said second phase.

178. The method of claim 177, further comprising (e) in a fourth phase, sequentially bringing said plurality of nucleic acid molecules in contact with up to said four types of nucleotides, wherein a sequential order of nucleotides in said fourth phase is different than a sequential order of nucleotides in said first phase, said second phase, and said third phase.

179. The method of claim 178, further comprising (f) repeating (b), (c), (d), (e), or any combination thereof.

180. The method of claim 176, wherein said phase lag or phase lead is at most 4 bases.

181. The method of claim 180, wherein said phase lag or phase lead is at most 3 bases.

182. The method of claim 181, wherein said phase lag or phase lead is at most 2 bases.

183. The method of claim 182, wherein said phase lag or phase lead is at most 1 base.

184. The method of claim 176, wherein said first set of signals or said second set of signals are associated with an impedance, conductivity, charge, or change thereof, associated with said plurality of nucleic acid molecules.

185. A method of performing a stepwise extension of a plurality of primers hybridized to a plurality of nucleic acid molecules as part of a clonal population, comprising:

(a) contacting, in a first phase, said clonal population with each of four types of nucleotides under conditions sufficient to extend said primers in a template directed synthesis; and

(b) contacting, in a second phase, said clonal population with fewer than each of four types of nucleotides.

186. The method of claim 185, further comprising (c) contacting, in a third phase, said clonal population with fewer than each of four types of nucleotides wherein a sequential order of nucleotides in said third phase is different than a sequential order of nucleotides in said first phase and said second phase.

187. The method of claim 186, further comprising (d) contacting, in a fourth phase, said clonal population with fewer than each of four types of nucleotides wherein a sequential order of nucleotides in said fourth phase is different than a sequential order of nucleotides in said first phase, said second phase, and said third phase.

188. The method of claim 187, further comprising (e) repeating (b), (c), (d), or any combination thereof.

189. The method of claim 185, further comprising detecting signals from said plurality of nucleic acid molecules to generate a plurality of sequences of said plurality of nucleic acid molecules.

190. The method of claim 185, wherein a sequence of said plurality of nucleic acid molecules has a phase lag or phase lead of at most 5 bases with respect to another sequence of said plurality of nucleic acid molecules.

191. The method of claim 190, wherein said phase lag or phase lead is at most 4 bases.

192. The method of claim 191, wherein said phase lag or phase lead is at most 3 bases.

193. The method of claim 192, wherein said phase lag or phase lead is at most 2 bases.

194. The method of claim 193, wherein said phase lag or phase lead is at most 1 base.

195. The method of claim 185, wherein a sequential order of nucleotides in said first phase is different than a sequential order of nucleotides in said second phase.

196. The method of claim 185, wherein said sequencing comprises sequencing via sequencing- by-synthesis.

197. The method of claim 196, wherein said sequencing comprises measuring one or more signals associated with sequencing-by-synthesis.

198. The method of claim 197, wherein said signals associated with an impedance, conductivity, charge, or change thereof, associated with said plurality of nucleic acid molecules.

199. A method for sequencing a template nucleic acid molecule, comprising:

(b) in a first phase, bringing said plurality of nucleic acid molecules in contact with fewer than each of four types of nucleotides; and

(c) in a second phase, bringing said plurality of nucleic acid molecules in contact with said four types of nucleotides, to obtain sequences of said plurality of nucleic acid molecules, wherein a sequence of said plurality of nucleic acid molecules has a phase lag or phase lead of at most 5 bases with respect to another sequence of said plurality of nucleic acid molecules.

200. The method of claim 199, wherein said second phase is subsequent to said first phase.

201. The method of claim 199, wherein said second phase is prior to said first phase.

202. The method of claim 199, further comprising (d) in a third phase, bringing said plurality of nucleic acid molecules in contact with up to said four types of nucleotides, wherein a sequential order of nucleic acid molecules in said third phase is different than a sequential order of nucleic acid molecules in said first phase and said second phase.

203. The method of claim 202, further comprising (e) in a fourth phase, bringing said plurality of nucleic acid molecules in contact with up to said four types of nucleotides, wherein a sequential order of nucleic acid molecules in said fourth phase is different than a sequential order of nucleic acid molecules in said first phase, said second phase, and said third phase.

204. The method of claim 203, further comprising (f) repeating (b), (c), (d), (e), or any combination thereof.

205. The method of claim 199, wherein said phase lag or phase lead is at most 4 bases.

206. The method of claim 205, wherein said phase lag or phase lead is at most 3 bases.

207. The method of claim 206, wherein said phase lag or phase lead is at most 2 bases.

208. The method of claim 207, wherein said phase lag or phase lead is at most 1 base.

209. The method of claim 199, wherein said obtaining sequences comprises sequencing via sequencing-by-synthesis.

210. The method of claim 209, wherein said obtaining sequences comprises measuring one or more signals associated with sequencing-by-synthesis.

211. The method of claim 210, wherein said signals are associated with an impedance, conductivity, charge, or change thereof, associated with said plurality of nucleic acid molecules.